M Brian Oliver

Rapporteur Mme

Julia Cordero

Rapporteure M Georges Nemer

Rapporteur M Ziad

CRIBBS M David

Examinateur Mme Laure

Examinatrice E L Chamy

M Dani Osman

Directeur De Thèse

PAYRE M François

Brian Oliver

Laure El Chamy

David Cribbs

Sandy Al Hayek

Ahmad Alsawadi

Zakaria Kambris

Jean-Philippe Boquete

Jérome Bohère

Brice Ronsin

Serge Plaza

Bruno Lemaitre

François Payre
email: francois.payre@univ-tlse3.fr

Dani Osman
email: dani.osman@ul.edu.

Affiliations

OvoL/Shavenbaby transcriptional switch orchestrates the self-renewal versus differentiation of intestinal stem cells Authors

Keywords: Adult midgut progenitors  AMPs, Anti-microbial peptides  APC, Adenomatous Polyposis Coli  Arm, Armadillo

Razzak for his valuable help at the beginning of my thesis.

Je tiens également à remercier les membres de mon comité de suivi de thèse :

François Leullier et Allison Bardin pour leurs conseils et leurs encouragements.

C'était un grand plaisir pour moi de pouvoir discuter avec vous de mon projet de thèse.

Un énorme merci à François : mon directeur de thèse. Je te remercie du plus profond de mon coeur de m'avoir accueillie dans ton labo ainsi que pour ton aide et ton support tout au long de ma thèse. Merci pour toutes les discussions interminables, les réunions (longues mais productives). Tu m'as aidée à acquérir une maturité scientifique et surtout à croire en mes capacités ! Tu étais là pour les hauts comme pour les bas. Tu m'as vue craquer, rigoler (et râler). Tu m'as appris à être patiente, même si c'était un peu dur parfois ! Je suis également fascinée par ta façon de tout gérer et je vais prendre exemple sur toi pour la suite de ma carrière ! Dani, mon co-directeur de thèse, tu étais là dès le début pendant mes recherches de thèse et tu n'as jamais lâché l'affaire ! Si j'en suis là aujourd'hui c'est en grande partie grâce à toi. Tu m'as confié un projet qui t'est très cher ! Tu m'as poussée à aller plus loin. J'étais ta première étudiante après ton retour définitif au Liban et j'ai eu l'occasion de participer (un tout petit peu) à tes enseignements. Tu m'as appris à bien réfléchir et à bien analyser mes données (sans sur-interpréter les résultats). Choukran !!! Dani et François je vous remercie infiniment de m'avoir donné la chance de présenter mon projet au meeting Américain de Recherche en Drosophile. C'était une opportunité en or que je n'oublierai jamais.

« A mind that is stretched by a new experience can never go back to its old dimensions. » (Oliver Wendell Holmes) Je tiens également à remercier les membres de l'équipe sans qui ces quatre années de thèses n'auraient pas été si merveilleuses.

Un vif remerciement à ma meilleure amie française, ma voisine de bureau et ma complice Alex (ça risque d'être un peu long). Je ne me souviens pas comment nous sommes devenues si proches l'une et l'autre mais crois-moi sur paroles que ça a bouleversé ma vie de doctorante, en mode stress continu ! Merci d'être toujours là pour me remonter le moral, pour essuyer mes larmes et de faire de ton mieux pour me faire sourire ! Bon, passons aux choses rigolotes !! Merci Alex de m'avoir aidée à améliorer mon Français en ayant le courage et surtout la patience de me reprendre (gentiment). Tu as toujours apprécié ma volonté à vouloir apprendre encore plus. Et d'ailleurs ce fameux bouquin d'expressions françaises que tu m'avais conseillé m'a permis de bien briller en société ! Je n'oublierai jamais les soirées cinéma (les lundi soir ces derniers temps), les petites escapades et pleins d'autres belles aventures !!! Bon courage pour la suite de ta carrière ma star en Bio-informatique !! Cédric, merci d'avoir été mon « confident expérimental » au début de ma thèse.

Je te remercie aussi pour tes conseils, ta bonne humeur et ta positivité. Merci également pour les journal clubs rigolos et la bonne ambiance au bureau !! PhiPhi, la star de la génétique, merci pour ton humour et tes blagues sans cesse, qui m'ont permis de déstresser !! Je ne serais malheureusement pas là pour ta soutenance de thèse mais je suis sûre que ça va être un énorme succès ! et « Ce qui est fait, ce n'est plus à faire ;) » Hélène, merci pour ton encouragement « je veux voir des plus partout les jeunes » ! Merci pour toutes les discussions aux mouches : « pas plus que deux gosses Sandy ;) » ; Pour tes retours sur mon article ainsi que pour tes commentaires constructifs sur tous mes topos ! Damien, le doctorant le plus jeune du labo, je ne me souviens pas d'un seul instant où je t'ai vu fâché ou même énervé ! Merci pour ta positivité, ta gentillesse et ton soutien et encore pour les expressions françaises que tu m'as apprises !! Je te souhaite un avenir aussi beau que tes cheveux bien coiffés et tes belles introductions ;) !!! Maleaume, nous avons commencé nos thèses en même temps et tu m'as toujours épaulée. Merci pour ta confiance en moi, les fou rires, les discussions (sérieuses et rigolotes), mais également pour les Jeudi Subway (et cookie Macadamia), les exercices de prononciations : « Les chaussettes de la duchesse sont-elles sèches, archi sèches ? » ainsi que pour ta patience « Sandy comprends super vite mais il faut qu'on lui explique très longtemps ». Merci énormément d'avoir chouchouté mes mouches pendant mes vacances et évidemment pour la « Super macro de la mort qui tue », d'ailleurs elle a rendu mes analyses beaucoup plus rapides. DyDy te souhaite un grand succès ! Tu vas y arriver ! Maylis, Je te remercie surtout pour ta positivité, ta bonne humeur et la bonne ambiance ! Et contrairement à Damien, je trouve que t'as une très belle voix que tu pourrais en profiter plus tard ! Anne, j'ai toujours apprécié nos discussions scientifiques et personnelles ! Tu voulais toujours prendre de mes nouvelles tout en ayant peur de poser des questions qui fâchent (du genre : « il est parti ton papier Sandy ? ») !! Merci pour Figure 1: Adult Drosophila gut organization ............................................................................ Figure 2 : Adult midgut progenitors at the origin of ISC .......................................................... Figure 3 : Drosophila adult midgut regionalization ................................................................. Figure 4 : Notch signaling pathway in Drosophila adult midgut .............................................. Figure 5 : EE cell fate regulation ............................................................................................... Figure 6 : Canonical Wnt signaling in Drosophila .................................................................... Figure 7 : Canonical JAK/STAT signaling in Drosophila ............................................................ Figure 8 : Overview of the Hippo pathway in Drosophila ......................................................... Figure 9: esg TF regulates both stemness and ISC differentiation ........................................... Figure 10 : ISC regulation by neighboring cells and tissues ..................................................... Figure 11 : The mammalian intestinal epithelium ................................................................... Figure 12 : ISC implication in intestinal cancer ......................................................................... polished-rice ....................................................................................................... 22 : svb target genes in ISC ........................................................................................... Figure 23 : The effect of desiccation on Pri-A and Pri-J activity in ISC .....................................

List of figures

Stem cells in health and in diseases 1.Emergence of the stem cell concept

The term "stem cell" (German stammzelle) emerged with the German biologist Ernst Haeckel in 1868 to designate the origin of all cell types. After Darwin, who at the same time proposed his theory on the common origin of species, Haeckel used the term stem cells to refer to the fertilized egg that is the source of all the organism's cell types. In 1892, Theodor Boveri and Valentin Hacker expanded the definition of stem cells to include primordial germ cells (PGC), since germ cells maintain unaltered genetic material and transmit it to the next generation. At the same time, the hematologist Pappenheim used the word stem cell to denote common precursors that can generate both myeloid and lymphoid cells. Hematopoietic stem cells were then established as a prototype of stem cells according to two criteria: the capacity to proliferate indefinitely and the ability to generate differentiated cells [START_REF] Ramalho-Santos | On the origin of the term "stem cell[END_REF].

Adult stem cells

Tissue homeostasis is the process by which a robust structural organization and function are maintained, in the face of varying internal and environmental conditions. This stability is accomplished by the activity of tissue-specific populations of non-differentiated cells, called adult stem cells (SCs), which have three distinguishing features. First, compared to the shortlived differentiated cells that insure tissue function, adult stem cells are long-lived to regenerate tissue damages throughout the adult life. Second, they divide to generate new SCs and maintain their pools. Third: they are able to differentiate into a limited range of cell types that build the tissue in which they reside; hence, adult stem cells are multipotent.

The rate of tissue renewal is very variable depending on the intrinsic characteristics of each organ. For instance, it is very high for blood cells [START_REF] Seita | Hematopoietic stem cell: self-renewal versus differentiation[END_REF] or the gut epithelium [START_REF] Eastwood | Gastrointestinal epithelial renewal[END_REF][START_REF] Leblond | The life history of cells in renewing systems[END_REF] which is entirely renewed every week in humans. Some other tissues display a low rate of cell renewal, such as the brain [START_REF] Ihunwo | The dynamics of adult neurogenesis in human hippocampus[END_REF] , kidney [START_REF] Little | Mammalian kidney development: principles, progress, and projections[END_REF] or heart [START_REF] Doppler | Mammalian Heart Regeneration: The Race to the Finish Line[END_REF]. In the case of lesions or other stresses (infections, injuries), these organs yet display increased regenerative capacities necessary to replace dead cells. In the heart, an organ less exposed to external aggressions, cell renewal can occur by the dedifferentiation of mature cells that will proliferate to repair the injury [START_REF] Szibor | Remodeling and dedifferentiation of adult cardiomyocytes during disease and regeneration[END_REF]. The study of adult stem cell behavior has become a major topic for the following reasons:

1. Given their potential to differentiate into various cell types, SCs have tremendous potentials for the treatment of human diseases [START_REF] Kiskinis | Progress toward the clinical application of patient-specific pluripotent stem cells[END_REF].

2. Reduced SC function and/or homeostasis can trigger degenerative diseases and is involved in ageing [START_REF] Clevers | The cancer stem cell: premises, promises and challenges[END_REF][START_REF] Jones | Emerging models and paradigms for stem cell ageing[END_REF].

3. Deregulated growth of SCs may lead to tumors and adult stem cells share many similarities with the so-called cancer stem cells (CSCs) [START_REF] Visvader | Cancer stem cells in solid tumours: accumulating evidence and unresolved questions[END_REF].

Cancer stem cells The discovery of cancer stem cells

The development of tumors and their metastatic potential depend on the activity of rare cancer stem cells (CSCs), or tumor-initiating cells. As for adult stem cells, CSCs have the capacity to proliferate in order to produce more CSCs and to give rise to committed malignant cells.

The concept of CSCs has first emerged in the thirties, from the work of Furth and Kahn [START_REF] Furth | Transmission of Myeloid Leukemia of Mice : Its Relation to Myeloma[END_REF] showing that individual cells derived from mouse leukemia could generate new tumors when engrafted in a healthy recipient mouse. Lapidot et al later hypothesized that a population of CSCs sustains leukemic clones, since myeloid cancer cells have a restricted proliferative capacity [START_REF] Lapidot | A cell initiating human acute myeloid leukaemia after transplantation into SCID mice[END_REF]. Cancer cells were sorted based on the differential expression of cell surface markers and then transplanted into immune deficient mice. Cells that are able to initiate new tumors in recipient mice were considered as CSCs [START_REF] Lapidot | A cell initiating human acute myeloid leukaemia after transplantation into SCID mice[END_REF].

Furthermore, CSCs were also identified in solid tumors, such as in brain [START_REF] Singh | Identification of human brain tumour initiating cells[END_REF] and breast cancers [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF] using the same transplantation approach. Subsequently CSCs were identified in many solid cancers including colon, pancreas and lung among others [START_REF] Tirino | Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization[END_REF].

Origin of cancer stem cells

Despite numerous evidence of the importance of CSCs in tumors, their origin remains controversial. It was hypothesized that transformed cells can fuse with normal stem cells; the resulting fusion cells thus possess both self-renew and transformed abilities. The fusion may be accompanied by chromosomal loss further favoring CSC formation [START_REF] Ogle | Biological implications of cell fusion[END_REF]. In addition, mutations in somatic cell's DNA lead to apoptosis and DNA fragmentation. The fragmented DNA can be taken up by a recipient cell leading to nuclear reprogramming and tumor initiation [START_REF] Camargo | Stem cell plasticity: from transdifferentiation to macrophage fusion[END_REF][START_REF] O'malley | Stem cell fusion confusion[END_REF][START_REF] Pomerantz | Nuclear reprogramming: a key to stem cell function in regenerative medicine[END_REF].

The resulted genomic instability such as chromosomal rearrangement and mutations within stem cells thus lead to their transformation to CSCs [START_REF] Bergsmedh | Horizontal transfer of oncogenes by uptake of apoptotic bodies[END_REF][START_REF] Holmgren | Horizontal transfer of DNA by the uptake of apoptotic bodies[END_REF]).

An alternative model suggests that CSC arise from tumor cells that undergo multiple changes that favor a stem-like phenotype, including loss of cell-cell adhesion molecules such as Ecadherin, alteration of apical basal polarity, and the gain of motility. This reversible process known as epithelial to mesenchymal transition (EMT) permits CSCs invasion to new sites where they seed micro tumors by reverse mesenchymal to epithelial transition (MET) [START_REF] De Craene | Regulatory networks defining EMT during cancer initiation and progression[END_REF]. In addition to generating CSCs and inducing metastasis, EMT governs multiple normal processes including tissue remodeling during embryogenesis, organ development and tissue regeneration [START_REF] Thiery | Epithelial-mesenchymal transitions in development and disease[END_REF].

The EMT program is induced by a series of transcription factors (TFs) mainly Snail, Slug, Twist and Zinc finger E-box binding Homeobox 1 (Zeb1 and Zeb2) collectively called EMT-inducing factors. The expression of these TFs is induced by signaling cues emanating from tumorassociated stroma, including Transforming Growth Factor Beta (TGF-β), Notch and Wnt signaling [START_REF] Peinado | Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?[END_REF][START_REF] Polyak | Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits[END_REF].

Under normal conditions, micro-RNA composed of 20-22 nucleotides silences the expression of EMT-TF by binding to 3'UTR of target mRNA [START_REF] Lewis | Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[END_REF]. miR-200 and miR-205 act as EMT repressors by targeting Zeb1 and Zeb2 mRNA, thus their downregulation facilitates early steps of metastasis [START_REF] Cano | Non-coding RNAs take centre stage in epithelial-to-mesenchymal transition[END_REF]. Moreover, the downregulation of Zeb1 in some cancer types lead to an upregulation of miR-200 expression, pointing to a negative feedback loop between Zeb and miR-200. miR-200 also acts in a zeb-independent manner, through the regulation of various stemness-related genes, and Zeb factors are regulated by additional micro-RNAs less characterized than miR-200. Another regulatory feedback loop also exists between miR-34, miR-203 and the EMT-inducing TF Snail. Conversely, both Zeb and Snail target miR-34 expression providing a tight crosstalk between the miR-200/Zeb and miR-34/Snail networks [START_REF] Diaz-Lopez | Role of microRNA in epithelial to mesenchymal transition and metastasis and clinical perspectives[END_REF].

Since the discovery of CSCs in solid tumors, many studies have focused on the mechanisms that induce EMT, based on the hypothesis that this process is at the origin of metastasis (Nieto et al., 2016). In contrast, recent findings suggest that tumor cells that contain both mesenchymal and epithelial features have the strongest metastatic potential. The screening for the factors that inhibit EMT have led to the identification of the transcription factor OvoL, as a crucial gatekeeper of the epithelial state [START_REF] Roca | A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial -mesenchymal cell reprogramming and cancer progression[END_REF]Watanabe et al., 2014). However, most studies that characterized OvoL functions in EMT-TF inhibition were performed using cultured cells. Thus, it is crucial to find a suitable in vivo model system to decipher OvoL mechanisms of action, in both physiological and pathogenic conditions.

The fly gut as a model system of stem cell function and dysfunction

For over a century, the "fruit fly" Drosophila melanogaster has been a powerful model for many areas of research, such as genetics, development, evolution and diseases. Notably, six Nobel prizes were awarded to Drosophila researchers. Starting from 2006, Drosophila has now emerged as an attractive model system to study intestinal stem cells (ISCs) and gut regeneration.

The intestine is one of the largest and most active organs of the adult body. Beside its function in food processing and nutrient absorption, the gut epithelium constitutes an efficient line of defense against a wide variety of ingested pathogens in both mammals and Drosophila [START_REF] Buchon | Immunity in Drosophila melanogaster--from microbial recognition to whole-organism physiology[END_REF][START_REF] Chassaing | Mammalian gut immunity[END_REF]. The gut is also a major source of hormones that modulate the functions of other organs, such as pancreas and brain in mammals [START_REF] Bewick | Bowels control brain: gut hormones and obesity[END_REF]. Thus the gut is not simply a passive digestive tract but it is a vital organ implicated in multiple physiological functions.

The Drosophila adult midgut epithelium is a simple structure that shares anatomical and functional similarities with its mammalian counterpart, the small intestine [START_REF] Liu | Drosophila as a model for homeostatic, antibacterial, and antiviral mechanisms in the gut[END_REF].

Adult flies feed on decayed fruits and are therefore continually exposed to ingested pathogens, which can have detrimental impacts in the absence of an effective defense mechanism. To deal with these challenges, damaged cells are constantly replaced by new cells, through the activity of intestinal stem cells (ISCs) (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006).

In the following section, I will describe the structural and cellular organization of the fly digestive tract. I will focus on ISCs of the posterior midgut, the region of interest in my PhD work. In a second part, I will present key findings regarding the molecular mechanisms that regulate both ISC differentiation and proliferation in steady state and stress conditions. In the final part, I will summarize similarities between Drosophila and mammalian digestive tracts, in both structural and cellular organization, as well as the key role of signaling pathways in normal homeostasis and tumor development.

Structural and cellular organization of Drosophila adult digestive tract 2.1.1 The structural organization of the adult fly intestine

The adult Drosophila digestive system is subdivided into three main regions: the foregut, the midgut and the hindgut [START_REF] Royet | Epithelial homeostasis and the underlying molecular mechanisms in the gut of the insect model Drosophila melanogaster[END_REF]. The foregut originates from the ectoderm. It is a short tube-like structure composed of the esophagus, which connects to the salivary glands and to the crop, a bi-lobed structure responsible for temporary food storage before its transition into midgut lumen (For review see (Miguel-Aliaga et al., 2018)). The midgut represent the functional equivalent of the mammalian small intestine. It originates from the endoderm and is responsible for food digestion and absorption. The anterior midgut extremity, called proventriculus, is responsible for the initiation of the mechanical breakdown of the ingested food [START_REF] King | Cellular organization and peritrophic membrane formation in the cardia (proventriculus) of Drosophila melanogaster[END_REF]. The proventriculus synthesizes a chitin-containing membrane called peritrophic matrix (PM) that lines the midgut epithelium and separates it from the food bolus.

The PM is thus analogous to mucous secretion in the mammalian intestine and constitutes a physical barrier that protects the epithelium from harmful microbes and food particles [START_REF] Kuraishi | Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster[END_REF]. The midgut is enclosed by visceral muscles (VM), nerves, as well as trachea which communicate with the epithelium by signaling molecules regulating intestinal homeostasis. The hindgut derives from the ectoderm and is in charge of water and electrolytes uptake, before discharging the waste into the rectum. The boundary between the midgut and the hindgut is where branching tubules called Malpighian tubules (MTs) are connected. MTs the Drosophila renal system are responsible for adjustment of hemolymph ionic concentrations, through the absorption of electrolytes and their subsequent release into the hindgut. It is interesting to note that distinct populations of stem cells residing in each region maintain the overall integrity of the digestive tract throughout adult life [START_REF] Zeng | Stem cells in the Drosophila digestive system[END_REF] (Figure 1).

Figure 1: Adult Drosophila gut organization

The Drosophila adult gut is folded into a 3D structure within the abdomen. It is composed of three mains regions: the foregut containing the esophagus, the proventriculus and the crop; the midgut, the main site of digestion and the hindgut. Malpighian tubules (MT) are attached to the midgut/hindgut boundary. The midgut epithelium is composed of Enterocytes (blue), Enteroendocrine cells (pink), intestinal stem cells (green) and enteroblast precursor cells (orange).

The midgut cellular composition and molecular markers

The Drosophila midgut epithelium is a cell monolayer, composed of two types of differentiated cells, responsible for its physiological functions, and stem cells and progenitors cells that ensure the replacement of damaged differentiated cells (Figure 2).

Enterocytes (ECs) are large polyploid absorptive cells that constitute 90% of the midgut epithelium. ECs are positive for the transcription factor Pdm1, also known as Nubbin (Beebe et al., 2010;[START_REF] Lee | Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation[END_REF][START_REF] Mathur | A transient niche regulates the specification of Drosophila intestinal stem cells[END_REF][START_REF] Tang | The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation[END_REF], which is homolog to the mammalian Pou/Oct proteins [START_REF] Holland | Classification and nomenclature of all human homeobox genes[END_REF][START_REF] Tantin | Oct transcription factors in development and stem cells: insights and mechanisms[END_REF]. Enteroendocrine cells (EE), are small secretory cells interspersed between ECs, and characterized by the expression of the panneural TF Prospero (Figure 2c). Spradling labs (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006). In order to identify proliferating cells within the adult midgut, the researchers traced cells that incorporate Bromodesoxyuridine (BrdU) and thus synthetize DNA. They showed that both small diploid cells and large polyploid cells are BrdU-positive, but only diploid cells are positive for phosphorylated Histone 3 (on a serine at position 10, PH3), a specific marker of mitotic cells.

Lineage tracing approaches demonstrated that PH3+ cells are able to both self-renew and give rise to differentiated intestine cells (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006). The small diploid cells are thus intestinal stem cells (ISCs) and larger BrdU-cells represent their daughter post-mitotic progenitors, called enteroblasts (EBs).

ISC and EB are characterized by the expression of the zinc finger TF escargot (Esg), the Drosophila counterpart of the mammalian genes Snail/Slug, and are found as duplets along the basement membrane (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006). In addition, ISCs specifically express Delta (Dl), a ligand of the Notch signaling pathway that binds to the Notch receptor present at the cellular surface of the newly formed EB. Consistently, activation of Notch within EB cells is attested by the expression of the Su(H)-Gbe-LacZ reporter (Ohlstein and Spradling, 2007) (Figure 2c) .

The midgut development: origin of ISCs

During embryonic development, the endodermal primordia from both sides of the embryo invaginate and some cells undergo epithelial to mesenchymal transition (EMT) to form loosely organized mesenchymal mass containing Adult Midgut Progenitors (AMPs) [START_REF] Hartenstein | The function of the neurogenic genes during epithelial development in the Drosophila embryo[END_REF]. Mesenchymal cells then migrate toward each other's and undergo reverse MET transition in order to produce EC. This is initiated when endodermal cells enter in contact with mesodermal cells. At this stage, the midgut is composed exclusively of ECs and AMPs closely associated to visceral muscles (Jiang and Edgar, 2009). The embryonic midgut is conserved until pupal stage. During metamorphosis, AMP daughter cells fuse together to form the adult midgut (Jiang and Edgar, 2009;[START_REF] Mathur | A transient niche regulates the specification of Drosophila intestinal stem cells[END_REF]. Lineage tracing demonstrated that AMPs

give rise to all cells of the adult midgut, including ISCs, ECs and EEs [START_REF] Mathur | A transient niche regulates the specification of Drosophila intestinal stem cells[END_REF].

During midgut development, AMPs undergo either symmetric or asymmetric division. During early larval stages, dispersed AMPs divide symmetrically to increase their number as the midgut increases in size. Later at mid-larval stage, AMPs undergo one asymmetric division generating a differentiated cell called Peripheral cell (PC), which wraps around clusters of AMP and inhibits their differentiation [START_REF] Mathur | A transient niche regulates the specification of Drosophila intestinal stem cells[END_REF]. AMPs continue to divide within islets under the control of mitogenic signaling such as epidermal growth factor Receptor (EGFR) (Jiang and Edgar, 2009). AMPs behavior is also regulated by the ecdysone hormone during larval stages [START_REF] Micchelli | Identification of adult midgut precursors in Drosophila[END_REF]. During pupariation, PCs are lost by apoptosis and AMPs become free to differentiate (Mathur et al., 2010) (Figure 2a-b). progenitor, that will further differentiate into EE or EC cells, respectively.

The Drosophila adult midgut regionalization

The midgut was initially subdivided into three regions namely the anterior, the middle and the posterior midgut [START_REF] Demerec | Biology of Drosophila[END_REF][START_REF] Hakim | Regulation of midgut growth, development, and metamorphosis[END_REF]. This subdivision was based on the discovery in the middle region of acid secreting cells called "copper cells" [START_REF] Dubreuil | Copper cells and stomach acid secretion in the Drosophila midgut[END_REF].

These cells confer a high acidity for the middle midgut, similarly to the mammal's stomach. In contrast, the luminal content of both the anterior and the posterior midgut are mildly alkaline [START_REF] Dubreuil | Copper cells and stomach acid secretion in the Drosophila midgut[END_REF].

Recently, work from two independent groups have resulted in the establishment of a nose-totail atlas of the Drosophila adult midgut compartmentalization (Buchon et al., 2013;Marianes and Spradling, 2013). Morphometric analyses conducted by Buchon et al. have revealed the presence of six constrictions that split the midgut into five main chambers named R1-R5. These domains were further subdivided into eight subregions based on EC morphology. Next, they performed transcriptome analysis that unveil an unprecedented compartment-specific gene expression and led to a fine-grained division of the midgut into 14 subregions. Interestingly, this high level of regionalization seems to be very robust since it remains intact independently of diet fluctuations or gut damage. This regionalization is governed by a complex regulatory network involving TFs that were used for midgut development during embryogenesis (Buchon et al., 2013). Marianne and Spradling also subdivided the midgut into 10 regions contained within the 14 regions identified by Buchon et al. They also revealed the existence of regionspecific expression profiles by performing transcriptome analysis, region-specific microarrays and RNA-seq experiments (Buchon et al., 2013;Marianes and Spradling, 2013). Marianne and Spradling also uncovered a partial role for ISC in establishing midgut regionalization. They showed that ISC of different subregions have both different morphology and mitotic activity.

ISCs of the posterior midgut region P1-P3 (R4) are the most rapidly dividing cells, with a division rate of once per day, the rate of division is less important in the anterior part.

Importantly, ISC daughter cells residing near a region boundary always give rise to the same cell lineages and, even in rare cases where they cross the boundaries , they keep cellular memory of their region of origin (Marianes and Spradling, 2013) (Figure 3).

The Drosophila adult Malpighian tubules

In addition to digestion and nutrient absorption, ionic concentrations should be tightly regulated in order to maintain a healthy organism. This function is achieved by the Malpighian tubules (MT) attached to the midgut/hindgut junction. MT are also renewed by adult stem cells called Renal Nephric Stem cells (RNSC). In the following section, I will describe MT structural and physiological organization with emphasize on RNSC origin and regulatory mechanisms. I have to note that the first paper of this manuscript concern RNSC maintenance.

