Orianne Sara

Laure Lea

Gus Arthur

Joao Chiara

Norus Ben

Lena

Caitlin Victor

Alyona, Francis, Herve, Stephanie, los muchachos Eva

and Bearded during furrow invagination in Drosophila

First of all I would like to thank Francois for all these years helping me to ask the right questions, always one at a time. I really felt that, for you, it was more important to teach me the subtleties of scientific reasoning, than the results I could produce. And for that, I am deeply, deeply grateful... Then, I would like to acknowledge all of the other people with whom I worked: Vanessa, with one 'n' and two 's'. It was pure pleasure to work with you. I couldn't find a word in english to describe how much you complained each time an experiment failed, but I saw it as a sign of involvement! Khallil, for your 'magic hands' for molecular biology and your ability to talk and understand my nonsense, a little bit everywhere, a little bit all the time. Giulia, the Italian student. I'm still somehow shocked that you were scared of me and thought that I was strict. My two collaborators, Vito and Francis, who are experts in completely different subjects, and yet share the same uncontainable enthusiasm. I don't know what would happen if you ever worked together. A lot of enthusiasm in the air. And Chloe, the soul and heart of the morphogenesis club, and an incredible coffee partner.

I will also like to express my gratitude to the other members of the lab: Mateusz, there were so many good moments. The beer, any live sport, the dubious scientific discussions. I'm sure we discovered the third ligand of Notch, several times. Lydie, always smiling, always stressing. Such a weird combination. I cannot believe I went to yoga with you several times, such a waste of time. Elodie, the Illustrator teacher (at least the figures look professional) and always an incredible guest for super-organised 'aperos'. I'd like to state that your cat made me hate cats. Maria, so full of dutchcaribbean attitude, and always willing to help.

Preface

Epithelial cells serve many biological functions; they act as a barrier to separate the interior from the exterior, and form a mechanical continuum through the junctions that interconnect them. During development, they undergo dramatic changes to shape the embryo; they change their shape, modify their relative position or lose their epithelial integrity. Most of these changes rely on the regulation of the proteins that define and maintain the epithelial apical/basal polarity, and on the modulation of cortical and junctional actomyosin. Thus, understanding how epithelial apical/basal polarity is established and maintained and how epithelial tissues are remodeled by the modulation of actomyosin, is essential for understanding embryogenesis.

The introduction of this PhD is subdivided into three chapters: the first, describes how epithelial apical/basal polarity is established, maintained and lost in Drosophila; the second, presents gastrulation as an example of epithelial remodeling involving the modulation of actomyosin contractility; and the third chapter introduces the E3 ubiquitin ligase Neuralized as a regulator of epithelial apical/basal polarity and of epithelial remodeling.

Chapter 1

Epithelial Polarity 1.1 The establishment of epithelial polarity in Drosophila [1] The drosophila epithelia can be distinguished as primary or secondary epithelia by the way in which they are established. The primary epithelia arise from of a blastodermal stage in a process termed 'cellularization' (described below in detail). It encompasses all of the cells derived from the ectoderm, such as the embryonic and larval epidermis. Secondary epithelia, on the other hand, are formed after a mesenchymal to epithelial transition and include tissues derived from the endoderm, such as the intestine and the heart.

This section describes how primary epithelia polarity is established during cellularization and how apical polarity can be lost in a primary epithelia to allow the mesenchymal to epithelial transition required for the formation of secondary epithelia.

Cellularization

In drosophila, fecundation of the oocyte triggers a series of 14 synchronous divisions without cytokinesis. Therefore, at early stages of development the embryo is a single cell containing 5000 nuclei at the vicinity of the membrane and sharing the same cytoplasm. This is called a syncitial blastoderm and is schematised in Figure 1.1a. Cellularization describes the process by which membrane furrows will appear around each of the nuclei and grow deeper until single cells are formed (Figure 1.1).

At the onset of cellularization, the membrane shows an asymmetric configuration with an actin-rich cap on top of the nuclei that forms microvilli and a flat membrane in between the nuclei containing a series of proteins, such as E-cadherin, Par3/Baz, Patj ,Dlg and Septins [2], [3] [4] (see Figure 1.1a). A contractile ring of actomyosin forms in the internuclear domain by the stabilisation of a myosin flow in a dunk dependent manner [5]. Then, the furrow canal deepens progressively as the cellular membrane stored in the microvilli is actively transported and added into the furrow [6] (Figure 1.1 c and c'), and as myosin is recruited into the furrow in a slam dependent manner [5].

During the early stages of furrow formation a basal Cadherin-Catenin complex (shown in Figure 1.1 b and b') is stabilised in a nullo dependent manner [7]. As the canal deepens, more Cadherin-Catenin clusters and Par3/Baz proteins are recruited to the lateral membrane. It is important to note that at this stage, the localisation of Cadherin and Par3/Baz are not interdependent and embryos lacking either of these proteins are still able to localise the other [8].

Progressively, in a Par3/Baz dependent manner, the different Cadherin-Catenin junctions recruited to the lateral membrane and termed spot junctions, coalesce to form a single junction at the adherens domain, known as the zonula adherens. [3], [9], [10]

Junctional proteins

The zonula adherens is a type of adherens junction that mechanically connects cells by forming a rigid ring-like structure below the apical domain. It is composed of electrondense plaques of actin filaments connected by E-cadherin (E-cad) proteins [START_REF] Harris | Adherens junctions: from molecules to morphogenesis[END_REF]. Ecadherin is a transmembrane protein, which is highly conserved during evolution. It can create homophilic interactions, in a calcium-dependent manner, through the interaction of its cadherin domains. Its intracellular domain binds to a set of cytoplasmic proteins, such as p120 and β-Catenin/ Armadillo, to establish a complex signaling centre that feeds back to the cytoskeleton and to other proteins that define and maintain epithelial polarity [START_REF] Baum | Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. en[END_REF] (see Figure 1.2. The formation of an adherens junction is directed by the recruitment of Cadherin-Catenin complexes by the scaffolding protein Par3/Baz [10]. Baz directly binds to the junctional protein β-Catenin/Armadillo, through its PDZ domain, and regulates the position of the adherens junctions. This is exemplified in the formation of the dorsal fold, where a basal shift of Par3/Baz is sufficient to shift the position of the adherens junctions and create a fold [START_REF] Wang | Differential positioning of adherens junctions is associated with initiation of epithelial folding[END_REF].

In the Drosophila embryo, Par3/Baz is the initial cue that establishes apical polarity, by driving the formation of the zonula adherens [9] and by directing the establishment of a set of membrane specific protein-scaffolds composed of proteins termed the polarity proteins.

Overview of the different players of epithelial polarity

The different polarity proteins are highly conserved among Metazoa. They can be divided by their cellular localisation into two groups: the members of the apical domain and the members of the baso/lateral domain. They include the proteins from the Par, FIGURE 1.2: The Cadherin-Catenin complex establishes a signaling center. From Baum and Georgiou, 2011 [12].

Crumbs (Crb), and the Scribble and Coracle complexes or group of proteins. In addition, RhoGTPases, PIP 2 and PIP 3 also play a role in the regulation of the apical-basal polarity. These different regulators are summarised in the following sections. In addition, a scheme of some of the different polarity proteins and their physical interactions is presented in Figure 1.3. The Crb complex, which is of special interest for this thesis, is presented in more detail in the next section.

Par complex

A screen in C.elegans for maternal genes required for early partitioning of the zygote, allowed the identification of several genes called the partitionate defective (Par) genes [START_REF] Kemphues | Identification of genes required for cytoplasmic localization in early C. elegans embryos[END_REF]. Further studies revealed that all but one of the c.elegans Par genes were conserved in other species [START_REF] Goldstein | The PAR Proteins: Fundamental Players in Animal Cell Polarization[END_REF], and that several of them played different roles in the establishment and maintenance of epithelial polarity. The Par proteins involved in apical basal polarity are: the kinases Par1 and Par4 (also called LKB1 or SKT11 in mammals), the phospho binding protein Par5 (an isoform of 14-3-3) and the scaffolding proteins containing PDZ domain Par3 (or Bazooka in drosophila see Figure 1.3) and Par6 (see Figure 1.3). Moreover, Par6 associates with the atypical protein kinase C aPKC (see Figure 1.3) and the small GTPase CDC42 to form one of the complexes of the apical domain.

Scribble and Coracle group of proteins

Other protein modules, conserved across Metazoa that genetically interact with the Par proteins and that are implicated in apical basal polarity are; the Crumbs complex and the Scribble and Coracle group of proteins. The Scribble group (see Figure 1.3); composed of Scribble, Disc large (dlg) and lethal giant larvae (lgl) [START_REF] Bilder | Cooperative regulation of Cell Polarity and Growth by Drosophila Tumor Suppressors[END_REF], and the Coracle group; composed of the FERM (four-point-one, ezrin, radixinmoesin) domain protein, Adapted from St Johnston et Ahringer 2010 [START_REF] St Johnston | Cell Polarity in Eggs and Epithelia: Parallels and Diversity[END_REF].

Moesin and Yurt, Neurexin IV, Na + , K + -ATPase and Coracle [START_REF] Laprise | Coracle, Neurexin IV and the Na+,K+-ATPase form a novel group of epithelial polarity proteins[END_REF], are two groups of genes that act at different developmental times to specify baso/lateral identity [START_REF] Laprise | Coracle, Neurexin IV and the Na+,K+-ATPase form a novel group of epithelial polarity proteins[END_REF].

RhoGTPases

RhoGTPases are evolutionarily conserved small proteins that cycle between an active GTP bound state and an inactive GDP bound state. They play many roles in cell biology, ranging from regulating myosin contractility to regulating adhesion and lamellipodia formation. The three RhoGTPases that are best characterised are Rho1, CDC42 and Rac, all of which are implicated in the regulation of apical basal polarity [START_REF] Mack | The interdependence of the Rho GTPases and apicobasal cell polarity[END_REF]. In MDCK cysts, Rac has been shown to be crucial to establish the location of apical domain formation, probably by signaling downstream of β1-integrin to promote surface laminin assembly [START_REF] O'brien | Rac1 oreintates epithelial apical polarity through effects on basolateral laminin assembly[END_REF], [START_REF] Yu | beta1-integrin orients epithelial polarity via Rac1 and laminin[END_REF]. CDC42 is recruited to the apical domain in a PIP 2 and annexin-2 dependent manner, and recruits Par6/apKC to promote apical basal polarity [START_REF] Martin-Belmonte | PTEN-Mediated Apical Segregation of Phosphoinositides Controls Epithelial Morphogenesis through Cdc42[END_REF]. Moreover, the tight regulation of Rho1, CDC42 and Rac is required for the establishment and maintenance of both tight and adherens junctions.

PIP 2 and PIP 3

Phosphoinositides are membrane lipids that are always localised on the cytosolic face of membranes. They can be phosphorylated in three different locations, allowing different specificities in their subcellular location and in their molecular partners. Two of these forms, the PtdIns-4,5-bisphosphate (PIP 2 ) and the phosphatidylinositol-3,4,5trisphosphates (PIP 3 ), are known regulators of apical/basal polarity [START_REF] Krahn | Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex[END_REF] that are segregated in different parts of the cell. PIP 2 is enriched in the apical domain and PIP 3 is present in the apical and baso/lateral domain. The interaction of cytosolic proteins with the phosphoinositides is mediated by well-known lipid binding domains (such as the pleckstrin homology domain). These lipid binding domains can discriminate between the different phosphoinositides, and thus the spatial segregation of PIP 2 and PIP 3 allows the specific recruitment of proteins to the different domains and the establishment of the apical/basal polarity.

The Crb complex

Crumbs (Crb) is a transmembrane protein, conserved from sponges to mammals [START_REF] Bivic | Evolution and Cell Physiology. 4. Why invent yet another protein complex to build junctions in epithelial cells? en[END_REF], and is a key determinant for apical/basal polarity. It was first discovered in 1990 by the Knust lab as an essential protein in Drosophila for the maintenance of apical/basal polarity [START_REF] Tepass | Crumbs encodes an EGF-like protein expressed on apical membranes of drosophila epithelial cells and required for Organization of Epithelia[END_REF]. It is a transmembrane protein bearing, in drosophila, 29 egf-like and four laminin-A globular-domain-like repeats and a small intracellular domain with two highly conserved regions: an ERLI motif (a PDZ binding motif) and a FERM motif(4.1/ezrin/radixin/moesin).

The members of the Crb complex

Crb binds to several proteins that are separated into core-and accessory-partners of Crb [START_REF] Bulgakova | The Crumbs complex: from epithelial-cell polarity to retinal degeneration[END_REF], where the distinction between these two groups correlates with the historical discovery of the different partners. It is noteworthy to state that this division is completely artificial, as neither the composition of the Crb complex in different tissues and at different developmental times, nor the relative affinity of the different proteins to Crb, have been properly addressed.

Core partners of Crumbs

The core partners of Crb are the proteins Pals-1/Stardust, Lin7 and Patj (Protein associated with tight junctions). These three proteins can interact through their proteinprotein interaction domains, PDZ (PSD-P5/Discs-large/ZO-1) and L27 .

A scheme of the interaction between these three proteins can be found in Figure 1.4. This figure shows the core partners of Crb (Sdt, Lin7 and Patj) that form a complex with Crb via an interaction with Sdt. Crb is required for the apical localisation of Sdt, Lin 7 and Patj [START_REF] Bulgakova | The Crumbs complex: from epithelial-cell polarity to retinal degeneration[END_REF]. However, the requirement of Sdt, Lin7 and Patj for the stability of Crb is tissue specific. For example, Sdt is strictly required in the early embryo [27][28][29], however, its removal in mature epithelia reduces the levels of the core Crb complex but does not completely abrogate it [START_REF] Lin | AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust[END_REF]. In drosophila, Patj is dispensable for the localisation of Crb in the embryo but is required for the survival of the pupae [START_REF] Penalva | Tissue-specific function of Patj in regulating the Crumbs complex and epithelial polarity[END_REF], [START_REF] Zhou | Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability[END_REF] , and for the maintenance of the Crb protein in photoreceptor cells [START_REF] Richard | DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in theDrosophila eye[END_REF], [START_REF] Nam | Domain-specific early and late function of Dpatj in Drosophila photoreceptor cells[END_REF]. Finally, Lin-7 seems to be dispensable for the establishment of the core Crb complex as Lin-7 flies are viable and fertile, but they show light-induced retinal degeneration similar to that observed in crb mutant flies [START_REF] Bachmann | Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration[END_REF]. PatJ and Sdt do not always interact with Crb, and Sdt has been shown to bind to Par3/Baz in the early embryo. This interaction is broken by the phosphorylation of Baz by aPKCs, and has been proposed to be required for the exclusion of Baz from the apical domain once epithelial polarity is established [START_REF] Krahn | Formation of a Bazooka Stardust complex is essential for plasma membrane polarity in epithelia. en[END_REF]. The balance of Sdt bound to Crb and to Par3/Baz in different tissues, and the relevance of this balance, has never been properly addressed. Moreover, PatJ has been shown to interact and inhibit the Myosin phosphatase, thus suggesting a role of the Crb core complex in the positive regulation of myosin activity [START_REF] Sen | Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity. en[END_REF]. FIGURE 1.4: Crb core partners. Figure from Bulgakova et Knust 2009 [26].

Accessory partners of Crumbs

In addition to the Crb core complex, Crb interacts with other proteins. The accessory partners of Crb are Par6, aPKC, Moesin, Yurt and Expanded, presented in Figure 1.5. Par6 and aPKC are other members of the so called apical polarity proteins, as they define and maintain the apical domain.

APKC binds to Crb or to PatJ through Par6 [START_REF] Sotillos | DaPKCdependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. en[END_REF]. Moreover, the PDZ domain of Par6 has been shown to interact with the ERLI motif of Crb [START_REF] Kempkens | Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6. en[END_REF], and with two evolutionary conserved regions (ECR) of Std [START_REF] Bulgakova | Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila[END_REF], thus blocking Sdt-PatJ interaction [START_REF] Wang | Tight Junction Protein Par6 Interacts with an Evolutionarily Conserved Region in the Amino Terminus of PALS1/Stardust. en[END_REF]. Additionally, the PP1 domain of Par6 can bind to the PDZ domain of PatJ [START_REF] Hutterer | Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis[END_REF]. Thus, Par6 can bind to Sdt or PatJ, creating two different apical complexes with, most likely, different properties ( Figure 1.6.) FIGURE 1.5: Accessory partners of Crb. Of note, the interaction with Expanded is not shown. Image from Bulgakova et Knust 2009 [START_REF] Bulgakova | The Crumbs complex: from epithelial-cell polarity to retinal degeneration[END_REF].

Crb can physically interact with the FERM domain of Yurt [START_REF] Laprise | Coracle, Neurexin IV and the Na+,K+-ATPase form a novel group of epithelial polarity proteins[END_REF]. Yurt has been implicated in the regulation of several morphogenic events, such as dorsal closure and FIGURE 1.6: Different Crb complexes can be formed. Again, the complex with Expanded is not shown. Image from Bulgakova et Knust 2009 [START_REF] Bulgakova | The Crumbs complex: from epithelial-cell polarity to retinal degeneration[END_REF].

germ-band retraction [START_REF] Hoover | Drosophila Yurt is a new protein-4.1-like protein required for epithelial morphogenesis[END_REF], and its removal causes the expansion of the apical domain, in a phenotype similar to the overexpression of Crb. More recent work suggested that the negative regulation of the apical domain by Yurt is mediated through an inhibition of aPKC activity [START_REF] Gamblin | A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity. en[END_REF].

Crb has been immunoprecipitated with β-heavy-spectrin and with moesin, and shown to be required for the stability of β-heavy-spectrin [START_REF] Medina | Crumbs interacts with moesin and beta Heavy -spectrin in the apical membrane skeleton of Drosophila. en[END_REF]. Spectrins are long tetrametric proteins constituted of two α and two β subunits that form a branched cytoskeletal network called the spectrin-based skeleton. Moesin is a FERM domain protein and the proposed model [START_REF] Medina | Crumbs interacts with moesin and beta Heavy -spectrin in the apical membrane skeleton of Drosophila. en[END_REF] is that it acts by physically connecting Crb to β-heavy-spectrin [START_REF] Medina | Crumbs interacts with moesin and beta Heavy -spectrin in the apical membrane skeleton of Drosophila. en[END_REF] or by inhibiting RhoGTPases [START_REF] Flores-Benitez | Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila[END_REF]. Flies lacking the Crb FERM domain, and thus failing to interact with moesin, show defects in dorsal closure that are associated with the over-activation of a positive regulator of the actin network [START_REF] Flores-Benitez | Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila[END_REF]. Additionally, Sdt and Moesin bind to nearby regions of Crb and compete for its interaction. It is the phosphorylation of Crb that regulates which protein is recruited, as non-phosphorylated Crb binds preferentially to Moesin [START_REF] Wei | Structural Basis for the Phosphorylationregulated Interaction between the Cytoplasmic Tail of Cell Polarity Protein Crumbs and the Actin-binding Protein Moesin[END_REF].

Finally, Crb can bind to the Hippo Pathway regulator, Expanded, through an interaction between the FERM domains of both proteins [START_REF] Ling | The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded[END_REF] [START_REF] Robinson | Crumbs Regulates Salvador/Warts/Hippo Signaling in Drosophila via the FERM-Domain Protein Expanded[END_REF], [START_REF] Chen | The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila[END_REF]. Both the overexpression of Crb and the removal of crb in clones, affected the plasma membrane levels of expanded, and were associated with Yorkie-dependent tissue overgrowth [START_REF] Ling | The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded[END_REF] [START_REF] Robinson | Crumbs Regulates Salvador/Warts/Hippo Signaling in Drosophila via the FERM-Domain Protein Expanded[END_REF], [START_REF] Chen | The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila[END_REF].

The diversity of Sdt

Sdt is a key component in the Crb complex and is responsible for forming the scaffold between Crb and the other core proteins. This thesis explores the regulation of the isoforms of Sdt by the E3 ubiquitin ligase Neur.

Sdt is a member of the membrane-associated guanylate kinase (MAGUK) family, comprised of proteins containing a PDZ-SH3-GUK tandem in their domain organisation, a Hook domain, and putatively some LIN2-LIN7 (L27) domains. MAGUK proteins are scaffolding proteins which associate with membrane proteins to organise signaling in the cell. The PDZ, L27 and SH3 (SRC homology3) domains are proteinprotein interaction domains. Moreover, the GUK (Guanilate Kinase) domain of the MAGUK proteins is very similar to that of the Guanylate Kinases, proteins that catalyse the transformation of Guanosine-monophosphate (GMP) to Guanosine triphosphate or (GTP), but do not have any Kinase activity as they cannot bind to ATP [START_REF] Zhu | Mechanistic basis of MAGUK-organized complexes in synaptic development and signalling[END_REF]. Finally, the Hook domain is a flexible region that favours conformational changes.

Chapter 1. Epithelial Polarity

In drosophila, the gene Sdt could encode for 12 different isoforms, four of which are presented in Figure 1.7. N e u r FIGURE 1.7: Sdt is a scaffolding protein encoding for several isoforms. Adapted from Bulgakova et al 2010 [START_REF] Bulgakova | Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells[END_REF].

It has been proposed that different isoforms are expressed in a tissue dependent manner and at different points throughout Drosophila development. For instance, it is known that the Sdt-PB isoform is highly expressed during early development [START_REF] Bachmann | Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity[END_REF][29], [START_REF] Bulgakova | Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells[END_REF] but is absent from the retina. Conversely, other Sdt isoforms (Sdt-PH and Sdt-PD) which are absent in the early embryo are found to be expressed in the retina [START_REF] Bulgakova | Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells[END_REF]. It has been also suggested that different isoforms have different properties, as exemplified by the opposite effect of the overexpression of Sdt-PH and Sdt-PD on stalk membrane length in the retina. But again, this idea needs further exploration. However, where and when all of the different isoforms are expressed has not been addressed in detail.

Sdt is known to bind to several proteins. It binds to Par6 through its ECR (Evolutionary Conserved Repeats) repeats, Patj and Lin7 through the L27N and L27 domains, respectively, and Par3/Baz or Crb through the PDZ domain (see Figure 1.7). The SH3 and GUK domains have been shown by crystallography to favour the binding of Sdt to Crbs [START_REF] Li | Structure of Crumbs tail in complex with the PALS1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex[END_REF]. Whether the different Sdt isoforms form different complexes with these proteins, remains to be further studied.

Regulation of the Crb Complex stability

Various factors regulate the stability of the core Crb complex, for example the phosphorylation of Crb, its oligomerisation through Crb-Crb interactions or different mechanisms associated to its intracellular trafficking.

Phosphorylation

APKC/par6 can interact with the crb core complex, either through Patj or Crb. In vitro data shows that Crb can be phosphorylated by aPKC at threonines 6 and 9 of its 37 residue tail [START_REF] Sotillos | DaPKCdependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. en[END_REF]. This phosphorylation is thought to stabilise Crb in the membrane; follicular cells overexpressing Crb proteins which are not phosphorylated at these residues, are endocytosed and localise in endosomes [START_REF] Fletcher | Positive Feedback and Mutual Antagonism Combine to Polarize Crumbs in the Drosophila Follicle Cell Epithelium[END_REF].

Oligomerisation in cis and trans

Homo-or oligomerisation of the Crb protein is thought to increase the stability of the Crb complex. It has been proposed that Crb can homo or hetero oligomerise both in trans (between Crb proteins from two different cells) and in cis (in the same cell). Cis-oligomerisation of Crb was proposed by Pellikka et al. [55], and derives from the observation that Crb lacking its intracellular domain, and thus the ability to bind to protein partners, can only localise to the apical domain if endogenous Crb is present. This suggests that endogenous Crb can recruit the Crb lacking the intracellular domain via an interaction through their extracellular domain. Indirect evidence that Crb can be oligomerise in trans is put forward in three papers. The first is by Zou et al 2012 [START_REF] Zou | Crb Apical Polarity Proteins Maintain Zebrafish Retinal Cone Mosaics via Intercellular Binding of Their Extracellular Domains[END_REF] who studied the different crumbs homologs in the zebrafish and showed that different crb genes can form homophilic and heterophilic interactions in vitro and when overexpressed in cultured cells, can cause cells to aggregate.

Secondly, a study by Roper[57], of the formation of the salivary glands during drosophila embryogenesis, shows that the cells that invaginate to form these glands (the salivary placode) are surrounded by an actomyosin cable. This cable correlates with reduced levels of Crb. Roper suggests that the asymmetric distribution of Crb in the cells surrounding the placode is due to the trans-doligomerisation of Crb. Finally, [START_REF] Hafezi | Differences in levels of the transmembrane protein Crumbs can influence cell survival at clonal boundaries[END_REF] [START_REF] Hafezi | Differences in levels of the transmembrane protein Crumbs can influence cell survival at clonal boundaries[END_REF] studies the non cell-autonomous effects of overexpression or removal of Crb on cell death that can only be explained by trans-oligomerisation and by the role of Crb in regulating the hippo pathway.

Intracellular trafficking

The Crb complex stability is also regulated by intracellular trafficking. Three different examples are given which involve the retromer complex, Rab11 exocytic transport and Sdt mediated protection from an AP-2 dependent endocytosis. Retromer is a highly conserved complex of proteins, forming an endocytic machine that sorts cargo proteins in the golgi apparatus which had previously been selected for degradation, and recycles them back through retrograde endosome-to-Golgi transport [START_REF] Cullen | Sorting nexins provide diversity for retromerdependent trafficking events[END_REF]. VSP35, a protein of the retromer complex, can physically interact with the intracellular tail of Crb and the absence of this protein reduces Crb levels at the apical membrane [60] [61]. This finding lead the authors to propose that retromer mediated recycling is required for maintaining high levels of Crb at the apical membrane. The endocytic pathways involved in the relocalisation of Crb to the apical domain, remain unknown.

Additionally, the overexpression of a Rab11 dominant negative form of Rab11, or the removal of rab11 was sufficient to perturb the apical localisation of Crb and phenocopied embryos lacking crb. This supports a model where Rab11 exocitic transport is required for the localisation and stabilisation of Crb.

Finally, Lin and colleagues [START_REF] Lin | AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust[END_REF] proposed that the interaction between Sdt and Crb protected Crb from an AP-2 dependent endocytosis . These results stress the importance of the recycling pathways in the regulation of the Crb complex stability.

The Crb complex maintains epithelial polarity

Throughout embryogenesis, epithelial cells undergo extensive cellular rearrangements. During germ-band extension the exchange of neighbours of epithelial cells causes the tissue to elongate; during neuroblast formation cells delaminate basally; and during the formation of the renal tubules cells intercalate. These morphogenic events are Chapter 1. Epithelial Polarity accompanied by extreme junctional rearrangements, as junctions are eliminated and reformed.

Crb and Sdt are examples of proteins involved in the maintenance of epithelial polarity during junctional rearrangement. Indeed, crb and sdt embryos do establish initial epithelial polarity, but fail to develop the zonula adherens from spot junctions, and fail to maintain epithelial polarity during neuroblast delamination, germ-band extension [START_REF] Tepass | Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia ofDrosophila[END_REF] [START_REF] Grawe | The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions[END_REF] or during the elongation of the renal tubules [START_REF] Campbell | Crumbs stabilises epithelial polarity during tissue remodelling[END_REF]. Moreover, blocking cell rearrangements, by using a Kruppel mutant which fails to undergo germ-band elongation [START_REF] Campbell | Crumbs stabilises epithelial polarity during tissue remodelling[END_REF], by overexpressing the intracellular part of Notch to block neuroblast delamination [START_REF] Harris | Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis[END_REF], or by removing a gene essential for the cell intercalation of renal tubules is sufficient to rescue the loss of epithelial integrity seen in Crb embryos, or in embryos failing to properly localise Crb.

Thus, the role of the proteins from the Crb complex seems to be primarily in maintaining epithelial polarity and integrity in tissues undergoing extreme junctional rearrangement.

Positive and Negative interactions regulate the formation of different specific protein-scaffold domains

The different proteins involved in the regulation of apical-basal polarity form specific protein-scaffold domains by undergoing complex negative and positive interactions (summarised in Figure 1.8). Some of these interactions are presented next:

Crb Sdt Patj Lin7 Cdc42 aPKC Par6 Baz PTEN Moesin Yurt apical domain junctional domain baso/lateral domain

Baz/ Par3

Lgl Dlg Scrib Par1 Yurt Cora Nak-ATPase NrxIV PI3K Rac FIGURE 1.8: Antagonistic interactions between membrane specific protein-scaffolds establishes the apical-basal polarity domains. Scheme adapted from Tepass 2012 [START_REF] Tepass | The Apical Polarity Protein Network in Drosophila Epithelial Cells: Regulation of Polarity, Junctions, Morphogenesis, Cell Growth, and Survival[END_REF].

Negative Feedback Loops

Antagonistic interactions between proteins maintains the separation between the apical and the baso/lateral domains, and loss of function of proteins from one domain leads to the expansion of the other. This is exemplified by the overexpression of Crb (an apical protein), which leads to the expansion of the apical domain at the expense of the baso/lateral domain [START_REF] Wodarz | Expression of Crumbs Confers Apical Character on Plasma Membrane Domains of Ectodermal Epithelia of Drosophila[END_REF]. Moreover, the loss of apical/basal polarity seen in the absence of Crb is partially rescued by the removal of proteins of the Scribble (lateral domain) group of proteins [START_REF] Bilder | Cooperative regulation of Cell Polarity and Growth by Drosophila Tumor Suppressors[END_REF] [68] [START_REF] St Johnston | Cell Polarity in Eggs and Epithelia: Parallels and Diversity[END_REF].

1.3. Positive and Negative interactions regulate the formation of different specific protein-scaffold domains 15

The mutual exclusion of the polarity proteins from the two domains relies on the kinases aPKc and Par1. The aPKC/PAR6 complex is recruited apically by Cdc42 [START_REF] Harris | Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis[END_REF], where it phosphorylates any baso/lateral proteins that have 'invaded' the apical domain. Phospho-Regulated Basic and Hydrophobic Motif PRBH FIGURE 1.9: Model for aPKCdependent exclusion of membrane associated proteins.

Taken from Bailey and Prehoda 2015 [START_REF] Bailey | Establishment of Par-Polarized Cortical Domains via Phosphoregulated Membrane Motifs[END_REF].

In several proteins, a PRABH (Phosphoregulated Basic and Hydrophobic) motif interacts with phospholipids allowing the protein to bind the phospholipid membrane. aPKC phosphorylation of this motif perturbs its interaction with the phospholipid membrane, sending the protein to the cytoplasm and excluding it from the apical domain [START_REF] Bailey | Establishment of Par-Polarized Cortical Domains via Phosphoregulated Membrane Motifs[END_REF]. Par1 [START_REF] Hurov | Atypical PKC phosphorylates PAR-1 kinases to regulate localization and activity[END_REF] and Lgl [START_REF] Tian | Lgl and its phosphorylation by aPKC regulate oocyte polarity formation in Drosophila[END_REF] are two well documented examples of baso/lateral proteins which are regulated in this manner by aPKC.

The positioning of the junctional domain, mediated by the Par-3/Baz complex, is another excellent demonstration of the means by which antagonistic interaction segregates the domains. It was proposed that the position of Par-3/Baz is dependent on at least two mechanisms.

Firstly, Par3 is regulated by the lateral protein Par1 (see Figure 1.10a). Par3 proteins that diffuse basally are phosphorylated at Ser 151 and Ser 1085 by the Kinase, Par1. This triggers binding to Par5/14-3-3 and the removal of Par3/Baz from the membrane [START_REF] Benton | Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells[END_REF]. Concurrently, the phosphatase PP2A (recruited to the apical domain by its interaction with PatJ, which is bound to Crb through Sdt) removes the phosphate at position Ser 1085 to refine Par3/Baz at the apico/lateral position [START_REF] Krahn | PP2A Antagonizes Phosphorylation of Bazooka by PAR-1 to Control Apical-Basal Polarity in Dividing Embryonic Neuroblasts[END_REF], [START_REF] Flores-Benitez | Dynamics of epithelial cell polarity in Drosophila: how to regulate the regulators? en[END_REF], [START_REF] Sen | Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity. en[END_REF]. Secondly, the maintenance of the position of Par3/Baz is thought to depend on an interaction with Crb and aPKC (see Figure 1.10b). In other developmental contexts (oocyte antero posterior polarity and asymmetric cell division) Par3/Baz (junctional protein) forms a complex with aPKC and Par6 (apical proteins). In epithelial cells, the formation of this complex is inhibited by the phosphorylation of Par3/Baz by aPKC on Ser 980 and by Crb, which outcompetes Par6 for the binding to Par3/Baz [START_REF] Morais-De Sa | aPKC Phosphorylation of Bazooka Defines the Apical/Lateral Border in Drosophila Epithelial Cells[END_REF].

It remains unclear whether or not both mechanisms occur in all epithelial cells, nor whether they occur at the same developmental time.

Chapter 1. Epithelial Polarity Moreover, the above models fail to explain several points; for example, the requirement of Par3/Baz for the apical recruitment of aPKC (observed in Morais- de-sa et al. 2010 [75]), or why Baz is not always phosphorylated by aPKC and released into the cytoplasm. One possibility is that Par3/Baz works as a scaffold protein to recruit aPKC and that Par3/Baz interaction with the phosphatase PP1 allows its protection from aPKC [START_REF] Traweger | Protein phosphatase 1 regulates the phosphorylation state of the polarity scaffold Par-3[END_REF].

Positive Feedback Loops

The establishment of apical/basal polarity has been modeled by several groups. The models usually require the implementation of negative feedback loops between the apical and the baso/lateral domains (apical vs baso/lateral), complemented by the addition of a positive feedback loop in one of the two domains [START_REF] Goehring | PAR polarity: From complexity to design principles[END_REF]. It has been proposed that the apical domain established this positive feedback loop via the intermediary protein Crb [77][54]. This is based on the observation that the overexpression of Crb is sufficient to increase the size of the apical domain [START_REF] Wodarz | Expression of Crumbs Confers Apical Character on Plasma Membrane Domains of Ectodermal Epithelia of Drosophila[END_REF]. Additionally, in MDCK cysts, where the establishment of apical-basal polarity has been widely studied and manifests itself in the formation of a lumen, the first signs of polarisation appear in the form of Rab11-endosomes containing Crb. Furthermore, in MDCK cysts, the establishment of apical/basal polarity is blocked in the absence of Crb, and in the Rab11-endosomes Crb already colocalises with Sdt and PatJ [START_REF] Schluter | Trafficking of Crumbs3 during cytokinesis is crucial for lumen formation[END_REF].

Fletcher et al 2012 [START_REF] Fletcher | Positive Feedback and Mutual Antagonism Combine to Polarize Crumbs in the Drosophila Follicle Cell Epithelium[END_REF] proposed a model for a positive feedback loop of the apical proteins based on their self-recruitment (see Figure 1.11). In this model, phosphorylated Crb recruits the other members of the apical domain. Following this, Crb forms homophilic interactions through its extracellular domains that recruits other Crb proteins. The new Crb proteins are then phosphorylated by aPKC and the cycle restarts. 2012 [START_REF] Fletcher | Positive Feedback and Mutual Antagonism Combine to Polarize Crumbs in the Drosophila Follicle Cell Epithelium[END_REF].

The loss of polarity during epithelial to mesenchymal transitions

Epithelial apical/basal polarity is a cell property that can be acquired and also lost. This is the case during epithelial to mesenchymal transitions (EMT), where epithelial cells lose polarity and migrate. The remodeling of the posterior midgut between stages 9 and 10 is an example of EMT. The midgut is specified at the onset gastrulation at the anterior and the posterior end of the embryo (Figure 1.12 stage 5 in red). It invaginates during gastrulation (Figure 1.12 stage 7 in red) and the posterior midgut is moved anteriorly during germband extension (Figure 1.12 stage 9). At the end of stage 9, the midgut, which is a polarised epithelia bearing a zonula adherens and expressing Crb and the other polarity proteins, will undergo an epithelial to mesenchymal transition, lose polarity and become mesenchymal (Figure 1.12 stage 11). Anterior and posterior midgut will migrate toward each other, using the mesoderm as a support (Figure 1.12 stage 12). At around stage 13, both anterior and posterior midgut join and undergo a mesenchymal to epithelial transition, re-acquire epithelial characteristics and form the gut (Figure 1.12 stage 12).

The remodeling of the posterior midgut as an example of EMT

The initial steps of the EMT which occur at stage 10 in the posterior midgut are a particular focal point of this thesis. At the beginning of stage 9, the epithelium of the posterior midgut is polarised and adherens junctions connect the different cells, as shown in the Figure 1.13 a. At stage 10 epithelial apical/basal polarity is lost, [82][81], adherens junctions are disassembled and intercellular gaps are formed [START_REF] Jaglarz | The active migration of Drosophila primordial germ cells[END_REF], [START_REF] Callaini | Pole Cell Migration through the Gut Wall of the Drosophila Embryo: Analysis of cell interactions[END_REF] (see the asterix in Figure 1.13 b). These EMT are controlled by the GATA transcription factor Serpent [START_REF] Campbell | Specific GATA Factors Act as Conserved Inducers of an Endodermal-EMT[END_REF], [START_REF] Reuter | The gene serpent has homeotic properties and specifies endoderm versus ectoderm within the Drosophila gut[END_REF]; in serpent embryos the posterior midgut never loses apical/basal polarity and never migrates, and the midgut is Chapter 1. Epithelial Polarity not properly formed.

Moreover, chromatin-immunoprecipitation experiments showed that Serpent binds to several genes, including crb [START_REF] Campbell | Specific GATA Factors Act as Conserved Inducers of an Endodermal-EMT[END_REF], and RT-qPCR analysis of the expression of crb and sdt in the posterior midgut of wild-type versus serpent embryos, suggested that their transcription is inhibited by Serpent. Thus, at stage 9 serpent is not expressed and the epithelia of the posterior midgut are polarised on their apical/basal axis (Figure 1

The transepithelial migration of PGCs as a readout of epithelial remodeling

During the time that the posterior midgut loses its apical/basal polarity, the primordial germ cells (PGCs) migrate in between the posterior midgut in a process called transepithelial migration [START_REF] Jaglarz | The active migration of Drosophila primordial germ cells[END_REF], [START_REF] Seifert | Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm[END_REF]. The PGCs are the cells that will give rise to the gametes [START_REF] Santos | Germ Cell Specification and Migration in Drosophila and beyond[END_REF], [START_REF] Richardson | Mechanisms guiding primordial germ cell migration: strategies from different organisms[END_REF]. Prior to cellularization, around 35 germ cells are specified in the posterior end of the embryo by the presence of the germ plasm determinant, oskar [87] (Figure 1.14 stage 5).