Malpighian tubules structural organization

Each adult gut is attached to two pairs of MTs: one pair is projected toward the anterior part of the digestive tract whereas the second one is oriented posteriorly. Each MT is divided into two regions: the lower tubules attached to the midgut through the ureters, and the upper tubules distant from the gut and further divided into main, transitional and initial segments [START_REF] Sozen | Functional domains are specified to single-cell resolution in a Drosophila epithelium[END_REF]. As for gut regionalization, MT subregions can be defined based on differential genes expression [START_REF] Sozen | Functional domains are specified to single-cell resolution in a Drosophila epithelium[END_REF]. MT are composed of two types of polyploid differentiated cells:

principal cells (PC) distributed all along the tubules, and stellate cells (SC) located in the upper MT [START_REF] Denholm | Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarize to establish secretory function[END_REF].

Malpighian tubules functions

MT are immerged in the hemolymph, the circulatory system of Drosophila. They ensure water reabsorption, ionic and acid/base equilibrium, as well as the elimination of metabolic waste. In addition, MT participate to innate immunity by acting as autoimmune sensor. A deprivation of nutrients and water (desiccation) induces the expression of the peptidoglycan recognition protein-LC (PGRP-LC). PGRP-LC is known for its role in bacterial detection and induction of the immune response, with an increased production of anti-microbial peptides (AMPs) in order to protect the animal from bacterial loads [START_REF] Zheng | Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells[END_REF].

Renal Nephric stem cells: origin and regulation

Similar to the midgut, MT are renewed by stem cells called Renal Nephric stem cells (RNSC) known as "tiny cells" due to their small size and to their diploid nuclei [START_REF] Sozen | Functional domains are specified to single-cell resolution in a Drosophila epithelium[END_REF].

RNSC are located in the lower tubules and can divide to maintain their pool. They also give rise to committed progenitors called renalblasts (RBs) that further differentiate into PCs or SCs (Singh et al., 2007). In contrast to differentiated cells, already present in the MT during embryogenesis, RNSC are specific to the adult stage. At the pupal stage, intestinal AMPs migrate from the midgut and form the ureter in order to attach the midgut to the MT (Takashima et al., 2013). The remaining undifferentiated AMPs then become RNSCs (Takashima et al., 2013). RNSCs conserve the expression of esg, already present in AMP but express an additional TF called Krüppel (Kr) [START_REF] Denholm | Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarize to establish secretory function[END_REF]Singh et al., 2007), required for normal MT formation during embryogenesis. Thus, migrating AMPs are reprogrammed within MTs to acquire novel characteristics different from those of ISC. In addition, RNSC are well individualized and their number is reduced compared to this of ISC (350 RSCs compared to 1000 ISCs approximatively).

Although RNSCs were identified about ten years ago, the mechanisms that regulate their activity remains poorly understood. Similarly to ISC, RNSC express Dl, which activates the Notch pathway in the RBs to initiate their differentiation into reabsorbing cells, CPs and SCs (Li et al., 2014). RNSCs proliferation is regulated by both EGFR and JAK/STAT signaling pathways [START_REF] Li | EGFR/MAPK signaling regulates the proliferation of Drosophila renal and nephric stem cells[END_REF]Singh et al., 2007) .In contrast, Hippo pathway loss of function leads to an overproliferation of RNSCs [START_REF] Doggett | Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling[END_REF][START_REF] Verghese | Scribble acts in the Drosophila fat-hippo pathway to regulate warts activity[END_REF][START_REF] Yang | Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules[END_REF]Zeng et al., 2010).

Molecular mechanisms governing ISC behavior in the adult midgut

Following the ISC discovery, many studies have focused on deciphering the molecular mechanisms that determine the choice between EC and EE fates, as well as the regulation of ISC proliferation. In the following paragraph, I will present our understanding of ISC regulation by both signaling pathways and transcription factors. I will next discuss recent findings concerning mitogenic signaling pathways required for ISC proliferation in both normal and stress conditions.

ISC differentiation by Notch and JAK/STAT signaling pathways

The generation of absorptive enterocytes (EC) requires high levels of Notch activity within EBs, whereas weaker Notch activation leads to the production of EE cells (Ohlstein and Spradling, 2007). In the absence of Notch, ISC cannot differentiate and continue to proliferate leading to tumoral masses (Ohlstein and Spradling, 2007). Conversely, constitutive activation of the Notch pathway by expressing the Notch Intra cellular Domain (NICD) forces the differentiation of ISCs into ECs. Moreover, the activation of Notch within EB cells seems to be dependent of ISC-EB interaction mediated by E-Cadherin molecules, also required for cell shape maintenance [START_REF] Maeda | E-cadherin prolongs the moment for interaction between intestinal stem cell and its progenitor cell to ensure Notch signaling in adult Drosophila midgut[END_REF].

ISC identity is maintained, at least in part, by Daughterless (Da) a basic helix loop helix (bHLH) transcriptional activator that sustains Delta expression and self-renewal capacity (Bardin et al., 2010). In addition, Hairless (H) and Suppressor of Hairless (Su(H) act within ISCs to repress the expression of a main Notch target gene, Enhancer of split (E-(spl)-C, (Bardin et al., 2010)).

Notch activation in EBs triggers the expression of E(spl)-C that, in turn, represses Da expression thus enabling cell cycle exit to generate an EC cell (Bardin et al., 2010) (Figure 4).

Interestingly, the commitment of ISC to EB also requires the activation of Notch but at a very high level compared to level of Notch signaling required for terminal differentiation of EB to EC (Perdigoto et al., 2011). It was suggested that this differential level is required for the protection of ISCs from their loss through differentiation.

An alternative model for cell fate choice suggested that EE cells arise from another type of progenitors that do not activate Notch. This hypothesis is based on the fact that EE cell progenitors are negative of Su(H)-GBE reporter and that Notch mutant tumors still contain EE cells (Biteau and Jasper, 2014;Guo and Ohlstein, 2015;Zeng and Hou, 2015). This model is further refined by recent observations demonstrating that EBs are able to produce a small population of class II EE cells [START_REF] Beehler-Evans | Generation of enteroendocrine cell diversity in midgut stem cell lineages[END_REF].

In addition to Notch, the signaling pathway Janus Kinase/signal transducer and activator of transcription (JAK/STAT) is also required for terminal differentiation of both ECs and EEs.

JAK/STAT inhibition in progenitors leads to the accumulation of undifferentiated cells and Notch mutant clones lacking JAK/STAT components are devoid of EE cells (Beebe et al., 2010;Jiang et al., 2009). Epistasis genetic analyses revealed that Stat92E, the TF downstream of JAK/STAT, acts in parallel or downstream to Notch to induce both EC and EE specification (Beebe et al., 2010;Jiang et al., 2009). On the other hand, JAK/STAT induces Dl expression in ISCs (Buchon et al., 2009b;Jiang et al., 2009), suggesting that JAK/STAT can act both downstream and upstream of Notch. Additional functions for JAK/STAT signaling in the maintenance of the Drosophila midgut homeostasis are discussed below (section 2.2.3.)

Implication of Transcription factors in cell lineage

Besides signaling pathways, a couple of transcriptional factors are known to determine the cell fate choice between EC and EE cells. In the following, I will describe the positive and the negative regulators of the EE cell fate determination.

Specific EE fate regulation program

Bardin et al. have revealed a key role for the proneural bHLH factors of the Achaete-Scute complex (AS-C) in the generation of EEs. The deletion of AS-C leads to a complete depletion of EEs, whereas the overexpression of either Asense or Scute in ISC/EB is sufficient to expand the population of Prospero+ cells (Bardin et al., 2010). Moreover, it has been proposed that Scute, a mitogenic factor is transiently expressed in ISCs and leads to their asymmetric division producing a new ISC and an EE progenitor (EEP) (Chen et al., 2018). EEPs divide once before the accumulation of Prospero causes cell cycle exit and EE differentiation (Chen et al., 2018).

Numb, an inhibitor of Notch signaling pathway, is asymmetrically dispatched between ISC daughter cells during mitosis [START_REF] Salle | Intrinsic regulation of enteroendocrine fate by Numb[END_REF]. Notch signaling is inhibited in cells that inherits Numb, consequently Asense and Scute expression is derepressed and Prospero is expressed [START_REF] Salle | Intrinsic regulation of enteroendocrine fate by Numb[END_REF].

In addition to factors required for EE differentiation, negative regulators of this cell fate are also detected such as the transcriptional repressor Tramtrack 69 (Ttk69) that inhibits EE cell fate (Wang et al., 2015) and disrupts their hormone producing capacity (Wang and Xi, 2015).

Interestingly EE cells are also able to regulate their number autonomously. Newly formed EE expresses the ligand Delta and activates Notch signaling in the apically located ISC. This activity blocks EE cell production and maintains ISC multipotency thus they revert to producing EC (Guo and Ohlstein, 2015). EE cells autoregulate their number by secreting a ligand called Slit. Slit is transduced by Roundabout receptor/leak receptor (Robot2/leak) present at the cell surface of intestinal precursor cells. Slit/robot pathway inhibits Prospero expression within ISC specifically and then inhibits the generation of new EE (Biteau and Jasper, 2014) (Figure 5). The achaete-scute complex (AS-C) required for prospero expression and subsequently EE cell production is transiently activated in dividing ISC (green) and leads to the generation of a new ISC and enteroendocrine progenitor cell (EEP)(light pink). EEP divide once before starting to accumulate prospero protein. In addition, the newly formed EEP inherits the protein Numb that inhibits Notch signaling and thus depresses AS-C genes. EE cell generation is negatively regulated by the transcriptional repressor tramtrack69 (Ttk69) that inhibits AS-C in parallel to Notch. Newly formed EE (dark pink) cells express Delta ligand and activates Notch in the apically located ISC that switches to the production of EC cells rather than EE. EE autoregulate their production also by secreting the ligand Slit that activates Roundabout receptor/leak receptor (Robot2/leak) present at the surface of ISC leading to the inhibition of prospero expression.

Regulation of ISC proliferation and survival

In addition to a proper differentiation, midgut homeostasis requires a tight regulation of ISC proliferation in order to replace damaged cells, but also to prevent excessive ISC proliferation that can lead to tumor development.

In the following section, I will describe our current knowledge of the role of diverse signaling pathways in the regulation of ISC proliferation under both homeostatic and stress conditions.

The Wnt/Wingless signaling pathway in Drosophila adult midgut

In Drosophila midgut (Figure 6, for the canonical Wnt pathway), Wingless (wg) is the only ligand that has a functional role in intestinal homeostasis [START_REF] Cordero | Inducible progenitorderived Wingless regulates adult midgut regeneration in Drosophila[END_REF]Lin et al., 2008).

The first evidence about a central role for Wnt signaling in regulating ISC behavior in the posterior midgut was reported by Lin et al. (Lin et al., 2008). wg expression was initially detected in the muscle layer surrounding the midgut. By generating clones lacking components of the Wnt pathway such as the receptor Frizzled, they observed a reduction of ISC proliferation rate that continue to produce EC and EE (Lin et al., 2008). Consistently the overexpression of wg in esg+ cells was sufficient to induce an expansion of esg+ Dl+ cells. This study led to the conclusion that Wnt signaling is necessary and sufficient to maintain ISC self-renewal and that the surrounding VM may serve as ISC niche (Lin et al., 2008).

Other studies brought different results about the source of Wnt ligands and the site of Wnt signaling activation. Takashima et al detected Wg in epithelial cells at the midgut hindgut junction [START_REF] Takashima | The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling[END_REF]. Buchon et al. have observed an enrichment of Wnt signaling reporter genes expression at the different midgut boundaries (Buchon et al., 2013); this expression decreases within the different midgut compartments. Tian et al demonstrated that EC cells rather than ISC cells are the primary site of Wnt signaling activation within Drosophila adult midgut [START_REF] Tian | Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries[END_REF]. This activity is required to inhibit growth factors release from EC such as Upd2 and Upd3 that act as paracrine signals and activate ISC proliferation (Tian et al., 2016). The β-catenin protein known as Armadillo (Arm) in Drosophila is a key effector in the Wnt signaling cascade. In normal conditions, β-catenin is kept in the cytoplasm where it is phosphorylated by a "destructive complex" composed of four kinases: Axin, Adenomatous polyposis Coli (APC), Glycogen Synthase Knase 3 (GSK3) and Casein Kinase 1α (Ck1α) [START_REF] Kishida | Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin[END_REF]. This phosphorylation induces the ubiquitination of β-catenin that is then targeted to proteasome degradation. Upon Wnt binding to Frizzled (FZD) and lipoprotein receptor related protein 6 (LRP6), the receptor complex is endocytosed and the protein Dishevelled (Dvl) form polymers at the cytoplasmic domain of the receptors. Together, LRP6 FZD and Dvl form the Wnt signalosome that sequestrates and inactivates the destruction complex. β-cat thus reaches the nucleus where it acts in collaboration with Pangolin (a.k.a. TCF/LEF) to induce the expression of Wnt target genes [START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/colon carcinoma[END_REF][START_REF] Morin | Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC[END_REF] (adapted from Clevers et al; 2012).

Epidermal Growth Factor Receptor (EGFR) pathway

Under homeostatic conditions, EGFR signaling is activated in ISC/EB by three redundantly active ligands released from different sources (Biteau and Jasper, 2011;Jiang et al., 2011;[START_REF] Xu | EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells[END_REF]. Visceral muscles produce the ligand Vein, Keren is released from ECs, and Spitz is expressed in ISCs and EBs. Clones mutant for EGFR components do not proliferate (Biteau and Jasper, 2011;Jiang et al., 2011;[START_REF] Xu | EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells[END_REF] Capicua a major inhibitor of both cell cycle gene and Pointed, the transcription factor activated downstream of EGFR signaling pathway. Phosphorylated Capicua is then excluded from the nuclei, pnt and cell cycle genes repression is released and ISC proliferation is induced (Jin et al., 2015). Moreover, EGFR signaling levels are kept in check by chromatin modifiers called Kismet and Trr [START_REF] Gervais | Stem Cell Proliferation Is Kept in Check by the Chromatin Regulators Kismet/CHD7/CHD8 and Trr/MLL3/4[END_REF]. These two complexes cooperate to induce the expression of an EGFR regulatory gene called Cbl within ISCs. Trr and Kismet loss of function inhibits

Cbl expression and leads to abnormal ISC accumulation, associated with a significant increase in EGFR activity.

The JAK/STAT signaling pathway

In Drosophila, the JAK/STAT signaling pathway is a major player of tissue homeostasis. It is required for the coordination of stem cell proliferation and differentiation in many tissues. In addition, it is required for testis niche maintenance and digestive tract development [START_REF] Arbouzova | JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions[END_REF].

JAK/STAT signaling is initiated by binding of glycosylated Unpaired (Upd1) [START_REF] Harrison | Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway[END_REF] and Upd-like proteins (Upd2, Upd3) [START_REF] Agaisse | Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury[END_REF][START_REF] Gilbert | A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation[END_REF][START_REF] Hombria | Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand[END_REF] to the receptor Domeless (Dome) [START_REF] Brown | Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development[END_REF][START_REF] Chen | Inhibition of cytokine-induced JAK-STAT signalling pathways by an endonuclease inhibitor aurintricarboxylic acid[END_REF]. The signal is then transduced within the cells through the receptor associated JAK kinase Hopscotch (Hop) [START_REF] Binari | Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila[END_REF] and the transcription factor Stat92E [START_REF] Hou | The Jak/STAT pathway in model organisms: emerging roles in cell movement[END_REF][START_REF] Yan | Phosphorylated interferon-alpha receptor 1 subunit (IFNaR1) acts as a docking site for the latent form of the 113 kDa STAT2 protein[END_REF]. When activated, Stat92E dimerizes and translocates to the nucleus to induce the expression of target genes, including Socs36E a negative regulator of JAK/STAT and Stat92E

itself [START_REF] Arbouzova | JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions[END_REF]) (Figure 7).

Upd ligands are released from multiple sources, but JAK/STAT activity is only detected in ISC/EBs (Beebe et al., 2010;Jiang et al., 2009;[START_REF] Liu | JAK-STAT is restrained by Notch to control cell proliferation of the Drosophila intestinal stem cells[END_REF]. Osman et al. have reported that Upd1 is released from ISCs and acts in autocrine manner to induce ISC proliferation in baseline conditions [START_REF] Osman | Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division[END_REF]. In ageing flies, ISC proliferate abnormally, this feature is partially mediated by both Upd2 and Upd3 ligands released from EBs and ECs. In addition, these two ligands are also overexpressed upon bacterial infection in order to induce rapid epithelial turnover [START_REF] Osman | Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division[END_REF]. Upd ligands are also released from the inner circular muscles and the outer longitudinal muscles and activate JAK/STAT signaling within ISC/EB cells [START_REF] Lin | Paracrine unpaired signaling through the JAK/STAT pathway controls self-renewal and lineage differentiation of drosophila intestinal stem cells[END_REF]. Clones mutant for JAK/STAT components are less proliferative and lack mature cells, while elevated signaling levels accelerate the rate of ISC division. Hence, JAK/STAT pathway regulates ISC proliferation and differentiation in healthy and pathogenic conditions.

The Hippo pathway: autonomous and non-autonomous restriction of ISC proliferation

The Hippo pathway also plays a pivotal role in the regulation of ISC proliferation. Hippo pathway, which is regulated by mechanical forces applied on cells, ensure an adapted organ growth and an efficient tissue renewal by regulating both cell proliferation and apoptosis [START_REF] Harvey | The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis[END_REF][START_REF] Pantalacci | The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila[END_REF][START_REF] Thompson | The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila[END_REF][START_REF] Udan | Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway[END_REF][START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF].

The Hippo complex is composed of four kinases: Warts (wts) [START_REF] Justice | The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation[END_REF][START_REF] Xu | Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase[END_REF], Hippo (hpo) [START_REF] Harvey | The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis[END_REF][START_REF] Pantalacci | The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila[END_REF][START_REF] Udan | Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway[END_REF][START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF], mob as tumor suppressor (mats) [START_REF] Lai | Control of cell proliferation and apoptosis by mob as tumor suppressor, mats[END_REF] and Salvador (Sav) (Kango- [START_REF] Kango-Singh | Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila[END_REF][START_REF] Tapon | salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines[END_REF]. Hpo phosphorylates Sav and thus stabilizes their physical interaction [START_REF] Aerne | Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4[END_REF][START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF]. Hpo than phosphorylates Mats that interacts with Wts and potentiates its kinase activity [START_REF] Justice | The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation[END_REF][START_REF] Wei | Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila[END_REF][START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF]. The kinase cascade inactivates a unique nuclear effector called Yorki (Yki) (Huang et al., 2005;[START_REF] Oh | In vivo regulation of Yorkie phosphorylation and localization[END_REF][START_REF] Ren | Hippo signaling regulates Yorkie nuclear localization and activity through 14-3-3 dependent and independent mechanisms[END_REF], a transcriptional coactivator that induces the expression of antiapoptotic (DIAP1) and cell cycle genes (Cyclin E) [START_REF] Dong | Elucidation of a universal size-control mechanism in Drosophila and mammals[END_REF]Huang et al., 2005).

Within ISCs, Hippo inhibits Yki expression required for ISC proliferation (Huang et al., 2005;[START_REF] Karpowicz | The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration[END_REF]Ren et al., 2010a;[START_REF] Shaw | The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration[END_REF]. Yki gain of function (or wts loss of function) induces abnormal ISC proliferation by surexpression of DIAP1 and Cycline E [START_REF] Shaw | The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration[END_REF]. Moreover, Yki is required for ISC proliferation following pharmacological or immune stress (Ren et al., 2010a). Yki activity is dependent of its physical association to two proteins: Scalloped (Sd) and Brahma. The complexe Yki/Sd/Brahma binds to Yki target genes and promotes ISC proliferation [START_REF] Jin | Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling[END_REF]. Interestingly, in steady state conditions Hippo activates caspase proteins that induce programmed cell death. These caspases inactivate Brahma by inducing its proteolytic cleavage [START_REF] Jin | Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling[END_REF] (Figure 8).

In addition, Hippo signaling acts non-autonomously within EC cells in order to restrict ISC proliferation. In damaged ECs, Yki increases the expression of both Upd [START_REF] Shaw | The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration[END_REF]Staley and Irvine, 2010;[START_REF] Vodovar | Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species[END_REF] and Keren (Ren et al., 2010a). Subsequently, both JAK/STAT and EGFR signaling are activated within ISCs, inducing their proliferation.

Therefore, the Hippo signaling pathway in general -and Yki in particular-acts as a sensor of epithelia stress within ECs allowing them to signal to the ISC when damaged.

Regulation of ISC behavior in stress conditions

The intestine is an organ in continuous contact with the surrounding environment. Thus, in addition to the normal regeneration of the tissue, the gut has also to cope with external stresses like physical (lesions), chemical (oxidative stress) or infectious aggressions. To re-establish the integrity of the tissue and ensure a functional physiological state of the organ, ISCs are also actively solicited. A careful control of their proliferative potential is then indispensable and ensured by the same signaling pathways that are active in basal conditions. In the following section, I will describe some experimental approaches used to induce the fly midgut regeneration. In the second part, I will describe different signals that are communicated to ISC in stress conditions in order to activate mitogenic signaling pathways mainly EGFR, Wnt and JAK/STAT.

Experimental techniques used to study the midgut regeneration

In order to study ISC response to epithelial damage several approaches involving feeding flies different tissue-damage inducing agents have been used:

1. Oral infection of flies with different strains of pathogenic bacteria (Pseudomonas entomophila, Erwinia carotovora (Ecc15), Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli...) [START_REF] Apidianakis | Synergy between bacterial infection and genetic predisposition in intestinal dysplasia[END_REF]Buchon et al., 2009a;Buchon et al., 2009b;[START_REF] Cronin | Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection[END_REF][START_REF] Vodovar | Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species[END_REF].

2. Chemicals feeding with compounds like Hydrogen peroxide and Paraquat [START_REF] Choi | Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor[END_REF] that induce oxidative stress, dextran sodium sulfate (DSS) that disrupts the basement membrane, or Bleomycin, a cytotoxic antibiotic that causes damages to ECs [START_REF] Amcheslavsky | Tissue damage-induced intestinal stem cell division in Drosophila[END_REF].

3. The targeted expression of proapoptotic genes such as reaper (Rpr) within EC cells inducing their death (Jiang et al., 2011).

Signaling pathways in midgut regeneration upon damages

Differentiated ECs are the first target of bacterial infections and cytotoxic agents since these cells are the first to encounter the stressors. Indeed, pathogenic bacterial infection or Bleomycin treatment induce EC apoptosis and removal from the intestinal epithelium [START_REF] Amcheslavsky | Tissue damage-induced intestinal stem cell division in Drosophila[END_REF][START_REF] Apidianakis | Synergy between bacterial infection and genetic predisposition in intestinal dysplasia[END_REF][START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]Jiang et al., 2009). ISCs respond to EC loss by increasing their proliferative capacity in order to replenish the midgut. Interestingly, apoptosis inhibition within ECs abolishes ISC proliferation, whereas the apoptosis induction by the expression of Rpr in ECs is sufficient to induce ISC proliferation, even in the absence of infection (Jiang et al., 2011). Thus, EC apoptosis is a main signal for the activation of ISC proliferation upon gut damages.

At the molecular level, damaged ECs secrete the Upd3 ligand that activates JAK/STAT signaling within ISCs, therefore inducing their proliferation (Buchon et al., 2009a;[START_REF] Cronin | Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection[END_REF]Jiang and Edgar, 2009). Moreover, Upd3 released from damaged EC signals to the surrounding visceral muscles that transduce the JAK/STAT signaling and release Vein that also induces ISC proliferation [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]Jiang et al., 2011). The other EGFR ligand Keren is also released from damaged EC and further potentiates ISC proliferation [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]Jiang et al., 2011). Interestingly, EGFR signaling is also required for both damaged EC-detachment from the epithelium and their morphogenesis during regenerative episodes [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]. Indeed, EGFR inhibition within infected ECs blocks the disruption of cell-cell adhesion and their subsequent delamination from the epithelium, and abolishes ISC proliferation [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]. Recently, an additional function of EGFR within regenerating EC was also reported. In addition to ISC proliferation, endoreplication within EC provides a mechanism of infection-induced regeneration to compensate for lost cells. During regeneration, newly formed ECs inherit the activated EGFR signaling from EB, posttranscriptionally activate the translational factor E2F gene and undergo multiple rounds of endoreplication that lead to EC hypertrophy and higher ploidy [START_REF] Xiang | EGFR-dependent TORindependent endocycles support Drosophila gut epithelial regeneration[END_REF].

EBs also participate to the induction of ISC proliferation. Upon damage, EBs secrete the EGFR ligand Spitz and the JAK/STAT ligand Upd and activate these pathways within ISCs inducing their proliferation [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]Jiang et al., 2011;[START_REF] Tian | Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells[END_REF]. Similarly, following infection or DSS treatment, Wg is secreted by EBs and activates Wnt signaling within ISCs via the activation of Myc expression [START_REF] Cordero | Inducible progenitorderived Wingless regulates adult midgut regeneration in Drosophila[END_REF]. It has been shown that the RAL GTPase proteins (RAL) are expressed in ISC/EB and are required for membrane receptor endocytosis within ISCs to properly induce Wnt signaling. RALA acts downstream of the Wg ligand and upstream of β-catenin and its overexpression is sufficient to induce Wnt activation and ISC proliferation upon damages. Moreover, the same mechanism is conserved in murine intestine [START_REF] Johansson | RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes[END_REF].

Investigating the communication between signaling pathways, epistasis genetic analysis demonstrated that the Upd capacity to induce ISC proliferation upon infection is abolished in an EGFR mutant background. Thus, EGFR acts downstream of JAK/STAT in order to induce ISC proliferation upon acute damage [START_REF] Buchon | Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection[END_REF]Jiang et al., 2011;[START_REF] Xu | EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells[END_REF].

In the same line, Xu et al. showed that EGFR, Wnt and JAK/STAT act redundantly in the regulation of ISC proliferation in steady state condition and that simultaneous inhibition of the three signaling pathways induces ISC loss (Figure 10).

Despite the critical requirement for these signaling pathways in the regulation of ISC behavior, their activity is not exclusive to these cells. It is rather conserved in different type of organs among diverse species. Thus, it is important to look for intrinsic factors that specifically characterize adult stem cells. In the Drosophila adult midgut, escargot (esg) is expressed in ISC/EB and it is a hallmark of diploid cells including germline stem cells and cyst stem cells of the adult testis [START_REF] Kiger | Somatic support cells restrict germline stem cell self-renewal and promote differentiation[END_REF][START_REF] Voog | Escargot restricts niche cell to stem cell conversion in the Drosophila testis[END_REF] in addition to imaginal discs during development [START_REF] Fuse | Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot[END_REF].

The importance of EMT-inducing factors in ISCs

Clones lacking Esg or the zeb TF Zfh1 loose Dl-positive ISCs and they are solely composed of differentiated cells. It was shown that esg knockdown induces an upregulation of Pdm1 and Prospero, as well as other differentiation markers. Dam-ID profiling further showed that Esg represses the expression of differentiation genes by direct binding to their regulatory regions under basal conditions (Korzelius et al., 2014). In regenerating guts, when the production of new ECs is vital, Pdm1 represses the expression of esg in differentiating EBs thus ensuring a rapid turnover of lost cells (Korzelius et al., 2014;[START_REF] Tang | The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation[END_REF]. Within EBs, Esg is required at two different levels. First, it enables EBs to form long protrusions that establish contacts with differentiated cells. This mesenchymal-like phenotype allows EBs to sense environmental cues, pausing their differentiation until a signal is communicated from neighboring dying ECs (Antonello et al., 2015). Expression of miR-8, the fly homolog of the mammals miR200, represses the expression of esg and zfh1, the homologous gene of the EMT-TF Zeb, and induces final differentiation of EB into EC. On the other hand, esg plays a central role in the EC fate decision by directly inhibiting the expression of a negative regulator of Notch called Amun (Loza-Coll et al., 2014).