Somatic Gonadal Precursor (SGPs) Primordial Germ Cells (PGCs) The active migration of PGCs is regulated by different mechanisms. The GPCR receptor Tre1 is required to activate the RHoGTPase Rho1 during the transepithelial migration (Figure 1.15a) [88][89]. The ligand of Tre1 still remains to be discovered. After transepithelial migration, PGCs migrate in a Wunen and Wunen-2 dependent manner [START_REF] Zhang | The Drosophila protein Wunen repels migrating germ cells[END_REF], [START_REF] Starz-Gaiano | Spatially restricted activity of a Drosophila lipid phosphatase guides migrating germ cells[END_REF]. These two proteins are lipid phosphate phosphatases (they hydrolyse lipids), expressed in the posterior midgut that simultaneously generate a repellent and survival cue for the PGCs. Indeed, in the absence of the Wunen genes the PGCs die. Furthermore, the misexpression of the Wunen genes causes PGCs to migrate to the wrong location. (Figure 1.15b). The final step of migration, when the PGCs migrate towards the SGPs, involves the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR or columbus) enzymatic pathway. Mutation in this pathway leads to a defect in migration [92][93]. HMGR is upregulated in the SGPs and their surrounding tissues, and the ectopic expression of HMGCR is sufficient to attract the PGC. HMGR is an enzyme capable of isoprenylating proteins (a postranslational modification by the addition of lipids), and other proteins implicated in the isoprenylation pathway are also required for the migration of the PGC. Nonetheless, the molecule that is isoprenylated and that serves as a chemo-attractant for PGC migration remains to be identified(Figure 1.15c). Several papers [94] [95] suggest that this chemo-attractant could be the protein Hedgehog and that HMGCR aids to diffuse it. However, the role of hedghehog in the regulation of PGC migration is still controversial [96] [95]. The transepithelial migration of the PGCs is dependent on the epithelial remodeling of the posterior midgut. In embryos failing to remodel their midgut, for example serpent embryos, the PGCs are physically blocked during migration through the epithelium [START_REF] Jaglarz | The active migration of Drosophila primordial germ cells[END_REF], [START_REF] Seifert | Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm[END_REF]. Nonetheless, the failure to remodel the posterior midgut, does not affect the ability of the PGCs to migrate, which is exemplified by the double mutant for embryos for serpent and crb where transepithelial migration is rescued [START_REF] Seifert | Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm[END_REF]. Therefore, the transepithelial migration of PGCs between stages 9 and stages 10 is an excellent read-out of the early steps of the EMT occurring in the posterior midgut. Moreover, the fact that this migration can be scored, enables us to quantify the extent to which epithelial remodeling has been perturbed.

20

Chapter 1. Epithelial Polarity

Conclusions

Apical/basal polarity is an essential characteristic of epithelial cells. It is established very early on during embryogenesis by a complex interplay of polarity proteins that interact through positive and negative interactions. The Crb complex is a key component of this polarity, as it allows its maintenance during epithelial remodeling. Finally, once established polarity can be lost during Epithelial to Mesenchymal Transitions (EMTs), as exemplified by the epithelial remodeling of the posterior midgut occurring between stages 9 and 10.

Chapter 2

Epithelial remodeling during gastrulation

Eptihelial tissues are polarised structures interconnected through mechanical junctions. They undergo massive remodeling to shape an embryo: they change their neighbour during germ-band extension, modify their shape during apical constriction or divide during the formation of the wing. Thus, understanding the mechanisms behind epithelial tissue remodeling is key to understanding embryo morphogenesis. Drosophila gastrulation is one of the textbook models of epithelial remodeling. It is characterised by the invagination of the presumptive mesoderm. This chapter will summarise the current knowledge on the morphogenetic movements during drosophila gastrulation, focusing on the events that occur before the mesoderm has invaginated. Post mesoderm invagination events are out with the scope of this thesis.

Cell shape changes during gastrulation:

At the onset of gastrulation, the drosophila embryo is an ovoid structure composed of a single layer epithelium of around 5000 cells, surrounding a yolk and delimited by a rigid membrane, the vitelline membrane [START_REF] Leptin | Cell shape changes during gastrulation in Drosophila[END_REF]. Gastrulation is a morphogenetic movement that deeply impacts all embryonic cells: the cells from the mesoderm (a band of about 18 by 60 cells located ventrally) undergo a set of fast shape changes to end up inside the embryo [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF], and the cells from the ectoderm rearrange accordingly to accommodate this [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF].

Shape changes in the mesoderm:

[100], [START_REF] Leptin | Cell shape changes during gastrulation in Drosophila[END_REF], [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF], [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF], [101], [102].The cells located in the most ventral part of the mesoderm, and comprising an 8 to 10-cell wide region, will be referred to as the ventral mesoderm (Figure 2.1 in yellow). The two bands, of around 4-cell wide located at each side of the ventral mesoderm are referred to as the lateral mesoderm (Figure 2.1 in red). Gastrulation begins with the reduction of the apical surface of the cells from the ventral mesoderm (Figure 2.1a). As the apical areas of the ventral mesoderm continue to be reduced, the cells from the lateral mesoderm stretch toward the middle of the embryo and the cells of the mesoderm elongate along their apical/basal axis, resulting in a flattening of the ventral region of the embryo (Figure 2.1b). Once mesodermal cell elongation has reached a certain level, the cells from the ventral mesoderm (and not the cells from the lateral mesoderm), begin to shorten along their apical/basal axis and lengthen their basal sides. This, combined with the continued elongation of the cells from the lateral mesoderm, causes the buckling of the tissue and the initial formation of a furrow (Figure 2.1c). The ventral furrow grows in depth as cells in the Costa et al. 1993. [102]. Images from Conte et al. 2012[103]. See text for details.

ventral mesoderm continue to shorten along their apical/basal axis, and those in the lateral mesoderm begin to shorten also(Figure 2.1d). Mesoderm invagination is complete when the cells from the ectoderm, located at each side of the furrow, come into contact with each other(Figure 2.1e). To summarise, during gastrulation, cells in the ventral mesoderm first undergo apical constriction and apical-basal lengthening, followed by basal side area increase and apical/basal shortening. Meanwhile, cells in the lateral mesoderm undergo apical stretching and apical/basal elongation, followed by basal elongation and apical/basal shortening. The different mechanisms behind these shape changes and their interdependency will be discussed below.

Shape changes in the ectoderm:

The cell shape changes of the cells from the ectoderm were studied by Rauzi et al 2015 ([99]). To do so, gastrulating embryos were filmed using light sheet microscopy from four different angles which, after image processing, allows a view of the surface of the embryo to be displayed cartographically (Figure 2.2a). From this viewpoint, a kymograph of a cross section of the embryo allowed the authors to track apical cell shape changes over time (kymograph in Figure 2.2b). They found that the ectoderm can be divided into two regions with different behaviors: the lateral ectoderm (left lateral and right lateral, between the orange arrows in Figure 2.2b ) and the dorsal ectoderm (named dorsal and shown twice between the green arrows in Figure 2.2b). Moreover, the authors noted that cell shape changes in the ectoderm can be separated into two phases: the first commencing with the onset of gastrulation and up until 3 minutes after the formation of the ventral furrow, and the second from this point onward. In the first phase, cells in the ventral mesoderm constrict, and those in the lateral mesoderm stretch toward the furrow, during this phase the ectodermal cells do not move or change cell shape. During the second phase, after initial formation of the ventral furrow, cells in the lateral ectoderm "shift ventrally as a compact cohort", and those in the dorsal ectoderm stretch and elongate towards the ventral part of the embryo (this corresponds with the time points between Figure 2.1c and Figure 2.1e). Live imaging, laser ablation and cauterisation experiments show that the differences in behavior between the lateral and dorsal ectoderm correlate with differences in density in the actin cortical network and in the relative stiffness of the cells. Additionally, the authors suggest that the ectoderm doesn't play any role in the initial formation of the ventral furrow (phase 1), but is required for mesoderm invagination (phase 2). Cauterisation of the ectoderm causes the cells at the site of the injury (furthest from the ventral furrow) to cease to move or change shape. This suggests that the forces required for cell shape change in the lateral and dorsal ectoderm are generated in the mesoderm.

Molecular pathways involved in mesoderm invagination

Molecularly speaking, mesoderm invagination can be seen as regulated at three different levels [104]. Firstly, it is regulated by developmental signals comprising the molecules responsible for dorso/ventral patterning and the transcription factors, Twist and Snail; secondly, by surface signal ligands, such as Fog, T48 and RhoGEF2; and Chapter 2. Epithelial remodeling during gastrulation thirdly, by the conserved intracellular pathway modulated by Rho1 that positively regulates actin and myosin. Gastrulation is tightly linked to the dorso/ventral domains of the fly. Indeed, dorso-ventral patterning establishes the location of the mesoderm, and thus the cells that will invaginate.

Developmental signals

Dorsoventral prepatterning

Twist and Snail

Twist and Snail are two transcription factors, essential for gastrulation, which are under the control of Dorsal. Twist is a basic helix-loop-helix activator expressed as a . Moreover, snail expression delimits the mesoderm and hence the cells that are destined to be internalised. Embryos mutant for either twist or snail do not invaginate properly (but still form a small furrow), and invagination is completely blocked if both transcription factors are removed [START_REF] Leptin | Cell shape changes during gastrulation in Drosophila[END_REF]. CHIP on chip experiments identified 106 genes directly under the control of Snail, half of them being repressed by Snail and the other half activated[117], however, the mechanism by which these intermediary genes direct the cell shape changes seen in embryos lacking Twist remains unclear. On the other hand, it has been proposed that Twist acts through at least 6 zygotic genes [118]: Snail (which is essential and whose maintenance in the mesoderm is dependent on Twist

Cell surface signals

Fog and its receptors

Fog is a 78 kDa protein expressed as a gradient in the mesoderm [123], [125]. Absence of fog disrupts normal mesoderm invagination due to perturbation of collective apical constriction (see apical constriction, for more details). Fog is secreted [126] and acts paracrinally to activate the two G protein-coupled receptors (GPCR) mist [127] and smog [128]. Mist was discovered in a screen in S2+ cells. Manning et al [127] observed that the addition of fog to the medium of S2R+ cells was sufficient to cause a constriction of the cell. An RNAi screen for all of the drosophila GPCR receptors identified Mist as target receptor involved in the activation of the signaling pathway downstream of Fog. The gene mist is a direct target of Snail and, in the embryo, is expressed exclusively in the mesoderm. Smog was discovered in a genetic screen of chromosomal deletions that perturbed mesoderm invagination [129]. At the onset of gastrulation smog is expressed ubiquitously in the embryo and also plays a role in the regulation of other morphogenetic events, such as germ band extension [128]. Embryos lacking either Mist or Smog do not constrict normally, an effect which is greatly amplified when both GPCRs are inhibited or deleted [128]. It has been proposed that the binding of Fog to the GPCRs, Mist and Smog, ultimately leads to the activation of the small GTPase, Rho1 Figure 2.5.

T48 and the apical localisation of RhoGEF2

Activated Concertina, then binds to the Guanine exchange factor RhoGEF2 [136] and favours its dissociation from the microtubules [137]. The transmembrane protein T48, another direct target of Twist, physically interacts with RhoGEF2 and acts in parallel to Concertina and Fog, to apically concentrate RhoGEF2 [124]. Apically localised RhoGEF2 then activates the small GTPase Rho1 by catalysing the exchange of GDP to GTP.

Negative regulation of the GPCR receptors

The activity of a ligand-bound GPCR is tightly regulated by G protein coupled kinases (GPRK). GPRKs can phosphorylate the C terminus of an activated GPCR, leading to the recruitment of β-Arrestin. This can result in the endocytosis and/or degradation of the receptor, an Arrestin-scaffolded signaling cascade or the inhibition of GPCR binding to G proteins [138] [139], [140]. In drosophila there is only one non-visual GPRK GPRK2 [141] and one non-visual Arrestin Kurtz [142] (called non-visual as they are not associated with photoreceptors). Embryos maternally and zygotically lacking GPRK2 fail to properly invaginate, showing a phenotype that could be interpreted as an overactivation of Rho1 in the mesoderm [143]. Moreover, GPRK2 genetically interacts with fog as the GRPK2 increase in myosin recruitment is suppressed by the removal of fog. Furthermore, embryos lacking Kurtz show a cuticular defect characteristic of embryos failing to properly invaginate [144], but the role of Kurtz in mesoderm invagination has never been studied in detail.

Intracellular pathways:

The developmental and cell surface signals lead to the activation of conserved intracellular pathways that positively regulate actin and myosin.

Rho1

Genes from the Ras homolog gene family (Rho) are small GTPases involved in the regulation of the cystoskeleton in different contexts, such as cell migration, cytokenesis and epithelial morphogenesis. In mammals the family is composed of around 20 members, of which RhoA (Rho1 in flies), Cdc42 and Rac1 are the most well studied. All members of the Rho family of GTPases are regulated by cycling from a GTP bound active state to an inactive GDP bound state. Guanine nucleotide exchange factors promote the exchange of GDP to GTP to activate the Rho protein, and GTPase activating proteins (GAPs) hydrolyse the GTP bound to the Rho protein to GDP, thus inactivating the protein. This cycle of activation and deactivation can be regulated by Guanine nucleotide dissociation inhibitors (GDIs), which bind to the Rho protein to prevent the exchange of the GDP [145]. The small GTPase, Rho1, is involved in the regulation of mesoderm invagination and the expression of a dominant negative form of Rho1 shows a similar phenotype that embryos lacking RhoGEF2 [146], [147].Thus, it is widely accepted that RhoGEF2 is a guanine nucleotide exchange factor (GEF) of Rho1. The GAP and the GDI of Rho1, involved in the regulation of mesoderm invagination, remain to be identified. Activated Rho1 has different effectors that lead to the positive regulation of myosin activity and actin stability and to the invagination of the mesoderm. Some of these effectors are shown in Figure 2.6: Rho1 activates the kinase Rok. Activated Rok then activates myosin directly by phosphorylating the regulatory myosin light chain (MLC),or indirectly, by inhibiting the myosin phosphatase, and stabilises actin filaments by activating LIM kinase (the inhibitor of the actin severer, adf/cofilin). Moreover, Rho1 promotes actin polymerisation by activating the formin (a group of

Other intracellular pathways

A range of other proteins are implicated in the regulation of mesoderm invagination, but their regulation and the way by which they are integrated into the different molecular cascades which regulate invagination remains unclear. One example is the non receptor tyrosine kinase, Abelson kinase (abl). Embryos maternally and zygotically lacking abl, show an uncoordinated apical constriction similar to that observed in embryos lacking concertina or fog. Moreover, the double abl, concertina loss of function embryo showed a stronger phenotype than any of the individual loss of function embryos, and resembled the loss of function phenotype of RhoGEF2 [153]. This lead the authors to conclude that abl acts in a parallel pathway to concertina and fog. Abl was shown to bind to actin [154] and to phosphorylate and inhibit the actin regulator, Enabled [155]. Grevengoed and colleagues propose that Abl regulates actin dynamics through Enabled, which in turn regulates mesoderm invagination. However, the mechanism by which Abl is activated in this context remains to be elucidated.

Another protein of unclear function, but which is involved in mesoderm invagination, is tumour necrosis factor (TNF) receptor-associated factor 4 (Traf4). Traf4 is a direct target of Twist and has been shown to interact with the adherens junction protein, Armadillo, to favour its apical relocalisation during mesoderm invagination (discussed in more detail below) [118]. The regulation of Armadillo was shown to be independent of the TNF receptor and the Jun N-terminal protein Kinase (JNK).

The actin severer Cofilin (Twinstar in Drosophila) and its activator Slingshot, have also been implicated in the regulation of apical constriction [151] (see the subsection Actin network organisation and MyoII recruitment for more details). How these actin regulators are regulated by Rho1 activation has not yet been investigated, and it is not clear whether the inhibitor of Cofilin LIM kinase is required for the regulation of mesoderm invagination.

Conclusions

To summarise; during gastrulation, developmental signals (lead by the dorso/ventral patterning cascade and the transcription factors Twist and Snail) control cell surface signals. These cell surface signals, involving the GPCR receptors, Mist and Smog, activate a conserved molecular pathway containing Rho1, which regulates different proteins that favour on one side the activity of myosin, and on the other side the formation of an actin network.

The following section of this thesis will discuss the mechanism by which actin and myosin regulation drives the cell shape changes observed in mesoderm invagination.

Apical constriction

Of all the shape changes described at the beginning of this chapter, the most studied and best understood is apical constriction. Apical constriction is the process by which the apical area of a cell is reduced, and can be observed during the formation of the ventral furrow. From Martin et al. 2009 [156] and Martin et al. 2010. [157] Chapter 2. Epithelial remodeling during gastrulation Genetic analysis has shown that apical constriction relies on the activation of Rho1 (by the molecular pathways described previously) leading to the formation of an apical supracellular network of actomyosin. This apical actomyosin network (exposed by MyoII in Figure (2.8a) ) spreads among the cells of the ventral mesoderm and displays pulsatile dynamics with phases of coalescence (seen by the increase of MyoII intensity) and stabilisation (Figure 2 and it is therefore proposed that its contraction and stabilisation generate the driving forces for apical constriction, and its ratchet like dynamics (Figure 2.8 d).

The evidence for the apical actomyosin network driving apical constriction is strong, however it is noteworthy to state that the idea that apical constriction is driven by the pulses of actomyosin comes from the correlation between the rate of constriction of a cell and the rate of increase of the myosin intensity [156]. This correlation is on average 0.5 [156], and not present in all constricting cells. This low correlation might be due to experimental noise.

This section describes in detail the mechanisms that drive apical constriction in drosophila during mesoderm invagination.

Actomyosin

Myosin

Myosin II is a molecular motor which is involved in a myriad of cellular processes. It is a hexamer composed of two heavy chains, two essential light chains (ELC) and two regulatory light chains (RLC) (Figure 2.9a). The N-terminal part of the heavy chain contains a globular domain which can interact with actin and ATP, and the C-terminal part is composed of α helical coiled-coil coil domains that allow the interaction between two heavy chains. Two IQ motifs, flanking the globular domain, allow binding of the RLC and the ELC. The activity of myosin is linked to its conformation state, whether it is bound to ATP or ADP, and whether it is bound to actin. ATP-bound MyoII binds to actin. The hydrolysis of the ATP and the release of the phosphate cause a conformational change of the heavy chain which is responsible for the displacement of actin ("the powerstroke"). Finally, the replacement of ADP by ATP triggers return to the initial conformational state of the heavy chain and the unbinding and rebinding of MyoII and actin [160]. A single molecule of myosin has low processivity [161]. However, two MyoII molecules can associate through their tail to form highly processive bipolar filaments, that attach to and pull antiparallel filaments of actin [162] (see Figure 2.9c). The association of several of these actin and myosin filaments form what is called an actomyosin network. The mechanical properties of an actomyosin network depend on: the activity of the myosin, the structure of the actin network (branched or unbranched) and the presence of actin cross-linkers. It is the modulation of the mechanical properties of actomyosin that is responsible (directly or indirectly) for most of the cell shape changes seen during mesoderm invagination.

Moreover, actomyosin can be associated to junctional proteins, this actomyosin will be termed junctional myosin, or can underlie the plasma membrane, covering the apical area of epithelial cells (called medial myosin [163]).

The regulation of Myosin activity by phosphorylation

The activity of MyoII is regulated by the phosphorylation of two highly conserved residues in the RLC: T18 and S19 [164] [165] [150]. Unphosphorylated MyoII acquires a conformation state with doesn't bind actin, and is thus inactive (see Figure 2.9a). The phosphorylation of MyoII modifies the conformation of the hexamer, allowing it to bind to actin and be activated (see Figure 2.9b). The inactivation of MyoII is mediated by myosin phosphatase. The kinases that activate MyoII include Rok, the citron kinase, MLCK (myosin light chain kinase) and MRCK. These kinases are context specific and are activated by different molecular cues (Figure 2.9a). The kinase relevant to mesoderm invagination is Rok. Myosin phosphatase is composed of three subunits: a catalytic subunit (PP1cβ, or Flapwing in Drosophila), a 20Kd protein of unknown function called M20 [166] and a myosin binding subunit (MBS) that gives the target specificity to the phosphatase [167]. Of note, there is no apparent homolog of M20 in Drosophila.

Actin network organisation and MyoII recruitment

Myosin bipolar filaments pull on actin to generate forces, hence the structure of the actin filaments is essential for the mechanical properties of the actomyosin network. Formins [168] and the WH2 containing proteins, Spire [169] and , nucleate actin to form linear bundles (Figure 2.10a See unbranched actin networks). In unbranched actin networks, contraction of the myosin bipolar filaments can reorganise the actin filaments in an antiparallel manner. The Arp2/3 complex promotes the formation of three dimensional branched actin networks [168] Figure 2.10b see branched actin networks), which are linked with actin cross-linkers and thus stiffer [171], needing severing and unbranching to allow for remodeling and contraction. The distinction between these two types of network is not always clearcut, and the presence of proteins able to branch (such as cofilin [172] )or unbranch the network create networks that are a mixture of both types. In the case of mesoderm invagination it was first suggested that the apical actomyosin network that drives apical constriction Chapter 2. Epithelial remodeling during gastrulation was composed of only unbranched actin filaments [152]. However, the discovery of the involvement of cofilin regulators in mesoderm invagination [151], suggests that the actin network is more likely a composition of both branched and unbranched filaments.

Actin cross-linkers

The composition of the branched and unbranched actomyosin networks are also impacted on by a range of actin cross-linkers that regulate network stiffness, such as filamin [173], α-actinin [174] and MyoII [175].

Actomyosin pulsatility and cell shape change

In order to understand cell shape deformation in the context of mesoderm invagination, it is necessary to explore the role of the actomyosin network in this process. This section will focus on the mechanism by which the actomyosin network generates pulses of contraction and relaxation, and the involvement of these pulses in cell shape deformation.

The generation of the myosin pulses

Actomyosin pulsatility is not exclusive to apical constriction. Work from the Lecuit laboratory has shown that, at least in the drosophila embryo, this pulsatility is an emergent property of the actomyosin network [176] (An emergent property is a property that a complex system acquires once the parts that make this system are combined. )

To understand the current model for actomyosin pulsatility [176], we have to consider that beneath the apical surface of the cell lies an actomyosin network, called the actin cortex [163] [177]. As myosin activity is tightly linked to its binding to actin, we can make the assumption that myosin cycles from an active, actin-bound form in this network to an inactive, monomeric state underneath this network (See Figure 2.11).

The activation and recruitment of MyoII into the actin network, and conversely its inactivation and dissociation, is regulated by several different proteins. In Figure 2.11 the activation and recruitment of MyoII by Rok (orange circle) is represented by an orange arrow. Its inactivation and dissociation by the myosin phosphatase (blue circle) is represented by a blue arrow. The affinity of these MyoII regulators can be assumed to be different and thus, in contexts where both activators and inhibitors are present and compete to affect myosin, the outcome of this competition can be assumed to be dependent on the concentration of myosin and on the concentration of the regulators.

The model from Munjal et al 2015 [176], supposes that when the concentration of active myosin and its regulators is low, the activators outcompete the repressors to induce recruitment of active myosin (Figure 2.11a). Moreover, when the concentration of active myosin and its regulators is high, repressors outcompete the activators to disassemble active myosin (Figure 2.11b). However, Munjal and colleagues do not clearly mention the molecular basis for this difference. For example; does the activity of the inhibitors increase when they are concentrated (by some sort of cooperative mechanism), or does activator activity decrease when concentrated? To add another layer of complexity, active myosin pulls on actin filaments. This pulling creates a movement of advection (Figure 2.11 Purple arrow) that concentrates the actin filaments in contact with the contracting myosin and the myosin regulators bound to them.

At the beginning of the pulse (Figure 2.11a), active myosin is recruited into the actin network. Myosin contracts and creates and advection movement that favours myosin recruitment and concentration of neighbouring myosin and its regulators. This generates a positive feedback loop that increases the concentration of myosin in one part of the actin cortex: an actomyosin pulse is born.

The positive feedback continues until it reaches a threshold (Figure 2.11brepresents the peak of the pulse), after which, activated myosin and its regulators are so highly concentrated that the inhibitors outcompete the activators and myosin starts to be disassembled. This is followed by the relaxation and depolymerisation of actin filaments and the decay of the pulse (Figure 2.11c). Then, the cycle can restart. However, as previously stated, actomyosin pulsatility does not necessarily translate into cell shape deformation. Apical constriction mediated by actomyosin pulsatility is dependent on two elements: the pattern, or regime, of pulsation, and the connexion between actomyosin and adherens junctions.

The regime of actomyosin pulsatility during apical constriction

The regime of actomyosin pulsatility corresponds to the biophysical conditions that define this pulsatility, its frequency and its amplitude [176]. This regime is under the control of the developmental and surface signals present in the cells of interest, which explains why myosin in the mesoderm (during apical constriction), the ectoderm (during germband extension) or the amnioserosa (during dorsal closure) behaves differently [128].

In the context of apical constriction, the alteration of developmental and surface signals affect, to different extents, the pulsatility of myosin. For example, myosin stops pulsing in embryos lacking Snail [156] and pulses in an un-ratcheted and uncoordinated manner in embryos lacking Twist [178] (Nonetheless, the exact mechanism by which the genes downstream of Twist and Snail molecularly regulate actomyosin pulses has yet to be determined). Moreover, in embryos where the phosphorylation of the MLC is mimicked or inhibited, the amplitude and the frequency of myosin pulses is altered. The removal of the Myosin phosphatase, or the chemical inhibition of Rok, also impacts on myosin pulsatility [150] [176].

One of the characteristics of actomyosin pulses in the mesoderm is that it shows ratchet-like dynamics and does not disassemble completely at the end of each pulse. This stabilisation of actomyosin is controlled by the Radial Cell Polarity (RCP) of constricting cells [149]. The apical domain of a cell can be divided into two regions; one in which the cell is in contact with other cells, the junctional domain; and the other inside of this junctional domain, the medio apical domain. In constricting mesodermal cells, RCP manifests by the exclusion of E-cadherin to the junctional domain of the cell, and of Rok and Rho1 to the medio apical domain. RCP is dependent on the transcription factors Snail and Twist, without them, Rok is no longer excluded to the medio apical domain. Instead, it forms aggregates in the junctional domain (interestingly Rok and E-Cad do not overlap in the junctional domain). RCP is also dependent on Diaphanous (Dia) and the presence of Actin filaments; the partial loss of Dia or the inhibition of Actin polymerisation, by the drug Cytokalasin D, completely abrogates RCP. In the above cases, with the exception of the Snail mutant embryos, abrogation of RCP correlates with pulsatile myosin failing to stabilise. This finding lead the authors, Mason and colleagues[149], to conclude that RCP is essential for the stabilisation of pulses and for the regime of actomyosin pulsatility, characteristic of apical constriction. A model of RCP, showing the stabilisation of Rok in the medio apical domain and favouring actomyosin stabilisation, is presented in Figure 2.12. The means by which Rho 1 is stabilised in the medio apical domain is unclear, but may be dependent on the GPCR receptor Mist which is exclusively expressed in the mesoderm. Overall, the role of GPCR receptors in RCP remains a mystery.

The role of junctions in apical constriction

The coupling of actomyosin to adherens junctions is another element involved in the generation of apical constriction by myosin pulsatility. This coupling, termed molecular clutch [179], translates deformation of the actomyosin network occurring in the medio apical domain, into deformation at the level of the junction of the cell, resulting in a cell shape change. Initial experiments to study apical constriction in C. elegans showed that the molecular clutch was transient, and that a pulse of actomyosin didn't necessarily pull the cell junction [179]. In Drosophila, it was first suggested that the molecular clutch between actomyosin and adherens junctions was also transient [177]. However, detailed analysis of the actomyosin pulses and their effect on cell shape showed that, at least during mesoderm invagination, the molecular clutch is always present and a pulse of myosin always translates into a pulling of the junction and subsequent reduction of the apical area [178].

Armadillo zygotic mutant FIGURE 2.13: Electron microscopy of a gastrulating embryo lacking armadillo zygotically .

Figure from Martin et al. 2010 [157].

The integrity of adherens junctions is essential during the molecular clutch process to sustain the forces generated by the actomyosin contraction. In zygotic mutant embryos for armadillo (βcatenin in drosophila), the molecular clutch breaks under the effect of the contraction of actomyosin and membrane tethers appear (see Figure 2.13. These tethers correspond to the places where actomyosin is still connected to the adherens junction) [157]. The formation of tethers is also observed in embryos knocked down (RNAi) for slingshot and capulet [151]. Slingshot (Ssh) is a phosphatase that activates the actin severer, Cofilin [180]. Capulet (Capt, also known as cyclase-associated protein or CAP) is a protein that favours actin de-polimerisation [181] and Cofilin-mediated severing. Live imaging of embryos with reduced levels of Ssh or Capt, showed that cells try to repair the broken link between actomyosin and adherens junctions by reforming an actomyosin network [151]. Thus, a strong attachment between the actomyosin network and the adherens junction is required for proper apical constriction. This strong attachment is dynamic and is achieved by the turnover of filamentous actin.

Furthermore, adherens junctions (see Chapter I for a detailed description of adherens junction components) show a dynamic localisation during gastrulation.

At the onset of apical constriction, they localise slightly basally (see the grey arrowhead in Figure 2.13a) but as constriction progresses, prior to the formation of the ventral furrow, they undergo a shift toward the apical part of the cell (black arrowhead in Figure 2.13a). This basal to apical shift, was initially interpreted as a controlled junction disassembly, followed by reassembly in the most apical part of the cell: embryos lacking regulators of apical constriction, such as RhoGEF2 and T48, disassemble adherens junctions in a Snail dependent manner [124].

Live imaging allowed a more detailed analysis and indicated that junctions progressively shift apically without disassembling (Figure 2.13b). Furthermore, this shift showed pulsatile dynamics, correlated to a certain extend (correlation = 0.5) with the actomyosin pulses (Figure 2.13c) and was disrupted if myosin levels are reduced by RNAi. This suggests that myosin pulls on the junctions. An alternative model for the role of adherens junctions in apical constriction is as follows: The actomyosin network is connected to spot junctions, and pulses in a manner which pulls these junctions from the basal towards the apical domain. This shift in adherens junction localisation is followed by the reduction of the apical cell surface and cell constriction [182]. It is noteworthy to state that this model is based on a correlation between myosin increase rate and the rate of junction shift which is relatively low.

Additionally, basal shift of the junctions, via actomyosin contractility, appears to protect them from Snail mediated disassembly [182]. This observation might explain why in RhoGEF2 and T48 embryos, where there is no increase of actomyosin contractility in the apical part of the mesoderm, the junctions disassemble. Interestingly, the molecular target of Snail involved in junction disassembly (only when there is no actomyosin contractility) is yet to be identified.

The integration of apical constriction at the tissue level

To understand the mechanism behind ventral furrow formation, it is necessary to explore how individual cell contractions can be translated into whole tissue movement. Secondly, the constrictions are anisotropic on the ventro-lateral axis [156], [183], making constricting cells elongate along the antero-posterior axis (see (Figure 2.15). Anisotropy can be defined as the property of depending on direction, in contrast with isotropy. Guglielmi et al 2015 [185] used optogenetic tools to inactivate myosin contractility and change the shape of the region undergoing apical constriction. They showed that if the shape of the region containing the constricting cells is changed from a rectangle (with the length on the antero posterior axis, and the width on the ventro lateral axis) to a square, the constriction of the cells became isotropic. Dependence of anisotropic constriction on the topology of the constricting tissue was already predicted by the vertex model of apical constriction of Spahn et Reuter [183]. Effect of a constricting cell on its neighbour

Description of the constriction at the tissue level

In 1991, Sweeton et al. [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF] proposed a "two phase" model of cell constriction. Using electron microscopy they analysed fixed embryos at different stages of ventral furrow formation. They observed that prior to ventral furrow formation, constricting cells were limited to a random population; however, in later stages, they noted a collective and simultaneous constriction of mesodermal cells. Interestingly, in fixed twist mutants, constricting cells were limited only to a random sub population and collective constriction was never observed. These observations lead them to propose that there are two phases of constriction; a first in which a subpopulation of cells constrict Chapter 2. Epithelial remodeling during gastrulation randomly; and a second in which all the cells from the ventral mesoderm constrict together in a coordinated manner. Furthermore, they hypothesised that the passage from one phase to the other was dependent on Twist.

However, advances in imaging techniques, allowing for real-time imaging of ventral furrow formation, have since prompted the revision of this model. Live imaging and systematic analysis of the myosin pulses and apical constriction [178] have shown that there are no different subpopulations of mesodermal cells. (This doesn't mean that fixated twist embryos do not show constricted and unconstricted cells).

On the contrary, the systematic analysis of myosin pules and cell constriction suggest that the cells in the most ventral part of the mesoderm behave in a similar manner, with the regulation of collective apical constriction occuring at the cellular and at the multicellular level (Figure 2.17). (It is important to state that the work by Xie and colleagues doesn't take into account the gradient of constriction seen in Spahn and Reuter 2014 [183], as they only focus on the most ventral cells of the mesoderm). At the cellular level, Xie and colleagues propose that on average, the actomyosin pulses and the constriction seem to follow a specific pattern (Figure 2.17a); at first, actomyosin pulses are weak in amplitude and infrequent, but as time passes they tend to become stronger and more frequent. A pulse of myosin seems to be translated, almost every time, into a reduction of the apical area, but the reduction of the apical area is not always stabilised (or ratcheted).

Initially, myosin pulses are un-ratcheted (not stabilised) and tend to become progressively more ratcheted (stabilised). In wild-type embryos, the passage from a weak, infrequent, unratched pulse to a strong, frequent and ratcheted pulse tends to be continuous and unidirectional. In other words, once a cell is generating strong pulses, it will most likely not revert to generating weaker ones. However, in twist mutants, the directionality of pulse amplitude is perturbed, and cells can switch from the generation of strong to weak pulses. This results in a mixed population of constricting cells, some producing high amplitude, strong, frequent pulses; and others producing weaker, infrequent pulses-which ultimately leads to a lack of coordinated apical constriction.

Apical constriction is also regulated at a multicellular level (Figure 2.17b): weak unratcheted myosin pulses tend to inhibit pulses in neighbouring cells, whereas strong, ratcheted myosin pulses tend to favour production of similarly high amplitude pulses in neighbouring cells. The mechanisms behind this regulation are not known, but they point to some kind of regulation dependent on mechanotransduction.

The role of Mechanical Inputs for apical constriction

The idea that mechanotransduction could be implicated in apical constriction was first proposed in 1981 [186], and several more recent publications have demonstrated that it indeed plays a role, to a certain extent. Pouille et al. 2009 [187] reported that the defect in invagination, seen in snail embryos, could by rescued by generating a small indentation in the ventral part of the embryo. This indentation was, according to the authors, sufficient to cause an increase of myosin accumulation at the apical surface (See Figure 2.18a), suggesting that the generation of a deformation, or a tension increase, is sufficient to induce accumulation of myosin. However, a certain amount of bias was present in the way by which the snail embryos were selected (embryos were selected based on the phenotype they showed, as opposed to by genotyping). Moreover, it has been shown that the topology of the constricting area is important for the formation of the furrow, and it is somehow counterintuitive that the indentation in a specific part of the furrow is sufficient to rescue the constriction along the whole antero-posterior length of the furrow.

In a slightly different context, Fernandez-Gonzales [188] used a micropipette to pull on the vitelline membrane of embryos undergoing germ-band extension. They observed that the generation of a deformation induced accumulation of myosin (See Figure 2.18b).

The above work has prompted the development of several hypotheses which aim to explain the recruitment of myosin in response to a mechanical stimulus increase in tension [190]. For example, the Farge laboratory proposed that the change of tension of the apical membrane could reduce the endocytosis of the GPCR receptors, allowing the active receptor to signal for longer, and thus increasing the levels of activated Rho1 [191]. Another, non exclusive possibility, is that the increase of tension could affect myosin itself by increasing the duration to which it is bound to actin [192]. Finally, tension could promote the secretion of some chemical signal that would promote myosin contractility [193].

These papers suggest that at a cellular level, the regulation of myosin activity, and thus its pulsatility, can be influenced by mechanical inputs derived from neighbouring cells. However, the effect of a mechanical input on apical constriction at a tissue level requires further investigation. This is highlighted when considering the effect of a constricting cell on its neighbour.

In the context of apical constriction, it is conceivable that the constriction of a cell creates an increase in tension which could serve as a mechanical input on neighbouring cells, thus resulting in the recruitment of myosin and the generation of a pulse in the neighbouring cell. Although this theory could serve to explain the propagation of high amplitude pulses amoung neighbouring cells observed by Xie and Martin [178], the inhibition of weak myosin pulses between neighbouring cells in response to weak cell constrictions is more puzzling.

Other morphogenetic movements generated by apical constriction

Apical surface reduction in ventral mesodermal cells is not the only cell-shape change that occurs during mesoderm invagination, cells from the lateral mesoderm elongate apically towards the furrow, and cells from the ventral mesoderm elongate along their apical/basal axis [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF]. Current data suggests that these cell shapes could be generated as a consequence of the apical constriction occurring in the ventral mesoderm. However, it remains to be determined whether or not apical constriction is the only driving force behind mesoderm invagination.

Hydrodynamic flow

He et al. 2014 [194] investigate the effect of cytoplasmic fluid flow as an additional driving force during mesoderm invagination. To do so they injected fluorescent beads into the cytoplasm and observed a hydrodynamic flow in the cytoplasm of ventral cells, as shown in (See Figure 2.18a). Following this, they studied double mutant embryos for dunk and slam, that failed to cellularize. These embryos didn't have lateral membranes in any of their cells, but nonetheless were able to invaginate to a certain extent and to show a hydrodynamic flow ((See Figure 2.18b)).Furthermore, the flow was blocked in the absence of the genes upstream of apical constriction (T48 and concertina), and the acellular embryos were shown to form an apical actomyosin network and to apically constrict. From this, the authors concluded that apical constriction was responsible for the hydrodynamic flow, in a lateral-membrane independent manner, and it was suggested that this hydrodynamic flow was sufficient to generate the initial force for the buckling and the invagination of the mesoderm. This was termed the volume conservation principle [195] and secondly; apical constriction generates a cytoplasmic flow from apical to basal (last subsection [194]). From these two observations, Gelbart and colleagues proposed that this apical to basal cytoplasmic flow stretches the cells along their apical basal axis, allowing the cells to maintain a constant cytoplasmic volume [195].See Figure 2.21a. This cytoplasmic flow and volume conservation theory, could also explain the nucleus migration (from apical to basal) observed during cell elongation. [196], [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF].

The above is an appealing hypothesis, however, it is possible that other mechanisms (such as microtubule polymerisation, myosin sub-cellular relocalisation or the regulation of cell adhesion) could act in in parallel to regulate mesodermal apical/basal elongation [197]. For example, microtubule polymerisation (See Figure 2.20a) has been shown to control cell height in zebrafish [198] and to control medio apical actomyosin pulsatility during the apical constriction in the drosophila salivary gland [199]. Myosin relocalises laterally to promote apical/basal shortening in ascidian gastrulation [200] Additionally, the regulation of cell adhesion in the lateral membrane could, in theory, stimulate elongation or shortening of the apical/basal axis [201] .

Apical/basal shortening

Immediately after the cells from the mesoderm apically constrict and elongate, they start to shorten on their apical/basal axis and expand basally. Polyakov et al. 2014 [184] explored the mechanism behind apical/basal shortening. They propose that during apical/basal elongation, cells accumulate energy in their lateral membrane due to stretching. In other words, behaving as a tensile element (see the next chapter for more details), the stretched lateral membrane tries to go back to its original length, but cannot because the apical and the basal surfaces of the cells are stiff and non deformable. Taken from Kondo et al. 2015 [197] As apical constriction advances, the basal myosin is disassembled (the intensity of the basal myosin is measured in the paper and seen to decrease with time.) This reduction of basal myosin would relax the basal membrane, create an imbalance in the forces present in the different membranes of the cell, and cause a release of the energy of the lateral membrane that will shorten to go back to its original length. Based on the volume conservation principle, this would lead to a basal expansion. See Figure 2.21b

The elongation of the lateral mesoderm Cells from the lateral mesoderm do not apically constrict, but elongate or stretch towards the furrow (see yellow cells in Figure 2.1d) [98] [153] [143] [99]. Apical constriction seems to precede this stretching [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF], and the stretching is lost in embryos that do not apically constrict (reinterpretation of [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF]). So it would seem that the elongation is a consequence of the deformations caused in the ventral mesoderm by apical constriction. This remains to be directly tested. Moreover, Fuse and colleagues [143], showed that in the absence of the G protein receptor kinase GPRK2, the lateral mesoderm didn't elongate but started to apically constrict, and mesoderm invagination was perturbed. GPRK2 was shown to inhibit GPCR receptor activity [139],[140] and GPRK2 genetically interacted with fog to control mesoderm invagination. From this, the authors proposed that GPRK2 thresholds Fog activity in the mesoderm to restrict it to the ventral mesoderm and allow a proper ventral furrow formation and ventral invagination. It remains unknown whether or not GPRK2 blocks Mist, Smog or both.

Is apical constriction the only driving force? What is the role of the ectoderm?

Most of the cell/shape changes described so far seem to be primarily driven by apical constriction in the ventral mesoderm. However, some of the groups trying to model Kondo et al. 2015 [197] mesoderm invagination (see next section) suggested an active role of the ectoderm in the process. This idea was explored by Rauzi and colleagues [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF]. They observed that there were two phases in gastrulation, one of ventral furrow formation and a second one of mesoderm invagination. During the first phase the ectoderm doesn't move, and in the second phase it moves toward the ventral midline while the mesoderm invaginates.

Cell transplantation experiments [202], proved that all of the information to make a cell apically constrict and elongate is located in the mesoderm, and thus the first phase of gastrulation-ventral furrow formation -is only dependent on the mesoderm. Furthermore, the genetic ectopic expression of fog in the ectoderm[125], the ventralisation of the ectoderm [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF] or cauterisation of the ectoderm [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF] affected the movement of the ectodermal cells and impaired the second phase of mesoderm invagination.

Thus the second phase of gastrulation is dependent on both the mesoderm and the ectoderm.

These results raise the following questions: What is the role of the ectoderm during the second phase of gastrulation? Is the ectoderm playing an active role by pushing the mesoderm, or it is playing a passive role by being pulled by the mesoderm? Independent of the answers to these questions (which are not known), these observations suggest the existence of molecular pathways regulating the ectoderm to actively push or to regulate its stiffness while it passively deforms. These molecular pathways remain to be discovered.

Physical models of mesoderm invagination

Modeling is widely used to study morphogenic events and presents several advantages compared to experimental approaches: firstly, it allows us to clearly state all of the assumptions that are being made in the interpretation of a process. It grants the possibility to play with different parameters of the studied system in a way that would be impossible otherwise. Finally, it enables us to make non trivial predictions that can be directly tested in a biological sample. Due to the relative simplicity of drosophila gastrulation-a single layered ovoid structure undergoing extreme shape remodelling, where there is no cell neighbour exchange or cell division-ventral furrow formation has been widely modeled during the past years. All of these models have relied on physical concepts taken from the study of fluids and elastic material (The definition of Stress, Contractility, Elasticity, Viscosity and Stiffness is given in Figure 2.22) and on the use of different approximations (subdivision of the epithelium, yolk incompressibility...) The different approximations used in these modeling systems, along with a comparison of the different models and the manuscripts from which they derive, will be discussed in the following section. A recapitulative table, based on [203], is shown in Figure 2.23.

Stress:"A force per unit area. Stress can be active (e.g.contractile) or arise as a passive (e.g. elastic) response to deformation" [163]. Contractility:"Capacity of an object to contract autonomously, by opposition to compression in response to external forces" [159]. Elasticity:"Capacity of an object to recover its original shape after deformation". This often involves the capacity to store the energy of the deformation [159]. Viscosity:"Internal resistance to deformation due to friction among constituents of a material" [159]. In opposition to elastic objects the energy of the deformation is lost in a purely viscous object. Stiffness:"Resistance of an elastic object to deformation" [159].

General concepts Subdivided Epithelium:

The first difference between the models is whether or not the authors make a distinction between the mesodermal cells and the ectodermal cells, or whether they consider the epithelium as a whole. This has significant consequences on how the process has to be imagined. The definition of mesoderm and ectoderm is based on the final location of the different cell groups: ie the localisation of the mesoderm inside the embryo and the ectoderm outside. It is accepted that, at the onset of gastrulation, the expression of transcription factors molecularly distinguish the mesoderm from ectoderm. This translates into differences in contractility and stiffness between the two germ layers, and then into the generation of forces that drive invagination. Interestingly, Hocevar Brezavscek et al 2012 [204] observed that, in a model exploring the minimal conditions required for ventral furrow formation, distinction between the mesoderm and ectoderm was not necessary for furrow formation. They observed that instability of the cells was sufficient to cause invagination.

Kinematic vs dynamic description:

The second difference lies in how the deformations are studied. If the deformations are imposed on the initial conditions, without trying to study or understand the causes of these deformations, the model is considered a kinematic description. If, in contrary, the causes of the deformations are also studied, for example by studying the forces that generate them, the model is considered a dynamic description. For example, some groups [184], [205], model apical constriction by increasing the apical surface tension of the mesodermal cells. These models are then considered to be a dynamic description. Conversely, other authors [206], [207] reduce the apical surface of mesodermal cells and study the effect on the morphology of the gastrulae. These are kinematic descriptions.

Mechanical Behaviour:

During development, cells can be considered to have viscoelastic properties [163]. Over short time scales they behave as elastic materials, as they are able to return to their original form after a deformation. Over longer periods, they deform in a viscous manner, losing the energy of the deformation in the form of cell rearrangements and cell division. The different authors presented in this chapter considered the embryo as a purely elastic material, a purely viscous material or the mixture of both, thus integrating or not in their models the dissipation of energy and/or the storage of energy.

Depending on the model, the embryo is considered as a purely elastic material, a purely viscous material or the mixture of both. Thus, for each model it must be decided if the dissipation and/or the storage of energy, is integrated into the model.

Cytoplasm incompressibility:

Using biphoton microscopy and image processing, the Wieschaus lab [195][184] showed that, during ventral furrow formation, the cells of the mesoderm maintain the same volume regardless of modification to their shape, in other words, they are incompressible.The authors suggest that volume conservation of the mesoderm is crucial in the process by which forces generated by apical constriction, cause apical/basal lengthening of the mesodermal cells. However, the incompressibility of the cytoplasm is not implemented in all of the models.

Discrete or continuous medium:

The embryo can be modeled as the sum of discrete, interconnected cells (discrete medium) or as a continuous material (continous medium).

Bulk/Surface elasticity:

In the elastic models, the passive deformations can affect only the surface or the bulk of the material. If the epithelium is modeled as a system of interconnected springs where each spring is a cell membrane, then the passive deformations are associated with surface elasticity. On the other hand, if the epithelium is presented as a continuous body with elastic properties then the passive deformations are associated with bulk elasticity.

Yolk and vitelline membrane:

In nearly all of the models the vitelline membrane is considered as a non deformable barrier surrounding the embryo. Some papers [204][206][208] explore the consequences of removing this membrane or of altering its rigidity. One paper [186] ignores its existence.

As for the yolk, it is mostly treated as a non-compressible material, with the exception of [103]. In Conte el al.'s model, the yolk can be slightly compressed to account for a yolk flow from the centre toward the extremities of the embryo, and in the model of Polyakov et al. 2014 [184] the yolk generates an outward push to the ectoderm.

Two dimensions versus three dimensions:

Mesoderm invagination was modeled, for numerical simplicity, mostly in two dimensions and considering only a cross section of the embryo. Many of these 2D models give a close representation of the in vivo process, therefore the addition of a third dimension to the modeling technique is not necessarily required. Nevertheless, a yolk flow from the middle to the extremities of the embryo is only predicted in a 3D model [208].

Different models

An outline of the published ventral furrow formation models, along with their strengths and criticisms, will be presented in the following section.

Exitable epithelium

In 1981, Odell et al. [186] published the first model for ventral furrow formation. They considered only a cross section of the embryo, and each cell was represented by a trapezoidal spring-and-dashpot viscoelastic mechanical unit. The cells from the mesoderm had the property to be excitable; if their apical length exceeded a threshold they started to reduce their apical length. The reduction of apical length was firstly imposed in two cells in the middle of the mesoderm. This resulted in stretching of the neighbouring cells until their apical length exceeds a threshold and their reduction of apical length was fired. The reduction of apical length propagated this way among the cells of the mesoderm and caused the invagination of the mesoderm. The final shape of the embryo resembles that of which is observed in vivo, however the intermediary shapes do not. Nonetheless, this paper is of great value; the insights gained from this model allowed the author to (correctly) hypothesise that the cytoskeleton was modulated in the apical side of the mesoderm to generate apical constriction, several years before myosin was actually seen at this stage [209] or proven to generate active deformations [156]. Moreover, the authors proposed that the reduction of apical area is the only source of active deformation, a statement that is still being debated 45 years later. Finally, the authors suggested that the activation of apical constriction spreads between cells in the form of a wave, a hypothesis that turned out to be false.

Active epithelium

Several years later, Munoz et al. [206] created a model which focused on a different approach. Ignoring the causes of the apical deformation, they carried out a kinematic description of the process. The embryo was considered as an elastic, non-viscous continuum, neglecting cell boundaries. The authors divided the embryo into mesoderm and ectoderm cells, and imposed apical constriction and apical/basal lengthening in the mesoderm, and apical/shortening in the ectoderm. Their model succeeded in the reproduction of the shape of an embryo to a certain extent; however, the buckling, in the region that connects the ectoderm to the mesoderm in the yolk, doesn't resembled that of a fixed cross-section. The model presented by Munoz and colleagues predicts a pushing role from the ectoderm. It is not completely clear whether this fits with experimental data: in 1990 Leptin et al. claimed that ventralised embryos, where the ectoderm cells are transformed into mesoderm cells, do form a ventral furrow and invaginate [START_REF] Leptin | Cell shape changes during gastrulation in Drosophila[END_REF]. However, in 2015 Rauzi et al. [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF] re-analysed these embryos using live imaging and, although the formation of the ventral furrow was observed, a kymograph (of the process) suggested that the mesoderm was pulled out at later stages. In 2009, Conte et al.[210] used the same model to explore, in a systematic manner, the effect of varying the values of apical constriction and apical/basal lengthening/shortening on mesoderm invagination. The authors concluded that apical constriction alone was not sufficient to create a deep furrow, that ectoderm shortening was required for the proper formation of the furrow, and that the process attained its maximal robustness when the three forces generating active deformation (apical constriction, mesodermal apical/basal lengthening, and ectodermal shortening) were present in similar strengths.

3D model:

Building on the model described in the previous subsection, the Miodownilk group modeled mesoderm invagination in three dimensions [208]. Their work demonstrated that it is possible to use the same set of assumptions used in 2D modeling, to generate a 3D model. Their 3D model predicted a yolk flow inside the embryo. This prediction, which has never been confirmed in vivo, suggests that in 2D models yolk should be considered as compressible to account for the flow seen in three dimensions.

Framed mesoderm:

In 2010, another group modeled mesoderm invagination in three dimensions [207]. Taking a similar approach to that of the Miodownik group, Allena et al. considered the embryo as an elastic continuous material. However, their model focused solely on the mesoderm, and they imposed a non deformable ectoderm and vitelline membrane (hence the name framed mesoderm). Additionally, the model imposed apical constriction and apical/basal lengthening to the mesodermal cells to create an invagination which only partially resembles in vivo invagination. The discrepancy between the model and in vivo results, may be due to the consideration that the ectoderm in non deformable, and that it doesn't play a role in mesoderm invagination.

Flow assisted invagination:

Pouille and Farge, [205], took a modeling approach more similar to that presented by Odell et al. [186]: the embryo is studied as a 2D cross-section, cells are considered as trapezoidal springs and dashpot viscoelastic mechanical units, where the apical, basal Chapter 2. Epithelial remodeling during gastrulation and lateral sides of the cells are under a given tension. They added a system of active spring at the level of the adherens junctions. Apical constriction was simulated by increasing, 100 fold, the tension of the apical sides of the cells from the mesoderm. By adding a centripetal viscous flow to the cytoplasm of the cells and to the yolk, they succeeded in inducing the formation of a ventral furrow. However, this ventral furrow is much shallower than that of the in vivo embryo. The difference in cell shape in the furrow suggests the implication of other mechanisms (probably present in the mesoderm or in the ectoderm).

Video force microscopy:

Video force microscopy (VFM) was introduced by Brodland et al. [211] to study mesoderm invagination. This technique uses images of live embryos to infer the different forces generated. The embryo is subdivided into discrete cells and the displacement of the cell boundaries is measured. By combining the measurement of the displacement with a known value of the viscosity, the value of the physical forces can be extracted. The authors found that these forces were strongest in the apical and lateral membranes of the mesoderm and in the basal part of the ectoderm. In a model termed "forward model", Conte et al. [103] used an approximation of the measured forces in the Brodland paper to model mesoderm invagination in a purely viscous system. The authors made use of four forces which actively contribute to mesoderm invagination: apical constriction, apical/basal shortening of the mesoderm, ectoderm apical/basal shortening and ectoderm basal constriction. The result was an embryo which overall resembles an in vivo embryo, with the exception of the ectodermal cells in contact with the mesoderm, which deform in an unusual manner. In following experiments, alteration of the strength and relative timing of these four forces resulted in the accurate reproduction of mutant embryo shapes for genes implicated in mesoderm invagination. This model is of particular interest, as it could be used to explore the forces behind poorly understood embryo shapes observed in vivo. One downfall of this model is the use of approximate force values, measured by VFM. It is unclear whether or not these approximations are justifiable. For example, apical constriction has been shown to form a gradient in the mesoderm, with constriction at its strongest, closest to the ventral midline [184], [183]. However, in the model presented above this gradient is not represented, and the force of constriction is seen as constant throughout all mesodermal cells. It remains to be determined whether or not the absence of this gradient is of great importance.

Collective Instability:

Hocevar et al. [204] studied the minimal requirements for a mesoderm to invaginate. They took a 2D representation of a cross-section of an embryo in which every cell was represented by a trapezoidal structure containing elastic properties. All cells were considered as mechanically identical and no distinction was made between the mesoderm and the ectoderm. Tension was imposed on the basal, apical and lateral side of all of the cells and the system was allowed to evolve until it reached an equilibrium. When the relative apical-to-lateral and basal-to-lateral tensions are large the structure doesn't change much; however, when the relative apical-to-lateral and basal-to-lateral tensions are small the system becomes unstable and buckles in a random place. To generate the buckling in the mesoderm, an asymmetry in the system had to be created. From this, the authors observed that the cells from the mesoderm cellularize faster than the rest of the cells, and thus at the onset of gastrulation they are longer [START_REF] Sweeton | Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations[END_REF]. This asymmetry was implemented in their model and shown to be sufficient to generate an invagination of the cells from the mesoderm. The validity of this model still has to be studied in vivo, however, it shows that at least theoretically, the formation of an apical acto-myosin network generating apical constriction is not required for the buckling of a tissue. It may be that this collective instability ensures robustness of mesoderm invagination.

Apical constriction and passive basal forces

Finally, the team of Eric Wieschaus proposed a purely elastic two dimensional model, where the cells were represented as discrete mechanical units of incompressible material [184]. The only active deformation imposed was apical constriction in a graded manner, with the cells closest to the ventral part of the mesoderm constricting most tightly.

The model also incorporated a decrease in basal membrane stiffness over time and an increasing outward push derived from the yolk. The resulting model was able to generate embryo shapes that closely resemble those seen in vivo. The authors chose to reduce basal membrane stiffness in this model due to the observation that in immunostained cross-sections, basal myosin progressively decreases in the mesoderm [184]. The forces generated in this model seem to be consistent with those measured by VFM in the Brodland paper [211], with the exception of basal forces in the mesoderm. The discrepancy in the measurement of mesodermal basal forces might be due to the fact that the model by Conte et al. 2008 is based on a viscous system, whereas Polyakov's model is based on an elastic system [184]. Discrepancies could also derive from errors in the VFM measurements.

Conclusions:

Due to the relative simplicity of gastrulation, modeling of this process has become increasingly popular and various research groups have adopted a range of very different approaches and approximations to model this process.

Nonetheless, several important questions need to be addressed in order to further improve the modeling of gastrulation. For example: Is apical constriction the only mechanism generating active deformations in the system? Does the ectoderm play an active role, or is it passively deformed by the movements occurring in the mesoderm? And finally, what is the molecular basis of the ectodermal apical/basal shortening?

Independently of these open questions, mesoderm invagination is an excellent example of how modeling has been used to advance our understanding of a developmental process. 2.5. Concluding remarks 51

Concluding remarks

To conclude; the folding of the epithelia is a relatively simple process which is driven by a complex set of cell shape changes. Most of the molecular pathways behind these shape changes are known, but biophysical modeling has highlighted that there are still some unresolved points. Moreover, an interesting point highlighted by the above mentioned papers is that some of the pathways implicated in gastrulation are conserved molecular modules reused repeatedly throughout development to shape an embryo. Therefore, embryo morphogenesis can be seen as a set of developmental cues encoded by a genetic program, that regulates conserved molecular modules in space and time. Examples of conserved modules implicated in morphogenesis include actomyosin pulsatility, cell division, cell death or epithelial apical basal polarity.

Thus, by studying a conserved molecular module (actomyosin pulsatility), in a very particular developmental process (the folding of a tissue at the beginning of drosophila's development), we are probably learning much more than what we think about embryo development and morphogenesis in general. E3 ubiquitin ligase is an enzyme that catalyses the transfer of a free ubiquitin to a protein. This transfer is a multistep process involving several enzymes (Figure 3.2). Ubiquitin is activated by the enzyme E1, which hydrolyses ATP and promotes subsequent binding of ubiquitin to the "ubiquitin conjugating enzyme", E2. An E3-ubiquitin ligase, containing a RING (Really INteresting Gene) domain (such as Neur) then catalyses the transfer of the ubiquitin from the E2 enzyme to the -side chain of a lysine in a protein, or to another ubiquitin. E3-ligase containing HECT (Homologous to the E6-AP Carboxyl Terminus) domains behave slightly differently as they bind directly to ubiquitin before transferring it to the substrate. It is the E3-ligase that determines the specificity of the substrate for ubiquitination. Finally E4, or "ubiquitin chain assembly factor" can modulate the formation of ubiquitin chains. Ubiquitination is a reversible process and ubiquitin can be cleaved from its substrate protein by the activity of deubiquitins (DUBs).

The neur gene encodes two isoforms, PA and PC, which differ in their C terminus sequence and by the presence of a C-terminal Phosphoinositide-Binding (PIP2) Motif. This PIP2 motif allows the PA isoform to bind to the plasma membrane [219]. It was proposed that singular Neur proteins are held in an inactive conformation through NHR1-NHR2 interactions and that Neur is activated through NHR2-NHR2 interaction with another Neur protein [220].

Neur is a positive regulator of Delta-Notch signaling, and flies lacking neur show the same phenotype as those defective for Delta-Notch signaling [215]. Experimental Furthermore, it was shown that the endocytosis of Delta is dependent on Epsin and that Neur loss of function alleles are dominant enhancers of Epsin loss of function alleles [224]. As Epsin recognises mono-ubiquitinated proteins [225] and promotes their endocytosis, it was proposed that Neur mono-ubiquitinates Delta to promote endocytosis by Epsin. However, the direct ubiquitination of Delta by Neur has never been proven, nor whether it mono-ubiquitinates or polyubiquitinates its targets. Daskalaki et al. (2011) [226] carried out an ubiquitination assay which shows that Delta is ubiquitinated by Neur. However, the study lacks several controls and thus leaves room for questions. Lai et al. 2001 [215] proposed that Delta was being degraded after being ubiquitinated by Neur, based on the observation that the ectopic expression of Neur was sufficient to reduce the detectable levels of Delta in immunostainings. Whether the ubiquitination of Delta causes its relocalisation or its degradation has never been fully investigated.

The Free NICD relocates to the nucleus and binds to the transcription factor CSL (CBF1, SU(H),LAG-1) and to activators, to trigger the expression of Notch target genes. Of note, when NICD is not in the nucleus, CSL binds to repressors and inhibits the expression of the same target genes (reviewed in Schweisguth 2004 [228]).

The Brd family of genes inhibit Neur activity

The Brd family of genes is composed of eight genes: mα, m4, m6, BobA, BobA, Brd, Tom and Ocho, located in two gene clusters [229].

In 2000, Tom was the first of the Brd genes shown to give a phenotype in the drosophila notum when overexpressed. This result was interpreted as an antagonistic role for Tom on Delta-Notch signaling [230].

Some years later, a screen for protein interactors identified Tom as a putative binding partner of Neur [231] and in 2006, Bardin and Schweisguth [232] confirmed that all of the proteins of the Brd family interacted with Neur by using yeast-two hybrid and immunoprecipitations in HEK cells. Furthermore, using immunoprecipitations in HEK cells, the Brd proteins were shown to bind Neur through the NHR1 domain and to compete for the Neur-Delta interaction [232]. Bioinformatic analysis of the sequences of Delta and the different Brd genes in different species identified a common sequence, [E,D or Q],N, x, x, N, x, x, [L, M or V], in the regions required for the binding of the proteins to Neur. This sequence was proposed to be a Neuralized binding motif[233], [232].

Moreover, the ectopic expression of the Brd genes in the Wing Imaginal discs was seen to block Delta endocytosis, and the removal of all of the Brd genes in the early embryo resulted in an increased Delta endocytosis in the ectoderm, in a Neur dependent manner. [222]. Finally, in 2009, Chanet and colleagues [229] used an elegant genomic engineering system to remove the two gene clusters containing the Brd genes. From this work, they showed that the different members of the Brd family of genes were redundant, as flies lacking either mα m4 and m6 or BobA BobB, Brd, Tom and Ocho had Chapter 3. Neuralized and the Neuralized family of genes only reduced viability, and that it was necessary to remove all of the Brd genes to observe complete lethality.

The current model derived from these results is that the Brd genes act redundantly to inhibit Delta-Notch signaling by binding to Neur and blocking its interaction with Delta.

Neur activity in the early embryo, and the specification of the mesectoderm

The protein Neur is deposited maternally and neur transcription is positively regulated by Twist (Figure 3.4A). The transcription of the Brd proteins is inhibited by Snail, and so the Brd genes are expressed in the ectoderm (Figure 3.4B).

Background information

My thesis is a follow-up of the paper by Chanet and Schweisguth [222] and revolves around the questions that arise from the results of this paper. The principal results and discussions from that paper are presented in the following section.

Neur is a regulator of epithelial polarity

Inhibition of Neur activity is required for the maintenance of epithelial polarity Drosophila epithelia are established during early embryogenenesis, through a process where immature epithelia composed of spot junctions progressively become a mature epithelia containing a zonula adherens and an apical/basal polarity (See Figure 3.6a for an example of an epithelial showing apical/basal polarity in which the apical marker, aPKC, is restricted to the most apical part of the embryo and the baso/lateral marker, Dlg, is restricted to the lateral membrane). The epidermis of the larvae is composed of a single layer of cells that secrete a protective cuticule (Figure 3.6e). This cuticule doesn't form properly in embryos where epithelial apical/basal polarity has been disturbed, which makes the analysis of the cuticule an excellent readout of the epithelial apical/basal polarity. The Crb polarity complex is not required for the initial establishment of apical/basal polarity but is required for its maintenance. Hence embryos lacking crb, for example, lose epithelial polarity and do not secrete a proper cuticule 3.6i). The activity of Neur is inhibited in the ectoderm by the presence of the Brd proteins. In the absence of all of the Brd genes (referred to as Brd for simplicity), epithelial polarity is lost: Figure 3.6b shows an Brd embryo with a pluristratified epithelia and where the apical polarity marker, aPKC, and the lateral polarity marker, Dlg, are not excluded to different domains. Moreover, Brd embryos show a cuticular defect that is similar to the one seen in crb embryos (Figure 3.6f). This loss of polarity was rescued in the double mutant Brd neur (Figure 3.6d and h), but no defects were seen in neur embryos (Figure 3.6c and g). This shows that the Brd proteins are required to inhibit Neur, for the proper maintenance of epithelial polarity and suggests that the activity of Neur negatively regulates apical polarity through an unknown molecular target.

Chapter 3. Neuralized and the Neuralized family of genes

Neur regulates the remodeling of the posterior midgut

At the end of developmental stage 9, the posterior midgut undergoes an epithelial to mesenchymal transition (EMT). The early phase of the EMT is characterised by downregulation of the apical markers in the posterior midgut, as exemplified in Figure 3.6a and by the downregulation of Crb. At the same time, the primordial germ cells (PGCs) actively migrate in between cells undergoing EMT, traveling from the interior of the midgut towards its exterior. This can be quantified by measuring the number of PGCs that are no longer present in the pouch of the posterior midgut Figure 3.6c. Hence, the percentage of migrating PGCs serves as a readout for the remodeling of the posterior midgut. The results seen in the embryonic lateral epithelia and in the posterior midgut, suggest that Neur negatively regulates epithelial polarity, in a Delta-Notch independent manner.

The first aim of this thesis was to find the molecular target through which Neur regulates epithelial polarity.

The Brd genes and mesoderm invagination

Mesoderm invagination is driven by the formation of an apical actomyosin network, that constricts and pulls on adherens junction to reduce the apical area of the mesoderm. While the apical side of the mesoderm constricts, the adherens junction are relocalised apically.

At the onset of mesoderm invagination, Neur activity is restricted to the mesoderm by the Brd genes (Figure 3.5). In the absence of the Brd genes from the ectoderm, the junctions in the lateral ectoderm relocalised apically (compare Figure 3.9a with b), suggesting that the activity of Neur in the ectoderm is sufficient for this relocalisation. However, it was found that the double zygotic mutant Brd, neur does not rescue the relocalisation of the junctions (Figure 3.9d). This result could be interpreted in two ways: Brd acts through another protein, rather than Neur, to regulate junction relocalisation, or; the maternal contribution of neur is sufficient to relocalise junctions in the embryos zygotically mutant for Brd and neur.

Moreover, the ectopic expression of the Brd genes in the mesoderm, which blocks Neur activity in the mesoderm was sufficient to perturb mesoderm invagination (compare Figure 3.9h with g), suggesting that Neur activity might be required for the regulation of mesoderm invagination. However, neur zygotic embryos didn't show any defect in mesoderm invagination (Figure 3.9f). This could again be interpreted as Brd acting through another gene, rather than neur, or that the presence of maternal Neur could be sufficient for proper mesoderm invagination in the absence of zygotic neur.

Hence, at the beginning of my thesis it was unclear whether or not Neur could regulate mesoderm invagination, and if it could, how it was doing so.

The second aim of the PhD, was to test whether Neur is required for the regulation of mesoderm invagination.

Identification of E3 substrates

Chanet and Schweiguth [222] showed that, in addition to Delta and the regulation of Delta-Notch signaling, Neur has other molecular targets and functions. One of these functions is the regulation of epithelial polarity through an unknown target, but other functions might exist. Therefore, we decided to undertake a proteomic analysis to try to identify new substrates of Neur, in an unbiased manner. A range of different techniques have been developed to identify the protein targets of E3 ubiquitin ligases. [236], [237]. The majority of them rely on the general identification of ubiquitinated proteins or peptides by mass spectrometry (MS), after an initial purification step. For the identification of new Neur substrates we decided to focus on two purification approaches: Bio-Ubiquitins and the Peptide antibody. Bio-Ubiquitins: Figure 3.10(1). BirA is a bacterially derived protein that recognises and covalently binds biotin to a specific amino acid sequence, Avi-Tag (GLNDIFEAQKIEWHE). Avitag can be fused to the N-terminus of ubiquitin and this fusion can be used to identify ubiquitin conjugated proteins by exploiting the strong interaction between biotin and streptavidin, or avidin. Biotin-ubiquitin substrates can be purified by pull-down using streptavidin or avidin coated beads. The Hugo Mayor laboratory developed a construct where BirA was cloned with five avi-tagged ubiquitins in tandem. After translation, these ubiquitins are separated by DUBs and BirA covalently binds a biotin to them. Mayor's group then purified ubiquitinated proteins by taking advantage of the strong streptavidin-ubiquitin interaction. Using this method, they identified 103 proteins [238] [239].

Peptide antibody:

Figure 3.10(2). Trypsination of a ubiquitinated protein results in a characteristic modification to the substrate protein: two glycine molecules, originally belonging to the ubiquitin, remain bound to the substrate protein lysine after trypsination. A monoclonal antibody against this K-Gly-Gly signature was raised [240] and allowed three groups [241][242][243] to generate the biggest dataset of ubiquitinated proteins to date, with the identification of between 10 000 to 20 000 ubiquitinated sites. It is important to note that the extremely high number of uibquitinated sites detected is not only due to the use of the gly-gly antibody, but also to the development of MS techniques increasing the sensibility of detection.

The third axis of this thesis was to use these two methods to try to identify new substrates of Neur.

The Neuralized family of genes

The Neuralized family of genes is composed of proteins that contain Neuralized Homology Repeats (NHR). In drosophila the family is composed of 3 genes: Neuralized (Neur), Ozzy (Ozz) and Bluestreak (Blue). (See Figure 3 In the drosophila embryo, Bluestreak also localises at the level of the centrioles [249]. Homozygous flies for a loss-of-function allele of Bluestreak, are completely viable, however, they show a reduced number of primordial germ cells (PGCs) in the embryonic gonads. This correlates with increased levels of CP110, and CP110 dependent shape defects in the PGCs.

Moreover, PGCs are specified in the posterior end of the embryo prior to cellularization. Their specification is controlled by the presence of the protein Oskar. The localisation of Oskar is regulated by a complex pathway that ensures that its RNA, and subsequently the protein, is localised posteriorly in the oocyte (and the embryo) [87]. Bluestreak mutants show a reduction in the posterior Oskar levels in the oocyte, which might contribute, in part, to the reduction of PGC levels [249]. Again, the regulation of Oskar levels by Bluestreak remains unclear.

The Neur family of genes and epithelial polarity.

Several observations and preliminary results led us to hypothesise that the Neuralized family of genes could be implicated in the regulation of epithelial remodeling. Firstly, Neur was shown to regulate epithelial remodeling [222]. Secondly, we were able to show by immunoprecipitation, that Ozzy and Neur physically interact. Thirdly, we had the impression that homozygous mutant embryos for a null allele of Ozzy, showed epithelial folding defects (data not shown). Finally, Ozzy and Bluestreak were shown to regulate actin, myosin and microtubules (see previous pages) which are drivers of epithelial remodeling in some developmental contexts.

Hence the fourth axis of my thesis was to test whether the other members of the Neur family of genes also regulated epithelial remodeling.

3.3. Axes of the thesis 63

Axes of the thesis

This thesis revolves around four questions, each of which is addressed by a Chapter in the results section: i) What is the molecular target of Neur that allows the regulation of epithelial polarity? ii) Does Neur regulate mesoderm invagination? iii) Can new substrates of Neur be identified by using proteomic approaches? iv) Do the other members of the Neur family of genes also regulate epithelial remodeling?

The first two chapters (which address questions 1 and 2) are written in the form of papers ready to be submitted. Finally, a fifth chapter gathers additional results. remodelling, the apical polarity protein Crumbs (Crb), that acts as a conserved determinant of the apical domain in epithelia, is required for the stabilization of cellcell contacts (Bulgakova and Knust, 2009;Campbell et al., 2009;Grawe et al., 1996;St Johnston and Ahringer, 2010;Tepass, 1996;Tepass et al., 1990;[START_REF] Thompson | Sticking together the Crumbs -an unexpected function for an old friend[END_REF].

Part

The protein Crb has a large extracellular region and a short intracellular tail with a PDZ95/Dlg/ZO-1 (PDZ) Binding Motif (PBM) that recruits the scaffolding protein Stardust (Sdt in flies, Pals1 in mammals) (Li et al., 2014). Sdt/Pals1 is a Membrane Associated GUanylate Kinase (MAGUK) family protein (Bachmann et al., 2001;Hong et al., 2001;Li et al., 2014) that also interacts with Patj to form the Crb-Sdt/Pals1-Patj complex (Bulgakova and Knust, 2009;[START_REF] Thompson | Sticking together the Crumbs -an unexpected function for an old friend[END_REF]. One function of Sdt is to stabilize Crb at the plasma membrane, in part by inhibiting the endocytosis of Crb (Lin et al., 2015). Crb and Sdt are required to stabilize epithelia (Bachmann et al., 2001;Campbell et al., 2009;Grawe et al., 1996;Hong et al., 2001;Tepass, 1996) and to negatively regulate acto-myosin during tissue remodelling (Flores-Benitez and Knust, 2015;Roper, 2012). Consistent with this, changes in Crb protein levels, subcellular localization and/or gene expression correlated with various epithelial remodelling events (Campbell et al., 2011;Roper, 2012;[START_REF] Sherrard | The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC[END_REF]. Additionally, the Drosophila crb and sdt genes encode multiple isoforms. This complexity likely contributes to the stage and/or cell-specific regulation of Crb activity and may also confer functional diversity [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]Bulgakova et al., 2010;Campbell et al., 2009;Hong et al., 2001;[START_REF] Vichas | The Ski2-family helicase Obelus regulates Crumbs alternative splicing and cell polarity[END_REF].

Neuralized (Neur) is a conserved membrane-associated E3 ubiquitin ligase (Deblandre et al., 2001;Lai et al., 2001;Pavlopoulos et al., 2001;Yeh et al., 2001). It contains a C-terminal RING domain that is predicted to bind an E2 enzyme that transfers ubiquitin to protein substrates bound to Neur. The best known target of Neur is the Notch ligand Delta (Commisso and Boulianne, 2007;Daskalaki et al., 2011;Fontana and Posakony, 2009;Lai et al., 2001;Le Borgne and Schweisguth, 2003;Pavlopoulos et al., 2001) (Figure 1A). The ubiquitination of Delta by Neur regulates its endocytosis and Neur-mediated endocytosis of Delta promotes the trans-activation of Notch [START_REF] Weinmaster | Notch ligand ubiquitylation: what is it good for?[END_REF]. In Drosophila, this activity of Neur proteins is antagonized by proteins of the Bearded (Brd) family that compete Delta for interaction with Neur ( Bardin and Schweisguth, 2006;Chanet et al., 2009;De Renzis et al., 2006;Fontana and Posakony, 2009;Lai et al., 2000) (Figure 1A).

Interestingly, the synthetic deletion of all Brd family genes resulted in embryonic lethality associated with a complete loss of epithelium integrity similar to the one seen in crb mutant embryos. This phenotype was suppressed by the loss of neur activity, hence revealing that Neur negatively regulate epithelial polarity (Chanet and Schweisguth, 2012). This second activity of Neur was independent of Delta-Notch signaling, implying that Neur regulates epithelial morphogenesis via other molecular targets (Figure 1B). The mechanism whereby Neur antagonizes epithelial polarity is unknown.