Therefore, esg plays a pivotal role in both maintenance of stemness and the cell fate choice between EEs and ECs (Figure 9).

Evolutionary conservation between mammals small intestine and Drosophila adult digestive tract

Although there is a physiological divergence between vertebrates and insects, there is a great potential for modelling human intestinal diseases in Drosophila because of the high level of preservation between the two models with regard to the cellular lineage and the signaling pathways that regulate both ISC proliferation and differentiation.

In the following section, I will compare the Drosophila adult midgut to its mammalian counterpart, the small intestine. I will describe the small intestine structural organization, the cellular lineage and plasticity as well as the signaling pathways implicated in the gut homeostasis. In the final part, I will discuss the effect of signaling pathways deregulation in both intestinal cancer initiation and progression.

Comparison between structural and cellular organization

In contrast to the simple linear epithelium that lines the Drosophila adult midgut, the mammalian small intestine is ordered in crypt of Lieberkühn, tubular epithelium invagination that harbors ISC and thus constitutes a proliferative zone and the intestinal villi that project into the intestinal lumen and represent the differentiated segment [START_REF] Schmidt | Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine[END_REF][START_REF] Wright | Epithelial stem cell repertoire in the gut: clues to the origin of cell lineages, proliferative units and cancer[END_REF]. Despite these structural differences absorptive EC and secretory cells accomplish the mammalian small intestine major functions. The secretory cell lineage is further divided into EE cells that produce systemic hormone, and two additional cell types not found in Drosophila:

Goblet cells that secrete mucin and facilitates the transition of compact stools into the colon, and Paneth cells, the only crypt-resident differentiated cells secret protective antimicrobials products and constitute an important niche for ISC [START_REF] Barker | The intestinal stem cell[END_REF] (Figure 11). As mentioned earlier, Drosophila EE cells are subdivided into four different classes (Beehler-Evans and [START_REF] Beehler-Evans | Generation of enteroendocrine cell diversity in midgut stem cell lineages[END_REF]. Further investigations are required to test whether these populations resemble Goblet or Paneth cells.

In mammals, ISCs divide once per day and generate progenitors known as transient amplifying cells (TA), owing to their capacity to divide four-five times before terminally differentiating into Enterocytes (EC) or secretory cells while leaving the transient amplifying compartment toward the villi [START_REF] Heath | Epithelial cell migration in the intestine[END_REF]. In Drosophila, EB are post mitotic and thus give rise to EC without further division while EE progenitors conserve a restricted mitotic capacity similarly to TA cells (Guo and Ohlstein, 2015).

At the structural level, Evidences about region specific identity within mammalian small intestine are also emerging. The expression of genes encoding for digestive enzymes, ion transporters, and antimicrobial peptides among others is highly regionalized [START_REF] Bradley | The emerging role of the intestine in metabolic diseases[END_REF][START_REF] Cash | Symbiotic bacteria direct expression of an intestinal bactericidal lectin[END_REF][START_REF] Rindi | The "normal" endocrine cell of the gut: changing concepts and new evidences[END_REF]. This regionalization is also governed by regionspecific TF such as GATA and CDX2 . Moreover, the region-specific expression profile is maintained for a long time in the absence of environmental stimuli.

Cellular plasticity

Within the mammals small intestine there is two types of ISC: the +4 cells that occupy the 4 th position above the crypt basis [START_REF] Cairnie | Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic parameters[END_REF] and the Crypt base columnar cells (CBC) located between Paneth cells and named after their shape and their position [START_REF] Cheng | Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types[END_REF]. +4 cells are quiescent and characterized by the expression of transcriptional repressor Bmi1, required for the maintenance of their undifferentiated state [START_REF] Tian | A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable[END_REF] whereas CBC cells divide rapidly and are characterized by the expression of the Leucine-rich G protein-coupled receptor 5 gene (Lgr5) encoding for R-spondin receptor that potentiates the Wnt signaling pathway [START_REF] Barker | Identification of stem cells in small intestine and colon by marker gene Lgr5[END_REF]. Recently the Bmi1+ were named "reserve stem cells" due to their capacity to replace lost CBC cells [START_REF] Tian | A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable[END_REF]. On the other hand, the organoids generated from a single CBC contain Bmi1+ cells. Moreover, some types of progenitor cells located at +4 positions are also able to dedifferentiate in order to produce CBC upon their ablation [START_REF] Takeda | Interconversion between intestinal stem cell populations in distinct niches[END_REF]. Thus, the intestinal epithelial homeostasis relies on the plasticity of active ISC: the CBC, reserve stem cells: Bmi1+ and committed progenitors.

In Drosophila, evidences about different ISC types were brought by the work of many laboratories. the work of Marianes and Spradling has demonstrated that the ISC of the posterior midgut are the most active whereas those of the anterior and the middle midgut are relatively quiescent. Moreover they have proved the presence of ISC with different morphology and division rate (Marianes and Spradling, 2013).

Concerning the cellular plasticity in the Drosophila adult midgut, a recent study have demonstrated that in a situation of extreme stress (food deprivation for 2 days) ISC/EB cells are completely lost in posterior midgut and the gut is shortened [START_REF] Lucchetta | Amitosis of Polyploid Cells Regenerates Functional Stem Cells in the Drosophila Intestine[END_REF].

Surprisingly, after refeeding flies, the gut length is restored to wild type in one day and the number of ISC increases significantly. By performing lineage tracing analysis it was demonstrated that 4n polyploid pre-EC are able to dedifferentiate and give rise to diploid ISC by a process called amitosis.

Conservation of signaling pathways

In the following paragraph, I will briefly describe the regulation of mammalian ISC proliferation by both Wnt and Notch pathway and their differentiation by BMP.

The Wnt signaling pathway in homeostasis

In the mammalian intestine, Wnt signaling is the main mitogenic pathway. It forms a decreasing gradient from the crypt toward the villus, with the highest activity located at the basis of the crypt [START_REF] Batlle | Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB[END_REF][START_REF] Munoz | The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers[END_REF]. During homeostasis, Wnt is required for the maintenance of CBC, as well as for Paneth cell terminal differentiation. Wnt inactivation [START_REF] Van Es | A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal[END_REF] inhibits CBC proliferation in both normal and pathogenic conditions and thus leads to crypt loss [START_REF] Kuhnert | Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1[END_REF][START_REF] Pinto | Canonical Wnt signals are essential for homeostasis of the intestinal epithelium[END_REF]. Both Paneth cells and the surrounding stroma secrete Wnt ligands [START_REF] Sato | Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[END_REF][START_REF] Valenta | Wnt Ligands Secreted by Subepithelial Mesenchymal Cells Are Essential for the Survival of Intestinal Stem Cells and Gut Homeostasis[END_REF]. In Drosophila, both surrounding muscles and EB cells provide the Wnt ligand. Thus, the niche aspect of Wnt ligand as well as the function of Wnt signaling in ISC proliferation are both conserved in the Drosophila adult midgut.

The Notch signaling pathway

In contrast to its role in limiting ISC proliferation in Drosophila, Notch is a main mitogenic pathway in mammals. The inhibition of Notch signaling induces a complete loss of CBC and TA, associated with a large increase in the number of secretory cells [START_REF] Milano | Modulation of notch processing by gamma-secretase inhibitors causes intestinal goblet cell metaplasia and induction of genes known to specify gut secretory lineage differentiation[END_REF][START_REF] Van Es | Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells[END_REF]. In contrast, overexpression of NICD is sufficient to induce the expansion of the proliferative compartment and the loss of the secretory lineage [START_REF] Fre | Notch signals control the fate of immature progenitor cells in the intestine[END_REF].Hence, Notch signaling governs both ISC proliferation and EC cell fate choice in the mammal's intestinal crypt.

Interestingly, as mentioned earlier Notch signaling pathway is required for the production of EC in Drosophila adult midgut and the inhibition of Notch signaling in the midgut induces secretory cells accumulation (Ohlstein and Spradling, 2007). Thus, some aspects of Notch signaling are conserved between Drosophila and mammals intestine.

The Bone Morphogenetic Protein (BMP) signaling pathway

BMP proteins are growth factors, belonging to the TGF-β family [START_REF] Feng | Specificity and versatility in tgf-beta signaling through Smads[END_REF][START_REF] Massague | TGFbeta signalling in context[END_REF][START_REF] Miyazono | BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk[END_REF][START_REF] Moustakas | The regulation of TGFbeta signal transduction[END_REF].

Profile analysis of BMP signaling components in mammals small intestine revealed that the BMP ligand expression is enriched in the mesenchymal region surrounding villi [START_REF] Bitgood | Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo[END_REF][START_REF] Haramis | De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine[END_REF][START_REF] Hardwick | Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon[END_REF]. The inhibition of BMP signaling in mice intestine induces and ectopic formation of crypts in the villi region and ISC over proliferation, induced by Wnt signaling upregulation. Moreover, mice defective for Bmp receptor have altered intestinal morphology, in addition to a defective secretory lineage terminal differentiation [START_REF] Auclair | Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage[END_REF]. Thus, in contrast to Notch signaling, BMP signaling limits ISC hyper proliferation and is required for secretory cell lineage differentiation.

In Drosophila adult intestine, the function of BMP signaling is so controversial: An initial study by Tian et al in 2013 has demonstrated that BMP signaling is required for ISC self-renewal in both normal and stress conditions, by antagonizing Notch signaling pathway in basally locate ISC [START_REF] Tian | Intestinal epithelium-derived BMP controls stem cell self-renewal in Drosophila adult midgut[END_REF]. Later on, it was demonstrated that damaged EC cells secrete Bmp ligands that activates ISC pool expansion by favoring ISC symmetric division [START_REF] Tian | Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration[END_REF]. After recovery, BMP signaling is auto inhibited in EC cells in order to repress ligand secretion and to restore ISC proliferation to basal levels [START_REF] Tian | Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration[END_REF]. During regeneration, BMP activation within ISC cells limits their proliferation, providing an effective equilibrium between signals and an efficient response to damages [START_REF] Guo | Injury-induced BMP signaling negatively regulates Drosophila midgut homeostasis[END_REF]. The finding of both Guo et al and tian (after recovery) are in line with the function of BMP as growth inhibitory factor in mammalian intestine. An additional role of BMP in cell differentiation is also observed in the middle midgut where it is required for the specification of proton secreting cells called copper cells [START_REF] Guo | Injury-induced BMP signaling negatively regulates Drosophila midgut homeostasis[END_REF][START_REF] Li | Trachea-derived dpp controls adult midgut homeostasis in Drosophila[END_REF] as well as in the midgut for the differentiation and growth of EC cells [START_REF] Tian | Intestinal epithelium-derived BMP controls stem cell self-renewal in Drosophila adult midgut[END_REF][START_REF] Zhou | Dpp/Gbb signaling is required for normal intestinal regeneration during infection[END_REF].

The deregulation of ISC function: intestinal cancer initiation and progression

Given the great conservation between mammals and Drosophila adult midgut organizations, functions and regulatory mechanisms, in addition to the 70% similarity in disease genes, the Drosophila intestine has also been used to model colorectal cancers.

In both mammal's small intestine and Drosophila midgut, the main event leading to cancer initiation is ISC hyper proliferation, induced either by over activation of mitogenic signaling pathways or by inhibition of differentiation pathways [START_REF] Apidianakis | Synergy between bacterial infection and genetic predisposition in intestinal dysplasia[END_REF]Ohlstein and Spradling, 2007;[START_REF] Sansom | Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration[END_REF]. In human, Wnt constitutive activation by APC loss of function is the leading cause of colorectal cancers [START_REF] Sansom | Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration[END_REF]. In the Drosophila midgut, the Apc1 mutation mimics the tumor development observed in mammals [START_REF] Cordero | APC as a master regulator of intestinal homeostasis and transformation: from flies to vertebrates[END_REF][START_REF] Lee | Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation[END_REF]. In both cases, the proto-oncogene Myc (a downstream effector of Wg signaling) is upregulated and required for tumor maintenance [START_REF] Athineos | Myc heterozygosity attenuates the phenotypes of APC deficiency in the small intestine[END_REF][START_REF] Cordero | APC as a master regulator of intestinal homeostasis and transformation: from flies to vertebrates[END_REF][START_REF] Finch | Acute overexpression of Myc in intestinal epithelium recapitulates some but not all the changes elicited by Wnt/beta-catenin pathway activation[END_REF][START_REF] Lee | Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation[END_REF][START_REF] Sansom | Myc deletion rescues Apc deficiency in the small intestine[END_REF]. Following initiation by APC LOF and Myc upregulation, tumor progression from adenoma to invasive carcinoma in both mammals and Drosophila requires the mutation of the proto-oncogene K-RAS, encoding a small GTPase that transduces external signals to the nucleus and mutated in 40 to 50% of colorectal cancers [START_REF] Sansom | Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo[END_REF][START_REF] Wang | APC loss-induced intestinal tumorigenesis in Drosophila: Roles of Ras in Wnt signaling activation and tumor progression[END_REF] (Figure 12).

In the fly adult midgut, tumor initiation is accelerated by pathogenic bacterial infections.

Indeed, dying EC cells secrete mitogenic signaling molecules that potentiate ISC proliferation.

As it is known for tumor cells, proliferating ISC also secrete autocrine growth factors (Figure 14).

As discussed is this paragraph, an excess ISC proliferation can lead to tumor development, whereas a week ISC activity can lead to the gut degeneration. Thus, it is tempting to speculate the intrinsic factors that act downstream of the complex regulatory pathways in basal physiological conditions in order to maintain the pool of ISCs and to regulate their proliferation rate. In the following part, I will focus on the recent findings on the TF called Shavenbaby, the equivalent of the mammalian OvoL in Drosophila. In the following section, I will summarize current knowledge on svb/ovo, emphasizing its functions in Drosophila and I will discuss the functions of ovo/svb homolog in various species.

I will focus in more detail on its mode of action and the signaling pathways that regulate its expression in embryos. I will also present findings of François' lab concerning the mechanisms that provide a strict control to Svb transcriptional activity.

The ovo/shavenbaby locus

Shavenbaby (Svb) is a zinc finger transcription factor [START_REF] Mevel-Ninio | The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development[END_REF] encoded by the ovo/shavenbaby locus located on the X chromosome [START_REF] Busson | Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER[END_REF][START_REF] Oliver | Function of Drosophila ovo+ in germ-line sex determination depends on X-chromosome number[END_REF]. The locus encodes three main protein isoforms, from alternative promoters. There are two germline-specific proteins OvoA and OvoB, and Shavenbaby is the protein expressed in somatic tissues (Mevel-Ninio et al., 1995). All three isoforms share the same DNA binding and transcriptional activator domains. Whereas

OvoB is a transcriptional activator, OvoA possess an additional repression domain and thus acts as a transcriptional repressor (Andrews et al., 2000;Mevel-Ninio et al., 1995). Of note, the first exon of ovoB mRNA is non-coding, and the translation starts only at a very downstream AUG located in exon 2a, despite an open reading frame covering the whole exonic sequence.

Svb entirely overlaps OvoA and possesses an additional N terminal region, encoded by a somatic-specific exon called 1s (Delon et al., 2003) (Figure 13).

Ovo functions in the germline

The first ovo mutation was identified in Drosophila, in a screen of recessive X-linked mutations inducing female sterility [START_REF] Mohler | Developmental genetics of the Drosophila egg. I. Identification of 59 sex-linked cistrons with maternal effects on embryonic development[END_REF]. Later on, somatic genetic screens have identified mutations at the same locus, called shavenbaby, since mutant embryos are devoid of epidermal trichomes [START_REF] Wieschaus | Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : III. Zygotic loci on the X-chromosome and fourth chromosome[END_REF]. Mutations affect either ovo function or svb function, or both, suggesting that both functions are encoded by a common locus [START_REF] Oliver | The ovo locus is required for sex-specific germ line maintenance in Drosophila[END_REF]. The ovo/svb locus is therefore required during development for two distinct functions, one somatic (svb) in both sexes and the other in the female germline (ovo) (Mevel-Ninio et al., 1995).

Ovo proteins are implicated in the differentiation, survival, and sex determination of the female germ line [START_REF] Garfinkel | Multiple products from the shavenbaby-ovo gene region of Drosophila melanogaster: relationship to genetic complexity[END_REF][START_REF] Mevel-Ninio | The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development[END_REF][START_REF] Mével-Ninio | ovo, a Drosophila gene required for ovarian development, is specifically expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning[END_REF][START_REF] Oliver | The ovo locus is required for sex-specific germ line maintenance in Drosophila[END_REF]. A large number of ovo mutations have been identified and induce progressive defects, from defective oocyte maturation, oogenesis interruption, tumor formation and a complete loss of germ cells. Ovo proteins regulates the expression of ovarian tumor (otu), a gene required for ovarian differentiation and also leading to tumors [START_REF] Gans | Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster[END_REF][START_REF] King | Hereditary ovarian tumors in Drosophila melanogaster[END_REF][START_REF] King | The role of the otu gene in Drosophila oogenesis[END_REF]Oliver et al., 1993;[START_REF] Oliver | Suppression of distinct ovo phenotypes in the Drosophila female germline by maleless-and Sex-lethal[END_REF][START_REF] Oliver | The ovo locus is required for sex-specific germ line maintenance in Drosophila[END_REF][START_REF] Pauli | The role of the ovarian tumor locus in Drosophila melanogaster germ line sex determination[END_REF] as well as that of ovo itself, through direct binding to their proximal promoter region [START_REF] Lu | Drosophila OVO zinc-finger protein regulates ovo and ovarian tumor target promoters[END_REF][START_REF] Lu | Drosophila OVO regulates ovarian tumor transcription by binding unusually near the transcription start site[END_REF].

The two germinal isoforms, OvoA and OvoB are differentially expressed in the course of ovarian development. The ovoB promoter is active throughout oogenesis, whereas the ovoA promoter displays barely detectable activity until the very end of oogenesis (Andrews et al., 2000). Dominant sterile mutations have been identified (Ovo D ) and show gradual phenotypes that are reminiscent of the progressive loss of ovo function [START_REF] Busson | Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER[END_REF][START_REF] Campos-Ortega | Topological specificity of phenotype expression of neurogenic mutations inDrosophila[END_REF][START_REF] Kerridge | Lethal bithorax complex mutations of Drosophila melanogaster show no germ line maternal effects[END_REF][START_REF] Oliver | The ovo locus is required for sex-specific germ line maintenance in Drosophila[END_REF][START_REF] Perrimon | X-linked female-sterile loci in Drosophila melanogaster[END_REF]. Molecular analysis of ovo D mutations revealed that they result from the creation of a new AUG codon upstream of the normal translation start site of the OvoB protein. OvoD mutations thus lead to the premature production of OvoA, which antagonizes OvoB for the control of germ line (Andrews et al., 1998;[START_REF] Mevel-Ninio | The three dominant female-sterile mutations of the Drosophila ovo gene are point mutations that create new translation-initiator AUG codons[END_REF]. Thus, the fine regulation of ovoA and ovoB expression is critical for the formation and differentiation of the germline.

At the beginning of embryogenesis, OvoB mRNA is uniformly distributed in the syncytial blastoderm (Mevel-Ninio et al., 1995) then it accumulates within polar cells, also known as primordial germ cells (PGC,), which give rise to germline cells (Hayashi et al., 2017). Within PGCs, OvoB activates the expression of genes required for germline specification, including vasa, nanos and piwi. The knockdown of OvoB or the expression of OvoA specifically within PGC leads to the formation of agametic ovaries and testes in adults. A similar function is also observed in mice, with OvoL2 being expressed in PGCs and required for their development (Hayashi et al., 2017). Moreover, OvoL1 is also expressed later in adult mice testes, within spermatocytes and spermatids and is required for proper germline maturation [START_REF] Li | Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression[END_REF].

ovo1 mutants were previously shown to display hypogenitalism and have reduced ability to reproduce (Dai et al., 1998).

Hence, ovo is an important regulator of the germ line in a broad range of animals.

Svb is required and sufficient for trichome formation

Drosophila embryos are characterized by the alternance of epidermal cells with a smooth surface and cells that form apical extensions called trichomes (Figure 14). At the end of embryogenesis, epidermal cells secrete a complex exoskeleton called cuticle, which displays a stereotyped pattern of trichomes providing an exquisite readout of embryonic development, with specific trichomes organization along the anterior-posterior and dorsal-ventral axes (Payre, 2004). svb mutants have been isolated from the genetic screen performed by Nusslein and Wieschaus [START_REF] Nusslein-Volhard | Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : I. Zygotic loci on the second chromosome[END_REF][START_REF] Wieschaus | Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : III. Zygotic loci on the X-chromosome and fourth chromosome[END_REF], since they display an almost complete lack of trichomes (Figure 16). Consistently, svb is specifically expressed in trichome cells (Mevel-Ninio et al., 1995;Payre et al., 1999) and its ectopic expression, in smooth cells, is sufficient to force the formation of extra trichomes (Payre et al., 1999).

Ovo/svb functions across animal species

ovo/svb is specific to metazoans and well conserved throughout animals [START_REF] Kumar | Molecular phylogeny of OVOL genes illustrates a conserved C2H2 zinc finger domain coupled by hypervariable unstructured regions[END_REF].

In mammals, there are three genes called OvoL-1, -2 and -3 initially described for their conserved role in epidermal differentiation and germline development (Dai et al., 1998;Hayashi et al., 2017). In addition to these two tissues, studies in various species have disclosed a wide range of additional functions for ovo/svb orthologs.

For example, in Caenorhabditis elegans worms lin-48 is also required for the development of the hindgut, the male tail and the excretory duct cell [START_REF] Johnson | EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development[END_REF]. OvoL2 is an important regulator of kidney formation and homeostasis in mice [START_REF] Aue | A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion[END_REF]Dai et al., 1998). The murine renal collecting duct is lined by specialized epithelial cells, characterized by the expression of Grainyhead-Like homolog 2 (Grhl2). GrhL2 is a transcription factor that associates to the Histone 3 lysine 4 trimethylation (H3K4me3), a marker of transcriptionally active chromatin. OvoL2 is a direct target of Grhl2 in the renal collecting duct, and its reexpression is sufficient to compensate for the lack of Grhl2, otherwise leading to collapsed lumen and affected epithelial barrier [START_REF] Aue | A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion[END_REF]. A growing body of evidence implicates OvoL factors in the control of progenitors (Kitazawa et al., 2016;Li and Yang, 2014) and stem cells [START_REF] Kim | OVOL2 is a critical regulator of ER71/ETV2 in generating FLK1+, hematopoietic, and endothelial cells from embryonic stem cells[END_REF], in many cases through counteracting pro-EMT factors. Pioneering work from the Dai's lab has shown that OvoL1 and OvoL2 play key roles in the control of epidermal progenitors. Inactivation of OvoL1 and 2 blocks the terminal differentiation and promotes the expansion of embryonic epidermal progenitor cells [START_REF] Lee | Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells[END_REF][START_REF] Wells | Ovol2 suppresses cell cycling and terminal differentiation of keratinocytes by directly repressing c-Myc and Notch1[END_REF]. Conversely, overexpression of Ovol2 reduces the population of progenitors and leads to precocious differentiation (Lee et al., 2017).

Notably, Ovol1/2 deficient cells loose their epithelial characteristic and undergo EMT-like transformation. Further studies, showed that OvoL1/2 repress the activity of EMT-inducing factors, including Zeb1 [START_REF] Lee | Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells[END_REF]. A similar behavior has also been observed during mammary development, where OvoL2 is required for proper morphogenesis and regeneration (Watanabe et al., 2014). In mouse adult skin, OvoL2 function is required during both wound healing and hair regeneration. In this context, OvoL2 restricts the migration speed, optimizes the directionality of migrating cells and maintains their proliferative capacity, thus ensuring an efficient tissue repair (Haensel et al., 2019).

Moreover, OvoL2 is expressed in human corneal epithelial cells where it is required for their maintenance (Kitazawa et al., 2016) and Ovol2 mutations are the most frequent cause of corneal dystrophy (Davidson et al., 2016). In the planarian flatworm, ovo is also required for eye regeneration from pluripotent stem cells [START_REF] Cross | Control of Maintenance and Regeneration of Planarian Eyes by ovo[END_REF]Lapan and Reddien, 2012). Several studies have disclosed functions of Ovo-like factors in angiogenesis, heart formation and placental development in mammals [START_REF] Bai | Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium[END_REF][START_REF] Kusama | Regulation of human trophoblast cell syncytialization by transcription factors STAT5B and NR4A3[END_REF][START_REF] Renaud | OVOlike 1 regulates progenitor cell fate in human trophoblast development[END_REF][START_REF] Unezaki | Ovol2/Movo, a homologue of Drosophila ovo, is required for angiogenesis, heart formation and placental development in mice[END_REF]. It has been shown that OvoL factors regulate the fate of progenitor cells for trophoblast development, which normally involves differentiation into a syncytium that mediates exchanges between maternal and fetal blood [START_REF] Renaud | OVOlike 1 regulates progenitor cell fate in human trophoblast development[END_REF]. OvoL downregulation is essential to trophoblast attachment to the endometrial epithelium, probably to increase the expression of EMT-associated transcription factors Zeb1 and Snail. It has also been shown that OvoL2 interacts with the nuclear factor ER71 to promote the formation of hematopoietic and endothelial cells from embryonic stem cells [START_REF] Kim | OVOL2 is a critical regulator of ER71/ETV2 in generating FLK1+, hematopoietic, and endothelial cells from embryonic stem cells[END_REF]. Finally, both OvoL1 and OvoL2 were recently identified as regulators of induced pluripotent stem cells (iPSCs) reprogramming towards epithelial lineages (Kagawa et al., 2019;Watanabe et al., 2019).

Unbiased profiling of human tumors have shown a broad implication of OvoL factors in the metastatic potential, and thus morbidity, of prevalent cancers. This includes colorectal (Ye et al., 2016), renal [START_REF] Ricketts | Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma[END_REF], breast [START_REF] Hong | An Ovol2-Zeb1 Mutual Inhibitory Circuit Governs Bidirectional and Multi-step Transition between Epithelial and Mesenchymal States[END_REF]Roca et al., 2013;Watanabe et al., 2014), prostate (Roca et al., 2013;[START_REF] Roca | A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial -mesenchymal cell reprogramming and cancer progression[END_REF], lung cancers (Wang et al., 2017), as well as nasopharyngeal carcinoma and osteosarcoma [START_REF] Liu | Ovol2 induces mesenchymal-epithelial transition via targeting ZEB1 in osteosarcoma[END_REF]. Tumor cells with reduced OvoL levels, display increased expression of EMT-inducing factors such as miR-200

and Zeb [START_REF] Gregory | The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[END_REF] favoring the invasion of healthy tissues where they seed secondary tumors (Roca et al., 2013;[START_REF] Roca | A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial -mesenchymal cell reprogramming and cancer progression[END_REF] [START_REF] Hong | An Ovol2-Zeb1 Mutual Inhibitory Circuit Governs Bidirectional and Multi-step Transition between Epithelial and Mesenchymal States[END_REF][START_REF] Jia | OVOL guides the epithelial-hybrid-mesenchymal transition[END_REF].

Recent data support that the most aggressive tumor cells with high metastatic capacity are those with a dual E/M potential. Both OvoL and GrhL2 have been identified as phenotypic stability factors that stabilize the hybrid state [START_REF] Biswas | Stability and mean residence times for hybrid epithelial/mesenchymal phenotype[END_REF].

To conclude, OvoL/Svb transcription factors have been strongly conserved during evolution and are required for the regulation of various epithelial functions, including the regulation of somatic and germline precursor cells from Drosophila to mammals.

The regulation of shavenbaby developmental expression

svb expression in embryonic epidermal cells is controlled by multiple signaling pathways including Wnt, EGFR, Notch and Hedgehog (Payre, 2004) (Figure 15). In the ventral epidermis, svb integrates both EGFR and Wnt signaling to determine the subset of cells that will form trichomes. EGFR activates svb expression in trichome cells, whereas Wnt/Wingless (Wg) inhibits its expression to generate smooth cells (Payre et al., 1999).

Combined efforts between our lab and that of David Stern have shown that the cis-regulatory landscape of svb spans over 100kb upstream the svb promoter and comprises at least seven enhancers (see [START_REF] Stern | The structure and evolution of cis-regulatory regions: the shavenbaby story[END_REF] for review) (Figure 16). Three large cis-regulatory regions driving svb epidermal expression were initially identified and named proximal, medial and distal elements (McGregor et al., 2007). Further work delineated smaller enhancers called DG2, DG3, Z, A, E, and 7 driving expression in largely overlapping populations of trichome cells and, together, they recapitulate the complete pattern of svb expression in the embryonic epidermis (Frankel et al., 2010;Frankel et al., 2011). The E element was further dissected into two smaller enhancers, E3 and E6, driving expression in ventral and dorsal cells respectively (Frankel et al., 2010;Frankel et al., 2011). Alexandre Ahmad Al Sawadi, a former member of our lab, has next restricted the E3 292 bp region named E3-N [START_REF] Crocker | Low affinity binding site clusters confer hox specificity and regulatory robustness[END_REF]. In a same vein, he delineated the shortest version of the 7 (proximal) enhancer to a small region of 247 bp. This pretty exhaustive functional characterization of svb enhancers reveals the prevalence of apparently redundant enhancers driving similar pattern of expression. Similar cis-regulatory organization was also noticed for key developmental regulators [START_REF] Hong | Shadow enhancers as a source of evolutionary novelty[END_REF], its functional role remained, however, to be investigated. svb has provided an exquisite model to address the function of such apparently redundant, or shadow, enhancers. Genomic engineering allowed the generation of a small deletion that removes three remote enhancers and the fine readout of trichome implantation revealed no defects on the trichome pattern in normal conditions (Frankel et al., 2010). However, when embryos develop under non-optimal conditions, such as lower or higher temperature, or with slightly comprised genetic backgrounds, the lack of these enhancers leads to partial trichome loss. Therefore, the existence of apparently redundant enhancers confers robustness to developmental program in the face of challenging internal (genetic) or environmental conditions (Frankel et al., 2010;[START_REF] Frankel | Conserved regulatory architecture underlies parallel genetic changes and convergent phenotypic evolution[END_REF].

Following the identification of svb enhancers, Ahmad has searched for transcription factors that may directly regulate svb expression. He used an RNAi approach to screen more than 250 TF expressed in the embryo at the time of epidermis differentiation (Menoret et al., 2013) and he tested the effect of their depletion on the activity of the smallest versions of E3 (E3-14) and 7

(9CJ2) enhancers. This was complemented by "one-hybrid assay" that enables automated identification of TFs that bind a given regulatory regions when introduced in yeat. These two Therefore, svb embryonic expression is controlled by multiple signaling pathways that directly regulate the activity of svb enhancers.

Svb governs the expression of trichome effector genes

In order to identify effector genes directly responsible for the remodeling of epidermal cells that underlies trichome formation, the lab has undertaken the identification of Svb target genes.

Candidate approaches succeeded in the identification of a dozen of Svb direct targets and in the elucidation of their individual function for trichome formation (Chanut-Delalande et al., 2006;[START_REF] Fernandes | Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes[END_REF]. Genome-wide approaches combined mRNA profiling and in vivo ChIPseq of Svb bound regions in epidermal cells provided a broader picture (Menoret et al., 2013).

Together, these data demonstrated that Svb directly activates the expression of approx. 150 genes, encoding various regulators of epithelial cell organization (actin remodeling, extracellular matrix, junctional/membrane complexes) and cuticle composition (Menoret et al., 2013). To better understand the mechanism by which Svb regulates the expression of its target genes, enhancer reporter assays were realized in the embryos. Svb downstream enhancers contained one or more Svb binding sites (ACaGTTAgag) and individual mutation of these sites compromised the activity of target gene enhancers.

These data demonstrated that Svb directly regulates the expression of a whole set of effector genes that are collectively responsible for the formation of trichomes (Figure 17).

Pri temporally regulates Svb transcriptional activity

pri has been initially identified as a long non-coding RNA (lncRNA) [START_REF] Tupy | Identification of putative noncoding polyadenylated transcripts in Drosophila melanogaster[END_REF]. One main result from unbiased profiling of cellular RNAs has been the unexpected prevalence of uncharacterized transcripts. Although they often share similarities with mRNAs (spliced, polyadenylated, etc..) these transcripts were called lncRNAs, since they do not present classical open reading frames, besides small ORF (smORFs) that were expected to occur just randomly.

However, recent evidence, shows that smORF can encode bioactive peptides, from viruses to humans [START_REF] Albuquerque | small ORFs: A new class of essential genes for development[END_REF][START_REF] Couso | Finding smORFs: getting closer[END_REF]Couso and Patraquim, 2017;[START_REF] Hashimoto | Lilliputians get into the limelight: novel class of small peptide genes in morphogenesis[END_REF][START_REF] Kastenmayer | Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae[END_REF]Payre and Desplan, 2016;Plaza et al., 2017;Zanet et al., 2015). The pri gene produces a 1.5kb poly-adenylated RNA with four smORFs and compelling evidence have shown that they encode four functionally redundant peptides of 11 to 32 amino acids (Galindo et al., 2007;[START_REF] Kondo | Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA[END_REF][START_REF] Savard | A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides[END_REF]. Pri is dynamically expressed during Drosophila embryogenesis, including in the epidermis (Galindo et al., 2007;[START_REF] Kondo | Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA[END_REF]. Similar to svb depletion, pri loss of function leads to the absence of trichomes [START_REF] Kondo | Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA[END_REF]. In collaboration with Yugi Kageyama's group, the lab thus tested possible relationships between these two genes. While pri loss of function does not affect the expression of svb (and vice versa), it suppresses the expression of svb target genes suggesting that Pri regulates the transcriptional activity of Svb rather than its expression (Kondo et al., 2010).

Unexpectedly, the lab showed that Pri peptides induce the elimination of the N-terminal region of Svb protein, which contains exon 1s and a large part of the repressive domain. This posttranslational modification thus leads to the conversion of Svb from a long repressive form (SvbREP) to a short transcriptional activating form (SvbACT) (Kondo et al., 2010;Zanet et al., 2016).

To elucidate the molecular mode of action of Pri peptides, Emilie Benrabah performed a genome-wide functional RNAi screen in cultured cells, to identify the cellular factors required for Svb maturation. Emilie Benrabah and Jennifer Zanet demonstrated that the conversion of SvbREP into SvbACT results from a limited proteasome degradation (Zanet et al., 2015). Pri peptides bind to Ubr3, an E3 ubiquitin ligase, to allow its association with Svb via three conserved lysine residues in the Svb N-terminus. The recognition of Svb by Ubr3 allows its ubiquitination and targeting to the proteasome, which ensures degradation of the N-terminal part of Svb containing the repressive domain (Zanet et al., 2015). This degradation is specific to the somatic form Svb and the couple Pri/Ubr3 does not affect OvoA and OvoB which do not contain the N-terminal region bound by Ubr3 (Kondo et al., 2010) (Figure 20).

By regulating Ubr3 binding specificity, Pri peptides induce partial proteasome degradation of Svb and thus its conversion from a long repressive form to a short transcriptional activator.

Figure 18 : Pri peptides induce proteasome-mediated processing of the Svb protein In theIn the absence of pri expression, Svb is translated as a long transcriptional repressor. Following the production and binding of Pri peptides on Ubr3, it undergoes a conformational change allowing it to interact with Svb via its N-terminal part. Ubr3 recruits the E2 ligase Ubcd6 which ubiquitinates Svb thus ensuring its targeting to the proteasome where its Nterminal part is degraded. Svb is then released and acts as a transcriptional activator to trigger the expression of trichome effector target genes (Zanet et al., 2015).

The ecdysone hormone times epidermal differentiation by inducing pri expression

The deciphering of Svb activation highlights a very sophisticated mechanism (Zanet et al., 2015) and one may ask what it the rationale for this multistep activation process. An explanation has come from deciphering the regulation of pri expression, showing that a main role of Pri peptides is to provide a temporal regulation of Svb activity.

In insects, the steroid hormone ecdysone controls major developmental transitions, namely larval molting and pupal metamorphosis [START_REF] Yamanaka | Ecdysone control of developmental transitions: lessons from Drosophila research[END_REF]. The production of ecdysone is regulated by various internal and external cues, such as tissue growth [START_REF] Colombani | Antagonistic actions of ecdysone and insulins determine final size in Drosophila[END_REF][START_REF] Delanoue | The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells[END_REF], nutrition [START_REF] Andersen | Coordination of organ growth: principles and outstanding questions from the world of insects[END_REF] or light [START_REF] Yamanaka | Ecdysone control of developmental transitions: lessons from Drosophila research[END_REF]Yamanaka et al.). In larvae, ecdysone is produced by the prothoracic gland from food cholesterol that, following its uptake, it undergoes a cascade of modifications by metabolic enzymes. Inactivation of any of these enzymes leads to similar defects, with very thin undifferentiated cuticle, a phenotype called "Halloween" [START_REF] Niwa | Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond[END_REF]. Once synthesized, ecdysone is secreted into the hemolymph and target tissues locally transformed it into 20-hydroxy-ecdysone (20E), the active form of ecdysone. 20E binds to a heterodimeric nuclear receptor EcR/Usp (ecdysone Receptor and Ultraspiracle) to activate the expression of a temporal cascade of many genes [START_REF] Hill | Ecdysone receptors: from the Ashburner model to structural biology[END_REF].

To identify additional major regulators of trichome development, Hélène Chanut performed an unbiased genetic screen using overlapping small deficiencies that, together, cover the whole second chromosome (Chanut-Delalande et al., 2014;[START_REF] Dubois | Genetic dissection of the Transcription Factor code controlling serial specification of muscle identities in Drosophila[END_REF]. The screen identified a novel uncharacterized gene, which displayed a striking trichome-less phenotype. This gene encodes Gst-E14 and further characterization demonstrated that this protein is required for In conclusion, ecdysone provides a systemic control that temporally turns ON, via the activation of pri expression, the genetic program governed by Svb and allowing the formation of trichomes (Figure 18). T he family of OvoL/Ovo/Shavenbaby (Svb) transcription factors has been strongly conserved across evolution and is characteristic of animal species. Initially discovered in flies for a dual function in the development of the germline and of epidermal derivatives (Ovo/Svb) 1,2 , mammalian orthologs (OvoL1-3) have soon been identified 3,4 . OvoL/svb genes produce several protein isoforms and the existence of three partially redundant paralogs in mammals complicates their genetic analysis. There is a single gene in Drosophila, which expresses germline-(ovo) and somatic-specific (svb) transcripts from different promoters. Previous work has well-established the role of Svb in the development of embryonic epidermal tissues 2 , where it triggers a tridimensional cell shape remodeling for the formation of actin-rich apical extensions, called trichomes. Svb expression is driven by a large array of cis-regulatory regions, which have become a paradigm for elucidating the function 5,6 and evolution 7-9 of developmental enhancers. Svb enhancers directly integrate multiple inputs from upstream regulatory pathways 2,5,10 and often drive similar patterns 5,6,11 , together conferring robustness to epidermal development in the face of varying environmental conditions and/or genetic backgrounds 5,6 . During embryogenesis, the Svb transcription factor directly activates a battery of >150 target genes 12-14 collectively responsible for actin and extra-cellular-matrix reorganization that underlies trichome formation. Recent studies have unraveled a tight control of the Svb protein activity in response to Polished rice peptides (Pri, also known as Tarsal-less), which belongs to a fast-growing family of peptides encoded from small open reading frames (smORF) hidden within apparently long noncoding RNAs 15,16 . Svb is first translated as a long-sized protein that acts as a repressor (Svb REP ) 17 . Pri smORF peptides then induce a proteolytic processing of Svb REP leading to the degradation of its N-terminal region and releasing a shorter activator form, Svb [ACT 17,18 . Further work has demonstrated that pri expression is directly regulated by periodic pulses of steroid hormones 19 , allowing a functional connection between hard-wired genetic regulatory networks (svb expression) and systemic hormonal control (pri expression) for a proper spatio-temporal control of epidermal cell morphogenesis 15 .

Recent studies suggest that OvoL/Svb factors display broader functions throughout epithelial tissues in both normal and various pathological situations. Molecular profiling of human tumors has revealed that OvoL deregulation is a feature of many carcinomas, directly linked to the metastatic potential of morbid cancers 20-23 , including kidney 24 . OvoL factors have been proposed 25,26 to counteract a conserved core of regulators composed of Snail/Slug and Zeb1-2 transcription factors, as well as the micro RNA mir200, well known to promote epithelial-mesenchymal transition (EMT) 27 . The activity of OvoL might help stabilizing a hybrid phenotype between epithelial and mesenchymal states 25 , providing many advantages for both tumors and normal stem cells 28 . Indeed, recent data show that, like adult somatic stem cells, the most aggressive tumors often display a hybrid E/M phenotype 27 , and the expression of specific OvoL isoforms can annihilate the metastatic potential of mammary tumors 20,29 . In addition, OvoL/Svb factors have been linked to the control of various progenitors/stem cells, from basal invertebrates 30 to humans 31-33 . Therefore, whereas a large body of evidence supports a key role for OvoL/Svb in the behavior of somatic stem cells, a functional investigation of their mode of action in vivo remains to be undertaken.

Here, we built on the knowledge and tools accumulated for the study of Svb function in flies to investigate its putative contribution to the behavior of somatic stem cells in the adult. We show that in Malpighian tubules, which ensure renal function in insects 34,35 , svb is specifically expressed in the adult renal/nephric stem cells (RNSCs). We further find that the main function of Svb in the kidney is to protect RNSCs from apoptosis by controlling the expression of the inhibitor of apoptosis, DIAP1, in interaction with Yorkie (Yki), a nuclear effector of the Hippo pathway.

Results

svb is expressed in RNSCs and controls their maintenance. To assay whether svb might be expressed in the adult, we tested large genomic reporter constructs that cover each of the seven enhancers contributing to svb expression 7,8 . We found that one enhancer, svb E 8 , drove specific expression in tiny cells of the Malpighian tubules (Supplementary Figure 1a,b).

Malpighian tubules are mainly composed of two types of differentiated cells 35 . The principal cells-characterized by the homeodomain Cut protein (Fig. 1a,b)-express the vacuolar-ATPase (V-ATPase) that establishes an H + electrochemical potential promoting trans-epithelial secretion of Na + and K [+ 34 . The second main population of Malpighian tubules are termed stellate cells, featured by the expression of the Teashirt (Tsh) transcription factor (Fig. 1a), and that regulate the transport of Cl -and water 34 . While both principal and stellate cells display large-sized polyploid nuclei, a third population of small diploid cells, originally referred to as tiny cells with putative myoendocrine and/or neuroendocrine activity 36,37 , are located in the lower tubules (Fig. 1a,b). Accumulated evidence now supports that these tiny cells ensure the renewal of at least some populations of kidney cells 38,39 and correspond to adult RNSCs (see also Supplementary Figure 4). It has been shown that RNSCs derive from a subpopulation of intestinal stem cell precursors that migrate in Malpighian tubules during post-embryonic development 40 . RNSCs are characterized by the expression of Escargot (Esg), a transcription factor of the Snail/SLUG family that is also expressed in intestinal stem cells 41 where it acts to prevent stem cell differentiation 42,43 . Co-localization with an esg-LacZ reporter confirmed that the svb E enhancer was active in RNSCs (Fig. 1c and Supplementary Figure 1). To define the minimal region of svb responsible for the expression in RNSCs, we assayed a collection of overlapping constructs 5 . This identified two independent elements, the svb E3N and svb E6 enhancers 5,7 , which despite having distinct activities during embryogenesis 7 both drive similar expression in adult RNSCs (Supplementary Figure 1c).

Having established that two enhancers drive specific expression of svb in the adult stem cells of the renal system, we next assayed consequences of depleting svb function in RNSCs. We used a well-controlled genetic system, hereafter referred to as esg ts . esg ts ensures RNAi-mediated gene depletion, specifically in stem cells 44 , as monitored by GFP expression (Fig. 2a,b and Supplementary Figure 2a). In addition, the expression of esg tsdriven transgene(s) and/or RNAi is tightly regulated in a temporal manner by a temperature shift, ensuring that stem cell manipulation is turned ON only in adults (3 days after eclosion) to rule out any earlier developmental defects (Fig. 2c). We also developed an image analysis pipeline, allowing automated quantification of the whole population of RNSCs (see Methods). In control conditions, the number of esg-positive RNSCs remains stable after adult hatching, with approx. 350 cells per tubules (Fig. 2a-c). We only noticed a weak reduction of RNSCs after 1 month. In contrast, esg ts -driven RNAi depletion of svb in adults led to a progressive loss of RNSCs, which were completely absent after 32 days of treatment (Fig. 2a-c). Although not affecting RNSCs before (day 0, Supplementary Figure 2d)-or shortly after (2 days, Fig. 2c)-inducing transgene expression, the effects of svb depletion in the adults were already strong following 8 days of treatment, with a two-fold reduction in the number of RNSCs (Fig. 2a-c and Supplementary Figure 2). Similar results were observed when using either a second RNAi construct (Supplementary Figure 2b), or an independent driver of RNSCs (dome-MESO-gal4) to knockdown svb (Supplementary Figure 2e). We identified another transcription factor, Hindsight (Hnt), as being specific of RNSCs within Malpighian tubules (Supplementary Figure 2c); and the loss of RNSCs upon svb depletion was confirmed by staining against Hnt (Supplementary Figure 2e). Finally, the key role of svb in the maintenance of adult RNSCs was further demonstrated by results from genetic mosaics (MARCM 45 ), showing that mutant cells bearing strong alleles of svb were unable to maintain RNSCs (Fig. 2d and Supplementary Figure 2f,g). Taken together, these data thus reveal that svb is specifically expressed in RNSCs and critically required for the maintenance of the adult stem cell compartment.

Svb processing is essential for its activity in RNSCs. In the epidermis, Svb activity relies on a proteolytic processing that causes a switch from a repressor to an activator form 15 (see Fig. 3a). This processing is gated by Pri smORF peptides, which bind to and activate the Ubr3 ubiquitin E3-ligase that, in turn, triggers a limited degradation of Svb operated by the proteasome 18 . Thereby, pri mediates a systemic control of Svb maturation since the expression of pri is directly regulated by the ecdysone receptor (EcR) 19 .

To assess whether the function of Svb in Malpighian tubules also required its proteolytic maturation, we investigated a putative function of pri and ubr3 in RNSCs. We screened a collection of pri reporter lines 19,46 and identified two cis-regulatory regions driving expression in RNSCs (Fig. 3b and Supplementary Figure 3a,b). Consistently with the expression of pri in RNSCs, pri knockdown strongly impacted the population of RNSCs (Fig. 3c-e and Supplementary Figure 3c). The effects of RNAi-mediated depletion on RNSCs were even stronger for pri than for svb, as previously observed in the epidermis 17 . The lack of Pri peptides indeed leads to the accumulation of the Svb repressor 14,17,19 , which may explain a stronger downregulation of Svb target genes when compared to svb mutants 14 . In the case of RNSCs, it is also possible that pri-RNAi depletion is more efficient (smORF peptides are likely very unstable when compared to typical protein half-life), and/or that Pri peptides fulfil Svb-independent functions as also reported during embryogenesis 17 . In addition, a dominant negative form of the ecdysone receptor (EcRDN) that abolishes pri expression during both embryonic and postembryonic development 19 was sufficient to reduce the number of stem cells when specifically expressed in adult RNSCs (Fig. 3c-e). Furthermore, we found that ubr3 was also required for RNSC maintenance, as deduced from results of RNAimediated depletion (using three non-overlapping constructs) or genetic nullification 18 of ubr3 activity (Fig. 3c-e, Supplementary Figure 3d). Finally, the expression of OvoA (see Fig. 3a) that behaves as a constitutive repressor isoform of Svb 17,47,48 mimicked the effects observed in svb loss of function conditions (Fig. 3c,e). Reciprocally, the expression of OvoB (see Fig. 3a) that acts as a constitutive activator isoform of Svb 17,47,48 was sufficient to rescue the lack of ubr3 function (Fig. 3c,e), demonstrating that Svb function in RNSCs relies on its matured transcription activator form.

These results provide compelling evidence that the whole regulatory machinery discovered for its role in the development of epidermal cells 17-19 is also at work in adult RNSCs. We therefore concluded that the post-translational maturation of the Svb transcription factor is essential for the maintenance of RNSCs.

Svb protects renal nephric stem cells from apoptosis. The loss of RNSCs observed following the lack of svb function or maturation could theoretically result from at least three different causes: (i) lack of proliferation, (ii) precocious differentiation, or (iii) increased cell death. We therefore assayed the putative contribution of each of those aspects in the loss of stem cells resulting from svb inactivation.

Consistent with the quiescent behavior of RNSCs, we observed a low frequency of RNSC division in controls, as deduced from staining with the mitotic marker phospho-Histone H3 (Supplementary Figure 4a,b) and as previously noticed 38 . Additional cell lineage analysis using the esg-Gal4 and dome-MESO-Gal4 drivers confirmed that adult renal stem cells/progenitors give rise to a progeny of large differentiated cells (Cut positive) in lower tubules (Supplementary Figure 4c,d). Similar analyses using the Alkaline-Phosphatase 4 driver (Aph4-Gal4) that is specific of differentiated principal cells 49 in lower tubules showed no progenitor/progeny figures (Supplementary Figure 4c), supporting that only RNSCs are able to sustain cell renewal. Therefore, even a complete block of stem cell division cannot account for the disappearance of RNSCs observed in the absence of svb.

We next investigated a putative influence of svb on RNSC differentiation, making use of the lineage-tracing system called ReDDM that has been recently developed for intestinal stem cells 50 . Based on differences in the stability of two fluorescent proteins, ReDDM allows marking renal progenitors that express both mCD8::GFP and H2B::RFP, while their progeny only maintain the very stable H2B::RFP (see Fig. 4a). In control conditions, we detected rare H2B::RFP progeny (Fig. 4a) confirming a low rate of cell renewal in Malpighian tubules 38,39 (Supplementary Figure 4b). Recent work has shown that the expression of mir-8 (the fly homolog of mir-200 in vertebrates) downregulates the expression of EMT-inducing factors Escargot and Zfh1 (the homolog of Zeb1), triggering a strong burst of stem cell differentiation in the intestine 50 . Similarly, we found that mir-8 expression in RNSCs forced esg+ cells to differentiate and only rare RNSCs persisted after 8 days of treatment (Fig. 4a, Supplementary Figure 5). Upon mir-8 expression, the progeny (H2B::RFP-positive, GFP-negative cells) of RNSCs present in lower tubules also expressed Alkaline Phosphatase 4 confirming that the depletion of RNSCs upon mir-8 overexpression was caused by their premature differentiation (Supplementary Figure 5). In contrast, no significant modification of the progenitors/progeny ratio was observed in svb-RNAi conditions when compared to controls, showing that svb depletion did not trigger RNSC differentiation (Fig. 4a, Supplementary Figure 5).

Finally, we tested whether svb-depleted RNSCs were lost because they underwent apoptosis. As a first step, we assayed consequences of blocking programmed cell death by expressing the viral caspase inhibitor p35 51 . Although the expression of p35 had no detectable effect by itself on RNSCs, it rescued the number of RNSCs when svb was depleted (Fig. 4b). Next, we stained for cells undergoing apoptosis using the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL), an assay allowing the detection of apoptotic DNA fragmentation. While apoptotic figures were almost absent in control conditions, expression of the pro-apoptotic gene reaper induced a strong increase in the number of TUNEL-positive cells (Fig. 4c). Similarly, knocking down svb in stem cells led to frequent apoptotic figures in RNSCs (Fig. 4c) consistently with the progressive decrease in RNSC number observed throughout adult life (Fig. 2c).

Taken together, these data show that the loss of RNSCs observed upon svb loss of function is primarily due to stem cell death, indicating that a main role of Svb is to protect adult stem cells from undergoing apoptosis.

Svb acts downstream of Hippo. Previous work has shown that the Hippo pathway is a key regulator of the Drosophila gut homeostasis, controlling proliferation and survival of stem cells for tissue regeneration 52,53 . Since the Hippo pathway 54,55 is a key sensor of various stresses renowned to induce apoptosis 56,57 , we then investigated its function and putative interplay with Svb in the control of RNSC behavior.

The core Hippo complex is composed of two kinases, Hippo (Hpo) and Warts (Wts) and two scaffolding proteins, Salvador and Mob As Tumor Suppressor 54,55 . Activation of Hippo leads to the phosphorylation of the co-transcription factor Yorkie (Yki), preventing its positive action on the transcription of target genes such as DIAP1 and bantam, favoring resistance to apoptosis and proliferation, respectively 54,55 . Consistently, we found that activation of the pathway via Hpo overexpression induced a strong reduction in the number of RNSCs (Fig. 5a,b). Similarly, knockdown of Yki resulted in dramatic RNSC loss (Supplementary Figure 3d and6a). Conversely, increased Yki activity was sufficient to induce a remarkable increase in the number of RNSCs (Fig. 5), providing additional evidence of the proliferative potential of RNSCs. These data showing the role of the Hippo pathway in the regulation of RNSC survival/proliferation, we next assessed how it interacted with Svb function. Co-expression of OvoB-mimicking a constitutive activator form of Svb (see Fig. 3a)-together with Hpo was sufficient to rescue the loss of RNSCs (Fig. 5). These results therefore suggested that Svb was acting downstream of Hpo. Indeed, the strong increase in the number of renal stem cells observed following increased Yki activity was entirely suppressed upon simultaneous expression either of svb-RNAi, or of the constitutive repressor OvoA (Fig. 5). Quantification indicated that esg+ cells overexpressing Yki were even more sensitive to svb loss-of-function than otherwise normal RNSCs (Fig. 5b). Of note, Yki over-proliferating cells showed aberrant cell morphology reminiscent of tumors derived from intestine stem cells, which also display increased sensitivity to cell death when compared to normal stem cells 58 , and that might explain why RNSCS with high Yki levels cannot survive upon Svb knockdown. Hence, the function of Yki in RNSCs requires Svb, suggesting that Svb was interacting with this nuclear effector of the Hippo pathway. Supporting this view, we found that RNSC survival could not be rescued either by expression of OvoB in the absence of Yki (Supplementary Figure 6a), or by the overexpression of Yki in the absence of Svb (Fig. 5). In contrast, the artificial re-expression of a key target gene of Yki, DIAP1, was sufficient to compensate for svb-depletion, including in most extreme conditions, i.e., following 32 days of treatment (Figs. 2 and5a).

In sum, both Svb and Yki are required for RNSC homeostasis, functionally interacting for the survival of adult stem cells. We Svb as a nuclear effector of the Hippo pathway. Having established that Svb and Yki functionally interact, we sought to decipher the underlying molecular mechanisms and how these two regulators contribute to the control of relevant effector gene expression, e.g., DIAP1.