Here, we identify Sdt as a molecular target of Neur. We find that a specific subset of the Sdt isoforms physically interacts with Neur via a Neur Binding Motif (NBM). CRISPR-mediated deletion of the alternative NBM-encoding exon showed that Sdt is a key in vivo target of Neur. We propose that Neur destabilizes epithelia by interacting with specific Sdt isoforms and down-regulating Crb. effect on aPKC, E-Cad and Bazooka (Baz; Figure S1). While these data confirmed that Crb, Sdt and Patj are candidate targets of Neur, it remained unclear which of these proteins, if any, is a direct target. Indeed, since Crb, Sdt and Patj appear to be mutually dependent for their stable accumulation at the cortex, at least in some epithelia (Bachmann et al., 2001;Bulgakova et al., 2008;Hong et al., 2001;Lin et al., 2015;Penalva and Mirouse, 2012;Sen et al., 2012;Zhou and Hong, 2012), lowering the levels of one was sufficient to lower the levels of the two others (Figure 1J and

Neur interacts with the NBM-containing isoforms of Sdt

The sdt gene encodes twelve predicted protein isoforms produced by alternative splicing (Figure 2A) [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]Bulgakova et al., 2010). Two classes of Sdt isoforms can be defined by the alternative exon 3 (numbering according to [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]Hong et al., 2001)) that encodes 433 amino acids with no clearly identifiable motifs or domains (region noted here Exon3). Isoforms containing exon 3 are represented here by Sdt-PB (Bulgakova et al., 2010) (noted Sdt-A in [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]) whereas the other class is represented by Sdt-PF (noted Sdt-B1 in [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]; Figure 2B). When over-expressed in wing imaginal disc epithelia, both Sdt-PB and Sdt-PF stabilized Crb (Figure 2C,E,G). This stabilisation of Crb is consistent with both isoforms interacting via their SH3-GUK-PDZ domains with the PBM of Crb (Li et al., 2014). In contrast, we found that Neur (Lai et al., 2001)). In this assay,

Neur physically interacted with Sdt-PB but not Sdt-PF (Figure 2I). Moreover, this interaction was reduced when a stabilized version of the Brd protein, Brd R , was coexpressed (Figure 2I). Thus, Brd appeared to compete Sdt-PB for interaction with

Neur. Both Brd proteins and Delta interact with the NHR domain of Neur via related Neur Binding Motifs (NBMs), thus providing a simple molecular explanation for the competitive binding of Brd and Delta to Neur (Bardin and Schweisguth, 2006;Daskalaki et al., 2011;Fontana and Posakony, 2009;[START_REF] Gupta | Crystal structure, biochemical and biophysical characterization of NHR1 domain of E3 ubiquitin ligase Neuralized[END_REF]He et al., 2009). Since Brd appeared to compete Sdt-PB for Neur binding, we wondered whether Exon3 contains a NBM. Sequence analysis of Exon3 suggested the existence of two putative NBMs close to each other (Figure 2H). Consistent with this, deletion analysis of Exon3 indicated that the region encompassing these two NBMs is important for Neur-Sdt interaction (Figure S2). Additionally, deletion of these NBMs abolished the effect of Neur on Sdt-PB accumulation in vivo (Figure 2J-M). We conclude that Neur directly interacts with the NBM-containing isoforms of Sdt and that this interaction is required for the down-regulation of Sdt by Neur.

Regulation of endogenous Sdt by Neur

We next addressed whether Neur targets specific isoforms of endogenous Sdt. New reagents were therefore needed to specifically detect the NBM-containing isoforms of Sdt. Three GFP-tagged versions of Sdt were produced, as BAC transgene (Sdt-GFP) or as GFP knock-in at the locus (Sdt-GFP3 and SdtΔ3-GFP).

First, to detect all Sdt isoforms, GFP was inserted within an exon shared between all isoforms in the context of a 69 kb BAC transgene to produce Sdt-GFP (Figure 3A).

Genomic rescue showed that this BAC encodes a fully functional version of the sdt gene (see Methods). Second, to specifically detect all NBM-containing isoforms, we used CRISPR-mediated homologous recombination (HR) to insert GFP within exon 3 at the locus. This produced sdt GFP3 flies that were viable and fertile (Figure 3B). We collectively refer to the GFP-tagged isoforms of Sdt produced by these flies as Sdt-GFP3. Third, exon 3 was replaced at the locus by an exon encoding GFP using CRISPR-mediated HR to produce the sdt Δ3GFP allele (Figure 3C). The sdt Δ3GFP mutant flies were viable and fertile (see detailed analysis below). In these flies, the splicing events incorporating exon 3 into mature mRNAs generate GFP-tagged proteins lacking the sequence of exon 3, hence lacking the NBM. These are collectively referred to as SdtΔ3-GFP. Western blot analysis indicated that the predicted GFP-tagged isoforms were produced from these modified loci (Figure 3D).

Additionally, Sdt-GFP, Sdt-GFP3 and SdtΔ3-GFP were detected at the apical cortex of wing imaginal cells (Figure 3E,G,I). We therefore conclude that the NBMcontaining isoforms of Sdt are endogenously expressed in wing imaginal cells and that the apical localization of Sdt did not depend on exon 3.

We could then test whether Neur targets the endogenous NBM-containing isoforms of Sdt. First, ectopic Neur strongly down-regulated Sdt-GFP (Figure 3E,F)

and Sdt-GFP3 (Figure 3G,H; Figure S3). Of note, a weak Sdt-GFP signal remained detectable at the cortex whereas Sdt-GFP3 was not detectable. Thus, Neur appeared to have a stronger effect Sdt-GFP3 relative to Sdt-GFP. We therefore interpret the weak Sdt-GFP signal seen upon Neur expression as reflecting the persistence of Sdt isoforms lacking a NBM, e.g. Sdt-PF. By contrast, Neur had no significant effect on SdtΔ3-GFP that has no NBM (Figure 3I,J; Figure S3). This We also observed that ectopic Neur down-regulated Crb in the presence of the NBM-containing isoforms but not in their absence (Figure 3H,J; Figure S3). This showed that the effect of Neur on Crb is indirect and is mediated by the NBMcontaining isoforms of Sdt. We conclude that the NBM-containing isoforms of Sdt are direct in vivo targets of Neur and that Neur acts via these Sdt isoforms to downregulate Crb.

Neur acts via Sdt to disrupt epithelial integrity in Brd mutants

We have previously shown that the increased activity of Neur in Brd mutant embryos led to a rapid loss of epithelial integrity associated with a strong reduction in the level of Crb, Sdt and Patj (Chanet and Schweisguth, 2012). Our data above suggested that these effects of Neur on epithelial polarity could be mediated by the NBM-containing isoforms of Sdt. Consistent with this hypothesis, we found that Sdt-GFP3 was expressed during germ-band extension (Figure 4C), indicating that NBMcontaining isoforms are endogenously expressed in the early embryo. Moreover, the accumulation of Sdt-GFP3 was severely reduced in Brd mutant embryos (Figure 4D

Neur and Sdt regulate epithelial morphogenesis

We next studied the functional significance of the regulation of Sdt by Neur in embryos with normal Brd gene activity. As mentioned above, sdt Δ3GFP mutant flies were viable and fertile. This indicated that the NBM-containing isoforms of Sdt are not essential. To further test this, we generated a small deletion of the exon 3 at the sdt GFP3 locus using FLP-mediated excision. This produced sdt Δ3 mutant flies that were also viable and fertile. We therefore conclude that the regulation of Sdt by Neur is largely dispensable during development. However, since key morphogenetic processes often involve redundant regulatory inputs to achieve efficient and timely output (see for instance gastrulation in the fly embryo (Kolsch et al., 2007)), we decided to investigate further a known Neur-dependent epithelial remodelling process (Chanet and Schweisguth, 2012).

During gastrulation, cells of the posterior endoderm invaginate to form the posterior midgut (PMG; Figure 5A). As endoderm cells invaginate, they carry along the primordial germ cells (PGCs) that adhere to their apical surface. Once in the lumen of the PMG, PGCs migrate across this epithelium at stage 10 to eventually (Seifert and Lehmann, 2012). The latter appears to rely on two concomitant processes. First, it involves the delamination into the lumen of a specific subset of endodermal cells called the interstitial precursor cells (ICPs) that are selected through Notch-mediated signalling (Seifert and Lehmann, 2012;[START_REF] Tepass | Neurogenic and proneural genes control cell fate specification in the Drosophila endoderm[END_REF]. Second, it involves a neur-dependent down-regulation of apical polarity proteins, including Crb, Par6 and aPKC (Chanet and Schweisguth, 2012). In neur mutant embryos, both loss of apical Crb and migration of the PGCs appeared to be delayed (Chanet and Schweisguth, 2012) which cannot be easily explained by the specification of an excess number of ICPs due to loss of Notch signalling. Moreover, a loss of apical Crb appeared to be both necessary and sufficient to promote epithelial remodelling and PGC migration (Campbell et al., 2011;Seifert and Lehmann, 2012).

We How does Neur down-regulate Sdt? One possibility is that Neur targets Sdt for proteasome-mediated degradation and that the loss of Sdt results in the endocytosis of Crb by AP-2 (Lin et al., 2015). If so, one would expect the overexpression of Sdt-PB to suppress the effect of Neur on Crb. However, the effect of Neur was enhanced, not suppressed (Figure 2C'). This argues against Neur simply targeting Sdt for degradation. This observation is best interpreted by Neur providing a signal that targets the Sdt-Crb complex towards endo-lysosomal degradation, a major regulatory mechanism for Crb in both flies and mammals (Pocha et al., 2011;[START_REF] Rodriguez-Fraticelli | Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs[END_REF][START_REF] Schottenfeld-Roames | Seamless tube shape is constrained by endocytosis-dependent regulation of active Moesin[END_REF][START_REF] Sherrard | The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC[END_REF]. Thus, we speculate that Neur promotes the endocytosis of Crb associated with NBM-containing isoforms of Sdt. that Whether the NBM-containing isoforms of Sdt are ubiquitinated in vivo by Neur and whether ubiquitinated Sdt serves as a signal for Crb endocytosis remain to be investigated.

Previous studies have highlighted the functional diversity amongst the various Sdt isoforms [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]Bulgakova et al., 2010). Our data provide a molecular interpretation for this diversity by showing that specific Sdt isoforms are direct targets of Neur that are involved in Crb-mediated polarity remodelling. Here, we have shown that this regulation of Sdt by Neur contributes to the remodelling of the PMG epithelium. Previous studies had shown that regulated disruption of PMG epithelial architecture is important for PGC migration and that this process is developmentally regulated by the GATA factor Srp (Campbell et al., 2011;Chanet and Schweisguth, 2012;Seifert and Lehmann, 2012). Since the loss of Crb restored epithelium remodelling in srp mutant embryos, Crb was proposed to be a key target of Srp (Campbell et al., 2011). Thus, it would be interesting to examine whether Srp also inhibits the expression of the Brd genes to regulate Crb via a Srp-Brd-Neur-Sdt-Crb regulatory cascade in parallel to its regulation of Crb at the gene expression level (Campbell et al., 2011)

Methods

Transgenes

The BAC CH321-22E06 (Venken et al., 2009) was used to generate the Sdt-GFP BAC transgene using recombineering mediated gap-repair (Venken et al., 2006).

First, a 34 kb fragment located 3' to the sdt gene was deleted using the following primers:

Sdt3'partrpsl-F: aaatacaatcagaaatgcgaatgatggggggacgggacctggcctggtgatgatggcggg Sdt3'partneo-R: accgaaatcagcctgagtgagagagagagagagagcgctatcagaagaactcgtcaagaa Sdt3'part-F: agaaatgcgaatgatggggggacgggaccttagcgctctctctctctctctcactcaggc

The sequence of the sfGFP was then inserted within the sdt ORF at amino acid 599.

This site was chosen as it displayed low sequence conservation amongst various fly species. This strategy should help maintain function in the fusion protein (see also [START_REF] Couturier | Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila[END_REF][START_REF] Couturier | Endocytosis by Numb breaks Notch symmetry at cytokinesis[END_REF]). The following primers were used: Additional cloning details will be provided upon request. A mix of donor template (300 ng/µl) and gRNA plasmids (150 ng/µl) was injected into 900-1,200 embryos from the PBac{vas-Cas9}VK00027 stock (BL-51324). 1-3 independent RFP+ founder flies were selected. In both cases, proper recombination was checked by PCR.

Flies

Wild-type controls were w 1118 flies. The Brd mutant genotype corresponds to the combination of two deficiencies, Df(3)Brd-C1 and Df( 3)E(spl)δ-6 as described in (Chanet and Schweisguth, 2012;Chanet et al., 2009). Otherwise, the following stocks were used: sdt K85 (from E. Knust); UASt-FLAG-Sdt-PB and UASt-FLAG-Sdt-PF (described as UAS-Sdt-A and UAS-Sdt-B1 in [START_REF] Berger | Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration[END_REF]; from E.

Knust), UASt-Neur13.4 (Neur-PA isoform; from C. Delidakis), UAS-dsRNA sdt (BL-33909),UAS-dsRNA crb (BL-40969) and UAS-dsRNA patj (BL-35547).

Immunostaining

The following antibodies were used: aPKC (rabbit; 1:500; Santa-Cruz), Patj (rabbit;

1:100; gift of K. Choi), Crb (rat; 1:1000; gift of U. Tepass), Baz (rabbit; 1:500; gift of This chapter is composed of two sections: the manuscript of the paper and a text written by Vito CONTE, our collaborator, on his work modeling ventral furrow formation.

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Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila

Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila

Gantas Perez--Mockus 1,2,3 , Khalil Mazouni 1,2 , Giulia Corradi

INTRODUCTION

Embryo--scale morphogenesis arises from patterned mechanical forces that are produced by acto--myosin contractility (Lecuit et al., 2011;Martin and Goldstein, 2014).

During development, genetic information provides the spatial and temporal cues that pattern acto--myosin contractility to generate shapes in a precise and reproducible manner [START_REF] Leptin | Drosophila gastrulation: from pattern formation to morphogenesis[END_REF]. Shape changes also depend on the mechanical (material) properties of the tissue, e.g. elasticity/stiffness and viscosity, which also depend in part on actin filaments and myosin motors [START_REF] Humphrey | Active fluidization of polymer networks through molecular motors[END_REF][START_REF] Kasza | The cell as a material[END_REF]. Thus, understanding how genetic information regulate force generation and how cells and tissues respond to mechanical forces during morphogenesis are major challenges.

The early Drosophila embryo is an outstanding model to study how contractility regulates epithelial morphogenesis at the embryo--scale. Gastrulation is a classic example of how acto--myosin contractility drives apical constriction and formation of a furrow [START_REF] Leptin | Drosophila gastrulation: from pattern formation to morphogenesis[END_REF]Leptin and Grunewald, 1990;Martin and Goldstein, 2014;Martin et al., 2009;Rauzi et al., 2015;Sweeton et al., 1991). At the onset of gastrulation, the blastoderm embryo consists in a single sheet of ~5000 cells. Dorsal--ventral (DV) patterning of the embryo culminates into the activation of a G Protein--Coupled Receptor (GPCR)--Rho signaling cascade in ventral cells (Figure 1a) (Barrett et al., 1997;Costa et al., 1994;Kanesaki et al., 2013;Kolsch et al., 2007;Manning et al., 2013). This leads to the recruitment of Myosin II (MyoII) and formation of contractile acto--myosin meshworks at the medial--apical cortex of the ventral cells (Figure 1b). At the cellular level, these cortical acto--myosin meshworks undergo cycles of assembly and disassembly, thereby generating contractility pulses (Martin et al., 2009;Roh--Johnson et al., 2012). At the tissue--scale, these meshworks are interconnected via E--Cadherin to form a supracellular network, thereby ensuring tight mechanical coupling of the ventral cells (Martin et al., 2010;[START_REF] Sawyer | The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction[END_REF]. Contraction of this supracellular tensile network causes rapid and collective apical constriction of the ventral cells (Martin et al., 2010;Martin et al., 2009). Apical constriction of the ventral--most (central) cells causes the apical stretching of ventral cells flanking these constricting cells (Leptin and Grunewald, 1990;Sweeton et al., 1991). It also creates a hydrodynamic flow of the cytoplasm, with lateral membranes following the flow. This flow appears to mediate the lengthening of the ventral cells, which may contribute to furrow invagination (He et al., 2014) (Figure 1b). Indeed, once The Drosophila Bearded (Brd) genes encode a family of small proteins (collectively refereed here as Brd) that interact with the conserved E3 ubiquitin ligase Neuralized (Neur)

and inhibit its activity by competing substrates for binding (Bardin and Schweisguth, 2006;De Renzis et al., 2006;Fontana and Posakony, 2009;He et al., 2009;Lai et al., 2000;[START_REF] Leviten | The Drosophila gene Bearded encodes a novel small protein and shares 3' UTR sequence motifs with multiple Enhancer of split complex genes[END_REF]. Neur has at least two functions: it promotes Notch receptor activation by regulating the endocytosis of its ligand Delta (Daskalaki et al., 2011;Lai et al., 2001;Le Borgne and Schweisguth, 2003;Pavlopoulos et al., 2001) (Bardin and Schweisguth, 2006;De Renzis et al., 2006;[START_REF] Morel | Snail is required for Delta endocytosis and Notch--dependent activation of single--minded expression[END_REF]Morel and Schweisguth, 2000) (Figure 1b). Whether Neur also acts in the mesoderm to regulate gastrulation is unknown.

Here, we show that Neur is required to promote the recruitment of apical MyoII in ventral cells, thereby regulating collective constriction of ventral cells and rapid furrow formation. We further show that Brd is required in the ectoderm to lower contractility and that this regulation is critically required for ventral furrow invagination. We propose that the inhibition of Neur by Brd in the ectoderm contributes to establish a difference in mechanical properties between mesodermal and ectodermal cells. Thus, embryo--scale regulation of contractility and stiffness by Neur and Brd promotes furrow invagination in Drosophila.

RESULTS

Ectopic Bearded inhibits apical recruitment of Myosin II

We previously showed that the early and ubiquitous expression of two Brd family members, mα and Tom, was sufficient to perturb ventral furrow formation. This raised the possibility that Neur, the known target of mα and Tom, may regulate this process (Chanet and Schweisguth, 2012). However, since this observation was based on fixed samples, it remained unclear how these Brd proteins perturbed this dynamic process. In particular, it was not known whether MyoII was properly recruited at the apical cortex. To address this, we performed live imaging on embryos expressing a membrane targeted RFP (Gap43--Cherry) and a GFP--tagged MyoII (MyoII--GFP) and measured apical constriction and MyoII levels. In wild--type embryos (n=7), furrow invagination was observed ~ 6--7 minutes (min)

after the onset of constriction (defined as t=0). Analysis of apical constriction in the ~6 ventral--most cells (blue--shaded in Figure 2d; Figure S1a,b) confirmed that these cells undergo rapid and collective constriction (Figure 2g). By contrast, over--expression of mα and Tom under the control of a maternal--Gal4 driver (mat>mα+Tom) delayed furrow formation and invagination (Figure 2e,h and Figure S1l). To test whether ectopic expression of Brd in the mesoderm is sufficient to cause this delay, a stabilized version of Brd, Brd R , was specifically expressed in the mesoderm using the snail (sna) promoter (Figure S1c--h). Live imaging of sna>Brd R embryos showed that ectopic Brd in the mesoderm delayed furrow invagination (Figure 1f,i). Additionally, only a subset of the ventral cells underwent apical constriction while others failed to constrict in both mat>mα+Tom and sna>Brd R embryos (Figure 1g--I and Figures S1i--k). Thus, ectopic Brd inhibits collective apical constriction.

We next asked whether ectopic Brd acts by inhibiting the recruitment and activity of MyoII at the apical cortex of ventral cells. As described previously (Martin et al., 2009;[START_REF] Roh--Johnson | Triggering a cell shape change by exploiting preexisting actomyosin contractions[END_REF], a pulsatile supracellular network of MyoII was observed in wild--type embryos as ventral cells constrict their apex (Figure 1j,k and Figure S1n). By contrast, the acto--myosin network appeared less dense in mat>mα+Tom embryos and lower levels of apical MyoII were measured (Figure 1l,m and Figure S1m). Additionally, the number, amplitude, frequency and duration of the MyoII intensity pulses varied greatly from cell to cell upon ectopic Brd expression (Figure S1o--q). We conclude that ectopic Brd can interfere with the recruitment of medial--apical MyoII in ventral cells.

Neuralized is required for apical constriction

The E3 ubiquitin ligase Neur is the only known target of the Brd proteins (Bardin and Schweisguth, 2006;De Renzis et al., 2006). If ectopic Brd acts by inhibiting Neur in the mesoderm, then a loss of neur activity should mimick the Brd overexpression phenotype.

Since loss of zygotic neur showed no ventral furrow phenotypes (Chanet and Schweisguth, 2012), we hypothesized that maternal neur might compensate for the lack of zygotic neur activity. Indeed, the neur gene is expressed both maternally and zygotically under the control of Twist so that Neur is present in all cells with higher levels in ventral cells, as shown here using a functional GFP--tagged Neur (GFP--Neur; Figure 2a and Figure S2a--c). Since it was not possible to obtain eggs from neur germ--line clones due to the essential function of neur in the female germ--line (Lopez--Schier and Johnston, 2001), we used the deGradFP method [START_REF] Caussinus | Fluorescent fusion protein knockout mediated by anti--GFP nanobody[END_REF] to deplete the maternal and zygotic pools of Neur. This method relies upon a genetically encoded anti--GFP nanobody (Vhh) fused to the recognition subunit of a Cullin--based E3 ubiquitin ligase complex (Slmb). Conditional expression of this Slmb--Vhh fusion protein results in the degradation of GFP--tagged proteins (Figure 2b). We applied this method to Neur by expressing Slmb--Vhh in the germ--line of females carrying a trans-heterozygous combination of neur mutant alleles but rescued by the GFP--Neur BAC transgene. Thus, these females lacked endogenous Neur and only expressed GFP--tagged

Neur. These neur degrad females laid eggs for about a week before becoming sterile. Of note, sterility in old females correlated with developmental defects associated with defective Neur--Delta--Notch signaling (Figure S2h). Additionally, the neur degrad embryos laid by young females showed a neurogenic cuticle and defects in Delta endocytosis in the mesoderm (Figure 2c,c'), indicative of Neur depletion. Thus, Neur levels appeared to be optimally reduced down to a threshold level at which no egg is produced.

We then used live imaging to study furrow formation in neur degrad embryos expressing the Gap43--cherry marker (Figure 2). While the duration time of furrow invagination was variable, about a third of the embryos significantly deviated from the wild--type controls (Figure 2f and Figure S2d; this variability possibly resulted from varying residual levels of GFP--Neur depending on the mother's age). In the neur degrad embryos that showed delayed invagination, we observed that only a subset of the ventral cells constricted (Figure 2f with Crumbs (Crb), might be the target of Neur in the mesoderm. However, three lines of evidence strongly argued against this possibility. First, the zygotic expression of the sdt gene is restricted to the ectoderm (Bachmann et al., 2001) and Sdt was not detectable in the mesoderm during gastrulation (G. P.--M., unpublished). Second, live imaging analysis of sdt mutant embryos produced from mutant germ--line clones revealed no gastrulation defect (Figure S3). Thus, Neur appeared to regulate furrow invagination via another target that remains to be identified.

Neur is required for apical contractility in the mesoderm

To further study how low Neur activity in the mesoderm might result in defective furrow formation, we computationally investigated possible biomechanical scenarios that could explain the phenotypes seen in cross--section views of the neur degrad (Figure 2e) and sna>Brd R embryos (Figure 3j). To do so, we used a viscous finite--element 2D model described in (Conte et al., 2012) (see also Sup Info). Briefly, we assumed that biomechanics in wild--type embryos is governed by apical, lateral and basal forces that act along edges of individual cells to drive the motion of nodes. The distributions of these forces over time were previously obtained using Video Force Microscopy (VFM) (Brodland et al., 2010). While distributions were previously discretised over broad regions (Conte et al., 2012), we used here the complete force data set reported in (Brodland et al., 2010) and simply symmetrized and regularized distributions. Also, since low--quality imaging of MyoII signal deep into the embryo likely under--estimated basal forces, we tuned the topology of the VFM force distributions to better replicate in silico the gastrulation of wild--type embryos (see Sup Info for details). Using these distributions, our 2D viscous model nicely reproduced furrow invagination as seen in wild--type embryos (Figure 3a--c).

We then used simulations to explore biomechanical changes that would be sufficient in our model to reproduce the deformations seen in the neur degrad and sna>Brd R embryos. By keeping all model's parameters constant but changing the relative magnitude, pattern and timing of forces obtained from VFM to match the deformations seen in fixed cross--sections of these mutant embryos, we identified two potential physical mechanisms. For both, based on our observation that ectopic Brd lowered MyoII levels in the ventral cells, we introduced slower increases in apical forces in the mesoderm to reach lower maximal values. The Sigmoid mechanism uses a double--sigmoid shape distribution of apical forces over the mesoderm (Figure 3d--e) whereas the Gaussian mechanism uses a Gaussian bell--shaped over a domain wider than the mesoderm (Figure 3f--g) with force maxima being decreased down to 25% (Figure 3d) and 50% (Figure 3f) of the wild--type maximum. Thus, the phenotypes seen upon Neur depletion and inhibition could be reproduced in silico by primarily reducing the magnitude and profile of apical forces in ventral cells.

To test this interpretation for defective furrow formation, we analyzed the distribution of MyoII in cross--section views of wild--type and sna>Brd R embryos (Figure 3h,i).

Quantitative analysis showed that apical MyoII levels were reduced in sna>Brd R embryos with peak levels exhibiting a 3--fold decrease (Figure 3j). By contrast, basal MyoII levels were not detectably changed in the mesoderm (not shown). This suggested that defective invagination did not result from increased cortical tension at the basal cortex. Also, apical MyoII was detected over a similar width in wild--type and sna>Brd R embryos and the peak of MyoII accumulation appeared to be flattened in sna>Brd R embryos (Figure 3j). Thus, assuming that cortical MyoII accumulation closely relate to forces and that signal intensity measured here relate to MyoII levels, it appeared that the profile of MyoII seen in sna>Brd R

Furrow retraction in Bearded mutant embryos

We next investigated the functional significance of the inhibition of Neur by Brd in the ectoderm. Our previous analysis on fixed Brd mutant embryos failed to reveal invagination defects prior to disruption of epithelial polarity (Chanet and Schweisguth, 2012). Nevertheless, we had shown that Brd mutant embryos exhibit increased levels of apical MyoII in the ectoderm after furrow invagination in stage 6/7 embryos (Chanet and Schweisguth, 2012). This observation on fixed embryos raised the possibility that the activity of Brd may be required to prevent an early apical recruitment of MyoII as the ventral furrow invaginates. We therefore re--examined furrow invagination in Brd mutant embryos by live imaging. We first found that Brd mutant embryos invaginated nearly as fast as wild--type embryos (Figure 4a,b and Figure S5a). However, soon after the onset of furrow invagination, invagination seemed to stop before the furrow regressed as invaginated cells appeared to be pulled out. Thus a novel phenotype of furrow regression was observed in Brd mutant embryos (Figure 4a,b). We conclude that the activity of Brd is required in the ectoderm for mesoderm invagination.

We hypothesized that increasing apical forces in the ectoderm during furrow formation might trigger a retraction of the furrow. To test this hypothesis, we examined the level of apical MyoII using live imaging. In wild--type embryos, MyoII began to accumulate apically in the ectoderm 20 min after the onset of furrow invagination (Figure 4c,e) (Chanet and Schweisguth, 2012). MyoII accumulated mostly, but not exclusively, at junctions, leading to the formation of MyoII cables running along junctions oriented along the DV axis as reported earlier (Martin et al., 2009). In the absence of the Brd genes, apical MyoII levels increased to reach higher levels and apical MyoII accumulated not only at junctions but also Interestingly, Brd mutant embryos displayed more pulses of higher amplitude, indicating that Brd may act to limit the contractility of the apical cortex in the ectoderm. We therefore propose that an increase in apical cortex contractility in the ectoderm may cause the retraction of the invagination furrow in Brd mutant embryos. In support of this view, retraction of the furrow could be simulated in our 2D viscous model by a simple linear increase of apical forces in the ectoderm (see Sup Info)

Increased Rho signaling in the ectoderm induced furrow retraction

To test whether increased contractility in the ectoderm may be sufficient to block furrow invagination, we induced the expression of a constitutively active form of Rho1, RhoV14, in the ectoderm. RhoV14 was previously shown to increase acto--myosin contractility in the early embryo (Munjal et al., 2015;[START_REF] Simoes | Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension[END_REF]. The expression of RhoV14 was restricted to the ectoderm using the regulatory sequence of the Tom gene (Figure S6a the ectoderm is sufficient to suppress the Brd mutant phenotype. To do so, an active form of the myosin phosphatase Mbs, MbsN300 (Lee and Treisman, 2004), was specifically expressed in the ectoderm using the same strategy as for RhoV14 (Figure S6d). To achieve high levels of Mbs expression, two copies of the Tom>Mbs transgene were used (Figure S6e).

Live imaging revealed that Mbs expression in the ectoderm of Brd mutant embryos was sufficient to suppress furrow retraction (Figure 5h,I; of note, no suppression was seen with a single copy of the Tom>Mbs transgene). Thus, lowering MyoII activity in the ectoderm by regulating the dephosphorylation of MyoII was sufficient to counteract the effects of the Brd loss--of--function on cortical contractility. We conclude that decreasing contractility in the ectoderm restored furrow invagination in Brd mutant embryos. In summary, we have shown that the activity of Brd is required in the ectoderm to antagonize contractility, thereby permitting mesoderm invagination. The molecular mechanism whereby Neur regulates the activity of MyoII in the mesoderm remains to be determined. Indeed, Delta is unlikely to be a relevant target since zygotic Neur is required for the endocytosis of Delta and Notch activation in the mesoderm but is dispensable for furrow invagination, thereby uncoupling the two processes. Also, Sdt is not a relevant target since it is not detectably expressed in the mesoderm and it is not required for furrow invagination. Thus, Neur must have additional targets for the regulation of cortical contractility. One hypothesis, among others, would be that Neur regulates the endosomal trafficking of the GPCRs Mist and/or Smog (Kerridge et al., 2016;Manning et al., 2013), thereby regulating the intensity and/or duration of Rho1 signaling in ventral cells. Consistent with this, loss of the GPCR kinase GPRK2 led to the recruitment of apical MyoII in a broader ventral domain which may suggest that the regulated trafficking of GPCRs is critical to define a sharp peak of contractility in the ventral--most cells (Fuse et al., 2013). Further studies are needed to address how Neur regulates furrow formation at the molecular level.

We further showed here that the activity of the Brd proteins is required in the ectoderm to allow furrow invagination in the mesoderm. In Brd mutants, the furrow initially forms, albeit 122 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila at a lower speed, but rapidly regresses soon after its invagination, so that the mesoderm is pulled out at the embryo surface. Increased contractility in the ectoderm causes this phenotype. First, furrow regression correlated with increased apical MyoII in the ectoderm of Brd mutant embryos (of note, apical MyoII in Brd mutants correlated with apical relocalization of the junctions; these two processes apperead to be inter--dependent (Weng and Wieschaus, 2016)). Second, reducing contractility in the ectoderm of Brd mutant embryos using the MyoII phosphatase Mbs was sufficient to suppress this phenotype. Third, increasing contractility in the ectoderm of wild--type embryos using an activated version of Rho1 was sufficient to trigger furrow regression soon after its invagination. These experiments clearly establish that contractility and/or stiffness in the ectoderm matter for furrow invagination and that Brd is critically required to lower contractility of the ectoderm.

To our knowledge, this study provides the first experimental evidence showing that regulated contractility in the ectoderm is essential for furrow invagination. This finding further highlights that furrow invagination is an embryo--scale process (Rauzi et al., 2015).

In light of the role of Neur in regulating contractility in the mesoderm, we suggest that Brd that Snail defines a Neur ON/OFF limit along the DV axis. This limit was previously shown to define the mesectoderm as a single--cell row of Notch activation (Bardin and Schweisguth, 2006;Chanet et al., 2009;De Renzis et al., 2006). Recent studies have suggested that the

Materials and methods

Transgenes

The pUASt--Brd--3xHA and pUASt--Brd R --3xHA transgenes were obtained by subcloning the Brd--3xHA and Brd R --3xHA DNA fragments --produced by PCR amplification and gene synthesis, respectively --into a pUASt--attB plasmid. All ten lysines (K) of the Brd ORF were replaced by Arginine (R) in the Brd R sequence (primers and sequence can be obtained upon request).

Following sequencing, both plasmids were integrated at the same PB[y+ attP--9A] VK0013

(76A2) landing site.

The sna>Brd R BAC transgene was generated using recombineering mediated gap--repair (Venken et al., 2006) starting from the attB--P[acman]--based BAC CH322--18i14 (Venken et al., 2009). The snail ORF (and 30 nt of the 5' UTR) was replaced by a FRT--GFPnls--stop--FRT--BrdR--3xHA DNA fragment that was generated in a two--step PCR (primers and sequence can be obtained upon request). The resulting BAC was verified by sequencing of the recombined regions prior to phiC31--mediated integration at the PB[y+ attP] VK00033 (65B2) landing site.

The Brd--C BAC (Chanet et al., 2009)

Flies

To knock down the activity of neur in the female germline, we used the degradFP method [START_REF] Caussinus | Fluorescent fusion protein knockout mediated by anti--GFP nanobody[END_REF]. Two copies of a mat--Gal4 transgene were combined with two copies of the UAS--Nslmb--VhhGFP4 in neur trans--heterozygous females carrying the neur IF65 and neur GE mutations. We sequenced the strong hypomorphic neur IF65 allele and identified a A-to--T mutation in the splicing acceptor site at the cag/TTC intron2/EXON3 boundary. The lethality of these neur IF65 / neur GE mutant females was rescued using a GFP--Neur BAC transgene (Figure S2a--c FRT]82B neur IF65 flies were referred to here as control degrad embryos.

To study the complete loss of function of all eight Brd family members (BFM), we used the Df( 3)Brd--C1 and Df( 3)E(spl)δ--6 deletions (Chanet et al., 2009). These two deletions delete the Brd, BobA, BobB, Tom, Ocho, E(spl)mα--BFM, E(spl)m4--BFM and E(spl)m6--BFM genes.

Embryos homozygous for these two deletions are referred to here as Brd mutant embryos.

Note that the E(spl)--HLH genes of the E(spl)--C are also deleted in these embryos.

The UAS--Tom and UAS--mα transgenes used for over--expression are those used in (Chanet and Schweisguth, 2012). transgene copy number, accounting for phenotypic variability. To circumvent this issue, we produced and studied the sna>Brd R embryos.

The FRT--GFPnls--stop--FRT cassette was removed from the sna>Brd R , Tom>RhoV14 and Tom>Mbs BAC transgenes using a FLP expressed in the male germ--line provided by the P{ry+ betaTub85D--FLP}1 transgene . This resulted in a high frequency (80%) of FLP-- mediated excision of the stop cassette in the male germ--line. These males were used in crosses with females expressing maternally the Gap43--mCherry and MyoII--GFP markers.

Embryo imaging, PCR genotyping and image analysis

Embryos were dechorionated using bleach, glued on a slide, covered with halocarbon oil Voltalef 10S to avoid desiccation and flanked by a stack consistent of two 0.17 mm coverslips, on top of which a coverslip was added. Embryos were imaged at 22°C using a

Leica DMRXA microscope equipped with a CSU--X1 spinning disk, a back--illuminated Quantem 512C camera, 491 and 561 lasers and the Metamorph software.

Apical constriction was mostly studied on (Δt=1 min, Δz=2 mm, 11 z--sections) movies acquired using a 40x PlanApo NA=1.40 objective. To study the acto--myosin pulses, higher resolution movies were produced (Δt=12 sec, Δz=2 mm, 6 z--sections) using a 63x PlanApo NA=1.40 objective. In all movies, t=0 corresponds to the onset of apical constriction, as determined visually on the Gap43--mCherry channel.

For segmentation, the Gap43--mCherry (membrane) signal was smoothed using the Fiji smoothing function and contrast was increased. To increase the number of cells that can be segmented in a single focal plane, a basal confocal section was chosen. Segmentation was then performed using Packing Analyser [START_REF] Aigouy | Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila[END_REF]. Quantification of the Myo--GFP signal was performed following maximum projection of all confocal sections. Since the MyoII signal is maximal apically, this allowed us to analyze MyoII in cells that are slightly peripheral in the field of view. To do so, the segmentation mask produced using Packing Analyser was superposed on top of the MyoII--GFP images and the average signal intensity was measured from the raw GFP signal in each segmented cell using Fiji. For each movie, measured values were normalized relative to the initial values (corresponding to the GFP signal measured prior to the apical recruitment of MyoII; this signal varied with montage conditions). To measure the medial--apical pool of MyoII, the segmentation mask was eroded 3 times using the erode FIJI function, thereby creating an exclusion mask of 3 pixel wide (pixel size is n µm). Graphics and data were analyzed using R studio.

Wild--type controls were embryos maternally loaded with MyoII--GFP and Gap43--mCherry,

Immunostainings

For cross sections, embryos were dechorionated and heat--fixed as described in (Chanet and Schweisguth, 2012). These were then incubated with primary antibodies in PBS1x Triton 0.1% for 2 hr at 25°C (or overnight at 4°C), washed and incubated with secondary antibodies in PBS1x Triton 0.1% for 2 h at 25°C (or overnight at 4°C). Embryos were then post--fixed in PBS1x with 4% PFA for 1 hr. Embryos of the proper stage were then selected under the binocular scope, cut using a sharp scalpel blade and mounted in 4% N--propyl--galate, 80%

glycerol. Ovaries were fixed 20 min with 4% PFA and stained for 2 hr with Phalloidin--atto647N (1:1000; Sigma #65906) in PBS triton 0.1%. Wild--type controls were w 1118 embryos and ovaries.

The following primary antibodies were used: Armadillo (mouse N2 7A1 mAb; 1:500; DSHB), cytoplasmic MyoII (rabbit; 1:500; gift from D. Kiehart), intracellular Delta (rat anti--DlICD mAb 10D5; 1:1000; gift from M. Rand), GFP (goat; 1:500; Abcam #6673).

Images were acquired using a confocal Zeiss LSM780 microscope with a 63x (PL APO, N.A.

1.4 DIC M27) objective. f,g) Live imaging of a neur degrad embryo expressing Gap43--Cherry (white; ventral cells, blue; tracking, red). Furrow formation was delayed (see Figure S2d) g) Apical constriction (surface area values measure over time as in Figure 1g--i) was delayed in neur degrad embryos relative to wild--type (see Figure S2e). . Ventral cells appeared to constrict less rapidly than in wild--type embryos (see Figure S6f) and the furrow regressed 20--25 min after its initial invagination (see Figure S6c). Ventral cells could not be tracked during invagination (t=25) and were tracked again after furrow regression (t=35). c--e) A mutant version of Brd, Brd R , appeared to be more active in a gain--of--function assay.

Over--expressed Brd R appeared to antagonize the activity of Neur during bristle development more effectively than Brd. This difference in activity correlated with a difference in accumulation level. Since these two transgenes differed only by a few K--to--R mutations in the ORF of the Brd gene, we suggest that increased levels of Brd R results from increased stability rather than higher rate of synthesis. Indeed, mutating all K residues is predicted to prevent ubiquitin--dependent degradation.

f--h) Strategy to express Brd R in all mesodermal cells. The cis--regulatory elements of the snail gene encoded within a 22 kb Bacterial Artificial Chromosome (BAC) were used to drive the expression of Brd R in the mesoderm (f). Conditional expression was achieved using a FRT--GFP--stop--FRT cassette so that Brd R was expressed in the mesoderm only upon FLP--mediated excision of this stop cassette. Flp--mediated excision of the FRT--GFP--stop--FRT cassette produced a sna>Brd R transgene expressing Brd R (f). In the absence of recombination, GFP (green in g) was expressed in all mesodermal cells (Twist, red, in g). Single embryo PCR was used to genotype embryos carrying a recombined transgene, i.e. expressing Brd R and not GFP. Embryo genotyping was performed following live imaging (h). For each embryo, mean Myo--GFP intensities were calculated from ~ 30--40 ventral cells.

Ectopic Brd resulted in strongly delayed and slow increase of MyoII--GFP intensity. The observed variability in MyoII levels was likely due to embryo--to--embryo differences in transgene copy number (see Materials and Methods).

n--o'') single cell analysis of MyoII--GFP intensity (grey) revealing pulses of MyoII accumulation as cells constrict (area in black) in a wild--type embryo (yellow cell in Figure 1j). In mat>mα+Tom embryos, distinct patterns of MyoII pulses and constriction were observed.

Three individual cells are shown here (see insets; shown as color--coded cells in Figure 1l)

showing (cell highlighted in blue in o). MyoII pulses varied in number, frequency and amplitude but their presence still appeared to correlate with constriction. Neur targets the NBM-containing isoforms of Sdt, such as Sdt-PB, but not those lacking exon3, such as Sdt-PF (G. Perez-Mockus et al., submitted).

The sdt EH120 mutant allele carries a non-sense mutation within exon3. This mutation shoud therefore disrupt the synthesis of all isoforms that are targeted by Neur. c,d) Strategy for the live imaging of sdt EH120 mutant embryos derived from germ-line clone (GLC) and expressing Gap43-Cherry and MyoII-GFP (c). Following imaging, each embryo was individually genotyped using single-embryo PCR and sequencing of the PCR product (d).

Hemi/homozygous mutant embryos derived from germ-line clone were unambiguously identified from heterozygous mutants (d).

e) Live imaging of a sdt EH120 mutant embryos derived from germ-line clone (GLC) and expressing Gap43-Cherry (white) and MyoII-GFP (not shown). No defect in furrow formation and invagination could be observed. This indicated that these Sdt isoforms are not required for gastrulation, hence suggesting that Sdt is not a key target of Neur in the mesoderm. appeared to be slightly slower in Brd mutant embryos (n=3) compared to wild--type controls (same data set as in Figure S1l). b,c) Single cell analysis of the temporal evolution of both Myo--GFP intensity (grey curve) and surface area (black curve) suggested the existence of stronger pulses in the ectoderm of Brd mutant embryos compared to controls. (same data set as in Figure S1l). A similar effect was observed in Brd mutant embryos (Figure S5a).

Annex: In silico simulation of mesoderm invagination written by Vito CONTE

2D physical model of the Drosophila embryo

We studied the possible biomechanical scenarios that can explain the experimental phenotypes observed in wild-type and mutant embryos by developing a multi-scale 2D finite-element model of a cross-section of the Drosophila Gastrula along its anterior-posterior axis (Fig. S1). The role of the computational model is to create a coherent framework in which we can piece together the biomechanics of wild-type, Neur Degrad and Brd mutant embryos at the subcellular, cellular and epithelial scales. The model emulates the key biomechanical features of cells (Fig. S1,a-b) and the embryo structure (Fig. S1,e) by resorting to a mathematical formulation that the authors have previously detailed in Conte et al. [1] and have readapted in this section. According to this formulation, the initial configuration of the in silico model of the embryo consists of a circular epithelium of 84 cells. The finite element model of each single epithelial cell takes into account the subcellular effects of the complex force fields originating from molecular processes by assuming that they can be resolved into equivalent forces along cell edges, as shown in Figure S1,c [1].

Single cells in the model surround an incompressible inner cytoplasm, which was simulated by enforcing that the surface enclosed by the cell membrane remain constant over time (Fig. S1,c-d). Cell membrane was subdivided into 5 sections along the apical-basal edges and into 3 sections along the apical and basal edges respectively (Fig. S1,d-e), to allow individual cells to bend as observed in vivo [1].

The epithelium was assumed to respond viscously to deformations, in keeping with recent evidence from He et al. [2] demonstrating that during the lengthening phase of the Drosophila ventral furrow formation the entire epithelial bulk tissue below the apical cortex behaves very closely to a viscous fluid -despite this tissue being partitioned into cells with membranes containing a heterogeneous cytoplasm [2]. Although the epithelium seems to unlikely be elastic during the early stages of ventral furrow formation [2], the question of whether the elastic response of the embryo matters at the time scales of ventral furrow formation still remains partially unresolved experimentally [3,4]. For these reasons, we opted to give the cell cortex no elasticity and we assumed the cell cytoplasm to be purely viscous. Since the content of epithelial cells is contiguous to the inner yolk of the embryo before and during the epithelium's cellularisation process, the yolk viscosity was assumed to be equal to that of the cytoplasm [5]. The question of whether the yolk volume V remains constant throughout gastrulation (incompressibility of the fluid) or whether there is a residual pressure at the start of the invagination still remains unresolved experimentally [6]. Certainly, it is a common observation that the yolk squirts out when the embryo is pierced before or during gastrulation [6]. Also, the conservation of the 3D yolk volume does not necessarily grant constancy of the 2D crosssectional areas of the yolk. Therefore, we opted to model the 2D yolk as a compressible viscous fluid exerting a pressure on the inner (basal) side of the epithelium to a maximum of 10% of its initial value. The experimental value of the yolk pressure is not known, so this was a system variable chosen in such a way that surface variations of the inner yolk region are approximately of the same magnitude of in vivo yolk area variations [1]. Finally, the vitelline membrane has been modelled as a rigid boundary condition that prevents apical nodes from moving outside a bounding circle (Fig. S1,e). Consequently, the sum of the cross-sectional areas of the yolk, epithelial cells and ventral gap (the region where ventral cells pull away from the vitelline membrane) is constrained to be constant [1].

Biomechanics of the Wild type embryo

We assumed that the biomechanics of the Wild type embryo is governed by forces having spatial-temporal distributions that have been obtained in our previous Video Force Microscopy (VFM) study [7], as shown in Figure S2. This method assumed that the forces at work in an embryo can be decomposed into passive and active contributions [7]. Passive forces arise in subcellular systems (e.g. viscous cytoplasm) that dissipate the mechanical stress and mechanical energy generated in the cell system by the active forces [1]. Active forces, instead, originate from subcellular systems that do mechanical work to the cell system (e.g. contractile actino-myosin networks) and generate stress at the expense of biochemical energy sources within the cells (e.g. glycolysis, phosphorylation) [7]. We emulated the presence of biochemical sources of energy within the in silico cells of the model by computationally assigning the VFM force profile to the corresponding cell edges of the in silico geometry. We say nothing about the nature and the intensity of these energy sources in the in silico model, except that they exist and appear to produce forces shaping the observed Wild type phenotype [1]. The magnitude of the forces operating in silico is set by the viscosity of cellular cytoplasm. However, the experimental value of cytoplasmic viscosity of the early embryo available in the literature varies significantly [5]. We have circumvented this problem by normalizing the range of forces 'per unit of viscosity' that quantitatively reproduced in silico the invagination path observed in vivo [1]. Consequently, forces expressed in Newton are recovered by simply multiplying by a specific viscosity value. This approach does not change the location, timing or relative magnitude of the forces -the factors central to the biomechanics of the Wild type embryo [1].

We have furthermore symmetrised and regularised the force distributions obtained from our previous VFM study [7] (Fig. S2,e-g) -as shown in Figure S3,a-b -in order to take into account the symmetry of the 156 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila geometry of our 2D model and avoid numerical instabilities during the solution of the Finite-Elements problem (Fig. S1,d). Upon observing that symmetrised and regularised force distributions lead to the formation of a ventral furrow with abnormal epithelial thickness at the mesoderm's periphery (Fig. S3,c), we tuned the topology of the VFM force distributions to better replicate the wild type phenotype in silico (Fig. S2,a and S3,f). We could achieve in silico invagination perfectly resembling the in vivo one by doing the following: i) widening the ventral cellular domain of lateral cortex contraction (blue curves, Fig. S2,a3-a5) so that it could span the whole mesodermal domain (blue curves, Fig. S2,d); allowing ectobasal forces to gradually fade towards the central mesoderm. Differences between the symmetrised regularised VFM force distributions in Figures S2,a-b and S2,d-e shows represent an estimate of cell-tracking impresitions that may arise from multi-photon image clarity. For instance, poor visibility of the basal mesodermal domain at later stages of invagination (Fig. S2,a) might steer cell-tracking towards the underestimation of forces in the lateral and basal cortex of mesodermal cells. In this respect, it is worth noticing that previous in silico predictions by Pouille et al. [8] and by Poliakov et al. [9] suggested that both apical-basal cell elongation and shortening during invagination were passive mechanical consequences of processes that onset in other parts of the embryo. The 2D hydrodynamic viscous model proposed by Pouille and Farge [8] predicted that: i) an active process of increasing the apical membrane tension and flattening in mesodermal cells is needed to produce a shallow furrow formation; and ii) an additional force applied to the apical cell junctions and radially directed towards the centre of the 2D cross-section of the embryo is needed to achieve the complete internalisation of the furrow [8] -they postulated this force to be due to the curvature that in 3D characterises in vivo embryos along the anterior-posterior axis. Similarly, the 2D elastic model proposed by Poliakov et al. [9] predicted that: i) apically generated contractile forces are needed to increase the apical-basal length of ventral cells and to fan out the mesodermal domain towards the yolk's interior; and, ii) reduction of the basal rigidity of mesodermal cells is needed to trigger passive elastic forces that contract the lateral cortex of cells in the apical-basal direction to drive the internalisation and closure of the ventral furrow [9]. According to this physical mechanism, the actomyosin molecular processes leading to the constriction of mesodermal cell apices implicitly spend biochemical energy that is partly stored in the elastic potential energy of their lengthened lateral cortex. Softening of the basal cell cortex of elastic epithelium then sets the lateral cortex of mesodermal cells out of equilibrium and triggers the release of this elastic energy through a passive spring-like restoring force. The slingshot mechanism predicted by Polyakov et al. [9] provides the apical-basal shortening of mesodermal cells that is necessary to internalise the ventral furrow on the instantaneous times scales of a purely elastic epithelium and on condition that no viscous cell cytoplasm may dampen or delay the spring-like restoring force on the longer time scales of ventral furrow formation [2,4]. Finally, it is worth observing that the relative timing between active apical forces and lateral apical-basal shortening forces proved to have an impact on the process of ventral furrow formation in the Wild type [1]. This naturally raises the questions: i) what signal activate mesodermal radial shortening? [1] And, ii) are there any mechano-regulatory or other regulatory mechanisms involved in coordinating these two cellular active forces? [1] In our 2D viscous model of the wild type embryo we specify nothing in those respects as all active forces just follow each other in the temporal sequence defined through our previous VFM quantification [7] and shown in Figure S2e. The quantification and digitisation of the wild type invagination that we performed in Conte et al. [1] started during the fast phase of cellularisation and was timed with respect to the reference instant at which ectodermal cells appear fully cellularised as they reach their maximal apical-basal length. We assumed this reference instant to be the origin t=0min of all time measurements we performed [1]. This analysis showed that mesodermal cells begin to lengthen along the apical-basal direction at approximately t=-10min, before cellularisation is complete in the ectoderm Fig. S2,c. For these reasons, we assumed the phenotype and the VFM forces of the wild type embryo at t=-10.43min as the reference configuration for our in silico model. Consequently, the in silico model will simulate the in vivo wild type embryo in the intervals T1 and T2 shown in Figure S2,c-d) [1]. The interval T1 covers the monotonic increase of mesoapical forces towards their maximum at t=-2.18min, which precedes the maximal apical-basal extension of mesodermal cells by 45 seconds at t=-1.43min. Interval T1 also coincide approximately with the interval of Myosin-II relocation from the basal to the apical ends of mesodermal cells (Fig. S2,a1-a3). Mesolateral forces that contract mesodermal cells along their apical-basal direction begin increasing at rates faster than ectodermal ones at t=-6min, when apical forces are at approximately 80% of their maximum intensity (Fig. S2,c). This trigger and drives the rise of the furrow towards the yolk's interior, as shown in Figure S2,d (black lines).

Biomechanics of the Neur Degrad and Brd mutant embryos

Once our 2D viscous model had been tuned for the wild type simulation, see Fig. S3, none of the model's parameters were changed when we used the model to simulate the mutant prototypes -the only things that changed are the relative magnitudes and timing of forces (Fig. S2b) obtained from VFM quantification of in vivo experiment (Fig. S2).

Annex: In silico simulation of mesoderm invagination written by Vito CONTE 157

As for the Neur Degrad mutant embryo, the ectopic Bearded interfered with apical recruitment of Myosin-II and that Neur down-regulation delayed apical constriction. We tested under what mechanical conditions these in vivo processes would be sufficient to give rise to the Neur Degrad mutant embryo phenotype. We identified two potential physical mechanisms that are able to drive deformations as observed in the Neur Degrad mutant embryo phenotype. Both such mechanisms require the following mechanical conditions: 1) the Myosin-II relocation time-interval T1 (Fig. S2,c);gets four times longer in the mesoderm (Fig. S4,f); 2) the onset of apical-basal shortening of mesodermal cells delays from time-interval T1 to timeinterval T2 (see blue curves in Figure S4,a-b,f);

The first physical mechanism, referred to as Sigmoid Mechanism for the shape of it mesoapical force distributions, further requires (Fig. S4): B3) apical constriction force distributions that are double-Sigmoid-shaped over the mesodermal domain (see red curves in Figure S4,a-b,f) and 50% weaker with respect to the wild type embryo (Fig. S3c); B4) basal constriction forces fading to zero over a wider mesodermal domain (see green curves in Figure S5,a-b,f);

The second physical mechanism, referred to as Gaussian Mechanism for the shape of its mesoapical force distributions, further requires (Fig. S5): A3) apical constriction force distributions that are 50% weaker and Gaussian-bell-shaped over a wider mesodermal domain (see red curves in Figure S5,a-b,f) with respect to the wild type embryo (Fig. S3c); A4) basal constriction forces fading to zero over a wider mesodermal domain (see green curves in Figure S5,f);

As for the Brd mutant embryo, the increased apical Myosin-II activity observed in ectodermal cell correlated with retraction of the ventral furrow after its formation. We tested under what mechanical conditions this in vivo process would be sufficient to give rise to the Brd mutant embryo phenotype. A potential physical mechanisms leading to furrow retraction in mutant embryo phenotype requires mechanical conditions as follows (Fig. S6): 1) for t>-6min when furrow has started rising, the Myosin-II on the basal ectoderm start relocating towards the apical end of the epithelium, which correlates with a decrease in ectobasal tension forces and an increase in ectoapical constriction forces at a rate much higher than that of mesoapical ones (Fig. S5,f; 2) all other force relative timings and magnitude remain the same as in the wild type, except the analysis is allowed to run for t> 6min to allow for the full retraction of the furrow. We simulated in silico the wild-type embryo by using the complete in vivo force data set collected through the VFM [7], as opposed to our previous study detailed in Conte et al. [1] where we utilised discretised distributions varying in a simplified fashion with time but not with position over epithelial regions. (a-b) Symmetrised and regularised approximation of the VFM spatial force distributions quantified in vivo by Brodland et al. [7] were obtained by computing mean and characteristic statistical features of the in vivo trends. Time-dependence of the approximate distributions is equal to that of in vivo trends (Fig. S2,c). We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. -10.43,0] min to vary over wider interval [-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig. S2, c-d We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. -10.43,0] min to vary over wider interval [-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig. S2, c-d During my PhD I tried to identify new substrates of Neur. For this, I used two methods that relied on the purification and then the identification of all of the ubiquitinated proteins in embryonic extracts over-expressing Neur, or an inhibitor of Neur activity. The idea was that proteins identified in the extracts over-expressing Neur, but not in those over-expressing its inhibitor, could represent putative targets of Neur. Two methods were used for the purification of the ubiquitilome (all of the proteins that are ubiquitinated). Firstly, ubiquitinated proteins were purified by using biotinylated ubiquitins and taking advantage of the strong avidin-biotin interaction. Following this, protein identification was achieved by Mass Spectrometry, using the facility at the Pasteur Institute. In a second method, the purification of ubiquitinated proteins was achieved by using an antibody which recognises ubiquitinated proteins after trypsination. For this method, I collected the embryos, and Francis IMPENS from the lab of Pascale COSSART in the Pasteur Institute, performed the purification and the mass spectrometry analysis. Both methods and their results are presented below.

Bio-ubiquitin

In 2011, Hugo Mayor [238], developed a new technique to purify ubiquitinated proteins. He generated a construct containing 6 Avi-tagged ubiquitins and a protein called BirA encoded in tandem. The construct is called ubi6birA. After being translated, the DUBs separate the Avi-tagged ubiquitins from BirA, and BirA recognises the Avi-tag and catalyses the covalent binding of biotin to the tag. This produces biotinylated ubiquitins that are bound to the different E3 ligase substrates(Figure 6.1A) and that can be purified using avidin beads, and then identified by LC/MS-MS spectrometry.

This method purifies some proteins which naturally carry biotin, for example pyruvate carboxylase in Drosophila, shown by an arrow in the Figure 6.1B). Hence, pyruvate carboxylase is a good positive control in Drosophila extracts to confirm that the purification and the identification by mass spectrometry has worked.

Before doing the experiment, we set up the purification method in the lab. We managed to purify bio-ubiquitinated proteins from embryo extracts expressing the ubi6-BIA construct ubiquitously, by following the protocol described by Mayor and Peng 2012 [239] (data not shown). In this protocol, the proteins are extracted using mechanical lysis (douncer), very harsh denaturing conditions (8M Urea) and an inhibitor of the DUBs (NEM). The extract is then incubated with avidin beads and the beads are washed in harsh conditions (different concentrations of Urea and SDS) to remove unspecific binding. Finally the beads are boiled to release the bound proteins. For the purpose of identifying Neur substrates, we decided to co-express the Ubi6-BIA construct with Neur, or with Tom, an inhibitor of Neur, using the Gal4/UAS system and an ubiquitous promoter. The tubG80ts,UAS-ubi6BIRA/CYO; da-G4/TM6 line from the Mayor lab, was crossed with (1)UAS-NeurV5;DeltaGFP, (2)UAS-TOM;Delta-GFP or (3)DeltaGFP alone. Finally, wild-type flies (4) were used as a negative control. All the flies were allowed to lay eggs at 25 • C for 8h and the embryos were allowed to age at 29 • C for 8h, before being frozen with dry ice and ethanol and stored at -80 • C. Delta is a known target of Neur, and thus Delta-GFP was selected as a positive control.

Bio-ubiquitinated proteins were purified in each of the four conditions starting from 16 mg of protein extracts, and following the protocol described by Mayor and Peng (2012) [239]. 100 ul of avidin beads were used each time. Beads containing the different purified extracts were given to the mass spec facility, where they were trypsinised (instead of boiled) to release the bound proteins. This sample was then passed on the mass spectrometer to identify proteins. The results are presented in Figure 6.1C). A total of 54 different proteins were identified, compared to 103 in the original paper of Mayor[239]. Of these proteins, seven were identified in the four samples, including the biotin carrier, pyruvate carboxylase. Thus, the purification method had worked, at least to a certain extent. However, only seven proteins were identified exclusively in the sample over-expressing Neur. Furthermore, Delta-GFP was not picked in this screen and the most highly identified protein was Avidin (100 fold magnitude). These results suggest that avidin is being released from the beads, and thus the large amount of avidin in the sample, which is subsequently run in the mass spectrometry machine, decreases the sensitivity of the LS/MS-MS. To resolve this issue, we tried boiling the beads, instead of using trypsin to release the proteins, but found that the level of released avidin was still high (data not shown).

To summarise, this method was unsuccessful in the identification of a large number of ubiquitinated proteins. This was probably due to a high level of avidin in the sample, released by the purification method, that hampered the sensitivity of the mass spectrometer.

Cofilin, or Twinstar in drosophila, was an interesting candidate that was identified in this screen. It has been shown to be involved in the regulation of apical constriction during mesoderm invagination [151]. However, co-immunoprecipitation experiments carried out by Vanessa ROCA to determine whether cofilin was associated with Neur -RF in S2 cells, showed negative results. Moreover, the phenotypes published by Jodoin and colleagues [151] on embryos lacking twinstar or its activator slingshot were not at all similar with the phenotype of embryos lacking neur, or where its activity was blocked. Hence we decided to stop exploring the possibility that Cofilin was regulated by Neur. Mayor and Peng 2012.[239]. A construct expressing six tagged ubiquitins and the BirA protein is expressed in the tissue of interest. DUBs separate the tagged ubiquitins from the BirA protein and then BirA covalently binds a biotin to the ubiquitin tag. Bio-Ubiquitins are bound to different substrates by the E1, E2, and E3 ligases. Finally, the substrates are purified taking advantage of the biotin avidin interaction. B)Western Blots of purified extracts using avidin beads. From Mayor and Peng 2012 [239]. The Proteins are marked using a silver stain. The BirA lane shows the proteins purified from an extract expressing only BirA, while the Ubi6BirA lane shows the proteins purified from an extract expressing the construct presented in A. The smear in the Ubi6BirA shows that ubiquitinated proteins are purified using this method.The arrow marks the position of the Pyruvate Carboxylase a protein that binds biotin and that is hence purified in both extracts. The asterix shows free avadin. C) Results from the identification by MS/MS of proteins from 4 different conditions: Embryos overexpressing ubiquitously Ubi5BirA, Tom (in green), Ubi6BirA (in red), Ubi6BirA, Neur (in blue) and W1118 (in purple). 7 proteins were identified in all 4 extracts including avidin and the pyruvate carboxylase. 7 proteins were only present in the sample containing Neur and could be its putative targets.

Peptide antibody

The second method used to try to identify new substrates of Neur was based on the anti gly-gly antibody. This method is described in detail in the Chapter 3. It relies on an antibody recognising the molecular signature created when ubiquitin bound proteins are treated with trypsin. The use of this antibody allows the purification of ubiquitinated proteins after trypsination. Purification is followed by mass spectrometry to identify the different peptides.

For the experiment, three conditions were considered: (1) embryos ubiquitously expressing Neur (daG4 x UAS-Neur-V5), ( 2) embryos ubiquitously expressing two inhibitors of Neur (daG4 x UAS-mα, UAS-Tom) and ( 3) embryos ubiquitously expressing a catalytic death version of Neur (daG4 x UAS-Neur RF). The flies were allowed to lay eggs for 8 hours at 25 • C and the embryos to age for another 8 hours at 25 • C, before being collected and frozen.

Francis IMPENS treated the samples in two ways: Firstly, he listed all the identifiable proteins in the three samples without purification. Secondly, he listed all of the identifiable proteins after purification. A description of the protocol with the results (written by Francis IMPENS) is given in the following pages. The take home message from these experiments is that 1284 proteins were identified in all samples before purification and 417 proteins where identified after purification. This method is highly sensitive and allows the comparison of the relative abundance of the different proteins in the different samples. Interpretation of the results differs depending on the treatment criteria. For proteins identified without purification, it is assumed that the putative Neur substrates have been ubiquitinated and degraded. Hence, they should be less abundant in the samples containing active Neur. For the proteins identified after purification, it is supposed that the putative Neur substrates are ubiquitinated and then enriched by the purification method. They should be more abundant in the samples containing active Neur.

From the results after purification, the only protein that was clearly enriched in the samples containing Neur was the E2 CG7656. At present, Effete is the only known E2 for Neur [215]. CG7656 presents as a good candidate for another E2 of Neuralized. It may be that an actual target of Neur was purified with this method, but due to the large number of proteins identified it is hard to pin point any good candidate. Also, it is noteworthy to state that none of the 7 proteins found with the previous method, nor Delta or Sdt, appeared enriched. The different crosses were put in egg laying chambers and allowed to lay eggs on a petry dish for 8h.

Proteome

Then the embryos were left to develop for 8h before being collected, dechorionated, washed with PBS and frozen in dry ice and ethanol. They were stored at -80°C until further use.

2. Embryos from every condition were mixed and dounce-homogenized in 7 ml PTMScan lysisbuffer (20 mM HEPES pH 8.0, 9M urea, 1mM Na orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate).

3. Lysates were sonicated by four bursts of 15'' at an amplitude of 20%.

4. Lysates were cleared by centrifugation for 15' at 16000 x g at 15˚C.

5. The protein concentration was measured (Bradford):

-4.16 mg/ml (∆RF) -2.76 mg/ml (V5) -4.63 mg/ml (TOM)

6. From here onwards, the Cell Signaling Technologies (CST) protocol was followed to enrich for ubiquitinated (GG-modified) peptides for LC-MS/MS analysis.

7. The ∆RF and TOM samples were further diluted with lysisbuffer to the concentration of the V5 sample. Finally, aliquots of 6.6 ml of the lysates, each containing 19 mg of total protein were used for further analysis.

8. Proteins were reduced by addition of 5 mM DTT and incubation for 30' at 55˚C. 9. The solution was cooled down to room temperature and 20 mM chloroacetamide was added. The lysate was incubated in the dark for 15' at room temperature.

10. 20 mM HEPES pH 8.0 was added to the 7.26 ml lysate to a final volume of 29 ml (4 times dilution).

11. After dilution, 100 µg trypsin was added to each lysate and samples were incubated O/N at 37˚C.

12. The samples were acidified to 1% TFA, incubated on ice for 15' and centrifuged for 15' at 1780 x g.

13. The cleared lysates were purified on Sep-Pak columns as described in the CST protocol and peptides were recovered in 6 ml 40% acetonitrile, 0.1% TFA.

Peptide antibody 173

14. The eluate was frozen O/N at -80˚C, lyophilized for 48 hours and the dried peptides were stored at -20˚C until further use.

15. The lyophilized peptides were redissolved in 1.4 ml CST IAP buffer and the solution was centrifuged for 5' at 10,000 x g at 4˚C.

16. A 50 µl aliquot with 680 µg peptides was taken to analyze the input. 628 µl 0.15% TFA was added and 50 µl of this mixture (+/-100 µg peptides) was desalted on C18 OMIX tips as described below.

17. The antibody-bead slurry was washed three times with 1 ml of PBS.

18. The cleared peptide solution was transferred into the antibody tube which was incubated for 3 hours on a rotator at 4˚C.

19. The beads were collected and washed 2x with CST IAP buffer and 3x with water as stated in the CST protocol. 20. To elute immunoprecipitated peptides, 55 µl 0.15% TFA was added to the beads followed by incubation for 10 minutes at room temperature. A second elution step was performed and both eluates were mixed.

Desalting on C18 OMIX tips:

-tips were equilibrated by passing 5x 100 µl 50% acetonitrile, 0.1% TFA, followed by 10x 100 µl 0.1% TFA -the peptide samples were loaded by pippeting up and down 20 times -tips were washed by passing 5x 100 µl 0.1% TFA.

-peptides were eluted in 100 µl 40% acetonitrile, 0.1% TFA by pipetting up and down 5 times. A second elution step was performed in another aliquot of 40% acetonitrile, 0.1% TFA after which both aliquots were mixed and transferred to a mass spec vial.

20. Peptides were dried completely by vacuum drying.

21. Dried peptides were re-dissolved in 18 or 50 µl mass spec 2% acetonitrile, 0.1% formic acid of which 8 or 2 µl was injected on a Proxeon EASY-nLC 1000 UHPLC system in line connected to a Thermo Q-Exactive mass spectrometer, respectively. Samples were loaded on a 30 cm reverse-phase column (made in-house, 75 µm I.D. x 150 mm , 1.9 µm beads C18 Reprosil-HD, Dr. Maisch). Peptides were eluted by a increase from 5 to 60% 0.1% formic acid in acetonitrile over 150 minutes at a constant flow rate of 250 nl/min and MS and MS/MS spectra were recorded using an adjusted topN 5 method.

22. The LC-MS/MS reproducibility was excellent as observed from the ion chromatograms below:

22. Recorded spectra from both input and IP samples were searched with the MaxQuant software in a database with D. melanogaster proteins downloaded from NCBI (RefSeq entries, 30277 proteins downloaded on 02/09/2014). The enzyme was set to Trypsin/P allowing maximum 3 missed cleavages. GlyGly (K) / Oxidation (M) / Acetyl (protein N-term) were set as variable modifications, while Carbamidomethyl (C) was set as fixed modification. Labelfree Quantification was enables (LFQ setting)

and matching between runs was allowed.

23. In the input samples, 1724 proteins were identified. Of these, 1284 proteins were quantified in all three samples. A scatter plot comparing ∆RF/V5 versus ∆RF/TOM ratios revealed a subset of proteins that are specifically upregulated in the ∆RF sample, but not in the V5 and TOM samples as shown in figure 1. A list 122 proteins with log 2 (∆RF/V5) > 0.5 or log 2 (∆RF/TOM) > 0.5 showed the presence of 6.2. Peptide antibody several myosin-related proteins and proteins involved in contraction (such as myosin light chain, myosin heavy chain, paramyosin, tropomyosin, myofilin...). From these samples, without any enrichment, only 5 GlyGly-modified lysine residues were identified (16 out of 40635 peptide sequences, or 0.039 %). The data from the input samples is summarized in the excelfile input_data.xlsx.

24. In the GG-peptide IP samples, 417 GlyGly-modified lysine residues were identified (1396 out of 28606 peptide sequences, or 4.88%), resulting from a 125x enrichment of GG-modified peptides compared to the input samples. Of these, 332 sites were quantified in all three samples. A scatter plot comparing ∆RF/V5 versus ∆RF/TOM ratios revealed 4 subsets of regulated sites as shown in figure 2. The data from the IP samples is summarized in the excelfile IP_data.xlsx. -6 sites high in the ∆RF sample, but with exception of ribosomal protein S20 all protein levels were already upregulated in the input sample. Therefore, the higher presence of these sites in the ∆RF sample does not necessarily reflect an increase in ubiquitination: Results annex 1: CG3894/Ozz iv) Do the other members of the Neur family of genes also regulate epithelial remodeling?

In Drosophila, the Neur family of genes is composed of three genes containing NHR domains : Neur, CG3894/Ozz and Bluestreak. During my PhD I explored the possibility that Neur and CG3894/Ozz were forming a protein complex capable of regulating epithelial remodeling. This appendix details the ideas that led to this hypothesis and how it ended up being wrong. It also shows some tools developed for the study of CG3894's function. Bluestreak was not studied in this thesis due to the large size of the gene, making it difficult to clone.

CG3894/Ozz physically interacts with Neur and with Tom

The structure of Neur and CG3894/Ozz is given in

CG3894/Ozz is zygotically dispensable for embryonic development

As Neur and CG3894/Ozz could physically interact, and Neur is required for epithelial remodeling and Delta-Notch signaling, we wondered whether CG3894/Ozz also played a role in embryonic development. To assess this we studied the CG3894 17l null-allele generated in the Boulianne lab, which was generated by a P-element excision which removed most of the coding region of the gene Figure 7.1 C. It was not possible to obtain homozygote adult flies for the CG3894 17l allele, suggesting that the gene could be required during embryo development. A survivorship assay of 200 embryos laid by CG3894 17l /CyoRFP was performed. The homozygous CyoRFP embryos (25% of the progeny) are expected to die, and the heterozygous embryos (CG3894 17l /CyoRFP 50% of the progeny) are expected to hatch. The question was whether the remaining (25%) embryos, which are homozygous for CG3894 17l , will hatch. The survivorship assay for 200 embryos showed that 75% of the embryos hatched (Figure 7.2 A), and thus that the allele CG3894 17l was not embryonic lethal when it homozygous.

Moreover, L1s (the hatched embryos) containing the CyoRFP chromosome where marked RFP + , so homozygous CG3894 17l L1s could be selected based on the absence of RFP. RFP -L1s were then genotyped for the CG3894 17l allele using two couples of primers presented in Figure 7.2 C,D and E. The genotyping (Figure 7.2 B) showed that all of the RFP -L1s were homozygous for the CG3894 17l , thus confirming that the allele CG3894 17l was not homozygous embryonic lethal.

Pupae homozygous for CG3894 17l were never seen, suggesting that homozygous CG3894 17l larvae die between the L1 stage and pupation.

CG3894/Ozz is expressed in the testis but is dispensable for its morphological organisation

After discovering that the allele CG3894 17l was not homozygous embryonic lethal, we observed that it gave viable flies when combined with a chromosomal deficiency lacking the CG3894 gene, and so we decided to stop working on its role during embryonic development.

According to Flybase the transcript of CG3894/Ozz is highly expressed in the male testis. Moreover Neur has been implicated in the regulation of the stem cell niche in the testis [250]. We thus wondered whether CG3894/Ozz could play a role in the regulation of the stem cell niche in the testis.

First, Khallil MAZOUNI constructed a BAC containing a GFP tagged version of CG3894. This BAC was then combined with the CG3894 17l allele but was not able to rescue the allele, supporting the idea that there is a second mutation in the chromosome containing the CG3894 17l allele. The drosophila male testis is a tubular structure containing, the germ cells (Figure 7.3 E) which are located at one end around a group of compact cells called the hub. The germ cells differentiate into spermatozoids as they move away from the hub. Staining of the testes of males containing the CG3894-GFP BAC showed that CG3894-GFP is expressed in the cells from the tip of the testis and excluded from the hub (Figure 7. 3 A,B,C). Moreover, a strong signal was observed in the round spermatid cells Figure 7.3D and F.

Finally, to study the function of CG3894/Ozz, the testes of males of the genotype CG3894 17l /Df(CG3894), were analysed. No obvious defect was detected in the tip of the testis(Figure 7.3G and H) nor anywhere else (data not shown). Furthermore, flies lacking CG3894 showed no obvious defect in fertility. Thus, the role of CG3894 in the testis remains not known.

Tools generated in the lab to study the role of CG3894/Ozz

During the study of CG3894 several tools were developed in the lab: a BAC encoding a GFP version of it (already mentioned), a polyclonal antibody against CG3894 and a fly line expressing an UAS-CG3894-VSVG construct.

Antibody anti CG3894/Ozz

The antibody was made by the Chinese company Ab-Mart and allows the detection of two different UAS-constructs of CG3894 by western blot (Figure 7.4A) or the UAS-CG3894-MYC construct by immunofluorescence (Figure 7.4B) in red. The UAS-CG3894-VSVG was not tested in immunofluorescence. It recognises CG3894/Ozz in western blot -CG3894/Ozz is overexpressed in the wing disc or in HEK cells (A)-and in immunostainings; the protein was overexpressed in the dorsal compartment of a wing disc (B in the red chanel). Moreover, CG3894/Ozz seems to localise to the nucleus when overexpressed. Two drosophila lines contain a construct expressing CG3894/Ozz under the UAS promoter: the UAS-CG3894-Myc from the lab of Gabrielle Boulianne, and the UAS-CG3894-VSVG constructed in the lab. The two constructs code for proteins of different sizes (see C ) that are recognised by the antibody anti-CG3894 (see A the construct expressed in the HEK cells is the same that was inserted into the flies), and they both localise to the nucleus when overexpressed in the wing disk (D and E). The CG3894-VSVG correspond to the expected size of CG3894 according to Flybase.

The exact structure of the CG3894-Myc is not known.

Conclusions

To conclude, CG3894/Ozz physically interacts with Neur. But the relevance of this interaction is not known as CG3894/Ozz is not required for embryonic development and its function in the male testis is not clear. In mice CG3894/Ozz has been shown to regulate muscle development by ubiquitinating and degrading β-catenin/armadillo, the myosin heavy chain and the adaptor protein Alix, nonetheless the role of CG3894/ Ozz in the regulation of Drosophila's muscle development remains unexplored.

Candidate-based identification of Neur substrates

In the paper presented in the Chapter 4, the ectopic expression of Neur in the dorsal compartment of the wing disc was used as a fast way to screen for the effect of Neur on the levels of polarity proteins. This assay was also used to test for the role of Neur in the regulation of actomyosin contractility. It is noteworthy to state that this fast assay is far from being a perfect experiment, and a protein that doesn't seem to be affected by Neur in this assay should not be excluded as a putative Neur substrate.

Ectopic expression of Neur and actomyosin levels

The effect of ectopic expression of Neur on actomyosion levels was assessed in fixed and stained tissue (Figure 8.2). Neur didn't have a clear effect on the Myosin Light Chain (Squash or Sqh in Drosophila) and its effect on actin was variable. In some imaginal discs like the one shown in Figure 8.2B), the effect was not very clear, however in some other discs like the one in Figure 8.

Ectopic expression of Neur and T48

T48 is a transmembrane protein that also promotes apical constriction during mesoderm invagination, by recruiting RhoGEF2 [124]. The other known target of Neur is Delta, another transmembrane protein. So it could be conceivable that Neur could somehow promote T48 levels. T48-gfp showed grossly the same levels when coexpressed with Neur or when expressed alone in the dorsal compartment of wing imaginal discs (Figure 8.4). However, it cannot be excluded that Neur interacts with T48 and that for example the ubiquitination of T48 by Neur promotes its interaction with RhoGEF2.

Ectopic expression of Neur and apical/basal position of the junctions

A lot of proteins were tested using this assay, including the junctional proteins E-Cadherin and β-catenin/armadillo. The ectopic expression of Neur was never obviously able to affect the apical/basal position of junctions (data not shown). In the embryo Neur was proposed to regulate the apical relocalisation of junctions during mesoderm invagination [222]. This function of Neur seems to be context dependent. Manning et al. (2013) [127] used an assay based on S2R+ to identify Mist as one of the GPCR receptors of Fog. They allowed S2R+ cells to spread on a coated surface and then observed that the addition of cell medium containing the Fog ligand was sufficient to induce a contraction of the cells. Following this, they used RNAi to deplete the different GPRCr receptors and establish which one was required for this Fog-dependent contraction. We planned to use this assay to test whether or not Neur could regulate the pathway downstream of Fog. The results presented in Figure 8.5) are the work of Vanessa ROCA, a technician in the lab. Albeit some variability between experiments (which is not represented in the figure) she was able to successfully reproduce the above assay and show that, for S2R+ cells, the addition of a Fog enriched medium is sufficient to stimulate cell contraction (Figure 8.5A and C). We then wondered whether the expression of the Brd protein, an inhibitor of Neur could perturb this contraction. However, we found that the expression of Brd in S2R+ was sufficient to make them adopt a stellate morphology, independent of the presence, or absence, of a Fog enriched medium (see Figure 8.5B1 and D1). This result was confirmed in another experiment where Brd was expressed in S2R+ cells (see Figure 8.5E1 to E4). This meant that the effect of the inhibition of Neur by Brd, on the pathway downstream of Fog could not be tested with this assay. Hence, we decided to stop this experiment. Interestingly, Rogers et al.[253] and D'ambrosio and Vale 2010 [254] did RNAi screens looking for genes required for the spreading of S2 (not S2R+) cells on coated surfaces. They found that the absence of different members of the Arp2/3 complex (P16-ARC, Sop2, Arp14D, Arp66B, Arc-p34, Arc-P2), the WAVE complex (Abi, Sra1, Scar, Hem1) and some other genes (Rac1, Rac2, CK1alpha, Capping β) caused a similar phenotype to that of which we observed in cells ectopically expressing Brd ( see Figure 8.5F to see the image from the Rogers et al. paper [253] showing a S2 cell depleted from a subunit of the ARP2/3 complex). The Arp2/3 complex is responsible of nucleating branched actin filaments, and the WAVE complex is one of its activators.