A first piece of evidence emerged from the comparison of in vivo chromatin immuno-precipitation (ChIP-seq) datasets between Svb 14 and Yki 59 . We found that Svb and Yki share >1.300 common genomic binding sites (Supplementary Figure 7a and Supplementary Table 1) and statistical tests established the significance of this overlap (Supplementary Figure 7b). Interestingly, co-binding of Yki was rare for the direct target genes of Svb identified in the epidermis 12-14 , as illustrated by shavenoid or dusky-like that both lack Yki binding (Supplementary Figure 7c,d). In contrast, Svb was often bound to known Yki target genes, such as bantam, piwi, fat, or nanos 60 (Supplementary Figure 7e,f). Importantly, ChIP-seq also revealed that Svb binds in vivo to an enhancer of DIAP1 (Fig. 6a), previously identified as a Yki direct binding site 61,62 and that drives specific expression in intestinal stem cells 63 . We therefore tested if Svb might regulate DIAP1 expression in adult RNSCs. Although weak in control conditions, we observed specific expression of the DIAP1-LacZ reporter in RNSCs, which was strongly enhanced upon Yki overexpression (Fig. 6b). This induction of DIAP1 expression upon Yki overexpression was antagonized by OvoA (Fig. 6b). Similar results were obtained with the isolated DIAP1-4.3-GFP 62 enhancer that contains binding sites for Yki and Svb. Although OvoB did not show significant influence (Supplementary Figure 6b), the expression of DIAP1-4.3-GFP was again enhanced by Yki overexpression and abrogated upon counteracting Svb activity (Fig. 6b). These results thus strengthen the conclusion that Svb and Yki functionally interact in RNSCs to prevent apoptosis, at least in part through promoting DIAP1 expression.

The transcriptional activity of Yki/YAP/TAZ is well known to rely on the regulation of its subcellular localization between the cytoplasmic versus nuclear compartments. Assaying a putative influence of Svb on Yki distribution, we did not however detect any consequences of Svb activity (either Svb ACT or Svb REP ) on in vivo Yki nuclear accumulation within RNSCs (Supplementary Figure 6c). Yki is unable to bind DNA by itself and need to associate to sequence-specific transcription factors 55 . Hence we assayed whether Yki could associate with Svb to explain the functional interactions observed in RNSCs between these two nuclear factors. Biochemical assays showed that Svb bound to Yki, as seen by reciprocal co-immunoprecipitation in protein extracts prepared from cultured cells (Fig. 7a). Both the full length repressor and matured Svb activator interacted with Yki (Fig. 7a).

It has been shown that Yki contains two WW protein domains, mediating interaction with partners bearing PPxY motifs (such as Wts 61 or Mad 64 ). Of note, we detected two PPxY motifs within the Svb protein, at position 523 (PPFY) and 881 (PPSY), i.e., within the region common to Svb repressor and activator forms. We found that the mutation of Yki WW motifs was sufficient to abrogate the interaction with both forms of Svb (Fig. 7a). Furthermore, point mutations of Svb PPxY motifs also impaired interaction with Yki, as monitored using immunoprecipitation assays (Fig. 7a). We next investigated the in vivo consequences of impairing Svb/Yki interaction, investigating the functional behavior of the Svb mutant lacking Yki interaction motifs (Svb-PPxY). When expressed in the embryonic epidermis, Svb-PPxY retained the ability to induce ectopic trichomes, consistent with a function of Svb being independent of Yki activity for the terminal differentiation of epidermal tissues (Supplementary Figure 8a). In contrast, Svb-PPxY behaved as a potent dominant negative mutant within adult stem cells. Previous work has shown that Svb ACT and Svb REP display distinct patterns of intra-nuclear distribution, characterized by diffuse nucleoplasmic localization or accumulation in dense foci, respectively 17 . Svb-PPxY::GFP displayed a discrete accumulation in foci, reminiscent of that observed for the repressor (Fig. 7b). Importantly, expression of Svb-PPxY in RNSCs significantly reduced their number, as observed upon Svb or Yki knockdown (Fig. 7c). These data therefore indicate that Svb physically interacts with Yki (via PPxY and WW motifs, respectively) and impairing specifically this interaction alters adult stem cell behavior.

One important question was whether the interaction between Svb and the Hippo pathway also took place in other tissues. The function of Hippo has been initially described in larval imaginal discs, which give rise to most adult tissues 65 and Yki overexpression promotes cell proliferation in both wing and eye discs 61 . We tested Svb/Yki genetic interactions in the wing using collier-Gal4 that drives expression in the medial (L3-L4) intervein region. Yki expression resulted in the expansion of this region due to tissue overgrowth (Fig. 8a). In contrast, OvoA leads to both a reduction of the L3-L4 region and the absence of epidermal trichomes. As in RNSCs, OvoA was epistatic to Yki, since the wing region expressing both yki and ovoA was smaller than in controls and lacked trichomes (Fig. 8a). As expected for a mutant unable to interact with Yki, we found that expression of Svb-PPxY did not affect wing cell proliferation. Additional evidence of interaction between the Hippo pathway and Svb/Pri came from genetic assays demonstrating the importance of relative levels of their activity. While lowering wts activity causes a wing overgrowth phenotype (Fig. 8b), we noticed that reducing pri activity suppressed this phenotype (Fig. 8b,c). Furthermore, wts mutation can lead to dramatic defects in wing development when introduced in a given genetic background, and decreasing pri dosage was sufficient to almost completely restore normal wings (Supplementary Figure 8b).

In the eye, overexpression of Yki using the GMR-Gal4 driver promoted extra cell proliferation resulting in an increased eye size. Similar results were obtained following pri overexpression, and co-expressing pri and yki resulted in a synergistic eye growth (Supplementary Figure 9a). Northern blotting of RNAs extracted from adult heads revealed that DIAP1 mRNA levels were increased following pri overexpression (Supplementary Figure 9b), while there was no effect on yki or cycE mRNA. We interpret these results to imply that Svb functionally interacts with Yorkie, both in adult stem cells and in specific developing tissues, to regulate a subset of transcriptional targets of the Hippo pathway, including the activation of DIAP1 expression.

Discussion

Our results show that Shavenbaby is expressed and required for the maintenance of adult renal stem cells in flies, supporting the conclusion that the OvoL/Svb family of transcription factors plays a key and evolutionarily-conserved role in the behavior of progenitors/stem cells.

The role of Svb in adult stem cell maintenance in flies requires both a fine control of its expression and of its transcriptional activity. Svb expression in RNSCs involves at least two separable enhancers, driving similar expression patterns. Svb was one of the first cases to reveal the functional importance of apparently redundant (or shadow) enhancers in the phenotypic robustness of regulatory networks 5,6 across tissues and development stages 11 . Our data suggest that a similar cis-regulatory architecture is also underlying the control of adult stem cells.

RNSCs maintenance further requires a proper posttranslational maturation of the Svb protein, in response to Pri smORF peptides. During both embryonic 17 and post-embryonic development 18,19 , the main role of Pri peptides is to provide a temporal control of Svb activity, conveying systemic steroid signaling 19 . It is therefore possible that Pri smORF peptides also connect genetic networks to hormonal control for the regulation of adult stem cells. Recent work has shown that various smORF peptides contribute to the regulation of developmental pathways, muscle formation and physiology, etc… 15,66,67 , and our findings extend their influence to the control of adult stem cells. It has been proposed that the emerging field of smORF peptides may open innovative therapeutic strategies 16,68 , for example peptidomimetic drugs, which might also be of interest for regenerative medicine.

Our results establish that a main function of Svb in adult stem cells is mediated by a functional interplay with the Hippo pathway, well established for its roles in the control of adult stem cells 52-54 . Our results indicate that Svb behaves as a nuclear effector of Hippo, relying on a direct interaction with Yorkie in order to protect stem cells from apoptosis, at least in part through the regulation of DIAP1 expression. Analysis of genome-wide binding events further suggests that the Svb/Yki interaction is involved in the control of a broader set of Hippo-regulated genes, including during development. Since both Pri and Ubr3 are also essential for the survival of adult stem cells, it is interesting to note that Ubr3 protects the DIAP1 protein from degradation 69 , and direct binding of Ubr3 on the activated form of DIAP1 is elicited in the presence of Pri peptides 18 . Therefore, in addition to the control of DIAP1 expression (via Svb), Ubr3 and Pri could also stabilize the DIAP1 protein to protect stem cells from apoptosis. Although initially restricted to TEAD transcription factors, the number of Yorkie (YAP/TAZ) nuclear partners is rapidly growing 55 . Interestingly, recent work has shown a direct interaction of YAP/TAZ with the pro-EMT factors Snail/Slug, in the control of stem cell renewal and differentiation 70 . As previously reported for intestinal stem cells 42,43,50 , we show that pro-EMT regulators are also required for preventing premature differentiation of renal stem cells. While pro-EMT and OvoL factors are often viewed as antagonistic factors 20,25 , in vivo studies in Drosophila stem cells show that they both contribute to their maintenance, Svb/Yki preventing their apoptosis and EMT factors their differentiation. Since many studies have implicated the Hippo pathway, pro-EMT and OvoL/Svb factors in various tumors, new insights into their functional interactions in adult stem cells may provide additional knowledge directly relevant to understand their connections in human cancers.

Methods

Drosophila work. Flies were cultured (unless otherwise noted) at 25 °C, using standard cornmeal food (per liter: 17 g inactivated yeast powder, 80 g corn flour, 9 g agar, 45 g white sugar, and 17 ml of Moldex). Similar results were also observed using a richer medium (same composition except 45 g of yeast powder). Female adult flies were used in all analyses throughout the study and placed on fly food supplemented with fresh yeast, which was changed every 2 days. Conditional expression in RNSCs was achieved by maintaining tub-Gal80 ts expressing flies at 18 °C, until adulthood. Eclosed females aged for 3-days were shifted to 29 °C for induction of gene expression and were kept at 29 °C for the indicated period of time (in most cases 8 days). Virgin females bearing svb R9 , svb PL107 , or ubr3 B mutations 18,47 over FM0 balancers were mated with males of the following genotype: y, w, hs-FLP, tub-Gal80, FRT19A; UAS::mcd8-GFP; tub-Gal4/TM6B, Tb. Females of the correct phenotype (not B and not Tb) were heat shocked for 1 h at 37 °C. For G-TRACE experiments, females w; UAS-RedStinger, UAS-FLP, Ubi-p63E (FRT.STOP)Stinger/CyO were crossed with esg-Gal4, dome-MESO-Gal4 or c507-Gal4 males and raised at 18 °C until adulthood. Females were kept at 25 °C and dissected after 20 days. Flies were transferred on fresh food every 2 days and dissected at the indicated time. Detailed information about the genotype of each Drosophila stock is given in Supplementary Information.

Histology. Tissues were dissected in 1× PBS, fixed in 4% formaldehyde in PBS for 15 min at room temperature, washed for 5 min in PBS containing 0.1% Triton X-100 (PBT) and fixed again during 20 min. Following a 5 min wash in PBT, tissues were blocked for 30 min in PBT containing 1% BSA. Primary antibodies were incubated overnight at 4 °C. Anti-ß-Galactosidase (Cappel Cat#08559761) antibody was used at 1:1000, anti-Cut (DSHB Cat#2B10), anti-GFP (Acris Antibodies Cat#TP401) at 1:200, anti-phospho-Histone H3 (Upstate Cat#TAKMA312B), anti-Disc-large (DSHB Cat#4F3 anti-discs large) at 1:500, anti-Arm (DHSB Cat# N2 7A1) at 1:100, anti-Hnt (DHSB Cat# 1G9) at 1:30. AlexaFluor-488 or -555 secondary antibodies (ThermoFischer Scientific Cat#Z25302 and Cat#Z25205) were incubated for 2 h at room temperature at 1:500. After three washes, tissues were mounted in Vectashield (Vector Laboratories). For X-gal staining, adult tissues were dissected in 1× PBS, fixed in 1% glutaraldehyde in PBS for 15 min at room temperature and washed in PBS. The staining solution was warmed up at 37 °C for 10 min plus another 10 min after addition of 8% X-Gal (5-bromo-4- TUNEL assays. TUNEL staining was performed following a protocol kindly provided by A. Bergmann. Tissues were dissected in 1× PBS, fixed in 4% formaldehyde in PBS for 30 min at room temperature, washed for 3 times 15 min in PBS containing 0.3% Triton X-100 (PBT) and blocked 15 min in PBT + 5% Normal Goat Serum (PBNT). Anti-GFP antibody was incubated overnight at 4 °C. The following day, tissues were washed with WB (50 mM Tris/HCl pH = 6.8, 150 mM NaCl, 0.5% NP40, 1 mg/ml BSA) and incubated overnight with the secondary antibody. Tubules were then washed 3 times in WB and incubated 30 min at 65 °C with 99 µl of 100 mM Na-Citrate + 1 µl of 10% Triton X-100. Following quick washes with WB, tubules were incubated in 45 µl of labelling solution (in situ cell death detection TMR Red kit, Roche), for 30 min at 37 °C. 5 µl of the enzyme solution was added for 2 h at 37 °C. Tubules were finally washed in PBT and mounted in Vectashield.

Microscopy, image, and statistical analysis. Images of whole Malpighian tubules were acquired on a LSM710 confocal scanning microscope (×20 objective), using automated multi-position scan. After stitching, tiled images of individual pairs of tubules were analyzed with IMARIS 8.0 to quantify the number of GFP-positive cells. Data of at least three independent experiments (approx. 20 tubules) were combined. All statistical analyses were carried out using Prism 5 (GraphPad).

Comparisons between normally distributed groups were carried out using Student's t-test, unpaired, two-tailed and incorporating Welch's correction to account for unequal variances. One-way ANOVA with Bonferroni correction was used when multiple comparisons were applied. In all figures, ns indicates p ≥ 0.05, * indicates 0.05 > p ≥ 0.01, ** indicates 0.01 > p ≥ 0.001, and *** indicates p < 0.001. Close-up pictures were acquired using Leica SPE and Leica SP8 confocal laser scanning microscopes (×40 and ×63 objectives). Laser intensity and background filtering was set according to the control samples and remained the same for all subsequent samples. The intensity of pictures has been enhanced equally for all images within the same experiment using adjustments in Photoshop CS5. All images were processed using Adobe Photoshop and Illustrator CS5. Western blotting and immunoprecipitation. Drosophila S2 cells (Drosophila Genomics Resource Center,stock number: 6) were grown in Schneider medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Invitrogen) at 25 °C. We used either S2 cells or stable cell lines co-expressing the copperinducible constructs pMT-Svb::GFP and pMT-pri 17 . S2 cells were cultured in sixwell plates (1.75 × 10 6 cells/3 ml) and transfected in 100 µl of Opti-MEM (Invitrogen) with 3 µl of FugeneHD (Promega) and the indicated constructs. CuSO 4 (final concentration of 1 mM) was used to induce the expression of pMT plasmids. The following plasmids were used: pAc-Yki::HA and its related mutated version pAc-Yki-WW::HA, pAc-Svb::GFP, and pAc-Svb-PPxY::GFP. S2 cells were lysed on ice in 250 µl of ice-cold lysis buffer (150 mM NaCl, 50 mM Tris/HCl pH = 8.0), 0.5% NP40, 1 mM EGTA, and protease inhibitors (Roche). After clearing by centrifugation at 18,000g for 10 min, immuno-precipitations from transfected lysates were done in lysis buffer, using anti-GFP antibody (GFP-trap, Chromotek), or anti-HA antibody (Covance) and Fast-Flow Protein G-Sepharose (Sigma #P3296). Immuno-precipitated samples were separated by SDS-PAGE and transferred to PVDF membranes, then blotted by using the following antibodies: anti-GFP (Acris Antibodies, Cat#TP401, 1:10,000), anti-HA (Covance, Cat#BLE901513, 1:2000), and anti-Yki (1:4000, kind gift of K. Irvine). Secondary antibodies were anti-mouse or anti-rabbit IgG-HRP conjugates (Jackson Laboratory, Cat#515-035-003, 1:10,000), detected using the ECL Clarity (Bio-Rad). Uncropped scans of blots are supplied in Supplementary Information.

Northern blot analysis. Two days-old adults were frozen with liquid nitrogen and heads were sorted with sieves, followed by RNA purification with Isogen (Nippon Gene). 1 μg of RNA per lane was separated by formaldehyde-agarose gel electrophoresis and then transferred to a nylon membrane (Roche). Hybridization and wash procedures were carried out at 52 and 65 °C, respectively. The filters were reacted with an alkaline phosphatase-conjugated anti-DIG antibody (Roche) and chemiluminescent reactions with CPD-Star (Roche) were detected by LAS 4000mini (GE Healthcare).

Uncropped scans of Western and Northern blots Figure 7a

Full genotypes

ChIP-seq analysis

Raw data (.fq files) of ChIPseq obtained for Yki 6 (GSM945893 GSM945894) and Svb 7 (GSM1184656, GSM1184657, GSM1184658) were processed in parallel, using the same pipeline. Briefly, reads were aligned to the Drosophila genome release Dm6 with BWA 8 . SAM files were converted into BAM files using SAMtools 9-11 and further processed for peak calling. The quality of sequencing was checked using FastQC, and we used SAMTOOLS flagstat to calculate the percentage of mapped reads to estimate the quality of alignment from the percentage of mapped reads (as indicated below). (available under RBioconductor) was used to identify co-binding regions of Svb and Yki, and to perform permutation-statistical tests 13 . An UCSC genome browser is available at:

Number of reads

http://genome--euro.ucsc.edu/cgi--bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=amanchenoferris&hgS_otherUserSessionName=bohere_etal

DNA constructs and mutagenesis

The DNA region encoding point mutations to inactivate the two PPxY motifs of Svb (>PPxA)

has been optimized to be chemically synthetized (Integrated DNA Technologies). Resulting DNA was amplified by PCR and cloned into the pAc-Svb::GFP construct 4 , linearized with PmlI and HindIII restriction sites, using the In-Fusion HD Cloning kit (Clontech laboratories, Inc) to obtain the pAc-Svb-PPxY::GFP vector. The EcoRI fragment with both PPxA mutated sites from pAc-Svb-PPxY::GFP has been inserted into the pUASp-Svb::GFP 5 linearized with EcoRI. All construct sequences have been verified by sequencing.

NORTHERN blotting

Using adult total RNAs as a starting material, DNA fragments containing coding sequence of yki, CycE and DIAP1 were reverse transcribed and PCR amplified. An SP6 promoter sequence (CAAGC TATTT AGGTG ACACT ATAG) was attached to each reverse primer for in vitro transcription. DIG-labelled probes were prepared with SP6 RNA polymerase, according to the supplier's manual (Roche).

Oligonucleotides

Oligonucleotides PPxY mutagenesis

5-TCAGCTCAGCATGCCACGCGTG-3

Integrated DNA Technologies

INTRODUCTION

Living organisms are constantly exposed to aging and environmental challenges that disturb cell functions and ultimately lead to cell death. To maintain homeostasis, most adult organs are regenerated by self-renewing stem cells, which differentiate to replace dead cells and replenish damaged tissues. The highly regenerative digestive system is kept intact during adulthood by the activity of resident intestinal stem cells. Intestinal stem cells in flies have emerged as a powerful system to understand the signaling networks underlying stem cell biology and their implication in cancers (see [START_REF] Herrera | JAK/STAT signaling in stem cells and regeneration: from Drosophila to vertebrates[END_REF]Li and Jasper, 2016;Perochon et al., 2018) for recent reviews).

The adult fly midgut consists of a highly compartmentalized epithelium (Buchon et al., 2013;Marianes and Spradling, 2013), which shares anatomical and physiological similarities with its mammalian counterpart [START_REF] Casali | Intestinal stem cells in mammals and Drosophila[END_REF]. Drosophila intestinal stem cells (ISCs) are small diploid cells scattered along the basal surface of the epithelium (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006). In steady state conditions, ISC divide asymmetrically to generate a new stem cell and a progenitor cell called enteroblast (EB) (Ohlstein and Spradling, 2007); ISCs and EBs express Escargot (Esg), a transcription factor of the Snail/Slug family (Korzelius et al., 2014;Loza-Coll et al., 2014). EBs are post-mitotic cells that progressively acquire characteristics of either polyploid absorptive enterocytes (ECs) or hormone-secreting entero-endocrine cells (EEs) (Ohlstein and Spradling, 2007). It has been proposed that EEs arise from a separate pool of progenitors, called pre-enteroendocrines, which express markers of both ISCs and EEs (Biteau and Jasper, 2014;Zeng and Hou, 2015).

Al Hayek et al.,p: 4 The evolutionarily conserved Notch pathway establishes the asymmetry between ISC and EB (Bardin et al., 2010;Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2007;Perdigoto et al., 2011). ISCs express Delta, a ligand that activates the Notch receptor in daughter EBs, as seen by upregulation of Su(H) expression. The EC fate requires high levels of Notch, whereas lower Notch signaling lead to EEs that maintain Prospero expression. The JAK/STAT pathway also contributes to the differentiation of committed progenitor cells into ECs and EEs (Beebe et al., 2010;Jiang et al., 2009). Gut homeostasis relies on a tight regulation of ISC division through cooperative activity of additional signaling pathways (reviewed in [START_REF] Buchon | All for one and one for all: Regionalization of the Drosophila intestine[END_REF]). For instance, the epidermal growth factor receptor (EGFR), Wnt and JAK-STAT pathways contribute to ISC preservation and proliferation (Biteau and Jasper, 2011;Jiang and Edgar, 2009;Jiang et al., 2011). Despite the wealth of knowledge accumulated on the role of signaling pathways in regulating ISC maintenance, division and differentiation, the intrinsic mechanisms by which ISCs integrate these cues remain poorly understood.

During embryogenesis, the activity of Wnt and EGFR pathways in the epidermis is mediated by a common target gene, ovo/shavenbaby (svb), which controls differentiation of epidermal trichome cells (Payre et al., 1999). Svb is a transcription factor, which undergoes post-translational processing from a repressor (Svb REP ) to an activator (Svb ACT ) via limited proteasome degradation (Zanet et al., 2015). Svb maturation is triggered by Polished rice (Pri) peptides (Kondo et al., 2010), which are founding members of a fast growing family of peptides translated from small open reading frames (smORF peptides, see (Plaza et al., 2017;[START_REF] Saghatelian | Discovery and characterization of smORF-encoded bioactive polypeptides[END_REF] for review). pri steroid hormone in insects, which regulates -among others-reproduction, nutritional state and stress resistance in adults [START_REF] Uryu | Recent progress in understanding the role of ecdysteroids in adult insects: Germline development and circadian clock in the fruit fly Drosophila melanogaster[END_REF]. In addition to epidermal derivatives, the ovo/svb gene is also critical for germ cell maintenance and differentiation (Mevel-Ninio et al., 1995). It produces two germline-specific isoforms, called OvoA and OvoB, which are insensitive to Pri smORF peptides (Kondo et al., 2010) and act as "constitutive"

repressor and activator (Andrews et al., 2000), respectively.

Initially isolated in flies, Ovo/Svb was soon after identified in mammals (there are three paralogs called OvoL1-3) and shown to act in the epidermis and germline [START_REF] Dai | The ovo gene required for cuticle formation and oogenesis in flies is involved in hair formation and spermatogenesis in mice[END_REF]. Tumor profiling has recently identified OvoL factors as regulators of the metastatic potential of many human cancers, including the most prevalent ones such as breast (Roca et al., 2013), prostate [START_REF] Fu | Expression of Ovol2 is related to epithelial characteristics and shows a favorable clinical outcome in hepatocellular carcinoma[END_REF], lung (Wang et al., 2017), as well as colorectal (Ye et al., 2016) cancers. Other studies have disclosed key roles of OvoL in the repair of mammalian epithelial tissues, e.g. for epidermal and mammary regeneration (Haensel et al., 2019;Watanabe et al., 2014). Moreover, OvoL factors are required for the differentiation of neural progenitors and the maintenance of human corneal epithelial cells (Kitazawa et al., 2016); deregulation of OvoL2 is a common cause of inherited corneal dystrophies (Davidson et al., 2016). In the planarian flatworm, OvoL/Svb is also expressed in eye progenitors and required for eye regeneration from multipotent stem cells (Lapan and Reddien, 2012), suggesting a conserved role of OvoL/Svb in progenitors/stem cells across animals. Indeed, we recently found that Svb is required in flies for the survival of adult renal nephric stem cells (RNSCs). We showed that Svb activates the expression of the Drosophila anti apoptotic protein (DIAP1) by interaction with Yorkie (a.k.a. YAP/TAZ), the nuclear effector of the Hippo pathway (Bohere et al., Al Hayek et al., p: 6 2018). RNSCs derive from progenitors also producing intestinal stem cells (Xu et al., 2018), suggesting a broader function of Svb in stem cells.

Here, we demonstrate that Shavenbaby is an important regulator of the intestinal stem cell lineage, essential to adult midgut homeostasis. svb is expressed in both ISC/EBs progenitors and differentiated ECs, and its inactivation induces apoptotic cell death. Svb acts downstream of EGFR and Wnt mitogenic signaling in progenitor cells,

where it is processed into Svb ACT through the Pri/proteasome axis. In contrast, Svb REP

RESULTS

Svb is required to maintain Drosophila adult midgut progenitors svb expression is driven by a large array of enhancers, which collectively define at single cell resolution the pattern of epidermal differentiation in the embryo (Frankel et al., 2011;McGregor et al., 2007;Preger-Ben Noon et al., 2016;Sucena et al., 2003). To monitor svb expression in the adult midgut, we tested the activity of main svb enhancers. We found that the E svb enhancer (5 kb) drives specific expression in esg-positive cells, therefore identifying them as stem cells and enteroblasts (Figures 1A,B andS1A). Further dissection restricted the regions responsible for svb expression in progenitor cells to two separate enhancers called E3N (292 bp) and E6 (1kb).

As a first step to investigate the function of Svb in intestinal stem cells, we used targeted RNAi-mediated depletion, at the adult stage. The conditional thermosensitive driver esg ts (Micchelli and Perrimon, 2006) allowed svb knockdown in adult esg+ cells, resulting in a strong decrease in the number of progenitor cells (Figure 1C,C'). Using

Delta-lacZ and Su(H)-GBE-lacZ to discriminate ISCs vs EBs, respectively, we observed strong reduction of both stem cells and enteroblasts upon svb knockdown (Figure 1C-E').

The requirement of svb in ISCs and EBS was confirmed svb depletion specifically in ISCs by using the ISC ts driver [START_REF] Wang | EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells[END_REF] (Figure 1D,D'), or in EBs by using Su(H) ts (Zeng et al., 2010) (Figure S1B). Hence, svb depletion causes the loss of both ISCs and EBs, without detectable effects on enteroendocrine cells (Figure S1C).

This conclusion was further tested by lineage-tracing using the act ts F/O system, which led to random inactivation of svb in dividing intestinal cells and their progeny (marked by GFP). svb depletion leads to a strong decrease in both the number and size of GFP+ clones, when compared to controls (Figure 1F). This was also confirmed by the generation of genetically mutant cells for svb, using the MARCM technique (Lee and Luo, 2001). In contrast to controls, we obtained very few clones of intestinal cells bearing the null svb R9 mutant allele (Delon et al., 2003). In addition, svb mutant clones were unable to grow and often restricted to single cells (Figure 1G).

These results suggested that intestinal progenitors lacking svb undergo apoptosis, as recently shown for renal stem cells [START_REF] Bohere | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF]. To test this hypothesis, we stained for the apoptotic marker cleaved-Dcp1 in guts bearing clones of progenitor cells lacking svb, using esg ts F/O (Jiang et al., 2009) to drive svb-RNAi. While only rare Dcp1positive cells were seen in controls, we observed many apoptosis figures in GFP+ cells upon svb depletion (Figure 1H). In addition, the apoptosis inhibitor DIAP1 partially rescued the loss of ISC/EBs when expressed together with svb-RNAi (Figure 1I). Finally, ReDDM lineage tracing (Antonello et al., 2015) indicated that stem cell loss upon svb depletion did not result from premature differentiation (Figure S1D).