Brd and the spreading of S2R+ cells on coated plates

Based on our results and the screen carried out by D'ambrosio et al, I hypothesised that, in the context of the Brd expression when Neur is inhibited, one of the proteins found in the D'ambrosio screen could be inactive. This hypothesis lacks strength for several reasons. Firstly, the possibility that Brd acts directly on one of the proteins from the D'ambrosio screen cannot be excluded. Furthermore, it is possible that the spreading of the cells is an artifact caused by the ectopic expression of Brd. From the list of genes suggested, the gene Abi (Abelson interacting protein) is of particular interest. Indeed, Abi interacts and activates Abelson kinase [255], and the absence of Abelson kinase perturbs mesoderm invagination and apical constriction by regulating the levels of the actin regulator, Enabled [153]. Whether Neur can regulate the protein stability or the activity of Abi, Abelson kinase or Enabled, still needs to be properly tested. However in stained wing discs, the ectopic expression of Neur in the dorsal compartments did not affect the levels of Enabled (data not shown). The depletion of a subunit of the Arp2/3 complex in spreading S2 cells causes a similar defect. Image from Rogers et al. 2003[253]. RNAis directed against other proteins also caused the same stellate phenotype.

See the text for more details.

Neur and the activation of Rho1

During mesoderm invagination, apical constriction is driven by the activation of Rho1 by RhoGEF2. This leads to the activation of Rok and to the formation of an apical actomyosin network. In the absence of Neur the apical actomyosin network is perturbed, which raises two questions: Does Neur affect the activity of Rho1? Does Neur genetically interact with regulators of Rho1? The following experiments were carried out to address these questions.

Ectopic expression of Neur increases the accumulation of a Rho1 biosensor

The Parkhust laboratory developed several biosensors to test the activity of the Rho1 GTPase. One of these biosensors is composed of the part of Rok that binds to Rho1, bound to a GFP. When the biosensor is expressed the levels of the GFP signal are assumed to correlate with the activity of Rho1

Genetic interaction between Neur and RhoGEF2

In a second experiment, we wanted to test a putative genetic interaction between Neur and the activator of Rho1, RhoGEF2. The ectopic expression of Neur in the dorsal compartment of the wing imaginal disc caused no obvious morphogenic defect (Figure 8.7A). However, the ectopic expression of RhoGEF2 produced an increase in actin levels (marked with phalloidin) and perturbed the development of the disc (Figure 8.7B). Finally, the co-expression of Neur and RhoGEF2 increases the defect observed in the wing and causes overgrowth of the tissue, something that is not observed in the expression of either of the proteins alone (Figure 8.7B). The three wing discs are at the same scale. These results are hard to interpret as they don't show at what level the two proteins are interacting. It could be that both Neur and RhoGEF2 activate Rho1 in a parallel pathway, or that Neur somehow promotes RhoGEF2 activity. Both of these hypotheses fit with the results of the RokRBD biosensor experiment, suggesting that Neur promotes Rho1 activation. However, it could also be that Neur and RhoGEF2 are acting in two completely independent pathways that converge in the regulation of actin and/or cell growth.

The RING domain of Neur is required to downregulate Stardust

The ectopic expression of a version of Neur lacking the RING domain doesn't cause the downregulation of endogenous Sdt in the wing disc (Figure 8.8B). This shows that the E3 ligase activity of Neur is required for the downregulation of Sdt. Whether or not Sdt is ubiquitinated by Neur still remains to be explored.

Part III

Discussion and Perspectives

Chapter 9

Discussion

Sdt and Neur

A major focal point of this thesis is the regulation of the different crumbs complexes by Neur and the interaction between Neur and the crumbs complex protein, Sdt-PB. In chapter 4, we proposed that Neur regulates the Crumbs complex containing this isoform (Sdt-PB) in a different manner to those complexes containing the Sdt-PF isoforman isoform which does not interact with Neur. This finding introduces several interesting questions: Firstly, how does the interaction between Neur and Sdt-PB regulate the Crumbs complex and what other pathways may be involved in Crumbs regulation? And secondly, do complexes containing Sdt-PB share the same properties as those containing Sdt-PF and are they composed of the same proteins?

How does the interaction between Neur and Sdt-PB regulate the Crumbs complex?

There are several mechanisms by which Neur could regulate Crb through Sdt-PB: 1) Sdt-PB could act as an adaptor protein for Neur to ubiquitinate Crb. Ubiquitinated Crb would then be endocytosed in an Epsin dependent manner, similar to that of transmembrane protein Delta. 2) Sdt has been shown to protect Crb from and AP-2 dependent endocytosis. If Neur ubiquitinated Sdt to remove it from the complex, AP-2 would be able to induce Crb endocytosis. 3) Sdt-PB could be directly ubiquitinated by Neur and then whatever is bound to it endocytosed. 4) Sdt-Pb could act as an adaptor protein for Neur to ubiquitinate a protein that is part of the Crb complex, resulting in endocytosis of the whole complex. At present, the only evidence that allows to discriminate between these different possibilities, is that the ectopic expression of Neur and Sdt-PB has a cooperative effect and causes a stronger downregulation of Crb than Neur expressed alone. In light of this, the possibility that Neur acts by removing the 'protective' effect of Sdt over Crbs, resulting in AP-2 mediated exocytosis, can be excluded (possibility 2). However, the other possibilities are harder to prove wrong. A construct encoding a version of Sdt lacking the PDZ domain-and thus, in theory, not able to bind to Crb-has been shown to not localise to the apical domain when overexpressed in the wing disc. This suggests that it is not able to interact with Crb, or the other members of the Crb complex. Interestingly, Sdt PDZ is still downregulated when co-overexpressed with Neur (data not shown), suggesting that Neur acts on other proteins of the Crb complex, rather than Crb (Sdt PDZ still binds to Par6 and PatJ, thus it is possible that Neur could act on these proteins, using Sdt as a scaffolding protein). Daskalaki and colleagues [226] carried out in vitro experiments showing that Delta was ubiquitinated in a Neur dependent manner and mapped the site of Neur ubiquitination. However, the addition of further controls are needed to make these results more robust. Neur enhances the defects seen in epsin clones and epsin is required for the endocytosis of monoubiquitinated proteins and for the endocytosis of Delta. Therefore, it was proposed that Neur mono-ubiquitinates Delta [224]. However, mono-or poly-ubiquitination of Delta has never been shown. These same questions arise for Sdt-PB and Neur: it is not clear if Sdt-PB is ubiquitinated by Neur, or if it is, whether it is mono-or poly-ubiqitinated. There is also the possibility that Neur does not ubiquitinate Sdt at all, but rather serves as an adaptor protein for the ubiquitination of other proteins. Finally, it remains unknown as to whether the ubiquitination of Sdt (in case it is actually ubquitinated by Neur) causes its degradation.

Is Sdt-PB ubiquitinated by Neur?

Are there Crb complexes with different molecular constitutions?

There is a large gap in our understanding of epithelial polarity in regard to the composition of the different Crb complexes. For example, it is unknown if there exists only one type of Crb complex, or several composed of different proteins; or whether the different Sdt isoforms can be found in the same Crb complex, or whether the presence of one excludes the other. Similarly, it remains to be determined if Crb complexes containing the Sdt-PB isoform have the same properties as those containing Std-PF. Additionally, it is not known whether Neur could change the relative ratio between complexes containing Sdt-PB and containing Sdt-PF. The answers to these questions would require a deep biochemical analysis of the Crb complex.

How does Neur regulate other processes through Sdt?

The protein Sdt is not always bound to Crb and in the early embryo Sdt binds to Baz. In the paper by Krahn and colleagues [36] this binding is interpreted as a way to recruit Sdt apically prior to the expression of Crb. It is therefore possible than Sdt has other functions that are independent of Crb, and that these functions are also regulated by Neur.

Other mechanisms acting in parallel to Neur and Sdt-PB

Neur regulates the isoforms of Sdt containing the exon3. However, flies lacking the exon (Sdt 3 ) and where Neur is not able to bind Sdt are completely viable. This raises the possibility that Crb is regulated by other mechanisms in the absence of the Sdt-Neur interaction and that these mechanisms may, or may not be dependent on Neur. In the perspectives section of this thesis, I propose a screen to identify other proteins that may be involved in these mechanisms.

Neur, apical polarity and Delta-Notch signalling

In 2010 it was shown that, in the eye imaginal disc, the absence of Crb increases the endocytosis of Delta [258]. The discovery that Neur regulates Sdt-PB allows a novel interpretation of this data; that Sdt-PB buffers the levels of activity of Neur by recruiting it, and thus in the absence of Crb, Sdt is miss-localised and the fraction of Neur bound to Sdt could be released and start to ubiquitinate Delta, causing its endocytosis.

Regulation of Myosin contractility by Neur

203

Similarly, it's possible that Sdt could recruit Neur to specific parts of the cell undergoing Delta-Notch signalling and spatially regulate where Neur is acting. The connection between apical polarity and Delta-Notch signalling needs to be investigated in detail.

Regulation of Myosin contractility by Neur

In Chapter 5 we showed that Neur regulates Myosin contractility in the mesoderm. However, the molecular target of Neur for this function is unknown. Furthermore, the regulation of mesodermal myosin contractility by Neur raises other questions which are discussed below.

The molecular target of Neur

One potential molecular target of Neur is myosin; Neur could regulate Myosin by directly ubiquitinating some of the chains that constitute it. For instance it has been shown that the E3 ligases MuRF1 [259] and Ozz [245] , a protein of the Neur family of genes, can ubiquitinate and mark for degradation the Myosin Heavy Chain in the muscle. It is true that Neur overexpression in the wing disc doesn't noticeably affect the levels of the Myosin Light Chain, but it could be that the ubiquitination of Myosin by Neur doesn't cause its degradation but is important for the regulation of its activity.

In Chapter 7, some data is presented showing that the ectopic expression of Neur can stabilise a Rho1 biosensor, suggesting that Neur can positively regulate the activity of Rho1. There are several possibilities as to how Neur could do this.

Firstly, Neur could regulate either Smog or Mist, two GPCRs that are present in the mesoderm. This possibility is appealing as GPCRs are transmembrane proteins that, upon G protein binding, can trigger a signalling cascade. Furthermore, their endocytosis and trafficking is regulated by ubiquitination [260]. It is thus tempting to make an analogy between them and the transmembrane protein Delta, another known target of Neur which is endocytosed in a Neur dependent manner. However, co-immunoprecipitations assays in S2 cells, carried out by by Vanessa ROCA, between Mist and Neur RF showed no physical interaction in this context (data not shown). Furthermore, the ectopic expression of Neur in the wing disc didn't affect the levels of a BAC encoded GFP-fused version of Smog, suggesting that Smog, in this context, is not regulated by Neur. This data is difficult to interpret; it has been shown that, for some GPCRs, activation is required prior to their ubiquitination and endocytosis [260], or that their ubiquitination sometimes requires the presence of an adaptor protein which may not have been present in these two experimental conditions. Another possibility is that Neur regulates the activity of Smog and Mist indirectly, for example by regulating the proteins that inhibit their activity, such as GPRKs. GPRK2 has been suggested to limit GPCR activity in the mesoderm [143] and it is conceivable that Neur could regulate the effect of GPRK2 on the GPCR.

Mist and Smog bind and regulate the activity of a trimeric G protein composed of the sub-units Gγ1, Gβ13 and Concertina (also known Gα12/13). RNAi targeting of any of these three proteins perturbs apical constriction [128]. It has been shown that Concertina favours the recruitment of RhoGEF2 [136], but the role of the Gγ1, Gβ13 proteins is less clear. It is possible that they could help to recruit Concertina, as they do with Gαi in the neuroblast [132], or that they could signal independently of Concertina [261]. Moreover, in the neuroblast Gγ1, Gβ13 and Neur are all implicated in the regulation of asymmetric cell division. Even if they have never been linked, it would be interesting to study whether Neur could regulate Gγ1 and Gβ13 in the context of mesoderm invagination.

Finally, Neur could also regulate Rho1 downstream of the GPCRs, either through positive regulation of Rho1 GEFs, or negative regulation of Rho1 GAPs. RhoGEF2 has historically been the GEF that activates Rho1 in mesoderm invagination. However, it is possible that other GEFs could be implicated in the regulation of this process. The role of GAPs in mesodermal invagination has not been studied. It is therefore possible that Neur regulates some of the GEFs and GAPS of Rho1. Spahn and Reuters 2013[183] and Guglielmi et al. 2015 [185] have suggested that the topology of the region that is undergoing apical constriction is important for apical constriction to be anisotropic. This topology might be regulated by Neur activity. The transcription factor, Twist, is expressed in a gradient manner, peaking in the middle of the embryo and without a sharp boundary. Twist is thought to induce a gradient apical constriction, with cells closer to the centre of the embryo constricting more tightly. Conversely, Snail expression is constant and shows very precise boundaries. Brd is a target of Snail and, thus, the activity of Neur also shows very precise boundaries. Therefore, Neur could act to create a sharp boundary for myosin contractility. This would be important to define the topology of the cells undergoing apical constriction. Finally, by regulating Neur activity, Snail would define a sharp boundary for the region of activity of Twist.

Regulation of the topology of myosin contractility by Neur in the mesoderm

The role of the ectoderm during mesoderm invagination

Another open question in the field of mesoderm invagination is the role of the ectoderm. In silico models of mesoderm invagination [206] [208] have suggested that the ectoderm plays an active role in the invagination of the mesoderm and it has been suggested that apical/basal shortening of the ectoderm would be required for the mesoderm invagination. Moreover, mesoderm invagination can be divided into two phases. The first phase denotes apical constriction, leading to the formation of the ventral furrow, throughout which the ectoderm remains static. The second phase involves the movement of the ectoderm toward the furrow while the mesoderm internalises. Rauzi and colleagues [START_REF] Rauzi | Embryo-scale tissue mechanics during Drosophila gastrulation movements[END_REF], showed that the cauterisation of the ectoderm didn't affect the formation of the furrow but affected the internalisation of the mesoderm, suggesting that the ectoderm plays a role in the internalisation of the mesoderm. However, it remains unknown as to whether the ectoderm is actively pushing the mesoderm to favour its internalisation, or whether it passively deforms by being pulled by it.

The data presented in Chapter 5 does not allow for the discrimination between these two possibilities. Nonetheless, the observation that an increase in myosin contractility, and thus stiffness in the ectoderm, perturbs the internalisation of the mesoderm, fits better with the idea that deformation of the ectoderm is passive, as a result of being pushed by the mesoderm. It cannot be excluded that an increase of myosin contractility could somehow perturb the pushing ability of the ectoderm, but a molecular mechanism that is behind this pushing and that would be perturbed by an increase of medial myosin contractility still needs to be found.

Neuroblasts

Neur is able to regulate Delta-Notch signalling and apical constriction and it is possible that embryonic Neuroblast delamination is impacted on by both of these Neur functions. Neuroblasts are selected from proneural clusters through Delta-Notch signalling [262] and thus dependent on Neur activity.

Neuroblasts delaminate by undergoing an apical constriction [263]. There is suggestion that this apical constriction correlates with an increase of the medial pool of myosin (from an abstract of a drosophila conference of 2013 by Yan Yan and Chris Doe). We have shown that Neur is implicated in the regulation of apical constriction in the mesoderm by regulating medial myosin. Additionally, Neur seems to colocalise to the apical side of a delaminating Neuroblast (Figure 8.9), thus it is possible that Neur regulates its apical constriction.

As stated in the subsection on Sdt, Neur and Delta-Notch signalling, it is possible that Sdt buffers the activity of Neur. A possible mechanism for the activation of Neuroblast delamination, would be the reduction of Sdt-PB in the neuroblast, this would stop the buffering of Neur activity allowing Neur to induce apical constriction. Therefore, future work should investigate whether the reduction of Sdt-PB in the neuroblast precedes their delamination.

Conclusions

Neur can regulate several molecular pathways, for example cell fate through Delta, apical basal polarity through Sdt and actomyosin contractility in the mesoderm through an unknown target. These different functions of Neur were found independently, by studying different cellular processes, and future work will tell whether there is one process that requires all three molecular functions at the same time. Finally, discovering that Neur has three independent functions raises the exciting possibility that Neur has even more functions and targets which are yet to be discovered.

Chapter 10

Perspectives

The following Chapter proposes several experiments to answer to some of the questions presented in the discussion. Sdt is present in two forms, Std-PB and Std-PF (which lacks exon 3). In order to further explore the role of these two isoforms, it would be useful to be able to follow their localisation. The allele 3GFP-Sdt can be used to follow Sdt-PB. However, for now it is not possible to follow the Sdt-PF allele. Hence I propose to generate a tagged version of the Sdt-PF isoform. To do so, I plan to create a StdGFP in which the sfGFP sequence is split into two separate parts, flanking exon 3 (Figure 10.1B). The two parts of the sfGFP would only be transcribed together in the Sdt-PF isoform (when exon 3 is not present), allowing the generation of a complete sfGFP protein that is able to fluoresce and letting us track the Std-PF isoform. Of note, when determining where to split the sfGFP sequence, it is important to eliminate the risk of generating two parts of the sfGFP protein which are able to interact and fluoresce when they are in the same vicinity, as exemplified by the sfGFP truncated between the βstrands 10 and 11 [264]. One possible way of avoiding this would be to truncate the GFP in the middle of the strand 11, I speculate that a protein encoding half the strand 11 would not be able to interact with a protein encoding the stands 1 to 10 plus the other half of the strand 11, and cause its fluorescence.

Std-PB could be visualised by the addition of a second fluorophore at exon 3. This system would allow the visualisation of both the Std-PF and Std-PB isoforms at the same time, through the fluorescence of two different fluorophores. The selection of this second fluorophore should be done with extreme caution, as maturation time differs between different fluorophores [265][266] [267]. Therefore, a difference in the signal of proteins tagged with distinct fluorophores could reflect differences in the maturation time of the fluorophore and not in the subcellular location of the proteins. To counteract this, it would be best to select two fluorophores that mature at a similar speed, for example Cherry [267] and Venus [265] (both showing a maturation time of 40 minutes). In this example, the two parts of a split version of Cherry would be inserted in the exons flanking exon 3 and the Venus protein would be inserted into exon 3 (see Figure 10.1C). This would, in theory, result in the production of two fluorescent isoforms of Sdt; the Sdt-PB isoform tagged with Venus and the Sdt-PF isoform tagged with Cherry; and allow the observation of both proteins at the same time using live imaging. The different developmental stages in which the isoforms are expressed are not known. Therefore it would prove interesting to determine when during development, the different isoforms of Std are expressed. To do so, RT-qPCR could be used quantify the expression of mRNA containing the exon 3, Std-PB isoform, at different stages of embryonic development (for example every hour). Similarly, a boundary spanning primer for the sequence between the exons 2 and 4 and an opposite primer in a constitutive exon, could be used to identify the expression of Sdt mRNA lacking the exon 3, Std-PF isoform. One flaw in this experiment is that these two sets of primers would amplify more than two isofoms of Sdt (see Figure 10.1A. Several isoforms contain or lack the exon 3 marked in orange). However, this experiment would give a first indication of the developmental times where isoforms sensitive, or insensitive, to Neur are expressed at the same time. Following this, other RT-qPCR primers could be designed to better define the expression of the isoforms. It is possible that Sdt-PB and Sdt-PF form different protein complexes, with different properties, in the cell. In order to test the protein composition of Crb complex containing the Sdt-PB vs the Sdt-PF version and to do competitive binding experiments between the interactors of Sdt and the two isoforms.

Is the protein complex containing Sdt-PB different to the one containing the Sdt-PF isoform?

Sdt has been shown to interact with different proteins, summarised in Figure 10.1A)). However, it remains unclear as to whether the exon 3 can alter the affinity of Sdt for its interactors. To test this, we could perform co-immunoprecipitation in cell based assays for cells expressing, separately, each of the isoforms of Sdt and the different interactors: Par6, Crb, Bazooka, PatJ and Lin7. Following this, a pull-down of the different Sdt isoforms and their associated interactors would give information on the interactor affinities for each isoform. Sdt encodes two Par-6 binding sites which flank exon 3. Therefore, it is not unlikely that the affinity for Par-6 could differ between the Sdt-PF, lacking this exon, compared with Sdt-PB, with exon 3 present. Additionally, the PDZ domain of Sdt binds Crb or unphosphorylated Baz [START_REF] Krahn | Formation of a Bazooka Stardust complex is essential for plasma membrane polarity in epithelia. en[END_REF], thus it would be interesting to test whether or not the different Sdt isoforms show differential binding for these proteins. One could speculate that one isoform forms a complex preferentially with Crb and the other with Baz.

Finally, it would be ideal to be able to immunoprecipitate endogenous complexes formed by Sdt-PB or Sdt-PF. To do this, we could use the construct Sdt-Cherry-part1-Venus-Cherry-1-Par2, however, this would rely on the identification of an antibody specific to reconstituted Cherry, and not truncated Cherry. There are several alpaca nanobodies that are highly specific for RFP and GFP derived proteins. An initial test should be made to determine whether the nanobody that detects Cherry is specific to reconstituted, and not truncated, Cherry.

Do the two isoforms compete for the binding of their interactors?

To address this question, I plan to overexpress tagged versions of the two isoforms in the embryo, and then perform competitive immunoprecipitations, using known interactors as bait. For example, to test the Sdt-PB versus Sdt-PF affinity for Crbs, a pull-down of Crb in embryos of the genotype da>Sdt-PB-flag, Sdt-PF-flag would be performed. Supposing that both isoforms are overexpressed at the same levels, and that both bind Crb with the same affinity, we would expect to pull-down roughly the same amount of both isoforms when co-overexpressed. If this is not the case, that would indicate that one isoform has more affinity for Crb than the other. This could also be done in cell based assays by adjusting the amount of Sdt isoform that is expressed. For example to express the same amount of Sdt-PB and then progressively increase the amount of expressed Sdt-PF. Immunoprecipitate Crb and then look if by increasing the amount of Sdt-PB the amount of recovered Sdt-PF diminishes.

Neur and neuroblast delamination

One of the biggest limitations when working with Neur, is that its removal perturbs Delta-Notch signalling. In the case of Neuroblasts this translates into the complete transformation of all cells of the neurectoderm into neuroblasts, thus making the study of neuroblast delamination extremely difficult. To counteract this, we could construct a BAC containing all of the gene regulatory regions of Neur, and replace the Neur ORF by the one of the E3 ubiquitin ligases, Mindbomb. This would result in the expression Mindbomb in the place of Neur, while preserving the Neur regulatory sequences. Mindbomb is another E3 ligase that ubiquitinates Delta to promote Delta-Notch signalling. It has been shown that the ectopic expression of Mindbomb can rescue Neur mutant clones in the notum and in the wing disc [269]. Therefore, the BAC construct containing Mindbomb could rescue the Neur Delta-Notch function, but probably not its function in the regulation of apical basal polarity or the regulation of Myosin contractility. This would allow us to explore the role of Neur in the regulation of neuroblast delamination, without perturbing Delta-Notch signalling.

New Neur susbtrates

The following sections will discuss experiments that could be used to identify pathways acting redundantly to Neur, as well as strategies to identify new Neur substrates.

Sdt 3 and the deficiency kit

Neur regulates the isoforms of Sdt containing the exon 3. However flies lacking this exon (Sdt 3 ), in which Neur is not able to bind Sdt, are completely viable. This raises the possibility that other molecular pathways act in parallel to regulate Sdt in the absence of the Sdt-Neur interaction. To identify these parallel pathways I propose to screen for genes that increase lethality in a Sdt 3 context.

The Blomington Drosophila Stock Centre established a total of 417 fly lines containing genomic deletions covering 98.4% of the genes. Of these lines, 317 have deletions in chromosomes II and III [270],[271] males from these 317 lines could be crossed with Sdt 3 females (see Figure 10.2) and the viability of the male progeny assessed, by counting the number of males and female flies that develop to adulthood. Once a candidate region is identified, flies containing smaller deletions could be used to narrow down the genes involved. Finally, the candidate genes identified would be tested depending on the reagent available (RNAi, P-element excision null-alleles, or CRISPR mediated deletions could be considered).

Proteomics approaches for the identification of new Neur partners

The molecular target(s) that allow Neur to positively regulate actomyosin contractility in the mesoderm of early fly embryos remain unknown. During my PhD, I undertook two proteomic approaches to identify new Neur substrates. Unfortunately, these methods failed to give convincing results (presented in the results section). Nonetheless, there are two other proteomic approaches that could prove more fruitful. The difference between these new approaches and the previous ones, is that they rely on the purification of proteins interacting with or in the vicinity of Neuralized, whereas the previous approaches relied on the purification of the ubiquitilome (all ubiquitinated proteins) in different conditions. Hence, these new approaches are directed to identifying fewer proteins by mass spectrometry, and as a result, might work better.

GFP pull-down with nanobeads

Several papers have shown that the over-expression of a dominant negative version of an E3 ubiquitin ligase protects its target from degradation and forms stable complexes between the substrate and the E3 ligase. For example, the overexpression of a catalytically dead version of the E3 ubiquitin ligase Mindbomb, causes the formation of aggregates with its substrate Delta in the wing disk [272]. Similarly, the overexpression of a catalytically dead version of the E3 ligase Cullin increases the levels of it substrate, β-catenin in Xenopus embryos [273], and the overexpression of a catalytically dead version of Neur has been shown to increase the levels of Delta (its canonical substrate) also in Xenopus [214]. Moreover, the dominant negative form of E3 ligases have been used to identify E3 substrates [274].

Pull-down of GFP-tagged proteins in non denaturing conditions has been used extensively to detect protein complexes in vivo (for example in Keilhauer et al 2015. [275]) or to show interactions between an E3 ligase and its substrate (as exemplified in the paper from Shimizu et al. 2014 [276] where this tool is used to show a physical interaction between Notch and the E3 ligase, Deltex). Finally, the development of the single chain nanobody from Alpaca, which is able to recognise GFP and that can be coupled to agarose beads, allows better GFP immunoprecipitations (used for example in Shimizu et al. 2014 [276]).

I propose to use the GFP-tagged, catalytically dead version of Neur lacking the Ring Finger domain (Neur RF see Figure 10.3A)) to search for new Neur interacting partners. Flies containing an UAS-Neur RF-GFP construct, made by the Delidakis lab, are already available in the lab. The construct would be overexpressed in wing imaginal discs of Sdt 3 males, and beads coupled to the alpaca nanobody would be used to immonoprecipitate GFP in protein extracts from these wing discs. The purified product would then be sent to mass spectrometry.

Bio-ID based identification of substrates

In 2012 Roux and colleagues [277] developed a technique, called Bio-ID, which covalently links a biotin to the proteins in a complex with the protein a interest. For this they took advantage of a mutation of the E. Coli biotin ligase BirA, called BirA* , that is promiscuous and biotynilates all proteins in its vicinity. The authors speculated that the radius of the BirA* is of between 20 and 30nm, but were unable to obtain an actual measurement. The biotynilated proteins are then purified, taking advantage of the strong interaction between avidin and biotin, and identified by mass spectrometry. Coyaud and colleagues [278] used a flag-tagged BirA* fused to a subunit of the Skp Cullin F-box (SCF) beta-TrCP1/2 E3 ligase to identify its substates. In this paper, apart from purifying biotynilated proteins, they also performed an anti flag pull down to have a second method for detecting proteins interacting with the E3 ligase.

To detect Neur substrates, I propose the generation of two different versions of Neur, one where the RING domain is replaced by flag-BirA* (Neur RF-BirA*-Flag Figure 10.3A)) and another, in which flag-tagged BirA* is inserted into a region of Neur that is not conserved in Drosophilids (BirA*-Flag-Neur Figure 10.3A)). The logic behind insertion of flag-tagged BirA* into a non-conserved region, is that non-conserved regions are not under strong selective pressure, and thus perturbations in this region will probably not affect the function of the protein. These two Neur constructs will be encoded separately (1) replacing the open reading frame of a BAC containing an ubiquitously expressing gene (Figure 10.3B) or ( 2) under an UAS promoter. In total, four different lines should be made: two with the UAS promoter and two with the BAC.

For strategy number (1) a conditional cassette would be inserted prior to the ORF (Figure 10.3B) to control where and when the Neur RF-BirA*-Flag or BirA*-Flag-Neur will be expressed. Flies containing these constructs could then be crossed with a line expressing a flipase in the spermatozoids, so that the progeny of the males resulting from this cross contain the construct lacking the conditional cassette, and express Neur-BirA*-flag ubiquitously. The idea would be to purify the biotynilated proteins or the flag-tagged protein complex from embryos, and this construct could easily be combined with Sdt 3 flies.

For flies containing the Neur RF-BirA*-Flag or BirA*-Flag-Neur constructs under the UAS promoter (strategy 2), I propose to generate the stock presented in Figure 10.3A. This stock expresses the constructs ubiquitously only in embryos lacking the Brd genes, the inhibitors of Neur. I propose to use protein extracts of young, stage 6 to stage 8 embryos, to perform these experiments. These stages correspond to the time at which, in the absence of the Brd genes, the effect of Neur in the regulation of actomyosin contractility is observed.

FIGURE 1 . 1 :

 11 FIGURE 1.1: Cellularization and maturation of the adherens junctions. Bazooka is the drosophila homolog of Par3. Adapted from Laprise and Tepass 2011 [8].

FIGURE 1 . 3 :

 13 FIGURE 1.3: Main apical-basal polarity proteins. See text for details.Adapted from St Johnston et Ahringer 2010[START_REF] St Johnston | Cell Polarity in Eggs and Epithelia: Parallels and Diversity[END_REF].

FIGURE 1 . 10 :

 110 FIGURE 1.10: Possible mechanisms for the positioning of Par3/Baz. See text for details. Adapted from Flores-Benitez and Knust [74]

FIGURE 1 . 11 :

 111 FIGURE 1.11: Model for Crb self-recruitment. Figure from Fletcher et al 2012 [54].

FIGURE 1 . 12 :FIGURE 1 . 13 :

 112113 FIGURE 1.12: Formation of the larval gut. Image from the Atlas of Drosophila Development[START_REF] Hartenstein | Atlas of Drosophila Development[END_REF]

  .13 c), and at stage 10 Serpent is present in the posterior midgut and transcriptionally inhibits different polarity proteins, causing the apical/basal polarity to be lost (Figure 1.13 d).

FIGURE 1 . 14 :

 114 FIGURE 1.14: Stages of PGC migration. From Santos and Lehamnn 2004 [85] During gastrulation and germ-band extension they are passively carried as a cohesive group of cells, and at stage 8 they are located inside the posterior midgut (Figure 1.14 stage 8). At the end of stage 9, at the same time as the posterior midgut is undergoing the epithelial remodeling just described, they start to actively migrate through the intercellular space between the cells of the posterior midgut[80][83] (Figure 1.14 stage 9 and 10).

FIGURE 1 . 15 :

 115 FIGURE 1.15: Models for PGC migration. See text for details. FromRichardson and Lehamnn 2004 [86]

FIGURE 2 . 1 :

 21 FIGURE 2.1: Mesodermal shape changes during invagination. Schemes fromCosta et al. 1993. [102]. Images fromConte et al. 2012[103]. See text for details.

FIGURE 2 . 2 :

 22 FIGURE 2.2: Kymograph showing the apical shape changes in cross section of a gastrulating embryo. Taken fromRauzi et al. 2015 [99]

FIGURE 2 . 3 :

 23 FIGURE 2.3: Dorso/ventral patterning in drosophila. See text for details. Figure adapted from Haskel-ittah et al. 2012 [105] .

FIGURE 2 . 4 :

 24 FIGURE 2.4: Twist and Snail in situs at the onset of gastrulation. a) and d) Cross sections. b) and e) Surface view of the embryos. c) and e)Magnifications. Adapted fromLeptin 1991. [116] gradient peaking in the most ventral part of the embryo (Figure2.4c) and Snail is a zinc-finger repressor expressed homogeneously in the ventral part of the embryo with a sharp boundary of expression (Figure2.4f). The sharp boundary of snail expression is establish by the gradients of twist and dorsal[115]. Moreover, snail expression delimits the mesoderm and hence the cells that are destined to be internalised. Embryos mutant for either twist or snail do not invaginate properly (but still form a small furrow), and invagination is completely blocked if both transcription factors are removed[START_REF] Leptin | Cell shape changes during gastrulation in Drosophila[END_REF]. CHIP on chip experiments identified 106 genes directly under the control of Snail, half of them being repressed by Snail and the other half activated[117], however, the mechanism by which these intermediary genes direct the cell shape changes seen in embryos lacking Twist remains unclear. On the other hand, it has been proposed that Twist acts through at least 6 zygotic genes[118]: Snail (which is essential and whose maintenance in the mesoderm is dependent on Twist [119]), Tribbles and Fruhstart (two genes that play a permissive role by blocking cell division [120], [121], [122] ), the protein Traf4 [118], the ligand folded gastrulation (fog) [123] and the transmembrane protein T48 [124].

  [119]), Tribbles and Fruhstart (two genes that play a permissive role by blocking cell division [120], [121], [122] ), the protein Traf4 [118], the ligand folded gastrulation (fog) [123] and the transmembrane protein T48 [124].

FIGURE 2 . 5 :

 25 FIGURE 2.5: Activation of Rho1 by the Fog Signaling Pathway. Other names of the proteins appear in white boxes. Adapted from Manning et Rogers [130] GPCRs are coupled to a trimeric G protein composed of a Gα, Gβ and Gγ subunit. Embryos lacking any of these 3 proteins show defects in gastrulation [131],[132] [128]. Absence of the Gα protein, Concertina, phenocopies the absence of the Fog protein[133]. Moreover, the chaperone-like cofactor Ric-8 is required for the proper sub-cellular localisation of Gα proteins in neuroblasts and is implicated in gastrulation [134], [135]. The GPCRs, Mist and Smog, are predicted to interact with inactive, GDP-bound, Concertina. Thus, Fog binding to the GPCR results in a conformation change that promotes the exchange of GDP to GTP and the subsequent activation and release of Concertina[130], [128].

Chapter 2 .

 2 Epithelial remodeling during gastrulation proteins that are involved in the polymerisation of actin), Diaphanous (Dia) [148]. Rok[149] , myosin phosphatase[150], Cofilin[151], and Dia [152] have all been implicated in the regulation of apical constriction.

FIGURE 2 . 7 :FIGURE 2 . 8 :

 2728 FIGURE 2.7: Apical constriction in the ventral mesoderm during furrow formation.From Martin et al. 2009 [156]

  .8. b and c In Blue), that correlate with the phases of constriction and stabilisation of the apical area ((Figure 2.8 b and c. In Red). Perturbation of the apical actomyosin network disrupts apical constriction [156], [157],[149],

FIGURE 2 . 9 :

 29 FIGURE 2.9: Myosin II activation. Adapted fromLevayer et Lecuit 2012 [159]

FIGURE 2 . 12 :

 212 FIGURE 2.12: Radial cell polarity and actomyosin stabilisation.Figure adapted from Mason et al. 2013 [149].

  FIGURE 2.12: Radial cell polarity and actomyosin stabilisation.Figure adapted from Mason et al. 2013 [149].

FIGURE 2 . 14 :

 214 FIGURE 2.14: Junctions' basal to apical relocalisation. See text for details. Adapted fromKolsch et al. 2007 [124] andWeng and Wieschaus 2016 [182].

FIGURE 2 . 15 :

 215 FIGURE 2.15: Apical Constriction is graded and anisotropic. Adapted from Spahn et Reuter 2013 [183] Analysing the behaviour of cell constriction at the tissue level leads to two observations: Firstly, tissue constriction shows a gradient; cells constrict more, the closer they are to the ventral midline [183] [184] (see (Figure 2.15). This gradient seems to be directly under the control of Twist (Figure 2.4c); not only has Twist has been shown to direct apical constriction, but in the absence of certain Twist effectors, such as Fog, the constriction gradient is lost [123], [143].

  a) Constriction of a rectangular surface generates anisotropic deformation b) Constriction of a square surface generates isotropic deformation

FIGURE 2 . 16 :

 216 FIGURE 2.16: Vertex model of apical constriction showing that isotropic constriction can generate anisotropic deformation under some topologic conditions. Taken from Spahn et Reuter 2013 [183]

FIGURE 2 . 17 :

 217 FIGURE 2.17: Model for collective apical constriction. Apical constriction is regulated at a cellular and at a multi-cellular level. Figure from Xie et Martin 2015 [178]

FIGURE 2 . 18 :

 218 FIGURE 2.18: Tension increase recruits myosin in some developmental contexts. See text for more details. From Farge 2011 [189] and Fernandez-Gonzales et al. 2009. [188]

FIGURE 2 . 19 :

 219 FIGURE 2.19: Hydrodynamic flow in wild-type or acellular gastrulating embryos. Coming from He et al 2014 [194]

  elongation directed by polarized microtubules c) Strength of cell-cell adhersion b) Spatial regulation of Myosin II activity

FIGURE 2 . 20 :

 220 FIGURE 2.20: Cell height regulation in development. See text for details.Taken fromKondo et al. 2015 [197]

FIGURE 2 . 21 :

 221 FIGURE 2.21: Model for cell height changes during mesoderm invagination. See text for details. Ideas derived fromGelbart et al.2012 [195] andPolyakov et al 2014. [184]. Schemes adapted fromKondo et al. 2015 [197]

FIGURE 2 . 22 :

 222 FIGURE 2.22: Physical concepts used in the modeling of gastrulation.

FIGURE 2 . 23 :

 223 FIGURE 2.23: Comparative table of physical models of mesoderm invagination. See text for details. Figure adapted from [203].