Taken together, these data show that Svb function is required to protect intestinal stem cells and enteroblasts from apoptosis.

The Pri/Ubr3/proteasome axis controls Svb function in midgut progenitors

Svb is translated as a large (1354 aa) repressor (Svb REP ) that is processed into a shorter (909 aa) transcriptional activator (Svb ACT ) (Kondo et al., 2010). This switch is gated by Pri peptides that bind to and activate the E3 ubiquitin ligase Ubr3, triggering Ubr3 binding to Svb (Zanet et al., 2015). Ubr3 then induces Svb processing via limited proteasome degradation of its N-term repression domain (see Figure 2C). Originally described in the epidermis (Chanut-Delalande et al., 2014;Kondo et al., 2010), there is growing evidence that Pri-dependent processing underlies Svb function in other somatic tissues [START_REF] Pueyo | Tarsal-less peptides control Notch signalling through the Shavenbaby transcription factor[END_REF][START_REF] Ray | The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning[END_REF], including adult stem cells [START_REF] Bohere | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF].

To investigate whether Pri-mediated maturation of Svb was involved in ISC/EBs, we first examined pri expression in the adult midgut. Profiling of reporter lines (Chanut-Delalande et al., 2014) showed that three pri enhancers (priA, priJ and priH) drive specific expression in ISC/EBs (Figure 2A,B). pri expression was also confirmed by the activity of a gene trap (Galindo et al., 2007) in ISC/EBs (Figure 2B). We next assayed the consequences of pri knockdown and observed an acute loss of progenitor cells when pri-RNAi was driven by esg ts (Figure 2D), or of stem cells when using the ISC ts driver (Figure S2A). In the epidermis, ecdysone signaling times pri expression throughout embryonic and post-embryonic development (Chanut-Delalande et al., 2014). We reasoned that if this hormonal control of pri expression was occurring in the adult midgut, cell-autonomous disruption of the ecdysone pathway should affect the behavior of ISCs. Consistently with this prediction, downregulation of ecdysone signaling via a dominant negative receptor (EcR-DN, Figures 2E andS2D), or EcR-RNAi (Figure S2B,C), reduced the number of intestinal progenitor cells. Furthermore, pri expression was sufficient to rescue the loss of progenitors upon EcR inactivation (Figures 2E andS2D). These data thus support that pri is an important target of ecdysone required for the homeostasis of intestinal stem cells.

The role of Pri peptides in triggering Ubr3-mediated processing of Svb in ISC/EBs was demonstrated by additional lines of evidence. First, knockdown of Ubr3 in intestinal

Al Hayek et al.,p: 10 progenitor cells, or specifically in stem cells (Ubr3-RNAi driven by esg ts or ISC ts ), resulted in a strong reduction of their numbers (Figures 2F andS2E). Second, we generated clones of Ubr3-null mutant cells, which were very rare and unable to expand (Figure 2G).

Third, the loss of Ubr3 could be compensated by expression of the constitutive activator OvoB (Andrews et al., 2000;Kondo et al., 2010), indicating that a main function of Ubr3

in ISCs is to operate Svb maturation into the activator form (Figures 2F andS2E).

Taken together, these results establish that Svb undergoes Pri/Ubr3-dependent processing in intestinal stem cells to maintain the population of midgut progenitors.

The Svb activator form triggers ISC proliferation

Having shown that Svb ACT is required for the survival of ISC/EBs, we next investigated if it could be sufficient to influence adult stem cell homeostasis. Hence, we expressed the OvoB constitutive activator using esg ts and monitored the behavior of ISCs marked with Delta-lacZ. OvoB induced a strong increase in the number of ISCs (esg+, Dl-lacZ+), reaching up to four-fold the normal population (Figure 3A). This was due to ISC proliferation, as seen by increased levels of the mitotic marker phosphorylated-histone3 (PH3, Figure 3A'). Furthermore, while pri has weak if any effects when expressed alone in stem cells, it provoked ISC over-proliferation when expressed together with Svb REP to induce the production of Svb ACT (Figure 3B,B'). Similar results were obtained when pri and Svb REP were simultaneously expressed in esg+ cells, leading to increased numbers of progenitors and thus mimicking the phenotype of OvoB (Figure S3).

Al Hayek et al.,p: 11 These data thus show that the Svb ACT induced by Pri smORF peptides is sufficient to drive the proliferation of adult stem cells.

Svb acts downstream of Wnt and EGFR mitogenic pathways in the adult midgut svb acts as a functional integrator of signaling pathways, including Wnt, EGFR, Hedgehog and Notch, mediating their roles in the embryonic epidermis (Payre, 2004). Since these pathways are well-established to control ISC behavior [START_REF] Buchon | All for one and one for all: Regionalization of the Drosophila intestine[END_REF], we investigated whether Svb could also mediate their activity in intestinal stem cells.

As previously reported (Lin et al., 2008), inhibition of Wnt signaling by a dominant negative form of the nuclear effector TCF/Pangolin (TCF-DN) led to a marked decrease in the number of ISC/EBs (Figure 4A,A'). Co-expression of OvoB was not only sufficient to rescue the progenitor population, but could still induce ISC proliferation even when Wnt activity was compromised (Figure 4A,A'). Conversely, over-activation of Wnt signaling by expressing the Wingless ligand, or a constitutively active form of the nuclear effector ß-Catenin (Arm S10 ), promotes ISC division (Lin et al., 2008). Wnt-induced stem cell proliferation was further potentiated upon expression of OvoB and abrogated when svb function was inhibited (Figure 4A). Similarly, EGFR activation (esg ts F/O>Ras V12 ) that induces progenitor proliferation was counteracted upon svb depletion (Figure 4B).

Knocking-down EGFR signaling (esg ts >EGFR-DN) led to a loss of esg+ cells, and OvoB was also sufficient to bypass the depletion of stem cells (Figure 4B,B'). These results

show that Svb is epistatic to, in other words likely acts downstream, Wnt and EGFR pathways to control adult stem cell maintenance and division.

Al Hayek et al.,p: 12 Therefore, svb expression integrates inputs from Wnt and EGFR in both adult stem cells and embryonic epidermal cells (Payre et al., 1999); the underlying mechanisms yet remained to be elucidated. To identify in an unbiased manner transcription factors that control svb expression, we undertook functional screening in vivo, in the late embryonic epidermis, where Svb does not impinge on cell survival/ proliferation, as opposed to adult stem cells. We knocked down every TF showing detectable expression (Menoret et al., 2013) at the time of epidermal differentiation (227 candidates, see Table S1) and assayed whether it affected the activity of svb enhancers (Figure S4A). This identified a few factors the depletion of which altered E3N expression, including TCF, the nuclear effector of Wnt, and Pointed (Pnt), an ets effector of the EGFR pathway. Analysis of the E3N sequence predicted putative binding sites for TCF and Pointed (Figure S4B-C) and we generated lacZ reporters with mutations within TCF (E3N-TCF-mt) or Pointed (E3N-Pnt-mt) sites. In the embryo, E3N-Pnt-mt displayed a reduced activity when compared to wild type E3N, whereas the inactivation of TCF sites induced a weaker but ectopic pattern (Figure S4D).

To assay whether this altered pattern affected svb function, we generated transgenic lines driving svb under the control of E3N or E3N-TCF-mt and assayed their rescuing activity when introduced in svb mutants. While E3N-svb displayed clear rescue of trichomes consistent with previous report [START_REF] Crocker | Low affinity binding site clusters confer hox specificity and regulatory robustness[END_REF], the mutation of TCF sites abrogated its rescuing activity (Figure S4E). Accordingly, both E3N-TCF-mt and -Pnt-mt reporters displayed strongly decreased expression when assayed in adult stem cells (Figure 4C).

These data therefore show that the E3N svb enhancer integrates direct regulatory inputs from Wnt and EGFR pathways, to control the behavior of intestinal stem cells.

The Svb repressor promotes enterocyte differentiation

In addition to stem cells, we observed expression of Svb within enterocytes as deduced from complementary pieces of evidence. In situ hybridization to svb mRNA stained small doublet cells located close to the basement membrane, i.e. ISC/EBs, but also revealed svb expression in ECs, which are located more apically and display characteristic large polyploid nuclei (Figure 5A). The production of Svb in both ISCs and ECs was also supported by the expression of a svb::GFP mini-gene rescue construct (Menoret et al., 2013), while no expression was detected in EEs (Figure 5B,B') consistent with the absence of svb function in EEs (Figure S1). Expression of the svb::GFP mini-gene is driven by only two enhancers: E (active in progenitors, see Figure 1) and 7, suggesting that the latter was responsible for svb expression in ECs. Indeed, even the shortest version of the svb-7 enhancer, termed 9CJ2 (232bp), drove expression in differentiated cells with large nuclei (Figure 5C). Using our screening assay to identify upstream regulatory factors, we found that 9CJ2 activity was reduced upon knocking down Pdm1/Nubbin (Figure S5), which controls EC differentiation (Micchelli and Perrimon, 2006;Ohlstein andSpradling, 2006, 2007). We identified two putative Pdm1 binding sites within 9CJ2 (Figure S5B,C) and inactivated those by point mutations (9CJ2-Pdm-mt).

Knockout of Pdm1 sites prevented 9CJ2 expression, both in the embryonic epidermis and in adult intestinal stem cells (Figures 5C andS5D). We thus conclude that svb is expressed in ECs under the control of the 7 enhancer, likely directly regulated by Pdm1.

The switch in Svb transcriptional activity triggered by pri is associated with a marked change in Svb intra-nuclear distribution: whereas Svb ACT diffuses within the nucleoplasm, Svb REP accumulates in dense foci (Kondo et al., 2010;Zanet et al., 2015).

Al Hayek et al.,p: 14 Svb appeared diffuse in esg+ cells (that express pri), while displaying foci in ECs, which don't (Figure 5B'). Unlike ISCs that rely on Svb ACT , Svb REP might thus be involved in the differentiation of enterocytes. To test this hypothesis, we expressed the full size Svb protein (Svb REP ) in intestinal progenitor cells (esg ts ). The number of ISC/EBs was reduced and remaining esg+ cells displayed aberrant morphology, with enlarged nuclei reminiscent of polyploid ECs (Figure 5E). Moreover, Svb REP precluded the growth of esg ts F/O clones, limited to single cells with large nuclei (Figure 5D). These cells were negative for Dcp1 apoptotic staining and, accordingly, Svb REP phenotypes were not rescued by DIAP1 expression (Figure 5E,E'). These data thus suggested that Svb REP induces ISC/EB differentiation instead of cell death, a hypothesis we next tested using ReDDM lineage tracing. Indeed, the loss of progenitors seen upon Svb REP expression in esg+ cells was accompanied by a strong increase in differentiated cells with large nuclei (Figure 5F), showing that Svb REP induces differentiation towards the EC fate.

To investigate the role of Svb in differentiated enterocytes, we next knocked it down using the EC-specific driver MyoIA ts (Jiang et al., 2009). svb depletion in ECs altered midgut organization, with a thinner epithelium and enlarged lumen (Figure 6A).

We also noticed elevated Dcp1 levels (Figure S6A) and ultra-structural analyses confirmed that svb knockdown induced cell death, as seen by pyknotic nuclei and impaired cell contacts (Figure 6C). We then assayed which Svb form was required in ECs, through expressing either Svb REP or OvoB with MyoIA ts . Intestines expressing Svb REP in ECs did not show any detectable homeostatic or structural changes (Figure S6B). In contrast, OvoB induced striking defects of the midgut, with abnormal multilayered intestinal wall and a reduced lumen (Figure 6A). Furthermore, OvoB caused the loss of Myo1A expression, indicating deeply compromised differentiation (Figure 6B), as also manifested by brush border defects seen in electron microcopy (Figure 6C).

Since the deregulation of Svb caused drastic phenotypes upon two weeks of treatment, the role of Svb in ECs was investigated upon shorter manipulation times ( 6days) to capture early defects. In these mild conditions, OvoB still impaired epithelium organization, with multilayered cells showing reduced apical actin accumulation (Figure 6A,D). OvoB-induced extra-intestinal cells also displayed altered apico/basal polarity revealed by misdistribution of Scribble, which marks the lateral domain (Chen et al., 2018), as well as of Tsp2a and Coracle that are components of septate junctions (Figures 6D andS6). The conclusion that Svb function in ECs relies on the unprocessed Svb REP predicted specific consequences, which we assayed directly. First, unlike intestinal stem cells, Svb function in ECs should be insensitive to Ubr3; accordingly, we detected no defects upon MyoIA ts -driven Ubr3 knockdown (Figure S6B). Second, the endogenous Svb protein should be converted into Svb ACT upon expressing pri in ECs, and Pri was sufficient to induce prominent defects reminiscent of the OvoB phenotype (Figure 6D).

We interpret these results to imply that the repressor form of Svb is required to maintain the proper differentiation of mature ECs in the adult midgut.

Svb allows the growth of genetically-induced ISC tumors

A main determinant of intestinal stem cell differentiation is the activation of Notch, suggesting that Svb REP may synergize with Notch to direct EC differentiation. As previously reported (Ohlstein and Spradling, 2007), inhibition of Notch induces tumor-like

Al Hayek et al.,p: 16 expansion of ISCs (Figure 7A). Concomitant svb knockdown reduced the expansion of Notch-induced tumors. Strikingly, Svb REP was sufficient to suppress Notch tumors and force their differentiation (Figure 7A). Conversely, Notch activation by expressing the Notch intracellular domain (NICD) in esg+ cells caused an acute decrease in progenitors, likely differentiating in ECs (Figure S7A). The co-expression of OvoB with NICD restored progenitor populations, whereas Svb REP enhanced the NICD phenotype (Figure S7A). We also found antagonistic effects of Svb ACT vs Svb REP on the phenotype of a dominant negative form of Delta, the Notch ligand in ISCs (Figure S7B). We next tested whether Svb could as well suppress tumors induced by additional signaling pathways. Indeed, Svb REP suppressed ICS-derived tumors resulting from the inactivation of JAK/STAT, or the activation of Wnt, in esg+ cells (Figures 7B and S7C,D). In addition, the depletion of svb in those tumors was sufficient to block their expansion in the midgut (Figure 7B).

Hence, svb is required for the growth of ISC-derived tumors and Svb REP acts as a tumor suppressor, overriding different signaling pathways to promote differentiation.

DISCUSSION

Our data show that the OvoL/Shavenbaby transcription factor is a key regulator of adult intestinal stem cells and their progeny. In stem cells, Svb acts as an activator sufficient to promote stem cell division, and mediating the proliferative activity of EGFR and Wnt pathways. Svb is also expressed in enterocytes, where the repressor form of Svb promotes differentiation. The balance between Svb ACT and Svb REP is gated by Pri smORF peptides, which allow proteasome-mediated conversion of Svb transcriptional activity, in response to ecdysone signaling. Our results further suggest that the transcriptional switch of OvoL/Svb factors is a conserved mechanism for the control of stemness.

svb integrates multiple regulatory cues for the homeostasis of adult stem cells

Numerous studies have demonstrated the role of Wnt and EGFR signaling in adult stem cells and cancers [START_REF] Normanno | Epidermal growth factor receptor (EGFR) signaling in cancer[END_REF][START_REF] Zhan | Wnt signaling in cancer[END_REF], but little remains known on their specific molecular targets. We find that ovo/svb is a common target of Wnt and EGFR pathways mediating their activity in intestinal stem cells, as previously shown in the embryonic epidermis (Payre et al., 1999). Our data further indicate that the nuclear mediators of Wnt (TCF) and EGFR (Pointed) directly regulate a svb stem cell enhancer (E) (Figure 7C). Although the register of Wnt activity in the midgut remains a matter of debate (Perochon et al., 2018), the main EGFR mitogenic pathway is ON in ISCs and OFF in ECs (Jiang and Edgar, 2009;Jin et al., 2015), explaining restriction of svb E enhancer activity in stem cells. In addition, Pdm1, a key TF for the EC fate that is repressed in ISCs (Korzelius et al., 2014;Micchelli and Perrimon, 2006;Ohlstein andSpradling, 2006, 2007), activates a distinct enhancer (7) to drive svb expression in Al Hayek et al.,p: 18 enterocytes (Figure 7C). Of note, these two svb enhancers display similar expression in the embryo (Frankel et al., 2010;McGregor et al., 2007), supporting the notion that they harbor pleiotropic functions across the life-cycle (Preger-Ben Noon et al., 2018). Hence, the regulatory logic of svb expression is rewired in the adult intestinal lineage, involving separate enhancers and regulators between stem cells and enterocytes.

Svb ACT is required for the maintenance and proliferation of stem cells

Svb function relies on post-translational control of its transcriptional activity. Svb processing into an activator is indispensable for the maintenance of stem cells, which otherwise undergo apoptosis. Adult stem cells are particularly resistant to apoptotic cell death, as shown for both intestinal and renal stem cells [START_REF] Ma | Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila[END_REF]. In the latter, we recently reported that Svb physically interacts with Yorkie (YAP/TAZ in mammals), the nuclear effector of Hippo signaling (Staley and Irvine, 2012), to directly activate DIAP1 expression [START_REF] Bohere | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF]. Although renal stem cells are mostly quiescent [START_REF] Bohere | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF]Xu et al., 2018), intestinal stem cells self-renew under homeostatic conditions and proliferate in response to various challenges. Consistently, Svb ACT is required and sufficient to promote intestinal stem cell proliferation, high Svb ACT in ISCs leading to hyperplastic over-growth (Figure 7C).

Svb REP triggers the differentiation of enterocytes

Our results also show the expression and function of Shavenbaby in enterocytes, but not in enteroendocrine cells, consistent with an early separation between EC and EE lineages (Biteau and Jasper, 2014;Guo and Ohlstein, 2015;Zeng and Hou, 2015). In contrast to stem cells, Svb acts as a repressor within ECs where it is required for their maintenance and differentiation. Ectopic processing into Svb ACT in ECs disrupts epithelial organization, leading to multilayered cells that loose key features of mature ECs, including MyoI1A expression, brush border microvilli, as well as proper apical/basal polarity and junctions.

Junctional complexes are established during EC maturation and they are essential for differentiation. For instance, Tsp2A (a component of septate junctions) is required for turning OFF Hippo and JAK/STAT in ECs, showing the link between EC maturation and the restriction of ICS proliferation [START_REF] Xu | The Septate Junction Protein Tsp2A Restricts Intestinal Stem Cell Activity via Endocytic Regulation of aPKC and Hippo Signaling[END_REF]. Indeed, Svb REP is a potent tumor suppressor, abrogating the growth of Notch, JAK/STAT, Wnt or EGFR-induced tumors and forcing differentiation towards the EC fate (Figure 7C). Therefore, Svb ACT and Svb REP exert antagonistic functions within the adult intestinal lineage, Svb ACT promoting stem cell proliferation and progenitor survival, while Svb REP later acts to induce and maintain enterocyte differentiation.

Ecdysone function in intestinal stem cells

The switch between Svb REP and Svb ACT is triggered by Pri peptides throughout the fly lifecycle [START_REF] Bohere | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF]Chanut-Delalande et al., 2014;Kondo et al., 2010;Zanet et al., 2015). pri is expressed in ISC/EBs but absent from ECs, and Pri peptides are required, together with their target Ubr3 ubiquitin ligase (Zanet et al., 2015), for stem cell maintenance. A key role of Pri is to mediate ecdysone signaling for developmental timing (Chanut-Delalande et al., 2014). We find that disrupting ecdysone signaling affects the behavior of intestinal stem cells, decreasing proliferation and promoting differentiation, OvoL/Svb transcriptional switch for stem cell control across animals

Mounting evidence suggests a wide role of the regulation of OvoL/Svb transcriptional activity in progenitor/stem cells. Svb is expressed and required in the posterior growth zone, a group of embryonic precursors that endow the sequential formation of posterior segments in most insects, and this ancestral Svb function relies on Pri and Ubr3 activities [START_REF] Ray | The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning[END_REF]. OvoL factors are also known for their conserved role in germ cell precursors (Hayashi et al., 2017). In flies, the germline-specific OvoB activator and OvoA repressor are produced from two alternative promoters. OvoB is required for the maintenance of germ cells, while OvoA acts for their proper differentiation [START_REF] Andrews | OVO transcription factors function antagonistically in the Drosophila female germline[END_REF]Hayashi et al., 2017). Precocious expression of OvoA leads to germ line loss (Andrews et al., 1998) and other ovo mutations cause ovarian tumors (Oliver et al., 1993).

Although relying on different mechanisms between soma (post-translational processing)

and germline (alternative promoters), the REP-to-ACT switch appears central to the function of Ovo/Svb factors in Drosophila.

Human OvoL factors are associated with many cancers, in particular those of epithelial origin, which often involve deregulated Wnt and EGFR signaling [START_REF] Normanno | Epidermal growth factor receptor (EGFR) signaling in cancer[END_REF][START_REF] Zhan | Wnt signaling in cancer[END_REF]. Our studies in flies reveal opposing effects of Svb ACT vs Svb REP , which promotes or suppresses stem cell-derived tumors, respectively.

Interestingly, OvoL2 isoforms display strikingly different effects within tumor xenografts in mice, in which only the repressor suppresses tumor development (Watanabe et al., 2014).

Al Hayek et al.,p: 21 OvoL/Svb have been proposed to act as general epithelial stabilization factors, counteracting epithelial to mesenchyme transition (EMT) (Lee et al., 2017;[START_REF] Watanabe | Mammary morphogenesis and regeneration require the inhibition of EMT at terminal end buds by Ovol2 transcriptional repressor[END_REF]. They have recently been identified as key factors for reprogramming mesenchymal fibroblasts towards induced pluripotent stem cells (iPSCs) (Kagawa et al., 2019) and epithelial lineages (Watanabe et al., 2019). EMT is induced by a core of transcription factors, including Snail and Zeb1-2 in mammals (reviewed in (Nieto et al., 2016)) and their fly homologs called Escargot and ZFh1-2 prevent ISC differentiation (Antonello et al., 2015;Korzelius et al., 2014;Loza-Coll et al., 2014). According to the model of an antagonism between EMT factors and OvoL/Svb, Svb REP promotes intestinal stem cell differentiation. However, our results draw a more complex picture, where Svb ACT should also act together with Esg, ZFh1-2 for the maintenance of ISCs. EMT is not an allor-none process and instead progresses through a series of reversible intermediate states between the epithelial (E) and mesenchyme (M) phenotypes (Nieto et al., 2016).

Such hybrid E/M phenotypes are hallmarks of normal and cancer stem cells, and relative doses of EMT factors and OvoL/Svb isoforms may provide a tunable window of stemness (Jolly et al., 2015).

Taken together, these data show the importance of OvoL/Shavenbaby factors in the control of adult stem cell behavior, in both normal and tumorous conditions. We propose that OvoL/Shavenbaby factors are ancestral regulators of epithelial organization across animals, explaining their key roles in development, differentiation and adult homeostasis.

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, François Payre (francois.payre@univ-tlse3.fr)

EXPERIMENTAL MODEL AND SUBJECT DETAILS Animal Breeding and Maintenance

Drosophila melanogaster flies were kept at 25°C and grown on a standard cornmeal food medium (per liter: 17 g inactivated yeast powder, 80 g corn flour, 9 g agar, 45 g white sugar, and 17 ml of Moldex). Crosses involving targeted expression under the control of Gal4/Gal80 ts were maintained at 18°C until 3 to 4 days post hatching, and adult females were sifted to 29°C for 10-14 days for an optimal activity of the UAS/GAL4 system. Flies were transferred to fresh food vials daily. For F/O and MARCM clonal analyses, 3-4 days adult female flies of the indicated genotypes were heat shocked 1 hour at 37ºC and then shifted to 25ºC for 10 days. The full genotype of each Drosophila sample is detailed below. D-F) control: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+ D) pri-IR: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+; UAS-pri RNAi E) EcR-DN: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts / UAS-EcRDN B2w650A E) Pri: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+; UAS-pri /+ E) EcR-DN+Pri: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /UAS-EcRDN B2w650A ; UAS-pri/+ F) Ubr3-IR: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /UAS-Ubr3 RNAi F) Ubr3-IR+OvoB: esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /UAS-Ubr3-RNAi; UAS-ovoB/+ G) control: y, w, hs-FLP, tubP-Gal80, FR19A/ FRT19A; UAS-mCD8::GFP/+; tubulin-Gal4/ry 506 G) ubr3 null : y, w, hs-FLP, tubP-Gal80, FR19A/ y, w, ubr3 B, FRT19A; UAS-mCD8::GFP/+; tubulin-Gal4/+ S1) were crossed with virgin females of stock w; ptc-Gal4; E3N-lacZ or w; ptc-Gal4; 9CJ2-lacZ and eggs were collected for 12 hr at 28°C. Embryos were dechorionated, fixed and stained using standard protocols [START_REF] Fernandes | Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes[END_REF], with mouse anti-ß-galactosidase (ß-gal; 1:500, Promega) and biotinylated goat anti-rabbit (1:1,000) antibodies, revealed using VECTASTAIN ABC Peroxidase Kit (Vector Laboratories). After washing, stained embryos were mounted in Glycerol/PBS (80/20%) and imaged using a Nikon Eclipse 90i microscope using NIS-elements software (Nikon). Each experiment (typical 200 embryos per genotype) were performed at least three times and also included a UAS-w-RNAi as positive control and w embryos served as negative controls. Reporter patterns upon RNAi treatment were classified into "no change", "reduced" or "ectopic" expression, the two latter were kept for additional characterization (Table S1). For rescuing assays, males carrying pRSQsvb constructs were crossed with females of stock y 1 , w* btd 1 , svb 1 /FM7-kr>GFP, allowing unambiguous phenotypical identification of svb-mutant embryos. First instar larva cuticles were prepared in Hoyer's/lactic acid 1:1, imaged with phase-contrast microscopy, and trichomes were counted in the ventral region of A4 segments.

Full Genotypes as They Appear in Each

DNA constructs and transgenic lines

DNA fragments from svb enhancers were cloned into placZAttB for reporter constructs and into pRSQsvb for rescue constructs [START_REF] Crocker | Low affinity binding site clusters confer hox specificity and regulatory robustness[END_REF]Frankel et al., 2011;Menoret et al., 2013). All plasmids were integrated using the PhiC31 system into the same attP landing site (zh-86F) by Bestgene (Chino Hills, CA, USA) or in the Payre lab. Putative binding sites for transcription factors were identified using JASPAR (http://jaspar.genereg.net) and their evolutionary conservation was assayed using multiple sequence alignments (Clustal Omega and MUSCLE, https://www.ebi.ac.uk) with orthologous regions from several Drosophila species (http://flybase.org). Site specific mutations were introduced using DNA synthesis by Genscript (https://www.genscript.com) and modified enhancers were sub-cloned in reporter and rescue constructs using ligation-free cloning (In-Fusion, Takara) with the same oligonucleotides than respective wild type versions. All constructs were verified by sequencing and DNA sequences of wild type and mutated enhancers are given (Table S2).

Immunofluorescence

Stage-15 embryos were processed using standard protocols, using with mouse anti-ß-galactosidase (ßgal; 1:500, Promega), rabbit anti-Dyl at 1:400 [START_REF] Fernandes | Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes[END_REF] Adult midguts were dissected in 1% PBS and fixed for up to 1h at room temperature in a fresh 4% paraformaldehyde solution (Electron microscopy Science) prepared in PBS 1%. Following three washes of 15 min each in 0.1% Triton-PBS (0.1% PBS-T), samples were blocked in 1% BSA-Triton-PBS for 30 min at room temperature, prior to overnight incubation with primary antibody at 4°C. The following primary antibodies were used: mouse anti-GFP, rabbit anti-GFP at 1:500 both, rabbit anti-ß-Galactosidase (MP Biomedicals) antibody at 1:1,000, rat anti-RFP (5F8) at 1:800, cleaved Drosophila Dcp-1 (Asp216) rabbit antibody at 1:100 (Cell signaling), mouse anti-Prospero (DHSB) at 1:100, rabbit anti-Phospho-Histone 3 (Millipore) at 1:1,000, mouse anti-Coracle at 1:100 (DHSB), rabbit polyclonal Anti-Tsp2A and rabbit polyclonal anti-Scribble (gifts from D. St Johnston) at 1:1,000 both. F-Actin was stained with Phalloidin conjugated to Rhodamine (Invitrogen, at 1:500). The next day, samples were washed 3 times in 0.1% PBS-T for 15 min each. Samples were next incubated with AlexaFluor-488 or -555 secondary antibodies at 1:500 (Molecular Probes) for 2 hours at room temperature. After three washes, tissues were mounted in Vectashield mounting media containing DAPI (Vector Laboratories) for nuclear staining. Images of the whole posterior midgut were acquired on Leica SPE and Leica SP8 confocal microscopes (X40 objective). 3 to 5 images were acquired for each posterior midgut to cover the 4a to 5 regions (Lemaitre and Miguel-Aliaga, 2013).

In situ hybridization RNA probes were synthetized from svb cDNA (Delon et al., 2003) using a DIG RNA Labeling Kit (Roche). Guts were dissected in a 1XPBS and fixed in freshly-prepared 4% formaldehyde 5Mm EGTA fix solution in PBS. After two washes in PBS, guts were dehydrated in successive baths of methanol (5 times), ethanol (5 times), followed by 1 hour in 1:1 xylene/ethanol, and rinsed in methanol. Guts were post fixed for 30 min at room temperature in the same fix solution and washed in PBS-0.1% Tween 20 (PBST). Samples were treated by proteinase K at room temperature, and the reaction was stopped by a 5 min treatment in 2mgl/ml glycine, followed by washes in cold PBST. Samples were incubated in the fix solution overnight at 4°C and washed in PBST. After 2 hrs pre-hybridization at 60°C in hybridization buffer (HB: 50% formamide, 2X SSC, 1 mg/ml Tortula RNA, 0.05mg/ml Heparin, 2% blocking reagent (Roche), 0.1% CHAPS, 5mM EDTA, 0.1% Tween 20), guts were incubated overnight with svb anti-sense DIG-labeled RNA probe diluted in HB, at 60°C. After several washes in HB and PBST and PBST-1% BSA (blocking solution), samples were incubated with sheep anti-DIG primary antibody conjugated to alkaline phosphatase (Roche), at 1:2,000 in blocking solution. After washes in PBST, in situ hybridization signals were developed by incubating samples in a fresh staining buffer containing NBT/BCIP stock solution (Sigma Aldrich) diluted at 1:50. Finally, samples were washed and mounted in 50% glycerol-PBS for analysis.

X-gal staining assays

Adult females were dissected in 1% PBS and guts were fixed in 1% glutaraldehyde-PBS for 15 min at room temperature. Samples were washed 3 times for 15 min each. The Staining Buffer (SB, 10mM Na2HPO4, 1.6mM NaH2PO4, 150mM NaCl,1Mm MgCl2, 3.5Mm K3FeCN6, 3.5Mm K4FeCN6) was warmed up at 37°C for 10 min, an 8% X-Gal solution (5-bromo-4-chloro-3-indoyl-ß-D-Galactopyranoside, Sigma Aldrich) was added (final concentration 2.5% X-gal in SB) and kept for an additional 10 min at 37°C, before centrifugation (5' at 12,000g). Samples were incubated in staining solution overnight at 37°C, washed 3 times for at least 15 min with PBS and mounted in 50% glycerol-PBS. Bright-field pictures were acquired using a Nikon Eclipse 90i microscope.

QUANTIFICATION AND STATISTICAL ANALYSIS

Images were analyzed by using ImageJ, with automated macros we developed for quantification of indicated cell types (codes are available upon request). Briefly, images were acquired with same setting and transformed into multilayered TIFF files. To count the number of cells positive for a given marker (e.g. GFP), the corresponding channel was used to generate a ROI mask in which DAPI-labelled nuclei were automatically segmented and the number, size and morphometric features of each object was recorded in and xls file for further analysis. A similar analysis was performed for ReDDM assays, quantifying the number of nuclei in GFP+/RFP+ progenitors versus in GFP-/RFP+ differentiated cells. Data of at least three independent experiments were combined. Statistical analyses were carried out with Prism 8 (Graphpad), using nonparametric Mann-Whitney tests for comparison between two samples, and ANOVA Kruskal-Wallis tests with Dunn's correction for comparison between three or more samples. In each figure, graphs show all individual points. Images were processed and figures drawn using Adobe Photoshop CC.

The regulation of adult stem cell behavior is a key issue to insure organ homeostasis throughout the adult life and avoid illness. Since 2006 and the discovery of Intestinal Stem Cells (ISCs) in flies (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006), Drosophila has become a fruitful model system to address this question [START_REF] Jiang | Intestinal stem cell response to injury: lessons from Drosophila[END_REF]Li and Jasper, 2016;[START_REF] Naszai | Intestinal stem cell proliferation and epithelial homeostasis in the adult Drosophila midgut[END_REF][START_REF] Parasram | Intestinal Stem Cells Exhibit Conditional Circadian Clock Function[END_REF]Perochon et al., 2018). In the course of these investigations, it emerged that stemness and the differentiation of ISCs are controlled by a broad range of regulatory inputs. ISC behavior is regulated by many signaling pathways acting at different levels, from intrinsic cellular signals, extrinsic cues from the close micro-environment or niche, to the organismic level via systemic signals. Our studies on the function of Shavenbaby (Svb) in the Drosophila digestive track (gut and Malpighian tubules) now provide a fresh view of how these regulatory cues are integrated and mediated through Svb.

The activity of the Svb transcription factor is regulated by its posttranslational processing, mediated by Pri smORF peptides (Chanut-Delalande et al., 2014;Kondo et al., 2010;Plaza et al., 2017;Zanet et al., 2015;Zanet et al., 2016). Svb is first transcribed as a long repressor form (SvbREP) and following the expression of Pri, cleaved into a shorter activator form (SvbACT). This, in turn, allows the transcription of a battery of target genes required for epithelial remodeling and trichome differentiation (Chanut-Delalande et al., 2006;[START_REF] Fernandes | Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes[END_REF]Kondo et al., 2010;Menoret et al., 2013). While no specific function for SvbREP has been so far disclosed in epidermal derivatives where it mostly behaves as an inactive precursor of SvbACT, an additional outcome of this PhD is to provide first evidence of key and specific activities of both isoforms in the intestinal stem cell lineage. Our work shows that SvbACT is required in ISCs for their survival and to promote their self-renewal, while SvbREP is later indispensable for enterocyte differentiation and acts as a potent tumor suppressor.

Svb belongs to the metazoan-specific family of Ovo-like transcription factors [START_REF] Kumar | Molecular phylogeny of OVOL genes illustrates a conserved C2H2 zinc finger domain coupled by hypervariable unstructured regions[END_REF]. In the light of recent data on the importance of these factors in epithelial/mesenchymal equilibrium [START_REF] Bai | Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium[END_REF][START_REF] Biswas | Stability and mean residence times for hybrid epithelial/mesenchymal phenotype[END_REF]Haensel et al., 2019;[START_REF] Hong | An Ovol2-Zeb1 Mutual Inhibitory Circuit Governs Bidirectional and Multi-step Transition between Epithelial and Mesenchymal States[END_REF][START_REF] Jia | OVOL guides the epithelial-hybrid-mesenchymal transition[END_REF][START_REF] Lee | Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells[END_REF][START_REF] Liu | Ovol2 induces mesenchymal-epithelial transition via targeting ZEB1 in osteosarcoma[END_REF]Wang et al., 2017;Watanabe et al., 2019;Watanabe et al., 2014) and related diseases (Davidson et al., 2016;Kitazawa et al., 2016), as well as in the metastatic potential of many cancers [START_REF] Liu | Ovol2 induces mesenchymal-epithelial transition via targeting ZEB1 in osteosarcoma[END_REF]Roca et al., 2013;Wang et al., 2017;Watanabe et al., 2014;Ye et al., 2016), we believe that besides improving fundamental knowledge our studies may have impacts in biomedical research.

As several aspects of our results have been discussed in the two presented papers, the aim of this final part is to present additional data and put our results in a more general frame.

Shavenbaby as a regulatory hub

A key question to understand animal development and the homeostasis of adult functions is to decipher the mechanisms that shape the regulation of gene expression. Developmental genes contain cis-regulatory sequences, called enhancers, localized upstream or downstream of the transcribed region, and/or within introns, which plays a major role in directing specific patterns of gene expression [START_REF] Furlong | Developmental enhancers and chromosome topology[END_REF][START_REF] Levine | Transcriptional enhancers in animal development and evolution[END_REF]. Enhancers correspond to relatively short DNA regions comprising multiple binding sites for transcription factors (TFs).

Depending on chromatin accessibility, enhancers are thus the landing platform of TFs that help recruiting general transcription complexes to initiate gene transcription. Unlike coding regions, the identification of enhancers still rely on tedious experimental assays of their activity that involve the generation of transgenic reporter lines. The general strategy is to hook putative enhancers to a minimal promoter and a reporter gene, the activity of which reflecting all or part of the endogenous gene expression profile. In collaboration with the lab of David Stern, our team has extensively used this approach, leading to the identification of 7 enhancers spread over 150 kb that collectively regulate svb expression to pattern epidermal cell differentiation [START_REF] Stern | The structure and evolution of cis-regulatory regions: the shavenbaby story[END_REF]. Studies of svb enhancers has contributed to establish important concepts for the control of gene expression, including the developmental function and evolution of enhancers, how they participate to phenotypic robustness in the face of varying conditions, and their pleiotropic activity throughout successive stages of animal development [START_REF] Lagha | Mechanisms of transcriptional precision in animal development[END_REF]McGregor et al., 2007;Preger-Ben Noon et al., 2018;[START_REF] Stern | The structure and evolution of cis-regulatory regions: the shavenbaby story[END_REF].

In the mid 2000s, new approaches such as ChIPseq have helped predicting putative enhancers based on the genome-wide pattern of binding sites for a transcription factor. These data revealed that developmental genes often contain two or more prominent binding peaks for a transcription factor, suggesting that a same gene may comprise distinct enhancers that drive similar expression pattern [START_REF] Zeitlinger | Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo[END_REF]. This idea of apparently redundant enhancers, sometime referred to as "shadow" enhancers [START_REF] Hong | Shadow enhancers as a source of evolutionary novelty[END_REF], has rapidly received support from independent studies and is now viewed as a common feature of developmental regulators across animals. The question of the putative function of such shadow enhancers has however remained opened for a while. Functional assays of the activity of svb enhancers in building the pattern of epidermal differentiation have provided first insights into the role of this "redundant" cis-regulatory architecture (Frankel et al., 2010). svb expression in subsets of dorsal embryonic epidermal cells is directly responsible for the formation of thin hairs, called quaternary trichomes. We had previously shown that the dorsal pattern of svb expression is driven by at least four distinct proximal and remote enhancers that display overlapping expression within corresponding epidermal cells (McGregor et al., 2007).

Genomic engineering has allowed the deletion of two remote dorsal enhancers and thereby a direct assay of their developmental activity (Frankel et al., 2010). Puzzlingly, the lack of these remote enhancers has no detectable consequences on the pattern of dorsal trichomes when embryo develop under optimal conditions. However, when subjected to either environmental challenges (lower or higher temperature), or slight genetic alterations that do not produce by themselves any developmental defects (e.g., heterozygous recessive mutation), animals carrying the deletion of remote svb enhancers display a reduced number of dorsal trichomes.

These results have led to the conclusion that shadow enhancers provide phenotypic robustness in the face of varying genetic or environmental conditions (Frankel et al., 2010). This conclusion rapidly received independent support from a work of M. Levine's lab focusing on snail function during early development. snail encodes a transcription factor required for ventral furrow formation during gastrulation. Snail expression in the corresponding cell populations is driven by a proximal enhancer, as well as a more distant shadow enhancer.

Inactivation of the proximal enhancer does not alter snail function in normal condition meaning that the activity of the two enhancers, the distant and the shadow, are redundant to ensure snail expression [START_REF] Perry | Shadow enhancers foster robustness of Drosophila gastrulation[END_REF]. However, in challenging conditions (extreme temperatures), there is a drastic reduction of snail expression associated with a defective gastrulation [START_REF] Perry | Shadow enhancers foster robustness of Drosophila gastrulation[END_REF]. This shows that the activity of the two enhancers is required in case of stress to ensure the robustness of snail expression. It also shows that there is no obvious functional hierarchy between "main" and "shadow" enhancers.

In the course of our investigations to address the function of svb in adult stem cells, we identified two distinct enhancers that sustain the expression of svb in both ISCs and RNSCs. It is thus tempting to speculate that the collective activity of both svb enhancers may be required to protect ISCs from apoptosis and to support their proliferation, in particular under stress conditions. We have shown that the activity of one enhancer is directly dependent on both Wnt and EGFR signaling and we can speculate that the other can be regulated by the same and/or other cues such as JAK/STAT signaling pathway that is required for ISC proliferation specifically under stress conditions. Moreover, changes in svb enhancers have collectively evolved the pattern of svb expression in various insect species, resulting in evolutionary diversification of embryonic morphologies [START_REF] Delon | Evolution of larval morphology in flies: get in shape with shavenbaby[END_REF]Sucena et al., 2003). For example, in Drosophila Sechellia, a closely related species to Drosophila melanogaster, naked cuticle replaces most of quaternary dorsal trichomes (McGregor et al., 2007;[START_REF] Sucena | Divergence of larval morphology between Drosophila sechellia and its sibling species caused by cis-regulatory evolution of ovo/shaven-baby[END_REF]. It has been demonstrated that this evolutionary change was caused by multiple subtle modifications in svb enhancer sequences (Frankel et al., 2011;McGregor et al., 2007;[START_REF] Stern | The structure and evolution of cis-regulatory regions: the shavenbaby story[END_REF]. Further work has shown that a main svb dorsal enhancer contains binding sites for two transcriptional activators Arrowhead and Pannier. In Drosophila sechellia, four of Arrowhead binding sites were lost by point mutations, thus weakening svb expression. Moreover, the same enhancer in Sechellia has gained a binding site for a transcriptional repressor, Abrupt, leading to the loss of its activity in dorsal cells (Preger-Ben Noon et al., 2016). It will be interesting to further dissect the cis and trans regulatory mechanisms that control svb expression in adult stem cells. To address the question of the robustness of svb expression in ISCs, ongoing project in the lab aims at deleting by CRISPR-cas9 one or the other, or both, enhancers to test their function and possible redundancy regarding stem cells behavior.

One outcome of my work was to identify that the expression of svb in stem cells and ECs is controlled by distinct enhancers and pathways, i.e., Wnt and EGFR and Pdm1, respectively.

Interestingly, the two ISC enhancers in the adults display different activities in the embryo, while the EC enhancer shows overlapping epidermal pattern. These results reinforce the pleiotropy activity of svb enhancers first identified by (Preger-Ben Noon et al., 2018).

Ovo/Svb as an EMT regulatory factor

Epithelial-Mesenchymal Transitions (EMT) is the process by which epithelial cells become gradually transformed to mesenchymal cells, by passing through multiple intermediate and reversible states (Nieto et al., 2016). EMT is required for normal embryogenesis, including gastrulation, neural crest development, heart development, as well as for wound-healing in adults where epithelial cells dedifferentiate in order to replace lost cells. Deregulated EMT is also involved in various human pathologies such as fibrosis and cancers. EMT implicates a multitude of events that should be finely coordinated. First, cells need to lose their epithelial properties, including cell adhesion and cell polarity. Second, they need to acquire mesenchymal features such as motility, invasiveness and increased resistance to apoptosis. Of note, EMT is reversible by its reciprocal process the mesenchymal-epithelial transition (MET) supporting the fact that the EMT is not an all-or-not event. A conserved core of regulators (Snail/Slug, Zeb1/2 and miR200s) is well known to induce EMT [START_REF] Puisieux | Oncogenic roles of EMT-inducing transcription factors[END_REF] in response to signaling pathways (BMP, Wnt, Egf-r, Notch) [START_REF] Nieto | Epithelial plasticity: a common theme in embryonic and cancer cells[END_REF]Nieto et al., 2016). Results from recent studies converge towards a key role of OvoL factors (the orthologues of Svb in vertebrates) as stabilization factors that counterbalance EMT [START_REF] Jia | OVOL guides the epithelial-hybrid-mesenchymal transition[END_REF], thereby providing a tunable stemness window in normal and tumor cells (Jolly et al., 2015). Indeed, OvoL factors have been implicated in the fate of various progenitor/stem cells [START_REF] Aue | A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion[END_REF][START_REF] Kim | OVOL2 is a critical regulator of ER71/ETV2 in generating FLK1+, hematopoietic, and endothelial cells from embryonic stem cells[END_REF]Kitazawa et al., 2016;[START_REF] Lee | Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells[END_REF]Watanabe et al., 2014). In mammals, OvoL factors appear as critical protectors of the epithelial state by directly inhibiting the Snail/Slug, Zeb1/2 and miR200s core of regulators. The majority of cancer cells that loose OvoL expression become extremely dangerous, with invasive characteristics and high metastatic potential [START_REF] Hong | An Ovol2-Zeb1 Mutual Inhibitory Circuit Governs Bidirectional and Multi-step Transition between Epithelial and Mesenchymal States[END_REF][START_REF] Ricketts | Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma[END_REF][START_REF] Roca | A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial -mesenchymal cell reprogramming and cancer progression[END_REF]Wang et al., 2017;Ye et al., 2016).

In the fly gut, the Svb repressor (SvbREP) form likely fulfils a similar function within differentiated enterocytes (ECs) and is required to maintain their differentiated state.

Conversely, Svb-Act induces ISC proliferation and disrupts epithelial organization when expressed within ECs. Interestingly, the germinal forms of Svb, OvoA and OvoB also acts antagonistically within germ cells (Andrews et al., 2000). OvoB is required to maintain germinal stem cells, whereas OvoA may favor full differentiation. In line with previous findings (Antonello et al., 2015), we find that the expression of the EMT inhibitor miR-8 (miR-200 in vertebrates) in ISC/EBs induces a complete loss of progenitors caused by premature analysis identified EcR binding sites in several enhancers of pri during pupal development, as well as in cultured S2 cells (Chanut-Delalande et al., 2014). We tested the effect of ecdysone on pri enhancer activity by depriving adult female flies bearing Pri-A , Pri-H or Pri-J from both food and water (desiccation). Interestingly, we observed an increase in the Pri-A enhancer activity. These data are in line with the findings of Azza Dib a former PhD student of the lab who identified EcR binding sites in Pri-A, but not in Pri-J or Pri-H. We have to confirm the increase in ecdysone levels by looking for Ecdysone responsive genes expression in desiccated flies compared to control (Figure 24).

In Drosophila embryonic epidermis the binding of the 20E on its receptor EcR in association with the cofactor Ultraspiracle (USP) induces the expression of two subtypes of target genes, called "early" genes that then regulate the expression of a subset of genes called "late genes".

Thus, it will be interesting to test whether these target genes (or novel ecdysone target genes) are also required downstream of EcR in order to maintain ISC number.

Conclusion

We can then hypothesis that in Drosophila adult midgut, a systemic hormonal cue (Ecdysone) is sensed specifically within ISCs in order to activate the expression of Pri peptides. This activity is crucial to insure an efficient equilibrium between two isoforms of Svb and, consequently, to maintain adult midgut homeostasis. This ongoing work can shed light on the link between systemic signals and intrinsic genetic networks that control stem cell behavior.

Résumé

L'intestin adulte est un organe très dynamique chargé des fonctions vitales. Bien que les cellules qui composent l'intestin soient quotidiennement perdues, l'homéostasie intestinale est maintenue tout au long de l'âge adulte grâce aux propriétés d'auto-renouvèlement et de différenciation des cellules souches intestinales (CSI). Dans ce travail nous montrons que le facteur de transcription OvoL/Shavenbaby (Svb) est nécessaire pour l'homéostasie intestinale des mouches adultes. Des travaux récents ont montré que Svb est d'abord traduit en tant qu'une longue protéine qui agit comme facteur répresseur de transcription (Svb-REP), puis post-traductionnellement transformé en facteur de transcription activateur plus court (Svb-ACT) en réponse aux petits peptides appelés Polished rice (Pri). Nous constatons que Svb est spécifiquement exprimé et transformé en isoforme activatrice dans les progéniteures intestinales adultes, c'est-à-dire les cellules souches intestinales et les entéroblastes (ISC/EB). Svb-ACT est nécessaire pour protéger les cellules souches de l'apoptose et suffisant pour favoriser leur prolifération. En effet, les tests d'épistasie génétiques révèlent que l'expression de Svb dans les CSI est activée par deux voies mitotiques principales, EGFR et Wnt. Nous avons identifié une région régulatrice de svb qui active son expression dans les CSI et les EB et démontré que son activité repose sur la liaison directe des effecteurs nucléaires des voies EGFR et Wnt. En outre, nous avons délimité un second enhancer de svb nécessaire pour l'induction de son expression dans les cellules différenciées absorbantes, les entérocytes (ECs). D'une manière intéressante on a constaté que c'est l'isoforme Svb-REP qui est exprimée et requise au sein des EC. Svb-REP induit la différenciation de l'EB en EC et est nécessaire pour maintenir la survie et la différenciation adéquate des ECs. Enfin, nous montrons que Svb-ACT est nécessaire à la croissance des tumeurs dérivées de la prolifération excessive des CSI et que Svb-REP est suffisant pour bloquer le comportement tumoral et conduire à la différenciation des CSI en EC. L'ensemble de nos données sur les mouches démontrent donc que l'expression contrôlée et la maturation du facteur de transcription OvoL/Svb jouent un rôle clé dans l'équilibre entre le maintien/prolifération des cellules souches et la différenciation des entérocytes dans l'intestin adulte.

Abstract :

The adult gut is a highly dynamic organ in charge of vital functions. Although cells that make up the gut are daily lost, intestinal homeostasis is maintained throughout adulthood due to self-renewal and differentiation properties of intestinal stem cells (ISCs). Here we show that the OvoL/Shavenbaby (Svb) transcription factor is required for adult gut homeostasis in flies. Recent work has shown that Svb is translated as a large sized repressor (Svb-REP), and then post-translationally processed in a shorter activator (Svb-ACT) in response to small peptides called Polished rice (Pri). We find that Svb is specifically expressed and processed into the activator isoform in adult gut progenitors, i.e. intestinal stem cells and enteroblats (ISC/EB). Svb-ACT is required to protect stem cells from apoptosis, and sufficient to promote their proliferation. Indeed, genetic assays reveal that Svb expression in ISCs is activated by two main mitogenic pathways, EGFR and Wnt. We identified an enhancer driving svb expression in gut progenitors and demonstrate that its activity relies on the direct binding of nuclear effectors of the EGFR and Wnt pathways. In addition, we delineated a second svb enhancer driving expression in differentiated enterocytes (ECs). Strikingly, we find that this is the Svb-REP isoform that is expressed and required within ECs. Svb-REP induces the differentiation of EB to EC and is required to maintain proper differentiation and survival of ECs. Finally, we show that Svb-ACT is required for the growth of ISC-derived tumors and that Svb-REP is sufficient to override deregulated signaling pathways, blocking tumorous behavior and leading to enterocyte differentiation. Taken together, our data in flies therefore demonstrate that controlled expression and maturation of the OvoL/Svb transcription factor plays a key role in the balance between stem cell maintenance/proliferation and enterocyte differentiation in the adult gut.

  tout ça. Anne, tu es adorable ! Jenny, toujours souriante, toujours joyeuse et surtout positive !! Merci pour ton support et pour tes conseils et bonne chance dans tes recherches des petits peptides !!!! Simon, Merci pour ton sourire et ta bonne humeur ! Tu répands une énergie positive !! Une pensée aux anciens membres de l'équipe : Serge, Azza, Ahmad et Jérôme !! Je vous remercie pour votre aide et pour vos encouragements !! En plus d'une bonne équipe, avoir des bons amis c'est crucial ! Et pas que pour endurer une thèse ! Cyril, ta présence m'était toujours agréable à la pause déjeuner ! Merci d'avoir fait l'effort de me souhaiter mon anniversaire en arabe et d'apprendre quelques mots (haddi aasabak :D) !! Bonne chance pour tes recherches de post-doc ! Rim (Alice), ma compatriote Libano-Française. A toi je dois mes remerciements les plus sincères ! Merci pour les balades au bord du Canal (on voulait y aller plus souvent, mais enfin c'est l'intention qui compte !). Tu étais toujours à l'écoute et tu as toujours réussi à me réconforter et à me rassurer que tout allait bien se passer ! Merci pour les discussions très intéressantes ! Merci infiniment d'avoir été là pour le choix de ma robe de mariée ! C'était une belle aventure et t'as bien assuré !!!! Christelle, mes remerciements ne pourront jamais être à la hauteur de ton grand coeur qui m'a apporté beaucoup de soutien au moment où j'en avais besoin ! Merci d'être présente à mes côtés ! Rhoda, merci de m'avoir accueillie et d'avoir fait le guide touristique au début de mon séjour à Toulouse ! Encore merci pour les soirées (gâteaux, pizza…) entre filles ! Et bonne chance pour ta nouvelle aventure à Berlin (ou ailleurs). Katia, mon amie d'enfance et mon futur voisine au Liban !!! Je me souviens comment tes yeux brillaient à chaque fois que je te parlais de mon projet de thèse ! Même si on ne parle pas tous les jours je veux que tu saches que je pense toujours à toi ! Merci ma Katia d'amour pour ta tendresse et ta précieuse amitié !!! Je sais que ça va être un peu compliqué pour toi d'être en France pour ma soutenance de thèse mais je serais évidemment au Liban pour la tienne !!! Raymonda, mon adorable cousine ! J'ai vécu des périodes si pénibles que plus d'une fois j'ai cru que jamais je ne verrai le bout du tunnel. A chacun de ces instants, tu étais là pour me consoler et me rappeler que je suis forte et que je peux y arriver ! Sache que tu m'as aidé beaucoup plus que tu ne le penses et je ne te remercierai jamais assez pour tout ce que tu as fait pour moi !  TF : Transciption Factor  TGF-β : Transforming Growth Factor-β  Ttk69 : Tramtrack 69  Ubr3 : E3-Ubiquitin protein ligase  Upd : Unpaired like protein  Usp : Ultraspiracle  UTR : Untranslated region  VM : Visceral muscles  Wnt/Wg : Wnt/Wingless  Wts : Warts  Yki : Yorkie  Zeb : Zinc finger E-box binding Homeobox 1

Figure 13 :

 13 Figure 13: The ovo/shavenbaby locus encodes three different protein isoforms ....................

Figure 14 :

 14 Figure 14 : Svb is required for trichome formation in Drosophila ............................................

Figure 15 :

 15 Figure 15: svb integrates multiple signaling pathways to define its spatial register...............

Figure 16 :

 16 Figure 16 : svb embryonic expression is governed by the collective activity of 7 enhancers ...

Figure 17 :

 17 Figure 17 : Svb regulates the expression of effector genes for trichome formation ................

Figure 18 :

 18 Figure 18 : Pri peptides induce proteasome-mediated processing of the Svb protein .............

Figure 19 :

 19 Figure 19: Ecdysone regulates the temporality of trichome formation by activating the expression of polished-rice .......................................................................................................