FIGURE 3 . 1 :

 31 FIGURE 3.1: Neur structure and interacting proteins It contains two NHR domains (see Figure 3.11) and a RING domain, which provides its E3 ubiquitin ligase function[214] [215] [216] [216] [217].E3 ubiquitin ligase is an enzyme that catalyses the transfer of a free ubiquitin to a protein. This transfer is a multistep process involving several enzymes (Figure3.2). Ubiquitin is activated by the enzyme E1, which hydrolyses ATP and promotes subsequent binding of ubiquitin to the "ubiquitin conjugating enzyme", E2. An E3-ubiquitin ligase, containing a RING (Really INteresting Gene) domain (such as Neur) then catalyses the transfer of the ubiquitin from the E2 enzyme to the -side chain of a lysine in a protein, or to another ubiquitin. E3-ligase containing HECT (Homologous to the E6-AP Carboxyl Terminus) domains behave slightly differently as they bind directly to ubiquitin before transferring it to the substrate. It is the E3-ligase that determines the specificity of the substrate for ubiquitination. Finally E4, or "ubiquitin chain assembly factor" can modulate the formation of ubiquitin chains. Ubiquitination is a reversible process and ubiquitin can be cleaved from its substrate protein by the activity of deubiquitins (DUBs).The neur gene encodes two isoforms, PA and PC, which differ in their C terminus sequence and by the presence of a C-terminal Phosphoinositide-Binding (PIP2) Motif. This PIP2 motif allows the PA isoform to bind to the plasma membrane[219]. It was proposed that singular Neur proteins are held in an inactive conformation through NHR1-NHR2 interactions and that Neur is activated through NHR2-NHR2 interaction with another Neur protein[220].Neur is a positive regulator of Delta-Notch signaling, and flies lacking neur show the same phenotype as those defective for Delta-Notch signaling[215]. Experimental

FIGURE 3 . 2 :

 32 FIGURE 3.2: The ubiquitin destruction cycle. Figure from [218] . evidence from different studies shows that Neur regulates Delta. Firstly, the whole Neur protein or its NHR1 domain was shown to co-immunoprecipitate with Delta in S2 cells [221]; and secondly, loss of function alleles for Neur block Delta endocytosis in different developmental contexts, for example in the wing imaginal disc [215], the embryo [222] or the drosophila sensory organ precursor [223]. Finally, the ectopic expression of Neur is sufficient to cause the endocytosis of Delta in drosophila wing imaginal discs [215] and the expression of a Neur version lacking its RING domain and E3 ligase activity was shown to stabilise Delta [215]. Furthermore, it was shown that the endocytosis of Delta is dependent on Epsin and that Neur loss of function alleles are dominant enhancers of Epsin loss of function alleles [224]. As Epsin recognises mono-ubiquitinated proteins [225] and promotes their endocytosis, it was proposed that Neur mono-ubiquitinates Delta to promote endocytosis by Epsin. However, the direct ubiquitination of Delta by Neur has never been proven, nor whether it mono-ubiquitinates or polyubiquitinates its targets. Daskalaki et al. (2011) [226] carried out an ubiquitination assay which shows that Delta is ubiquitinated by Neur. However, the study lacks several controls and thus leaves room for questions. Lai et al. 2001 [215] proposed that Delta was being degraded after being ubiquitinated by Neur, based on the observation that the ectopic expression of Neur was sufficient to reduce the detectable levels of Delta in immunostainings. Whether the ubiquitination of Delta causes its relocalisation or its degradation has never been fully investigated. The current model of Neur action on the Delta-Notch pathway is presented in Figure 3.3. Neur binds Delta through its NHR1 domain [221] and ubiquitinates Delta [226], promoting its endocytosis in an Epsin dependent manner. In cultured cells, the endocytosis of Delta was shown to physically pull on Notch, changing its conformation and revealing cleavage sites [227]. The proteases, ADAM and γ-secretase, are then able to cleave Notch, in S2 and S3 sites respectively, triggering the release of the Notch intracellular domain (NICD).Free NICD relocates to the nucleus and binds to the transcription factor CSL (CBF1, SU(H),LAG-1) and to activators, to trigger the expression of Notch target genes. Of note, when NICD is not in the nucleus, CSL binds to repressors and inhibits the expression of the same target genes (reviewed inSchweisguth 2004 [228]).

  current model of Neur action on the Delta-Notch pathway is presented in Figure 3.3. Neur binds Delta through its NHR1 domain [221] and ubiquitinates Delta [226], promoting its endocytosis in an Epsin dependent manner. In cultured cells, the endocytosis of Delta was shown to physically pull on Notch, changing its conformation and revealing cleavage sites [227]. The proteases, ADAM and γ-secretase, are then able to cleave Notch, in S2 and S3 sites respectively, triggering the release of the Notch intracellular domain (NICD).

FIGURE 3 . 3 :

 33 FIGURE 3.3: Activation of the Notch pathway. From Soline CHANET's thesis.

FIGURE 3 . 4 :

 34 FIGURE 3.4: FISH for Neur and Tom. A and B From FlyFISH. C from Chanet et al. 2009 [222] Figure 3.5shows that in the stages 1 to 3, Neur is expressed and active in the whole embryo (shown in Figure 3.5). From stage 4, the Brd genes start to be expressed in the ectoderm, where they block Neur activity and exclude it to the mesoderm.

FIGURE 3 . 5 :

 35 FIGURE 3.5: Neur activity is restricted spatially in the early embryo. From Chanet and Schweisguth [222]. The mesectoderm is composed of a line of cells that is specified, in a Delta-Notch dependent manner, between the mesoderm and the ectoderm [234]. It can be identified by the expression of the gene sim. The Brd genes restrict the specification of the mesectoderm to a single line of cells[232] [235] [229], as embryos lacking the Brd genes show an increase of cells specified as the mesectoderm. The current model for the specification of the mesectoderm is that the Brd genes block Neur activity in the ectoderm, inhibiting Delta-Notch activity. Active Neur ubiquitinates Delta and activates Notch in one row of cells at the boundary of the mesoderm. This is presented in Figure 3.4C.

FIGURE 3 . 6 :

 36 FIGURE 3.6: Inhibition of Neur by Brd is required for the maintenance of epithelial polarity A to D Lateral view of wild-type, neur, Brd, Brd neur embryos. E to G Cuticule preps from wild-type, neur, Brd, Brd neur and Crb embryos. See text for details. From Chanet et al. 2009 [222]

FIGURE 3 . 7 :FIGURE 3 . 8 :

 3738 FIGURE 3.7: Neur is required for epithelial remodeling of the posterior midgut and for the trans-epithelial migration of PGCs. See text for details. Adapted from Chanet et Schweisguth 2012 [222].

FIGURE 3 . 9 :

 39 FIGURE 3.9: Brd genes are required to delay the relocalisation of junctions during mesoderm invagination. See text for details. Adapted from Chanet and Schweisguth 2012 [222].

FIGURE 3 . 10 :

 310 FIGURE 3.10: Enrichment strategies for ubiquitinated proteins. See the text for an explanation. Figure adapted from [236].

  Figures: 5 Supplementary Figures: 3

  Figures: 3 running title: Epithelial polarity regulation by Neur-Sdt-Crb Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 69 Introduction Epithelia form continuous sheets of interconnected and polarized cells that act as protective boundaries to separate inner from outer milieus and promote selective exchanges between these milieus. While structure and function need to be maintained over time, epithelia nevertheless can undergo dynamic remodelling through changes in the spatial arrangement of cell-cell contacts. During epithelial

Chapter 4 .

 4 Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 71

Chapter 4 .

 4 Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 73ResultsNeur down-regulates Crb complex proteinsWe previously showed that the inhibition of Neur by the Brd proteins is required to stabilize the primary epithelium during germ-band extension. High Neur activity in Brd mutant embryos resulted in the rapid loss of the apical Crb, Sdt, Patj, Par6 and aPKC proteins at stages 6-8(Chanet and Schweisguth, 2012). This suggested that the apical Crb complex is a target, direct or indirect, of Neur. To further investigate the regulation of Crb by Neur, we used an over-expression assay in third instar wing imaginal discs. Neur was ectopically expressed in the dorsal wing pouch cells that do not express Neur. As expected, ectopic Neur led to a downregulation of Delta. Interestingly, it also led to a strong down-regulation of Crb, Sdt and Patj (Figure 1C-H', I). Neur had also a weak effect on Par6 and no significant

Figure

  Figure S1). However, since the silencing of Patj had a weaker effect on the levels of Sdt and Crb than ectopic Neur (Figure1J), we could conclude that Patj is most likely an indirect target of Neur. Thus, Neur may act on Crb via Sdt, or vice versa. Since ectopic Neur had a stronger effect on Sdt than on Crb (Figure1I), we decided to further investigate whether Sdt could be a direct target of Neur.

  down-regulated Sdt-PB but not Sdt-PF (Figure 2C-G). This indicated that the downregulation of Sdt by Neur required the presence of exon 3. Moreover, we observed that Neur induced a stronger down-regulation of Crb when co-expressed with Sdt-PB (compare Figure 2F,G with Figure 1H-I).

Chapter 4 .

 4 Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 77 clearly indicated that Neur specifically targets the NBM-containing isoforms of Neur in vivo.

  ), indicating that increased Neur activity led to the down-regulation of these NBMcontaining isoforms. By contrast, SdtΔ3-GFP localized apically in wild-type embryos and was not detectably donw-regulated in Brd mutant embryos (Figure3E,F), confirming that the Sdt isoforms that lack a NBM are insensitive to high Neur activity levels. Finally, if the down-regulation of Sdt by Neur caused the Brd mutant phenotype, we then predicted that deletion of the exon 3 should suppress this polarity phenotype in sdt Δ3GFP Brd double mutant embryos. Using aPKC as an apical polarity marker and Neurotactin (Nrt) as a basal-lateral marker, we first confirmed that epithelial polarity was strongly disrupted in Brd mutant embryos (Figure4B,D)(Chanet and Schweisguth, 2012). We thene xamined sdt Δ3GFP Brd double mutant embryos and found that epithelial integrity and apical aPKC were remarkably restored (Figure4F). We therefore conclude that the down-regulation of the NBMcontaining isoforms of Sdt by Neur caused the loss of epithelial polarity in Brd mutant embryos and that the NBM-containing isoforms of Sdt are key in vivo targets of Neur.

Chapter 4 .

 4 Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 79 colonize the gonads. The timely migration of PGCs across the endoderm requires both activation of a G Protein-Coupled Receptor in PGCs and remodelling of the endodermal epithelium

81 Discussion

 81 therefore hypothesized that Neur down-regulates Crb via Sdt in the endoderm epithelium. Consistent with this hypothesis, the levels of apical Crb and Sdt were reduced in distal PMG cells at stage 10 (Figure5C,F; compare with stage 9 embryos, Figure5B,E) and this down-regulation was neur-dependent (Figure5D,G). Furthermore, while both Crb and the NBM-containing isoforms of Sdt showed reduced levels at stage 10 (Figure5H), deletion of the NMB in sdt Δ3GFP embryos resulted in the persistent apical accumulation of Crb and Sdt (Figure5I). Similarly, apical Crb was still detected at stage 10 in sdt Δ3 mutant embryos (Figure5J). This indicated that Neur prevents the apical accumulation of Sdt in an NBM-dependent manner and that the NBM-containing isoforms of Sdt are required for the neurdependent down-regulation of Crb. These results indicated that the NBM-containing 80 Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila isoforms of Sdt are key targets of Neur and that this regulation may contribute to control the level of apical Crb in this context. We next studied the trans-epithelial migration of PGCs across the endoderm epithelium. In both sdt Δ3GFP and sdt Δ3 mutant embryos, fewer PGCs had migrated through the PMG epithelium at stage 10 (Figure 5K,M-O). This migration delay appeared to be similar to the one seen in neur mutant embryos (Figure 5L,O). We therefore conclude that the regulation of Crb by Neur via specific isoforms of Sdt contributes to epithelial remodelling in this developmental context. Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila Crumbs is a key determinant of apical membrane identity that controls epithelial morphogenesis in a tissue-specific manner. Here, we have identified a molecular pathway that regulates its activity during Drosophila development. Indeed, our data revealed that Sdt is a molecular target of Neur, that Neur reduces apical Crb levels via specific isoforms of Sdt and that this regulation is important for epithelial remodelling in the embryo. Three lines of evidence indicate that specific Sdt isoforms are direct molecular targets of the E3 ubiquitin ligase E3 Neur. First, Neur physically interacts with Sdt.Interaction required a NBM present in a subset of the Sdt isoforms. Also, Brd inhibited this interaction. This is consistent with Sdt being the Neur target causing the loss of epithelial integrity in Brd mutant embryos, i.e. when Brd proteins no longer compete a key polarity regulator for binding Neur(Chanet and Schweisguth, 2012).Second, over-expression ofNeur led to reduced Sdt levels and the down-regulation of Sdt by Neur required a NBM. Thus, the weak Sdt signal that remained detectable upon ectopic expression of Neur (Figure 1F') is likely due to the Neur-resistant isoforms of Sdt that do not contain a NBM. These isoforms might contribute to stabilize Crb. Consistent with this interpretation, over-expression of both Neur and Sdt-PB, which likely competes endogenous Sdt, including the Sdt isoforms lacking a NBM, for Crb binding led to a nearly complete loss of Crb in this assay. Importantly, deletion at the locus of the NBM-coding exon showed that the effect of Neur on Crb required the presence of NBM-containing Sdt isoforms, demonstrating that Crb is an indirect target of Neur. Also, while increased Neur activity led to reduced levels of several apical proteins, low Sdt levels could account for the reduced accumulation of other apical proteins, suggesting that Sdt may be the only key target of Neur in 82 Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila epithelium morphogenesis. Third, the loss of epithelial integrity upon increased Neur activity, i.e. in Brd mutant embryos, required the presence of NBM-containing isoforms of Sdt. This clearly indicated that Sdt is a key in vivo target of Neur.

Chapter 4 .

 4 Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 83

  . Of note, this proposed Brd-Neur-Sdt-Crb regulatory cascade might contribute to other morphogenetic events in Drosophila, including during multicellular patterning in the developing retina (G. Perez-Mockus, data not shown).Nevertheless, since the genomic deletion of exon 3, i.e. disruption of the Neur-Sdt interaction, produced no major developmental defects, compensatory mechanisms must exist in the PMG and possibly in other tissues, e.g. the developing eye. For instance, other regulatory mechanisms could act in parallel, as exemplified by the transcriptional regulation of the crb gene by Srp(Campbell et al., 2011). Whether the same logic applies to Epithelial-Mesenchymal Transition (EMT) in mammals remains to be determined. Indeed, while it is known that Crumbs3, Sdt/Pals1 and Patj are transcriptional targets of the EMT transcription factors Snail[START_REF] Lamouille | Molecular mechanisms of epithelialmesenchymal transition[END_REF], whether Neur homologs act in parallel to regulate EMT remains to be investigated. Thus, morphogenesis may often involve redundant regulatory inputs to achieve efficient and timely output and the regulation of Crb by Sdt-Neur may represent one such developmental control mechanisms that contribute to morphogenesis.In mammals and flies, Neur regulates synaptic plasticity and memory[START_REF] Pavlopoulos | Neuralized is expressed in the alpha/beta lobes of adult Drosophila mushroom bodies and facilitates olfactory longterm memory formation[END_REF][START_REF] Pavlopoulos | Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage[END_REF]. Neur is expressed at high levels in the brain where it regulates dendritic spine morphogenesis. In the mouse, this function of Neur is in part mediated by the ubiquitination of CPEB3, which stimulates the local synthesis of AMPA receptors at the synapse[START_REF] Pavlopoulos | Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage[END_REF].Since the mouse homolog of Sdt, Pals1, regulates dendritic spine morphogenesis[START_REF] Zhang | The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase[END_REF], our study raises the possibility that one conserved function of Neur may be to regulate the activity of Sdt/Pals1 in the brain. Finally, our analysis suggests an intriguing connection between Notch signalling and apical-basal polarity. Indeed, in analogy with the inhibition of Neur by Brd, one may wonder whether the Sdt-Neur interaction may similarly compete with the Delta-Neur interaction. Consistent with this possibility, increased Delta endocytosis, hence Notch activity, was observed in crb mutant eyes (Richardson and Pichaud, 2010), a phenotype that might result from increased Neur activity due to the loss of Sdt in crb mutant cells. It is also conceivable that Sdt might positively contribute to Neur-mediated Delta signalling by promoting the apical localization of Neur. Reciprocally, high Neur in signal-sending cells might be associated with decreased epithelial stability and partial or complete delamination of neural cells, as seen in neuroblasts. Conversely, high Notch, hence high Brd, might contribute to stabilize epithelial polarity through Neur inhibition. The interplay between Notch/Delta and Crb/Sdt via Neur/Brd, linking epithelial polarity with Notch signalling, remains to be explored.Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 85

Sdtrpsl

  was verified by sequencing of the recombined regions prior to phiC31-mediated integration at the M[3xP3-RFP attP]ZH (51C) site. sdt K85 / Y flies carrying one copy of the corresponding BACs were viable and fertile. The DNA fragments encoding Sdt-PB/PF (from pUASt-Sdt-PB/-PF; gifts from E. Knust) were sub-cloned into pPac5.1 to generate pPac-3xFLAG-Std-PB/-PF. These plasmids were used to express FLAG-tagged isoforms under the control of the actin5C promoter in S2 cells. Starting from pPac-3xFLAG-Std-PB, a deletion analysis Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 87 of the sequence encoded by the exon 3 was performed using the Sequence and Ligation Independent Cloning (SLIC) method (Li and Elledge, 2012) (primers and cloning detail available upon request). The DNA fragments encoding 3xFLAG-Std-PB (from pPac-3xFLAG-Std-PB) and 3xFLAG-Sdt-PBΔNBM (from pPac-3xFLAG-Std-PBΔNBM) were sub-cloned into a pUASp-attB vector. These two transgenes were integrated at the PB[attP-3B y+]VK0033 (65B2) site. The pPac-Brd R -3xHA plasmid was produced by gene synthesis (to replace all ten lysines, K, of the Brd ORF by Arginine, R, hence producing the Brd R mutant version of Brd), PCR amplification andsubcloning into the pPac5.1 vector (primers and sequence can be obtained upon request). The Brd R mutant protein was found to be more stable, hence more active, than wild-type Brd (not shown).Injection was performed by BestGene Inc (Chinmo, USA).CRISPR-mediated HRCRSIPR-mediated HR was achieved by injecting three plasmids, a donor template with two gRNAs, into Cas9-expressing embryos. The following gRNAs were selected using the Optimal Target Finder tool (http://flycrispr.molbio.wisc.edu/tools):gRNA1: TTCCATTGGAGTCCACTAAG gRNA2: TTCGGCGGTGCAGGTCCGAC Oligonucleotides were cloned into pU6-BbsI-chiRNA (Addgene #45946) as described in www.addgene.org/crispr/OConnor-Giles/. The same gRNAs were used for both sdt Δ3GFP and sdt GFP3 . Donor templates for HR were first produced by BAC recombineering in E. coli using the sdt BAC CH321-22E06 and fetching into multicopy vectors as described in(Venken et al., 2006). The PAM sequences targeted by the gRNAs were mutated in the donor templates to avoid their Cas9-mediated cleavage. The 3xP3-RFP marker flanked by loxP sites was produced by gene synthesis. It was inserted in the 3' intron of exon 3. In sdt Δ3GFP , the sequence of the sfGFP precisely replaced the sequence of Exon3. In sdt GFP3 , GFP was inserted at amino acid 188 of Sdt-PB and the sequence of exon 3 was flanked with two FRT sites. Thus, GFP tags all six NBM-containing isoforms (Sdt-PB, PD, PE, PG, PM and PN) as well as 3 isoforms that contain only part of Exon3 excluding the NBM (Sdt-PI, PJ and PL; see flybase.org for detailed description of the 12 predicted isoforms of Sdt). Left and right homology arms flanking the target sites were 1.5 kb long.

Chapter 4 . 91 Figure legends Figure 1 :

 491legends1 Figure legendsFigure 1: Neur targets core Crb complex components A) Molecular activity and regulation of Neur. The E3 ubiquitin ligase Neur interacts with an E2 enzyme via its RING Finger (RF) and recruits its molecular targets, e.g. Delta, via its Neur Homology Repeats (NHR). Brd family proteins also bind the NHR and compete the Neur-target interaction. B) Neur negatively regulates epithelium polarity via unknown target(s). Brd antagonizes this activity of Neur. C-H') Ectopic Neur triggered the down-regulation of Delta (C,E), Sdt (D,F), Patj (G) and Sdt (H). GFP (C,D) and Neur (V5 tag, green in E-H) were expressed in the dorsal cells of third instar wing discs (see Figure S1 for negative GFP controls for

Figure 2 :

 2 Figure 2: Neur interacts with a subset of the Sdt isoforms A) Genomic organization of the sdt locus. Of the twelve isoforms, only four transcripts (RG, RB, RF and RD) are shown here. Exons are shown as boxes (ORF, black; exon 10, orange).

Figure 3 :

 3 Figure 3: isoform-specific down-regulation of Sdt by Neur

Figure 4 :

 4 Figure 4: loss of epithelial integrity in Brd mutants is caused by Neur-mediated down-regulation of Sdt A,B) Apical-basal polarity (aPKC, red; Nrt, white) and epithelial integrity were lost in Brd mutant embryos at stages 9-10 (B; wild-type control in A).C-F) The loss of epithelial polarity (aPKC, red; Nrt, white) seen in Brd mutants was

Figure 5 : 99 Legend 1 A

 5991 Figure 5: epithelial remodelling of the posterior midgut requires Neur-Sdt-Crb A) PGCs (yellow) form at the posterior pole of the embryo (stage 5) and adhere to the surface of the midgut primordium as it invaginates at stages 7-9. PGCs located in the lumen of the PMG migrate across the epithelium at stage 10. B-G) Crb (white, B-D; Nrt, red) and Sdt (white, E-G; note that anti-Sdt appeared to cross-react with yolk granules, yellow asterisk) were detected apically all along the PMG at stage 9 (B,E). At stage 10, Crb (C) and Sdt (F) were no longer detected in distal PMG cells (indicated with yellow arrows). In neur mutants, both Crb and Sdt remained detectable at the apical cortex of distal PMG cells (D,G).H-J) Crb (white) and Sdt-GFP3 (green) were not detected at the apical cortex of distal PMG cells (yellow arrows) in stage 10 sdt GFP3 embryos (H) whereas both Crb

Figure 2 AFigure 1 4 .

 214 Figure S2: Neur-Sdt interaction required a NBM-containing region of Exon3 connected to Figure 2 A,B) Structure of the deletion versions of Sdt-PB. The deletions introduced in Exon3 (A: cartoon of Sdt-PB; B: color-coded sequence of Exon3; the paired NBMs are boxed in black) were designed based on sequence conservation (plotted in purple in

Figure 2 :Flag 4 .Figure 3 4 .

 2434 Figure 2:

Chapter 4 .Figure 5 4 .Chapter 4 .Figure

 4544 Figure 4:

4 .

 4 Figure S3:

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 113elongated, these cells shorten along their apical--basal axis, possibly as the result of decreasing stiffness at their basal cortex, and this shortening may drive furrow invagination(Polyakov et al., 2014) (Figure1c). As most studies have focused on forces acting in the mesoderm, how ectoderm cells respond to apical constriction forces and contribute to furrow invagination and whether their contractility and stiffness are regulated during gastrulation remain largely unknown.

  and it regulates epithelial integrity and morphogenesis by targeting specific isoforms of the scaffolding protein Stardust (Sdt), a conserved MAGUK family protein interacting with Crumbs (Chanet and Schweisguth, 2012)(G. Perez--Mockus et al., submitted). During gastrulation, the Brd genes are expressed in the ectoderm, as a result of their repression by Snail in the mesoderm, whereas Neur is expressed both maternally and zygotically under the control of the transcription factor Twist. Since the Brd proteins inhibit Neur in the ectoderm, Neur is active only in the mesoderm where it regulates the endocytosis of Delta to promote Notch activation in the mesectoderm

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 115

  ,g). A similar phenotype was seen upon ectopic Brd expression (Figure1d--i). Furthermore, analysis 116 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila of fixed and immunostained embryos showed that some neur degrad embryos displayed a furrow formation defect characterized by an extended flat apical surface associated with a clear accumulation of apical MyoII (Figure 2d,e; note that defective endocytosis of Delta in the mesoderm revealed a strong loss of Neur activity). A similar furrow defect was previously seen upon over--expression of the Brd family proteins mα and Tom (Chanet and Schweisguth, 2012). It was also observed in sna>Brd R embryos (see Figure 3j below), strongly suggesting that Neur is the target of ectopic Brd in the mesoderm. This furrow invagination defect was seen in a small fraction of fixed neur degrad embryos (n = 3/x of stage 5--6 embryos), possibly due to the transient nature of this phenotype and/or to the incomplete loss of Neur in neur degrad embryos. Thus, we conclude that Neur is required for collective apical constriction and propose that ectopic Brd inhibits apical recruitment of MyoII and delays furrow formation by antagonizing Neur in the mesoderm. Neur was recently shown to target Sdt in epithelia (G. Perez--Mockus et al., submitted), raising the possibility that Sdt, a conserved scaffolding protein that interacts

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 117

  embryos might better match the Sigmoid force profile than the Gaussian one (which predicted a recruitment of apical MyoII beyond the mesoderm). If so, Neur would act to non--linearly regulate force generation in the mesoderm. In this view, the absence of Neur, 118 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila would result in low and uniform contractility in the mesoderm whereas in the presence of Neur, high MyoII activity in the most--ventral cells would favor rapid apical constriction in cells at the center of the mesoderm. Consistent with this view, the pattern of apical contractility appeared to be broader over the mesoderm in sna>Brd R , mat>mα+Tom and neur degrad embryos relative to wild--type controls (Figure S4). Together, simulations and experiments indicated that the activity of Neur is required in the mesoderm to regulate contractility in the ventral--most cells, thereby promoting collective apical constriction and rapid furrow invagination.

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 119 at the medial--apical cortex (Figure 4d,e). Thus, high Neur activity in the ectoderm, resulting from the loss of the Brd inhibitors, appeared to promote the recruitment of apical MyoII and to favor the formation of contractile apical--medial meshwork of MyoII. Single cell tracking and quantification of MyoII intensity levels confirmed that medial--apical MyoII underwent recruitment pulses indicative of assembly--disassembly cycles at the cortex (Figure S5b,c).

  ,b). Conditional expression was achieved by introducing a GFP reporter and a transcriptional stop signal, flanked by two FRT sites, upstream of the RhoV14 gene so that RhoV14 was expressed in the ectoderm only upon FLP--mediated excision of the stop cassette. Live imaging of Tom>RhoV14 embryos indicated that increased Rho signaling in the ectoderm was sufficient to slow down furrow invagination and to trigger furrow retraction following its invagination (Figure5a,e and FigureS6c,f). This correlated with increased apical MyoII levels in the ectoderm (Figure5b,d,g). Thus, increased contractility in the ectoderm is sufficient to trigger furrow retraction in Tom>RhoV14 embryos. Since furrow retraction was observed at similar apical MyoII levels but different time points in Tom>RhoV14 and Brd mutant embryos (Figure5c,d,g), we suggest that retraction occurred once a given threshold of contractility was reached in the ectoderm. Thus, furrow retraction in Brd mutant embryos appeared to result from increased contractility in the ectoderm.Decreased contractility in the ectoderm restored furrow invagination inBearded mutants 120 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila To further test this interpretation, we next tested whether decreasing contractility in

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 121DiscussionHere, we first found that the activity of Neur is required in the mesoderm for furrow formation. Specifically, we found that depletion of Neur, both maternal and zygotic, using the NeurDegraFP method results in defective collective constriction. Since inhibition of Neur in the mesoderm by a stabilized version of Brd yielded similar defects, we suggest that Neur is required in the mesoderm to regulate apical constriction in ventral cells. This interpretation is fully consistent with earlier results showing that Neur is only active in the mesoderm using Delta endocytosis as a read--out (Bardin and Schweisguth, 2006; De Renzis et al., 2006). The inhibition of Neur in the mesoderm led to a less efficient recruitment of MyoII at the apical cortex, taking longer to reach a lower maximum, producing embryos with a flat ventral surface. Moreover, we were able to reproduce this furrow formation defect in silico using a 2D viscous model by primarily lowering apical constriction forces acting in the mesoderm. Modeling and experiments further suggested that Neur promote the rapid onset of a peak of apical MyoII in the ventral--most cells. Together, our results strongly indicate that Neur is required in the mesoderm to positively regulate contractility in ventral cells.

  acts in the ectoderm by antagonizing Neur. However, both furrow retraction (data not shown) and high MyoII in the ectoderm(Chanet et al, 2012) were still observed in zygotic Brd neur double mutant embryos. We interpret these results to suggest that maternal Neur may be sufficient to sustain increased contractility in the ectoderm of Brd neur mutant embryos. Alternatively, Brd might regulate contractile independently of Neur. Assuming that Neur regulates contractility in the ectoderm of Brd mutant embryos, we speculate that Neur has the same target(s) in both ectoderm and mesoderm. Consistent with this, disruption of the regulation of Sdt by Neur in Brd sdtΔ19 double mutant embryos suppressed the Brd epithelial polarity phenotype (G. Perez--Mockus et al., submitted) but did not restore furrow regression (data not shonw), indicating that Sdt is not the Neur target in the ectoderm that mediates furrow regression.Our findings therefore suggest that the proposed inhibition of Neur by Brd in the ectoderm may contribute to establish a difference in mechanical properties between the mesoderm and the ectoderm. Since Neur is maternally provided, this difference is set by the expression of Brd along the DV axis, which is a direct read--out of the activity of Snail. Thus, we propose 5.1. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 123

  topology of the Snail domain contributes to define the shape and position of the invagination. In this model, the relative constriction forces produced within and outside the Snail domain are integrated over the whole surface of the embryo to define the extent of anisotropy in each apically constricting cells, hence the shape of the invagination. Thus, the pattern of contractility at the embryo--scale is used to read the topology of the embryo and define the shape of the invagination. Since Neur and Brd, as a read--out of Snail, contribute to delimit a domain of increased contractility, they should help define the topology of the invagination. In Brd mutant embryos, which have no Neur ON/OFF boundary downstream of Snail, increased contractility in the ectoderm is therefore predicted to affect anisotropic constriction, distribution of apical MyoII and, possibly, the shape of the invagination. Future studies will address the role of Neur and Brd in defining the topology of apical constriction at the embryo--scale. 124 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila

  was modified by recombineering mediated gap--repair to produce the Tom>RhoV14 and Tom>Mbs BACs. First, a 2.5 kb5' deletion (from --25,808 to -- 23,273 upstream the Tom ATG) and a 6.3 kb 3' deletion (from +2,734 to +9,004 downstream the stop codon of the Tom gene) were introduced to remove the Bob and Brd--Ocho genes, respectively, leaving intact the gene and intergenic sequences of the Tom gene. Second, the ORF of Tom was replaced by a FRT--GFPnls--stop--FRT--RhoV14 and a FRT--GFPnls--stop--FRT--Mbs1--300 DNA fragment obtained by PCR amplification to produce the Tom>RhoV14 and Tom>Mbs BACs , respectively. The sequences of the RhoV14 and Mbs1--300 genes were PCR amplified from genomic DNA prepared from UAS--RhoV14 (BL--8144) and UAS--Mbs1--300 (kind gift of J. Treisman) transgenic flies. The resulting BACs were verified by sequencing of the recombined regions prior to phiC31--mediated integration at the PB[y+ attP] VK00033 (65B2) and PB[y+ attP] VK00020 (99F8) landing sites.The GFP--Neur BAC was produced from CH322--162G17 BACs(Venken et al., 2009) was modified using recombineering mediated gap--repair to insert the EGFP flanked by GVG linkers after amino acid 46 in Neur--PA. This GFP--Neur BAC was integrated at the PB[y+ attP--3B] VK00037 (22A3) site. A control Neur WT BAC transgene was also was also produced by integrating the original CH322--162G17 BAC at the same landing site. 126 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila All primers and sequence can be obtained upon request. Injection was performed by BestGene Inc (Chinmo, USA).

  ). Thus, females of the following genotype: PB[y+ GFP--Neur]VK37 P[UAS--Nslmb--vhhGFP4]2 / P[mat--Gal4]67; P[neo, FRT]82B neur GE P[UAS--Nslmb--vhhGFP4]3 / P[mat--Gal4]15 P[neo, FRT]82B neur IF65 were crossed with male siblings and laid eggs maternally depleted of Neur. These embryos were referred to as neur degrad embryos. As a control for this complex genotype, we used the same flies but rescued with the control Neur WT BAC transgene. The progeny of these PB[y+ Neur WT ]VK37 P[UAS--Nslmb--vhhGFP4]2 / P[mat--Gal4]67; P[neo, FRT]82B neur GE P[UAS--Nslmb--vhhGFP4]3 / P[mat--Gal4]15 P[neo,

  Embryos produced by P[UAS--mα] / + or Y ; P[mat--Gal4]67 P[sqh--MyoII--GFP] / P[UAS--Tom] ; P[mat--Gal4]15 P[sqh--Gap43--Cherry] /+ parents were studied by live imaging. The level of expression of Brd proteins should vary with genotype, i.e.

5. 1 .

 1 Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 127

  i.e. laid by P[psqh--MyoII--GFP], [psqh--Gap43--mCherry] / + females. The presence of the Brd deficiencies at the homozygous state was unambiguously determined after imaging by single embryo PCR. Similarly, for all experiments involving the sna>Brd R , Tom>RhoV14 and 128 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila Tom>Mbs BAC transgenes, the excision of the FRT--GFPnls--stop--FRT cassette was determined after imaging using single embryo PCR. Embryos were allowed to develop overnight at 18°C prior to DNA extraction using Tris pH 8.2 10mM, EDTA 1mM, NaCl 25mM and Proteinase K 200mg/ml. All primers are available upon request.

5. 1 .FiguresFigure 1 :Figure 2 :

 112 Figure 1: Ectopic Brd inhibits apical constriction and recruitment of MyoII a) Genetic control of apical constriction: in response to the nuclear gradient of Dorsal, Twist regulates the expression of a small number of target genes in the ventral cells, including fog and T48. Fog is a ligand activating a GPCR/Gα/RhoGEF2/Rho signaling pathway whereas T48 promotes the apical localization of RhoGEF2. Rho signaling regulates apical constriction via the control of F--actin dynamics and MyoII activity. b,c) Furrow formation (b) and invagination (c) are shown in cross section views of embryos stained for MyoII (green), Armadillo (Arm, fly β--catenin; white) and Delta (red). Apical constriction, hydrodynamic flow of the cytoplasm and decreased stiffness at the basal cortex are indicated with arrows (green, orange and white, respectively). d--i) Live imaging analysis of apical constriction in wild--type (d), mat>mα+Tom (e) and sna>Brd R (f) embryos expressing MyoII--GFP (not shown) and Gap43--Cherry (white; tracking, red). Surface area values were measured for cells undergoing invagination (blue in d--f; values plotted in panels g--i). Surface value distributions in the wild--type embryo (shown in d) highlight the rapid and relatively synchronous constriction of ventral cells during furrow formation (g; each dot represents one cell; mean values are shown as a blue curve). Both maternal over--expression of mα and Tom (h) and zygotic expression of Brd R in the mesoderm (i) delayed furrow formation and led to the persistence of large non--constricting cells during furrow formation.j--m) Snapshots from movies of a wild--type (j) and mat>mα+Tom (l) embryos expressing MyoII--GFP (white) and Gap43--Cherry (membrane, red). A dense myosin meshwork interconnecting medial--apical pools of MyoII and running across cell--cell boundaries was observed in wild--type embryos (j). Total MyoII--GFP intensity (green) in Arbitray Units (A.U.;values measured in ventral cells from one embryo) increased sharply as ventral cells constricted (surface area, blue). Over--expression of mα and Tom resulted in reduced levels of apical MyoII organized in a loose network (l). A slow increase in apical MyoII correlated with asynchronous apical constriction and delayed furrow formation (m).Anterior is up; time is in min (d--f); t=0 (onset of apical constriction) is indicated with a grey bar (g--I, k,m).

Figure 3 :

 3 Figure 3: in silico simulation of furrow invagination in wild--type and neur degrad embryos a) Position landmarks of the embryo and color code for the apical (red), basal (green) and lateral (blue) force distributions. Color intensity increases with time. Time zero was set when dorsal cells reached a maximum apical--basal length (Conte et al., 2012). b,b') Symmetrised and regularised approximation of the VFM spatial force distributions (Brodland et al., 2010) modified to match the in vivo wild type phenotype in the invagination interval (b, from t=--10 to --2; b', from t=--2 to +6). Color codes and positions are defined in a). c) Selected frames of in silico sections of a simulated wild--type embryo using the force distribution shown in (b,b'). d--g) Force distributions proposed for the Sigmoid (d,d') and Gaussian (f,f') mechanisms and corresponding frames showing the invagination phenotype at t=23.

Figure 4 :

 4 Figure 4: Furrow retraction correlated with high MyoII in the ectoderm of Brd mutant embryos a,b) Live imaging of a Brd mutant embryo expressing MyoII--GFP (not shown) and Gap43--Cherry (white; tracking, red). Snapshots (a) showing that the furrow initially formed (t=25) then regressed (t=35). Plot (b) showing the surface area values measured in ventral--most cells (blue--shaded in a; mean values shown as a blue curve in b). Collective apical constriction was observed as the ventral furrow formed. The furrow appeared to form slightly slower than in wild--type embryos (Figure S5a). Ventral cells could not be tracked during invagination (t=25) and were tracked again after furrow regression (t=35). c,d) Live imaging of ectodermal cells in wild--type (c) and Brd mutant (d) embryos expressing MyoII--GFP (green) and Gap43--Cherry (red). MyoII localized at junctions to form supracellular cables along the DV axis in wild--type embryos (ventral furrow, right). Increased MyoII levels were detected at both junctions and medial--apical cortex in the ectodermal cells of Brd mutant embryos (yellow/green color--coded cells: see Figure S5 for MyoII dynamics at the single cell level). e) Elevated and increasing levels of MyoII--GFP were measured in the ectoderm of Brd mutant embryos (n=2; signal intensity was measured in cells tracked in c,d) relative to control embryos (n=2).

Figure 5 :

 5 Figure 5: Increased contractility in the ectoderm appeared to be necessary and sufficient for furrow retraction a,b) Snapshots from a movie showing the ventral surface of a Tom>RhoV14 embryo expressing MyoII--GFP (not shown) and Gap43--Cherry (white; tracking, red). Snapshots (a) showing that the furrow initially formed (t=25) then regressed (t=45). Plot (b) showing the

  Figure S1 (linked to Figure 1): Ectopic Brd inhibits apical constriction a,b) snapshots of a movie showing a wild--type embryo expressing a Gap43--Cherry (white; tracking in red) and GFP--MyoII (not shown) at t=--15 (a) and t=7 (b). Shape analysis of ventral cells showed that all cells have initially similar area, orientation and aspect ratio values (a) but that the ~6 ventral--most cells (blue--shaded) displayed smaller area values (reflecting apical constriction) and higher anisotropy values with their long axis aligned along the anterior--posterior, i.e. furrow, axis whereas the ventral--lateral cells (red) showed heterogeneous apical area values and displayed anisoptropy oriented along the DV axis.

  i--k) Plots showing the frequency distribution of surface area values measured in the ventral-most cells (blue--shaded in Figure1d--f) at mid--invagination in wild--type (i), mat>mα+Tom (j) and sna>Brd R (k) embryos. While most ventral cells have constricted in wild--type embryos, a more bimodal distribution was observed in mat>mα+Tom (j) and sna>Brd R (k) embryos with some cells undergoing apical constriction and others failing to constrict. 138 Chapter 5. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila l) Kinetics of apical constriction in wild--type (n=2), mat>mα+Tom (n=2) and sna>Brd R (n=2) embryos. Mean surface values of constricting ventral cells (blue--shaded in Figure 1d--f were plotted over time. Surface values were measured in individual cells after tracking as shown in Figure 1g--f. Apical constriction (on average) occurred at a slower rate upon expression of Brd proteins. m) Analysis of MyoII apical recruitment in wild--type (n=3) and mat>mα+Tom embryos (n=3).

Figure

  Figure S2 (linked with Figure 2): a--c) Simplified genomic structure of the neur locus encoded within the 162G17 BAC. The main neur--RA isoform is shown (exons are boxed, ORF is in black). GFP (green) was inserted within a poorly conserved region, N--terminal to the Neur Homology Repeats (NHR1 and 2) and the catalytic Ring Finger (RF). One copy of the GFP--Neur BAC fully rescued the phenotypes of neur IF65 / neur GE mutant flies (b,c). d) Box plots of time intervals from the onset of apical constriction to furrow invagination in wild--type (n=7), neur degrad (n =20), mat>mα+Tom (n=9), sna>Brd R (n=5) and control degrad (n=6) embryos (see Materials and Methods for detailed genotypes). Invagination was significantly delayed upon depletion of Neur, maternal over--expression of Brd family proteins and ectopic expression of a stabilized version of Brd in the mesoderm. The variability in delay seen between mat>mα+Tom embryos likely resulted from differences in transgene copy number. Also, the variability seen between neur degrad embryos possibly

Figure S3 :

 S3 Figure S3: Std is not a target of Neur in the mesoderm a,b) Genomic organization of the sdt locus (top ; 3 isoforms are shown) and domain structure of the Sdt-PB and Sdt-PF isoforms. The Exon3 (orange) encodes a Neur Binding Motif (NBM).

Figure S4 (linked with Figure 3 )

 3 Figure S4 (linked with Figure 3): Distribution of apically constricted cells along the DV axis a--d) Snapshots from wild--type (a; n= ), neur degrad (b; n= ), mat>mα+Tom (c; n= ) and sna>Brd R (d; n= ) embryos expressing MyoII--GFP (not shown) and Gap43--Cherry (white; tracking, red) at mid--constriction, i.e. when 3% of the ventral cells have constricted to reduce 5--fold their surface (wild--type, t= ~ 5; Neur perturbation experiments, t= ~ 14).e) Distributions of surface area values along the DV axis at mid--constriction showed a clear groove, indicating that apical constriction occurred mostly in ventral--most cells. This is consistent with the ventral--most cells undergoing collective apical constriction. By contrast, no clear minima were seen in neur degrade , mat>mα+Tom and sna>Brd R embryos, suggestive of a more widespread distribution of apical constriction accross the mesoderm.

Figure S5 (linked with Figure 4 )

 4 Figure S5 (linked with Figure 4): Apical constriction and MyoII dynamics in Brd mutant embryos a) Apical constriction (mean surface values of constricting ventral cells plotted over time)

Figure S6 (linked with Figure 5 )

 5 Figure S6 (linked with Figure 5) a,b) Strategy to express RhoV14 in the ectoderm. The Tom ORF was replaced by a FRT--GFP-stop--FRT--RhoV14 construct (a) to produce a Tom>GFP transgene. This transgene directed the expression of GFP (green; Twist, red) in the ectoderm (b). Flp--mediated excision of the FRT--GFP--stop--FRT cassette produced a Tom>RhoV14 transgene expressing RhoV14 in the

142 Chapter 5 .Figure 1 Chapter 5 .

 515 Figure 1:

2 :

 2 Figure 2:

Figure 4 2 .

 42 Figure 4:

Figure S1 .

 S1 Figure S1. Schematic of the multi-scale model of the 2D cross-section of the Drosophila embryo. This schematic was adapted from Conte et al. [1] along with the caption as follows: (a) Biological architecture of the Drosophila single epithelial cell. (b) Architectural constituent of the Drosophila epithelial cell. (c) The biomechanical role played by each subcellular component is resolved in net equivalent forces acting along the cell cortex (e.g. acto-myosin networks) and cell interior (e.g. incompressible viscous cytoplasm). (d) Individual cells (green) are subject to active edge forces (red arrows). These cells are broken into quadrilateral finite elements (blue) that are used to calculate the forces generated by their passive (viscous) components during each time step of the simulation. These passive forces set in motion the nodes at which finite elements are joined. The schematic shows a set of nodal forces (blue arrows) that would be needed to drive a particular set of nodal motions (magenta arrows) which are related to each other by the viscous motion equation of the model subject to a set of boundary conditions (e.g. inner yolk and outer membrane). (e) Reference configuration of the finite element model built by matching to in vivo cross-section of the wild type Drosophila embryo at the cellular level. 2D geometry has been sized on the in vivo dimensions of the wild type embryo at the reference initial instant t=-10.43 min (Fig.S2,c). Mesodermal region covers approximately 80 degrees across the ventral point V and is highlighted in dark green, whereas ectodermal region is highlighted in bright green.

5. 2 .

 2 Figure S2. Biomechanical quantification of the in vivo wild type Drosophila embryo. Quantitative data shown in this figure summarises the analysis carried out in Conte et al. [1]. (a) Selected multi-photon images (raw) of in vivo transverse cross-sections of a wild type embryo during ventral furrow formation, courtesy of Buzz Baum and Florian Ulrich [1]. The embryo is labelled with Sqh-GFP and the time interval between successive frames is 45 seconds. Time zero was set at the maximum apical-basal cell length on the dorsal side (blue curve in panel d), representing the end of ectodermal cellularisation. (b) The embryo is oriented with its dorsal surface upward. (c) Time-dependence of VFM force distributions quantified in vivo by Brodland et al. [7]. (d) Characteristic epithelial lengths (thick lines) and relative standard error of the mean (thin lines) [1]: mean of mesodermal ventral-most lengths (red); mean of ectodermal dorsal-most lengths (blue); ratio of the previous two lengths (green). (e-g) Space-dependence of VFM force distributions quantified by Brodland et al. [7]. Time goes with colour intensity according to legend in panel (b).

Figure S3 .

 S3 Figure S3. In silico simulation of the wild-type Drosophila embryo through VFM force distributions. We simulated in silico the wild-type embryo by using the complete in vivo force data set collected through the VFM[7], as opposed to our previous study detailed inConte et al. [1] where we utilised discretised distributions varying in a simplified fashion with time but not with position over epithelial regions. (a-b) Symmetrised and regularised approximation of the VFM spatial force distributions quantified in vivo byBrodland et al. [7] were obtained by computing mean and characteristic statistical features of the in vivo trends. Time-dependence of the approximate distributions is equal to that of in vivo trends (Fig.S2,c). (c) Selected frames (raw) of in silico transverse cross-sections of a wild type genotype corresponding to the in silico force distribution in panels (a-b) timed according to trends shown in FigureS2,c. (d-e) Symmetrised and regularised approximation of the VFM spatial force distributions shown in panels (a-b) have been modified to fully match the in vivo wild type phenotype in the invagination interval. Mismatching between distributions in (d-e) and (a-b) provide an estimate of the cell-tracking imprecisions arising from multiphoton image clarity. (f) Selected frames (raw) of in silico transverse cross-sections of a wild type genotype corresponding to the in silico force distribution in panels (d-e) timed according to trends shown in Figure S2,c. For the sake of clarity, colour codes and axes legends are shown in Figure S2,b only. (g-i) Comparison of the final wild-type phenotypes observed at t=6min and respectively shown in panels a5 and f5, and in Figure S2,a5.

  Figure S3. In silico simulation of the wild-type Drosophila embryo through VFM force distributions. We simulated in silico the wild-type embryo by using the complete in vivo force data set collected through the VFM[7], as opposed to our previous study detailed inConte et al. [1] where we utilised discretised distributions varying in a simplified fashion with time but not with position over epithelial regions. (a-b) Symmetrised and regularised approximation of the VFM spatial force distributions quantified in vivo byBrodland et al. [7] were obtained by computing mean and characteristic statistical features of the in vivo trends. Time-dependence of the approximate distributions is equal to that of in vivo trends (Fig.S2,c). (c) Selected frames (raw) of in silico transverse cross-sections of a wild type genotype corresponding to the in silico force distribution in panels (a-b) timed according to trends shown in FigureS2,c. (d-e) Symmetrised and regularised approximation of the VFM spatial force distributions shown in panels (a-b) have been modified to fully match the in vivo wild type phenotype in the invagination interval. Mismatching between distributions in (d-e) and (a-b) provide an estimate of the cell-tracking imprecisions arising from multiphoton image clarity. (f) Selected frames (raw) of in silico transverse cross-sections of a wild type genotype corresponding to the in silico force distribution in panels (d-e) timed according to trends shown in Figure S2,c. For the sake of clarity, colour codes and axes legends are shown in Figure S2,b only. (g-i) Comparison of the final wild-type phenotypes observed at t=6min and respectively shown in panels a5 and f5, and in Figure S2,a5.

Figure S4 .

 S4 Figure S4. In silico simulation of the Neur Degrad mutant Drosophila embryo -Sigmoid mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Sigmoid physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 75% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

  Figure S4. In silico simulation of the Neur Degrad mutant Drosophila embryo -Sigmoid mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Sigmoid physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 75% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

  Figure S4. In silico simulation of the Neur Degrad mutant Drosophila embryo -Sigmoid mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Sigmoid physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 75% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

Figure S5 .

 S5 Figure S5. In silico simulation of the Neur Degrad mutant Drosophila embryo -Gaussian mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Gaussian physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 50% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

  Figure S5. In silico simulation of the Neur Degrad mutant Drosophila embryo -Gaussian mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Gaussian physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 50% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

  Figure S5. In silico simulation of the Neur Degrad mutant Drosophila embryo -Gaussian mechanism.We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for: i) the in vivo quantification of impaired apical recruitment of Myosin-II and delayed apical constriction experimentally quantified for these mutants; and, ii) the phenotypic characterisation of a selection of fixed cross-sections of these mutants collected in vivo. (a) Selected frames (raw) showing the potential time-evolution of the Neur Degrad mutant phenotype according to the Gaussian physical mechanism detailed in panels b-d. (b) Timedependence of Neur Degrad force distributions operating in this physical mechanism referred to as Sigmoid for the characteristic shapes adopted for mesoapical force distributions (red curves in panels c-d). Time trend of force intensities was obtained by allowing wild-type forces of the interval T1=[-10.43,0] min to vary over wider interval[-10.43,23] min to emulate the delayed apical constriction observed experimentally -the wildtype interval T1 (Fig.S2, c-d) approximately corresponds to the interval of Myosin-II relocation from basal to apical side of mesodermal cells. Intensity of wild-type apical forces was decreased by 50% to emulate the impaired apical recruitment of mesodermal Myosin-II in the Neur Degrad mutant. (c-d) Spatial force distributions of the Neur Degrad mutant were determined to account for the in vivo phenotype of a selection of fixed cross-sections of these mutant embryos. (e-f) Comparison between the in silico and in vivo phenotype of the NeurDegrad mutant respectively.

Figure S6 .

 S6 Figure S6. In silico simulation of the Brd mutant Drosophila embryo. We simulated the simulated this mutant by altering the spatial distribution, the relative magnitude as well as the relative and absolute timing of forces in the wild-type embryo to account for the correlation between increased apical Myosin-II activity of ectodermal cells and the retraction of the ventral furrow after its formation in this mutant. (a) Selected frames (raw) showing the potential time-evolution of the Brd mutant phenotype according to the physical mechanism detailed in panels c-f. (b-c) Time trend of force intensities. (c,d-f) Spatial force distributions of the Brd mutant embryos.

Chapter 6 .

 6 Results annex 2: Identification of new Neur substrates

FIGURE 6 . 1 :

 61 FIGURE 6.1: Bio-Ubiquitin Strategy: A)Scheme of the strategy. FromMayor and Peng 2012.[239]. A construct expressing six tagged ubiquitins and the BirA protein is expressed in the tissue of interest. DUBs separate the tagged ubiquitins from the BirA protein and then BirA covalently binds a biotin to the ubiquitin tag. Bio-Ubiquitins are bound to different substrates by the E1, E2, and E3 ligases. Finally, the substrates are purified taking advantage of the biotin avidin interaction. B)Western Blots of purified extracts using avidin beads. FromMayor and Peng 2012 [239]. The Proteins are marked using a silver stain. The BirA lane shows the proteins purified from an extract expressing only BirA, while the Ubi6BirA lane shows the proteins purified from an extract expressing the construct presented in A. The smear in the Ubi6BirA shows that ubiquitinated proteins are purified using this method.The arrow marks the position of the Pyruvate Carboxylase a protein that binds biotin and that is hence purified in both extracts. The asterix shows free avadin. C) Results from the identification by MS/MS of proteins from 4 different conditions: Embryos overexpressing ubiquitously Ubi5BirA, Tom (in green), Ubi6BirA (in red), Ubi6BirA, Neur (in blue) and W1118 (in purple). 7 proteins were identified in all 4 extracts including avidin and the pyruvate carboxylase. 7 proteins were only present in the sample containing Neur and could be its putative targets.

  -wide identification of substrates of the E3 ubiquitin ligase Neuralized in D. melanogaster embryos By Francis IMPENS (from the laboratory of Pascale COSSART) 1. Gantas PEREZ-MOCKUS collected Drosophila embryos: -overexpressing a catalytic dead version of the E3 ligase (∆RF): (from the cross daG4 x UAS-Neur∆RF da stands for daughterless a ubiquitous promoter) -overexpressing the E3 ligase (V5) : (cross daG4 x UAS-Neur∆RF) -overexpressing the E3 ligase inhibitor (TOM): (cross daG4 x UAS-Neur∆RF)

6 .

 6 Figure 1

Figure 2 - 1

 21 Figure 2

Figure 7 . 1 AFIGURE 7 . 1 :

 7171 FIGURE 7.1: CG3894 Fig. 1 Fig. 1: CG3894/Ozz interacts with Neur and Tom. A) Neur and CG3894/Ozz protein structures. B) Result from Vanessa ROCA. Co-Ip of S2 cell extracts with CG3894 (anti-VSVG) showing that CG3894 physically interacts with Neur and with the Neur inhibitor Tom. Tom blocks the binding of CG3894 to Neur.

FIGURE 7 . 2 :

 72 FIGURE 7.2: CG3894 Fig. 1Fig. 1: CG3894/Ozz is not required for embryonic development. A) Survivorship assay for 200 embryos laid by wild-type females or by CG3894 17L /CyoTbRFP. 25% of the embryos laid by the CG3894 17L /CyoTbRFP do not hatch. These 25% area all RFP + and corresponding to homozygous embryos for Cyo. Hence the allele CG3894 17L is not embryonic lethal when homozygous. B) Genotyping of L1 larvae from the stock CG3894 17L /CyoTbRFP . L1 RFP -larvae are homozygous for CG3894 17L , confirming that this allele is not embryonic lethal. The different PCR products allowing the genotyping are described below. Note how the DNA extract of heterozygous individuals (marked with an asterix) produces two bands when amplified by the couple F+R2. C) Genomic structure of the gene CG3894. The location of the PCR primers F, R1 and R2 are represented by arrows. The PCR products of these primers are schematically represented. The couple F+R1 gives a product of 451 bp, and the couple F+R2 a product of 2148bp. D) Genomic structure of the allele CG3894 17L . PCR products of the primers F+R1 and F+R2 when this allele is present. The couple F+R1 gives no product and the couple F+R1 a product of 984bp. E)PCR products of the couples F+R1 and F+R2 when the wild-type CG3894 allele or the CG3894 17L allele are probed.

FIGURE 7 . 4 :Fig. 3 :

 743 FIGURE 7.4: Fig. 3: Tools developed in the lab for the study of CG3894/Ozz. An antibody from Ab-Mart was raised against CG3894/Ozz .It recognises CG3894/Ozz in western blot -CG3894/Ozz is overexpressed in the wing disc or in HEK cells (A)-and in immunostainings; the protein was overexpressed in the dorsal compartment of a wing disc (B in the red chanel). Moreover, CG3894/Ozz seems to localise to the nucleus when overexpressed. Two drosophila lines contain a construct expressing CG3894/Ozz under the UAS promoter: the UAS-CG3894-Myc from the lab of Gabrielle Boulianne, and the UAS-CG3894-VSVG constructed in the lab. The two constructs code for proteins of different sizes (see C ) that are recognised by the antibody anti-CG3894 (see A the construct expressed in the HEK cells is the same that was inserted into the flies), and they both localise to the nucleus when overexpressed in the wing disk (D and E). The CG3894-VSVG correspond to the expected size of CG3894 according to Flybase.The exact structure of the CG3894-Myc is not known.

FIGURE 8 . 1 :

 81 FIGURE 8.1: a Ommatidial development in the eye imaginal disc, the disc is stained with Crb. The morphogenic furrow is present in the part where Crb stops being highly expressed apically. b to b"' Patterning of an ommatidia from a type2-arc to a 5 cell pre-cluster. The formation of the ommatida includes a stage where the ommatidia precursor forms a rosette b', before excluding a cell (called the mystery cell) c to c"' Staining of eye disc of flies containing a GPP-tagged version of neur. Neur is expressed predominantly in the R3 and R4 cells. d At the 5cell precursor stage 3GFP-Sdt is excluded fro the R3 and R4 cells.e In absence of the exon 3, Sdt is no longer excluded from the R3 and R4 cells. g and e The exon 3 is required for the formation of a rosette stage during ommatidia development.

FIGURE 8 . 4 :

 84 FIGURE 8.4: Ectopic expression of Neur doesn't affect the levels of T48-GFP when both are overexpressed in the dorsal compartment of the wing imaginal disc.

8. 4 .FIGURE 8 . 5 :

 485 FIGURE 8.5: Work from Vanessa ROCA. A1 and A2) S2R+ cells spread on an α-tubulin coated surface. A1 and A2) Addition of medium containing the ligand fog induces a retraction of the cells . B1, B2, D1 and D2) The expression of Brd in S2R+ cells induces a stellate morphology independently of the addition, or not, of fog media. The cells positive for Brd are marked with a yellow asterisk. E1,E2,E3 and E4) S2R+ cells transfected with Brd-HA show a stellate morphology when plated on a α-Tubulin coated surface. The cells expressing Brd-HA are in red. F The depletion of a subunit of the Arp2/3 complex in spreading S2 cells causes a similar defect. Image fromRogers et al. 2003[253]. RNAis directed against other proteins also caused the same stellate phenotype.See the text for more details.

FIGURE 8 . 6 :

 86 FIGURE 8.6: Ectopic expression of Neur increases the accumulation of a Rho1 biosensor. A) Expression of a Rho1 biosensor (RokRBD-GTP from Abreu-Blanco et al. 2014[256]) in the dorsal compartment of a wing disc. The biosensor accumulates in the junctional and in the medial/apical part of the cells. B) Coexpression of the RHo1 biosensor and Neur stabilises the accumulation of the biosensor. C) Schematic representation of the junctional and the medial domain of a cell. D) Quantification of average GFP levels in the junctional and the medial part of cells expressing the Rho1 biosensor alone or with Neur. Neur co-expression increases the levels of the biosensor in both junctional and medial domain of the cell. A') and B') Segmentation of the cell boundaries using E-cad, shown in white.

FIGURE 8 . 7 :

 87 FIGURE 8.7: A) Expression of Neur in the dorsal compartment of the wing disc doesn't cause any obvious morphogenic defects. B) Expression of RhoGEF2 in the dorsal compartment of the wing disc perturbs its development C) Co-overexpression of Neur and RhoGEF2 enhances the effect of RHOGEF2 on disc morphogenesis. The three wing imaginal discs are at the same scale.

10. 1

 1 Neur and stardust 10.1.1 Tagging of the Sdt isoforms lacking the exon 3

FIGURE 10 . 1 : 3

 1013 FIGURE 10.1: Strategies to mark the Sdt-PF isofrom A)Schematic representation of Sdt splice variants, and two Sdt isoforms, differing by the presence of the exon 3 in orange.B)Schematic representation of a strategy to tag the Sdt with a sfGFP that only fluoresces in the Sdt-PF isoform but not in the Sdt-PB.C)Schematic representation of a strategy to tag the Sdt gene with a Cherry and Venus. The protein Sdt-PB would be marked with Venus and the Sdt-PF with Cherry.

10. 1 . 4

 14 Do the protein complexes containing Sdt-B share the same properties as those containing Sdt-PF?

FIGURE 10 . 2 :

 102 FIGURE 10.2: Fly cross scheme to identify pathways redundant to Neur/Sdt

FIGURE 10 . 3 :

 103 FIGURE 10.3: A) Different constructs that could allow the identification of new Neur binding partners compared with the wild-type Neur structure. B) Strategy to conditionally express Neur RF-BirA*-Flag or BirA*-Flag-Neur under the regulatory sequences of the ubiquitously expressed gene, daughterless. The addition of a cassette containing a RFP and a transcriptional stop ensures that the Neur constructs are only expressed when this cassette is removed by a flipase.B)Stocks that could be used to identify the targets of Neur in embryos lacking the Brd genes. These stocks express Neur RF-BirA*-Flag or BirA*-Flag-Neur exclusively in embryos lacking the Brd genes.

  The dorso/ventral axis of the fly is established during oogenesis[106],[107]. At stage 10 of oogenesis, the nucleus of the oocyte is located dorsally. It secretes the Gurken ligand, which activates the drosophila homolog of the epidermal growth In this way, the expression of Pipe results in the activation of Spz and formation of a Spz gradient. Active Spz triggers a protein cascade which results in the translocation of Dorsal to the nucleus. Thus, the Spz gradient is translated into a gradient of nuclear Dorsal. The transcription of different genes are activated depending on the concentration of Dorsal, and the dorsal/ventral domains are established [114] (Figure 2.3c).

factor (egf) receptor, Torpedo, in the most dorsal follicular cells

[108]

,

[109]

. Follicle cells in the top 40% of the most ventral part of the ovocyte, do not activate Torpedo, and hence express the gene pipe (Figure

2

.3a). The protein Pipe has sulfotransferase activity and modifies the vitelline membrane of the follicle cells in which it is expressed

[110]

. The modification of the membrane by Pipe is inherited by the embryo and results in the recruitment of different proteases to the perivitelline space, in contact with the modified vitelline membrane. These recruited proteases trigger a protease cascade, downstream of which the protease Easter (Ea) processes and activates the ligand Spatzle(Spz)

[111]

,

(Figure 2.3d)

. Active Spz forms a gradient which peaks in the ventral part of the embryo. This gradient is created as a result of inhibition of Spz activation, however, the precise mechanisms behind its formation remain unclear[106],

[105]

. Spz is a ligand for the receptor, Toll, which in turn activates the kinase, Pelle. Pelle phosphorylates the protein cactus [112] allowing the release of the NF-κB transcription factor, Dorsal, which is relocated to the nucleus

[113]

.

a) Unbranched actin b) Branched actin meshwork

	High contractility
		Key:
			ARP2/3
			Actin crosslinker
	Myo-II dependent actin lament rearrangement in an antiparallel array	-Limited actin rerrangement -Low contractility	Formin MyoII mini lament MyoII/actin sliding
	FIGURE 2.10: Actomyosin branching. From Levayer et Lecuit 2012
	[159].

  .11). Neuralized and the Neuralized family of genes domain. Ozzy has been implicated in the regulation of the myofibril organisation in the striated muscle during mouse development. Here, it binds and ubiquitinates β-catenin [244], the muscular heavy chain of myosin[245], and the actin regulator Alix/AIP1 [246] sending them to be degraded. Bluestreak was shown to associate with the centrosomal duplication regulator CPP110 and negatively regulate its levels by promoting its ubiquitination and degradation. The way in which Bluestreak promotes the ubiquitination of CPP110 is not clear, but the absence of Bluestreak leads to increased levels of CPP10 and the formation of ectopic microtubule organising centers [247],[248].

	Bluestreak
	Bluestreak, or Neuralized like 4 (Neurl4), is a scaffolding protein containing 6 NHR
	domains and no other domain or motif suggesting any molecular activity. In cultured
	cells,
	Neuralized
	NHR1	NHR2		RING
	Ozzy/CG3894
	NHR	SOCS
	Bluestreak
	NHR	NHR	NHR	NHR	NHR	NHR
	FIGURE 3.11: Neuralized family of genes

Ozzy

Ozzy (or CG3894) is another E3 ubiquitin ligase. In contrast to Neur, Ozzy has only one NHR domain (See Figure

3

.11) and has a HECT domain instead of a RING domain. Of note, transfer of ubiquitin to target proteins by E3 ligases bearing the HECT domain occurs in a slightly different manner than that of those bearing the RING Chapter 3.

  The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila 75 cells that were co-transfected to express Flag-tagged Sdt-PB (or Sdt-PF) and a Myctagged version of Neur deleted of its RING Finger (RF) domain (NeurΔRF; deletion of the RF domain resulted in increased levels of Neur

Thus, Sdt-PB appeared to synergize with Neur to down-regulate Crb. We interpret this observation to suggest that Neur acts together with Sdt-PB to down-regulate Crb. Finally, we found that Sdt-PF stabilized Crb despite ectopic Neur (Figure

2B

). This indicated that Sdt-PF can protect Crb from a down-regulation by Neur. We suggest that Sdt-PF may compete other Sdt isoforms for Crb binding, thereby blocking the effect of the Sdt isoforms synergizing with Neur, e.g. Sdt-PB, to down-regulate Crb.

We next investigated how Neur discriminated between Sdt-PB and Sdt-PF.

One hypothesis is that Neur specifically interacts with Sdt-PB via the Exon3. To test this possibility, we used a co-immunoprecipitation (co-IP) assay in Drosophila S2 Chapter 4. Paper 1:

4.2 Flies UAS-CG3894-VSVG Two

  fly lines expressing tagged versions of CG3894/Ozz under the UAS promoter are present in the lab: one made by Eric LAI (unpublished)-the UAS-CG3894-Myc -and another made by Vanessa ROCA in the lab -UAS-CG3894-VSVG. Both constructs localise to the nucleus when over-expressed in the dorsal compartment of the wing imaginal disc. They encode two proteins of different sizes, the construct made in the lab has the molecular weight predicted by Flybase (around 40 kDA), whereas the other construct encodes a protein of a bigger size. At present, we don't know why the UAS-CG3894-Myc construct encodes a protein of a bigger size, nonetheless the antibody against CG3894 does recognise this construct (Figure7.4A).

	A	DAPI	phalloidin	GFP	phalloidin
					GFP
	wild-type				63x
	B	DAPI	phalloidin	GFP	phalloidin
					GFP
	CG3894 17L ; CG3894-GFP				63x
	C	phalloidin	GFP	phalloïdin	E
				GFP
	CG3894 17L ; CG3894-GFP			40x
	D	phalloidin	GFP	GFP phalloidin	F	GFP
	CG3894 17L ; CG3894-GFP		40x		CG3894 17L ; CG3894-GFP	20x
	G	DAPI	phalloidin	vasa	DAPI	phalloidin
					vasa	vasa
	wild-type					63x
	H	DAPI	phalloidin	vasa	DAPI	phalloidin
					vasa	vasa
	CG3894 17L / DF(CG3894)					63x
		FIGURE 7.3: CG3894 Fig. 2: CG3894/Ozz is expressed in the testis
		but it doesn't seem to be required in the stem cell niche of the testis.A)
		and B) Immunostaining of testis from wild-type or CG3894 17L ;CG3894-
		GFP males, showing that CG3894 (in green) is expressed in the mi-
		totic cells but not in the hub. A) and B) Immunostaining of testis from
		CG3894 17L /+;CG3894-GFP males showing that CG3894 (in green) is ex-
		pressed in the mitotic cells and in the round spermatids. E) Schematic
		representation describing the different cells in the testis. E) Low magni-
		fication immunostaining of testis from a CG3894

17L

;CG3894-GFP male confirming that CG3894 (GFP) is expressed in the round spermatids. G) and H) Males lacking the CG3894 gene (CG3894 17L over a deficiency of CG3894) do not show any obvious defects in the testis stem cell niche.

Compare G) Wild type and G) CG3894 - 7.

2 Ectopic expression of Neur and Pog Pog

  3, filamentous actin was stabilised. The reason for this variability is not clear, and so whether or not Neur stabilises Factin still remains an open question.Also whether or not Neur can affect the cortical myosin pulses when over-expressed in the wing imaginal disc is not known, but remains a possibility. Effect of the ectopic expression of Neur on Sqh and F-actin in wing imaginal discsA)Phalloidin, MyoII and Sdt stainings in wing imaginal disc of the Sqh-GFP, apG4 genotypeB)Ectopic expression of Neur in the dorsal compartment (left part of the image) does not clearly affect F-actin and Sqh levels, but downregulates Sdt. The yellow arrow shows the limit between the dorsal and the ventral compartment. In some wing imaginal disks F-actin seemed up-regulated in the compartment expressing Neur, but this was variable. (poor gastrulation) or Smog, is a GPCr receptor involved in the regulation of apical constriction during mesoderm invagination[128]. Pog/Smog contains a sequence that could bind Neur, and the loss of function of neur and the loss of function of pog/smog both affect apical constriction. Hence Pog/Smog is a good candidate to be positively regulated by Neur. The lab generated a BAC encoding a GFP-tagged version of pog/smog. The ectopic expression of Neur didn't seem to affect the levels of pog/smog-GFP when over-expressed in the wing disc (Figure8.3), however as the activity of a receptor probably doesn't necessary correlate with its protein levels, it cannot be excluded that Neur could promote the activity of Pog/Smog without affecting its levels. Effect of the ectopic expression of Neur on Pog-gfp and Factin in wing imaginal discs. Ectopic expression of Neur doesn't affect the levels of Pog-gfp. In this wing imaginal disc the expression of Neur clearly increases the levels of F-actin. Pog seems to be expressed in the posterior wing compartment.

	merge	GFP	phalloidin
	squash-GFP,ap> squash-GFP,ap> Neur merge merge A B FIGURE 8.2: 8.3.pog-GFP,ap> Neur FIGURE 8.3:	phalloidin phalloidin	myoII myoII	Sdt Sdt

8.7 Neur-GFP accumulates in the apical membrane of what could be delaminating neuroblasts

  Ectopic expression of Neur lacking its RING doesn't downregulate Sdt. Cells expressing Neur RF-GFP grow bigger. Neur RF-GFP seems to recruit Sdt and Crb (data not shown), and this overgrowth might be dependent on the effect that Crb has on the hippo pathways Neuronal precursor cells, neuroblasts, basally delaminate from the neuroectoderm during drosophila development [257] (scheme in Figure 8.9A). A BAC encoding a GFP tagged version of neur was used to rescue two null alleles of neur. Immunostaining of the embryos containing the GFP-tagged version of Neur (rescuing the null alleles), showed a GFP signal in what could be the apical membrane of a delaminating neuroblast (Figure 8.9A)). Furthermore, as Neur was shown to regulate apical constriction during mesoderm invagination, this raises the possibility that Neur could also regulate neuroblast delamination. This hypothesis requires further exploration.

	8.7. Neur-GFP accumulates in the apical membrane of what could be delaminating neuroblasts 197
	Sdt GFP	GFP	Sdt
	ap>Neur-ΔRF-GFP
	Polarized epithelium FIGURE 8.8: Neur-GFP A B
	Delaminating neuroblast
	Metaphase neuroblast	Stage 10
	FIGURE 8.9: A) Scheme of neuroblast delamination, from [257]. B) Im-
	munostaining of a Stage 10 embryo containing a BAC encoding for a
	GFP tagged version of Neur rescuing two null alleles of Neur. An anti-
	GFP antibody is used. It marks the apical membrane of what could be
	a delaminating neuroblast.

In the absence of neur, the early phases of the EMT are perturbed and polarity markers, such as Crb, are no longer downregulated in the posterior midgut. This results in a blockage of the PGCs in the pouch of the posterior midgut (Figure3.6c). Hence, Neur is required in the posterior midgut to regulate its remodeling by negatively regulating epithelial polarity. This function is independent of Delta, as embryos lacking Delta do not show any defects in PGC migration (Figure3.6c). Moreover, some of the Brd genes which are expressed at stage 9 are no longer expressed at stage 10 (data not shown, fromChanet and Schweisguth 2012).The current model for the regulation of epithelial remodeling of the posterior midgut, based onCampbell et al. 2011[81] andChanet and Schweisguth[222], is that at stage 9 the transcription factor driving EMT in the posterior midgut, serpent is not expressed. The Brd genes, which are directly inhibited by Serpent, are expressed in the posterior midgut inhibiting Neur activity (Figure3.8 a). At this stage, the cells are polarised along their apical/basal axis, have apical polarity markers and form cohesive adherens junctions. At stage 10, Serpent is expressed in the posterior midgut and affects apical polarity in two different manners (Figure3.8 b)): Firstly, it blocks the transcription of the Brd genes allowing the activation of Neur, which downregulates the proteins of the Crb complex in a posttranscriptional manner. Secondly, it directly inhibits the transcription of Crb and Sdt. This leads to the loss of apical polarity, adherens junctions and the formation of intercellular gaps.

Chapter 4. Paper 1: The E3 ubiquitin ligase Neuralized targets specific isoforms of the apical polarity protein Stardust to regulate epithelial remodeling in Drosophila

Acknowledgements

Acknowledgements

We thank K. Choi, C. Delidakis, J. Knoblich, E. Knust, E. Lai, P. Lasko, M. Rand, U.

Tepass, A. Wodarz, the Bloomington Drosophila Stock Center, the Developmental Studies Hybridoma Bank and Flybase for flies, antibodies and other resources. We thank S. Chanet for her initial input. We thank L. Couturier for technical help. We thank A. Bardin and S. Chanet for critical reading and all lab members for discussion. This work was funded by an ANR grant (ANR12-BSV2-0010-01). G. P.-M. received fellowships from the MENRT and the FRM.

Ackowledgements

We thank M. Affolter, B. Aigouy, E. Caussinus, D. Kiehard, A. Martin, M. Rand, J. Treisman, the Bloomington Drosophila Stock Center, the Developmental Studies Hybridoma Bank and Flybase for flies, antibodies and other resources. We thank S. Chanet for her initial input. We thank D. del Alamo for molecular analysis of the neur IF65 allele. We also thank V. Roca and L.

Couturier for technical help. We thank NN for critical reading and all lab members for discussion. This work was funded by an ANR grant (ANR12--BSV2--0010--01). G. P.--M. received fellowships from the MENRT and the FRM. 5.1. Paper 2: Spatial control of contractility by Neuralized and Bearded during furrow invagination in Drosophila 125

Although not strictly essential, this regulation of Crb by Neur contributes to epithelial remodelling in the posterior midgut in the embryo and in the developing eye. Thus, our study uncovers a Neur-Sdt-Crb regulatory axis that contributes to epithelial morphogenesis in specific developmental contexts.

Chapter 8

Results annex 3: Other Results

During my thesis I performed some experiments that gave inconclusive, yet potentially interesting results. These results are presented in this appendix, with a special emphasis of why, even though they are far from being publishable, they are interesting.

Sdt, Neur and ommatidia morphogenesis

The development of ommatidia in the eye imaginal disc follows a predefined morphogenetic path [251]. Cells from the future ommatidia arise from the morphogenic furrow and arrange themselves, firstly into a Typ2-arc structure (Figure 8.1b) and then into a rosette of 6 cells where they all share a vertex (Figure 8.1b'). Later, a cell located between the R3 and R4 cells, called the mystery cell, is excluded from the rosette (Figure 8.1b") and a 5-cell pre-cluster is formed (Figure 8.1b"'). Neur has been shown to be expressed in the R3 and R4 cells [252]. This was confirmed (Figure 8.1c") by immunostainings of eye discs carrying a GFP-tagged construct of Neur encoded in a BAC.

Moreover, the location of GFP-Neur correlated with a zone were 3GFP-Sdt was downregulated at the 5 cell pre-cluster stage (Figure 8.1d). This downregulation was abrogated in eye discs of flies lacking the exon 3 (Sdt 3-GFP constructed by CRISPR mediated recombination), as shown in figure Figure 8.1e. Finally the absence of the exon 3 was sufficient to perturb the formation of the rosette stage (Figure 8.1g). However, in eyes discs of larvae lacking the exon 3, the rosette stage didn't appear to be required for the formation of the 5 cell pre-cluster at later stages. Finally, adult flies lacking the exon 3 didn't show any striking defect in the eye.

We propose that Neur downregulates Sdt-PB in the R3 and R4 cells to promote cell rearrangements during ommatidia development, but in the absence of the Sdt/Neur interaction some redundant mechanisms ensure the ommatidial development. . However, by closely looking at proteins that bind to Neur it is possible to see the last amino acid of the consensus sequence can also be R, K, Q or A. With this in mind, I performed a bioinformatic analysis to look at the proteins that contained the sequence [E,D or Q],N, x, x, N, x, x, [L, M, V, R, K, Q and A]. From the original 28828 proteins (isoforms included) present in the list downloaded from Flybase (the 3rd release from 2013), 2467, or 8.6% of the proteins, contained the consensus sequence. Hence this bioinformatic approach is not very helpful to identify new Neur targets.

Bioinformatic analysis of putative Neur substrates

List of Figures