Figure 20 :

 20 Figure 20 : svb's shadow enhancers ensure the robustness of the trichome formation program in extreme temperature variations conditions ..........................................................

Figure 21 :

 21 Figure 21 : Svb-Act is required for the maintenance of ISC/EB undifferentiated state ............

Figure

  Figure 22 : svb target genes in ISC ...........................................................................................

  EE cells can be subdivided in four different classes, marked by the expression of specific neuropeptides named Allatostatine (Ast), Tachykinin (Tk) and diuretic hormones (DH) (Beehler-Evans and Micchelli, 2015). Class I (AstB+, AstC+) and class II (AstC+) EE cells are present in the anterior part of the posterior midgut, whereas classes III (AstA+, AstC+) and IV (Tk+, Dh+) are localized in the remaining part of the midgut. Stem cells of the adult Drosophila midgut have initially been identified by the Perrimon and

Figure 2 :

 2 Figure 2 : Adult midgut progenitors at the origin of ISC (A) During early larval development Adult Midgut Progenitors (AMPs) express the Notch ligand Delta (Dl) and divide symmetrically in order to increase their number. Later on, AMPs divide asymmetrically and give rise to Su (H)+ peripheral cells (PC), which serve as niche. PC cells inhibits AMP differentiation and induce their self-renewal. (B) During metamorphosis, PC cells degenerate and AMPs start to differentiate. (C) AMPs give rise to ISC that divide asymmetrically to a generate a new ISC and a committed EB (orange) or EEP (pink) progenitor, that will further differentiate into EE or EC cells, respectively.

Figure 3 :

 3 Figure 3 : Drosophila adult midgut regionalizationBased on anatomical features the midgut is divided into 5 different regions (R1-R5). These regions are further divided to 8 subregions containing EC cells with different morphologies (histological level). Gene expressing patterning has led to an additional level of regionalization (14 subregions )(Buchon et al., 2013). Marianes and Spradling demonstrated the presence of 9 different regions contained within the 14 subregions found byBuchon et al. (Marianes and Spradling., 2013). (Adapted from[START_REF] Buchon | All for one and one for all: Regionalization of the Drosophila intestine[END_REF]

Figure 4 :

 4 Figure 4 : Notch signaling pathway in Drosophila adult midgut In ISC Suppressor of Hairless (Su(H)) in association with Hairless bind to the regulatory region of Enhancer of split (E(spl)-C) and inhibit its expression. In the absence of E(spl)-C the proneural factor Daughterless (Da) is expressed and induces the expression of Delta, thus maintaining ISC properties. Delta binds to the Notch receptor expressed at the cellular membrane of EBs. Notch intra cellular domain (NICD) translocate to the nucleus where it destabilizes Hairless and binds to Su(H), which leads to E(spl)-C derepression, Da repression and EB differentiation(Bardin et al., 2010).

Figure 5 :

 5 Figure 5 : EE cell fate regulationThe achaete-scute complex (AS-C) required for prospero expression and subsequently EE cell production is transiently activated in dividing ISC (green) and leads to the generation of a new ISC and enteroendocrine progenitor cell (EEP)(light pink). EEP divide once before starting to accumulate prospero protein. In addition, the newly formed EEP inherits the protein Numb that inhibits Notch signaling and thus depresses AS-C genes. EE cell generation is negatively regulated by the transcriptional repressor tramtrack69 (Ttk69) that inhibits AS-C in parallel to Notch. Newly formed EE (dark pink) cells express Delta ligand and activates Notch in the apically located ISC that switches to the production of EC cells rather than EE. EE autoregulate their production also by secreting the ligand Slit that activates Roundabout receptor/leak receptor (Robot2/leak) present at the surface of ISC leading to the inhibition of prospero expression.

Figure 7 :

 7 Figure 7 : Canonical JAK/STAT signaling in Drosophila[START_REF] Arbouzova | JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions[END_REF].

Figure 6 :

 6 Figure 6 : Canonical Wnt signaling in DrosophilaThe β-catenin protein known as Armadillo (Arm) in Drosophila is a key effector in the Wnt signaling cascade. In normal conditions, β-catenin is kept in the cytoplasm where it is phosphorylated by a "destructive complex" composed of four kinases: Axin, Adenomatous polyposis Coli (APC), Glycogen Synthase Knase 3 (GSK3) and Casein Kinase 1α (Ck1α)[START_REF] Kishida | Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin[END_REF]. This phosphorylation induces the ubiquitination of β-catenin that is then targeted to proteasome degradation. Upon Wnt binding to Frizzled (FZD) and lipoprotein receptor related protein 6 (LRP6), the receptor complex is endocytosed and the protein Dishevelled (Dvl) form polymers at the cytoplasmic domain of the receptors. Together, LRP6 FZD and Dvl form the Wnt signalosome that sequestrates and inactivates the destruction complex. β-cat thus reaches the nucleus where it acts in collaboration with Pangolin (a.k.a. TCF/LEF) to induce the expression of Wnt target genes[START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/colon carcinoma[END_REF][START_REF] Morin | Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC[END_REF] (adapted from[START_REF] Clevers | The cancer stem cell: premises, promises and challenges[END_REF] 2012).

Figure 8 :

 8 Figure 8 : Overview of the Hippo pathway in Drosophila Adapted from (Taha et al. ; 2018)

Figure 9 :

 9 Figure 9: esg TF regulates both stemness and ISC differentiationWithin ISCs (green) and EBs (orange), Esg activates the transcription of stemness genes (Dl) and inhibits that of differentiation genes, Pdm1, Prospero (pros). High Esg levels in EBs leads to the formation of cellular protrusions that sense the external environment. The balance between Esg and miR8 triggers EC terminal differentiation. Within EBs, Esg also inhibits the expression of Amun, a negative repressor of Notch. Esg expression is inhibited by Pdm1 within ECs.

Figure 10 :

 10 Figure 10 : ISC regulation by neighboring cells and tissues ISC receive signals from both epithelial (EC : Enterocytes, EE: Enteroendocrine, EB : Enteroblast) and non epithelial sources ( VM : visceral muscles , basement membrane and enteric neurons). Some cells such as the EE regulate ISC activities indirectly via the VM.

Figure 11 :

 11 Figure 11 : The mammalian intestinal epithelium The adult mammalian intestine is organized into crypts and villi. The crypt is the proliferative zone containing two types of ISC, LGR5+ stem cells and +4 cells interspersed between Paneth cells. ISC give rise to transient activating cells (TA) that proliferate and give rise to differentiated cells in the villus zone. Villi contains 4 types of differentiated cells: Enterocytes, Tuft cells, Enteroendocrine cells and Goblet cells.

Figure 12 :

 12 Figure 12 : ISC implication in intestinal cancer ISC tumors are genetically induced within ISC by : Notch inhibition, EGFR activation, Wnt activation (APC loss/ Wg overexpression ) in addition to tumor suppressor genes inhibition. Additional stress signals (infection) induces EC death that induces ISC proliferation and tumor initiation. Dividing cells secrete autocrine signals that sustain tumor growth. Growing tumors invade the neighboring EC and induces their detachment. Detached EC activates the stress signal JNK and inhibits Hpo pathway and subsequently secretes paracrine signals (Upd2,Upd3). VM also secretes growth factors. Wnt activation induces Myc expression within ISC/EB. Myc activates Spitz that signals to EC where it activates EGFR signaling. EGFR signaling within EC induces Upd3 ligand release. Upd3 activates JAK/STAT signaling within ISC. Spitz and Stat together induces intestinal hyperproliferation. K-Ras oncogene is required for both tumor initiation and growth following APC LOF.

Figure 13 :

 13 Figure 13: The ovo/shavenbaby locus encodes three different protein isoforms Schematic representation of the ovo/svb locus, with encoded mRNAs and corresponding proteins. Coding sequences (CDS) are in blue and non-coding regions in white. The three isoforms contain four zinc fingers (gray circles) in addition to a transcription activation domain (green). Svb and OvoA contain an additional repression domain (pink) and Svb comprises a specific N terminal extension (light blue) encoded by the 1s exon (Delon et al. 2003).

Figure 14 :

 14 Figure 14 : Svb is required for trichome formation in Drosophila (A) Cuticle of freshly eclosed larvae where trichome belts underlines the third thoracic (t3) and all abdominal segment (a1-a8). (B) Closeup view of the wildtype A4 ventral cuticle, with 6 rows of epidermal trichomes.(C) Closeup of the same region in a svb mutant, which lack most trichomes(Payre et al., 1999).

Figure 15 :Figure 16 :

 1516 Figure 15: svb integrates multiple signaling pathways to define its spatial register (A) In ventral epidermal cells, Wg activates engrailed (En) and Hh expression in two rows of cells (the posterior compartment), Rhomboid (Rho) activates EGFR and Serrate (Ser) the Notch pathway. (B) svb integrates these singling inputs through different enhancers.(C) The regulation of svb expression by Wg, Hh, Notch and EGFR establishes the epidermal pattern of svb that prefigures the differentiation of trichomes (adapted fromPayre, 2004)

  approaches have highlighted the role of multiple TFs in the regulation of svb expression by binding to the E3-14 or the 7 enhancers. The main TFs of interest are the following: Pointed (Pnt, EGF signaling pathway), Scalloped (Sd, Hippo pathway), SoxNeuro (SoxN) and Dichaete (Sox factors) and Pangolin/dTCF (Pan, Wg signaling pathway) and Pdm1/Nubbin.

Figure 17 :

 17 Figure 17 : Svb regulates the expression of effector genes for trichome formationSvb activates the expression of a set of genes directly implicated in trichome formation by regulating: the actin cytoskeleton (singed, forked, wasp, shavenoid,....), the extracellular matrix production (miniature,...), the production of cuticle components and enzymes implicated in cuticle pigmentation/sclerotization (yellow,...) as well as other genes whose function has not yet been defined(Chanut-Delalande et al., 2006)

  proper cholesterol uptake and/or handling within the prothoracic gland[START_REF] Chanut-Delalande | Pri peptides are mediators of ecdysone for the temporal control of development[END_REF][START_REF] Enya | A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila[END_REF] and, thereby, for ecdysone production. Consistently, all other mutants deprived of ecdysone showed a similar trichome-less phenotype, which was previously overlooked because of poor cuticle differentiation. In Gst-E14 or other Halloween mutants, svb is normally expressed, but the expression of trichome effector genes is abolished. Thus the absence of ecdysone prevents the expression of pri, leading to the accumulation of SvbREP that never matures and explains the absence of trichome effectors expression. Importantly, the expression of pri in a mutant lacking ecdysone is sufficient to restore trichome formation, even though cuticle differentiation remains compromised(Chanut-Delalande et al., 2014). Finally, combining systematic characterization of pri enhancers and EcR ChIP-seq, the team demonstrated that pri is a direct, and major, target of ecdysone signaling[START_REF] Chanut-Delalande | Pri peptides are mediators of ecdysone for the temporal control of development[END_REF].

  The result section corresponds to two articles exploring for the first time Svb and Pri functions in adult stem cells. The first paper, for which the majority of experiments were done by Jérôme, focuses on adult Malpighian tubules. The second preprint describes the major results I obtained during my thesis, concerning the function of Svb/Pri in the Drosophila adult midgut.

Figure 19 : 1 ARTICLE

 191 Figure 19: Ecdysone regulates the temporality of trichome formation by activating the expression of polished-rice (A) Transient peaks of ecdysone controls the temporality of developmental transitions for hatching at each larval stage and the initiation of metamorphosis. (B) Ecdysone bound EcR activates the expression of polished-rice(pri). This triggers the production of Pri peptides which allow Ubr3/proteasome-dependent maturation of Svb. Svb, transcribed as a long representative (SvbREP), is converted into a transcriptional activator (SvbACT) to initiate the genetic program of trichome formation (fromZanet et al., 2016)

Fig. 1 7 Fig. 2

 172 Fig.1svb is specifically expressed in renal stem cells. a Adult Malpighian tubules are composed of three types of cells. Principal cells (PC) are identified by immunostaining against Cut (cyan) and stellate cells (SC) by tsh-LacZ (red). RNSCs, located in the lower tubules, express esg-Gal4, UAS-GFP (green). b 3D reconstruction of the fork region of Malpighian tubules, with esg-positive RNSCs in green, immunostaining against Discs Large (Dlg) and Cut in yellow and blue, respectively. c Expression of svb and esg as monitored by co-staining for svb-E-GFP (green) and esg-LacZ (red) enhancers, respectively. Nuclei were counterstained with DAPI (blue). Scale bar is 30 µm

Fig. 3

 3 Fig. 3 Processing of Svb is essential for RNSC maintenance. a Schematic representation of Svb maturation, as well as the germinal isoforms OvoA and OvoB that act as constitutive (pri-independent) repressor and activator, respectively. b Expression of pri monitored by the activity of pri (tal)-Gal4 driving the expression of H2B::RFP (red). Nuclei (DAPI) are in blue. c Fork region of Malpighian tubules, with esg ts -driven expression of GFP and the indicated transgenes, after 8 days of induction. d MARCM clones (GFP-positive) of control and ubr3 B cells, 25 days after induction. e Quantification of the number of esg+ cells per tubule for the indicated genotype. Values are presented as average ± standard error of the mean (SEM) and boxes with whiskers (10-90 percentile). Unless otherwise indicated, p values correspond to comparison with control conditions: OvoB (p = 0.4899), OvoA (p < 0.0001), EcRDN (p < 0.0001), pri-RNAi (p < 0.0001); OvoB (p = 0.118), ubr3-RNAi (p < 0.0001), ubr3-RNAi vs ubr3-RNAi + ovoB (p < 0.0001). p-Values from Mann-Whitney test (ns, P > 0.05; ***P < 0.001). Scale bar is 30 µm

Fig. 4

 4 Fig. 4 svb protects RNSCs from apoptosis. a Lineage-tracing experiments (esg-ReDDM) at 8 days after induction. While stem cells (esg-positive) express both mCD8::GFP (green) and H2B::RFP (red), only the very stable H2B::RFP protein persists in their progeny (esg negative). Nuclei are in blue (DAPI). b Rescue of svb-depleted RNSCs by p35. esg ts was used to drive the expression of indicated transgenes (together with GFP), during 8 days. Quantification of esg+ cells is shown at the right. Ctrl vs p35 (p = 0.6613), svb-RNAi (p < 0.0001), p35 + svb-RNAi (p = 0.0006), svb-RNAi vs p35 + svb-RNAi (p < 0.0001). c TUNEL assays (red) revealing the increase in RNSC apoptosis upon expression of the pro-apoptotic gene reaper (Rpr, p < 0.0001), or RNAi depletion of svb (p = 0.0002), driven by esg ts > GFP (green). Renal stem cells undergoing apoptosis (green cytoplasm and red nuclei) are boxed, and better seen in closeups. The apoptotic index was calculated as the number of TUNEL-positive cells/GFP-positive cells (×100). Values are presented as average ± standard error of the mean (SEM) and boxes with whiskers (10-90 percentile). p-Values are calculated using Mann-Whitney test (***p < 0.001). n = number of observed tubules. Scale bar is 30 µm, except in closeup panels (10 µm)

Fig. 5

 5 Fig. 5 svb is a member of the Hippo pathway. a Pictures of Malpighian tubules with esg ts -driven expression of GFP (control) and indicated transgenes, at 8 days (8d) or 32 days (32d) after induction. b Quantification of esg-positive cells in corresponding genotypes. Values are presented as average ± standard error of the mean (SEM) and boxes with whiskers (10-90 percentile). Unless otherwise indicated, p values correspond to comparison with control conditions in a same series of experiments, as estimated by Mann-Whitney test (ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). Scale bar is 30 µm

Fig. 6 8 NATUREFig. 7 Fig. 8

 6878 Fig. 6 Svb interacts with Yki to regulate DIAP1 expression. a Drawing of the DIAP1 locus. Exons are represented in cyan, the 4.3 enhancer in dark green and the insertion site of the DIAP1-LacZ reporter is in red. In vivo ChIP-seq profiles and MACS peaks bound by Svb and Yki are indicated in green and magenta, respectively. b esg ts was used to drive the expression of yki, svb REP (ovoA), and yki together with svb REP in RNSCs. The expression of DIAP1 was followed by the activity of DIAP1-lacZ (X-Gal staining, top panel). Expression of the 4.3-GFP enhancer of DIAP1 (bottom panel) was followed by immuno-staining against GFP (green) and Hindsight (Hnt, red) revealing the influence of Yki and/or OvoA on RNSCs. Scale bar is 30 µm

  chloro-3indoyl-ß-D-Galactopyranoside). The X-Gal solution used to reveal the ß-Galactosidase activity was: 10 mM NaH 2 PO 4 •H 2 O/NA 2 HPO 4 •2H 2 O (pH = 7.2), 150 mM NaCl, 1 mM MgCl 2 •6H 2 O, 3.1 mM K4 (FeII(CN)6), 3.1 mM K3 (FeIII (CN)6), 0.3% Triton X-100. Bright-field pictures were acquired using a Nikon Eclipse 90i microscope.

  ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07569-0 10 NATURE COMMUNICATIONS | (2018) 9:5123 | DOI: 10.1038/s41467-018-07569-0 | www.nature.com/naturecommunications

Figure 1a :Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5

 1a5555555 Figure 1a: y, w/w; esg-Gal4, UAS-GFP/ tsh-LacZ Figure 1b: y, w/w; esg-Gal4, UAS-GFP Figure 1c: y, w/w; esg-LacZ/+; svb E3N -GFP/+

Figure 5

 5 Figure 5 yki+ ovoA: y, w/w; esg-Gal4, UAS-GFP/ +; tubulin-Gal80 ts / UAS-yki, UAS-ovoA.

Figure 5 FigureFigure

 5 Figure 5 DIAP1+ svb-RNAi: y, w/w; esg-Gal4, UAS-GFP/ +; tubulin-Gal80 ts / UAS-DIAP1, UAS-svb-RNAi

  expression is directly activated by ecdysone(Chanut-Delalande et al., 2014), the main AlHayek et al., p: 5

  promotes differentiation toward ECs, where it is required to maintain functional organization of mature cells. Two separate enhancers drive svb expression in the stem cell lineage, and we performed large scale screening to identify the nuclear factors required for their activity in ISCs versus ECs. Our data indicate that svb expression in stem cells integrate direct inputs from Wnt and EGFR pathways, while svb expression in ECs is controlled by the OCT/POU factor Pdm1, renowned for its key role in enterocyte differentiation. Our results show how the Svb/Pri axis integrates multiple signaling pathways and transcription factors to coordinate ISC survival, renewal and differentiation.AlHayek et al., p: 7

  i.e., as seen upon inhibition of Svb processing. The expression of pri, or of a constitutive Svb activator, can compensate compromised ecdysone signaling, showing that the regulation of Svb activity is a key target of hormonal control in intestinal stem cells. The role of ecdysone in midgut homeostasis remains to be fully elucidated (Miguel-Aliaga et AlHayek et al., p: 20 al., 2018), but it is tempting to speculate that ecdysone may contribute to adapt stem cell behavior in the face of various environmental conditions throughout the adult life.

Figure 1 :

 1 Figure 1: svb is expressed in ISC/EBs and is required for their maintenance (A) Schematic representation of the svb locus, with position of main enhancers (A, E, 7). (B) Expression of the E3N svb enhancer (GFP, green) in ISC/EBs, as shown by co-staining with esg-lacZ (β-Gal, red). (C,C') Expression of Dl-lacZ (purple) in control and esg ts >svb-RNAi. Close ups show separate channels for GFP (green) and Dl-lacZ. (C); quantification esg+ progenitors (C'). (D,D') Effect of svb-RNAi driven by ISC ts (D); quantification of YFP+ cells (D'). (E,E') esg ts >svb-RNAi in enteroblasts marked by Su(H)-lacZ (red) and esg ts >mCherry; (green) (E); quantification of Su(H)+ EBs (E'). (F) Act ts F/O clones (marked by GFP, green) of control and svb-RNAi cells. (G) Control and svb R9 null mutant MARCM clones, labeled by GFP (green). (H) Staining for the apoptotic marker DCP1 (red) in control and svb-RNAi esg ts F/O clones (GFP positive cells, green). (I) Quantification of GFP+ cells per posterior midgut in control esg ts and >DIAP1, svb-RNAi, svb-RNAi+DIAP1. In this and all other figures, non-parametric two-tailed Mann-Whitney tests were used to compare two conditions, and Kruskal-Wallis tests for three or more conditions. P values are ns: p>0.05, ***: p<0.001, ****: p < 0.0001. DAPI is blue, scale bars, 20µm, except in B, C and E close ups (10µm). See also Figure S1.

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3 AFigure 4 A

 34 Figure 3A) control: esg-Gal4, UAS-GFP.nls, tubP-Gal80 ts /+; Delta-LacZ/+ A) OvoB: esg-Gal4, UAS-GFP.nls, tubP-Gal80 ts /+; Delta-LacZ/UAS-ovoB B) control: esg-Gal4, UAS-YFP/+; Su(H)-GBE-Gal80, tubP-Gal80 ts /+ B) OvoB: esg-Gal4, UAS-YFP/+; Su(H)-GBE-Gal80, tubP-Gal80 ts /UAS-ovoB B) Svb+Pri: esg-Gal4, UAS-YFP/UAS-pri; Su(H)-GBE-Gal80, tubP-Gal80 ts /UAS-Svb

Figure 6 AFigure 7 A

 67 Figure 6A-C) control: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+ A-C) svb-IR: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-Svb RNAi /+ A-C) OvoB: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-OvoB/+ D) control: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-mCherry RNAi /+ D) svb-IR: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-Svb RNAi /+ D) OvoB: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-OvoB/+ D) Pri: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-Pri/+

Figure S6 A

 S6 Figure S6A) control: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+ A) Ubr3-IR: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /UAS-Ubr3 RNAi A,C) Svb REP : Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-Svb REP /+ A,C) Pri: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-pri/+ B) Svb-IR: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-svb RNAi /+ B) OvoB: Myo-Gal4, UAS-GFP, tubP-Gal80 ts /+; UAS-ovoB/+

Figure 20 :

 20 Figure 20 : svb's shadow enhancers ensure the robustness of the trichome formation program in extreme temperature variations conditions (A)Respective domain of activity of the "pioneer" enhancers E3, E6 and A as well as the "shadow" enhancers Z and DG2 in the fourth dorsal abdominal segment (quaternary domain).(B)Schematic representation of the effect of temperature fluctuationon the trichome formation program of the fourth abdominal dorsal segment of wild Drosophila larvae (wild type shavenbaby)and those whose DG2 and Z shadow enhancers have been deleted (Deletion of DG2 and Z in the svb gene) that have developed under normal conditions (25°C) or exposed to extreme temperatures (17°C or 32°C)[START_REF] Frankel | Conserved regulatory architecture underlies parallel genetic changes and convergent phenotypic evolution[END_REF]

Figure 21 :

 21 Figure 21 : Svb-Act is required for the maintenance of ISC/EB undifferentiated state Confocal images of the posterior midgut of control female flies , flies expressing mir-8, OvoB or both in esg+ cells. Green is GFP, Red is armadillo, Blue is DAPI. Scale bar : 20 µm.

Figure 22 :

 22 Figure 22 : svb target genes in ISC Representative images of three svb target genes expressed in adult ISC: CG4914, CG31022, SnE1 and SNE1 mutant for Svb binding sites.

Figure 23 :

 23 Figure 23 : The effect of desiccation on Pri-A and Pri-J activity in ISC Representative images of the Pri-A and Pri-J activities in normal (fed) or stressed conditions (starved), assessed by X-GAL assay.

  1,2,3 , Jennifer Zanet1 , Philippe Valenti 1 , Kohsuke Akino 4 , Yuya Yamabe 4 , Sachi Inagaki 5 , Hélène Chanut-Delalande 1 , Serge Plaza1,6 , Yuji Kageyama4,5 , Dani Osman 2,3 , Cédric Polesello 1 & François Payre 1To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

	DOI: 10.1038/s41467-018-07569-0	OPEN

1 Centre de Biologie du Développement (CBD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Bat 4R3, 118 route de Narbonne, F-31062 Toulouse, France.

2

Faculty of Sciences III, Lebanese University, Tripoli 1300, Lebanon.

3

Azm Center for Research in Biotechnology and its Applications, LBA3B, EDST, Lebanese University, Tripoli 1300, Lebanon.

4

Department of Biology, Graduate School of Science, Kobe 657-8501, Japan.

5

Biosignal Research Center, Kobe University, 1-1 Rokko-dai, Nada, Kobe 657-8501, Japan.

6

Present address: Laboratoire de Recherche en Sciences Végétales (LSRV), CNRS, UPS, 24 chemin de Borde Rouge, Auzeville, 31326 Castanet-Tolosan, France. Correspondence and requests for materials should be addressed to C.P. (email: cedric.polesello@univ-tlse3.fr) or to F.P. (email: francois.payre@univ-tlse3.fr) NATURE COMMUNICATIONS | (2018) 9:5123 | DOI: 10.1038/s41467-018-07569-0 | www.nature.com/naturecommunications 1 1234567890():,;

METHODS DETAILS In vivo screening of transcription factors We

ts /UAS-Dl-DN; UAS-OvoB/+ B) Dl-DN+Svb REP : esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts /UAS-Dl-DN; UAS-Svb REP /+ B) Dl-DN+svb-IR: esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts /UAS-Dl-DN; UAS-svb RNAi /+ C) Wg: esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts /UAS-Wg C) Wg+OvoB: esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts /UAS-Wg; UAS-ovoB/+ C) Wg+Svb REP : esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts UAS-Wg; UAS-Svb REP /+ C) Wg+Svb-IR: esg-Gal4, UAS-mCD8::GFP, tubP-Gal80 ts /UAS-Wg; UAS-svb RNAi /+ D) Arm s10 : UAS-Arm s10 /+; esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+ D) Arm s10 +pri-IR: UAS-Arm s10 /+esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+; UAS-pri RNAi /+ D) Arm s10 +Svb REP : UAS-Arm s10 /+; esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+; UAS-Svb REP /+ D) Arm s10 +svb-IR: UAS-Arm s10 /+; esg-Gal4, UAS-mDC8-GFP, tubP-Gal80 ts /+; UAS-svb RNAi /+ selected transcription and nuclear factors showing expression in stage-15 embryos

(Menoret et al., 2013)

or enrichment in dorsal trichome cells

(Preger-Ben Noon et al., 2016)

, resulting in a list of 227 candidate factors. 273 representative UAS-RNAi lines were obtained from Bloomington and VDRC stock centers, taken from the TRIP or VDRC collection, respectively. Males from each UAS::RNAi carrying line (Table

  , rabbit anti-Min 1:200 (Chanut- Delalande et al., 2006) antibodies, AlexaFluor-488 or -555 secondary antibodies at 1:500 (Molecular Probes). Embryos were mounted in Vectashield mounting media (Vector Laboratories) and imaged using X20 and X40 objectives on a Leica Spe confocal laser scanning microscope with Leica Application Suite software, or a Zeiss 710 confocal microscope using the ZEN software (Zeiss).

NATURE COMMUNICATIONS | (2018) 9:5123 | DOI: 10.1038/s41467-018-07569-0 | www.nature.com/naturecommunications

Acknowledgements

We are grateful to FlyBase, Bloomington and Vienna stock centers, Developmental Studies Hybridoma Bank, Drosophila Genomics Resource Center, supported by NIH grant 2P40OD010949, as well as N. Tapon, J. Colombani, K.F. Harvey, D.J. Pan, J. Dow, H. Skaer, M. Crozatier, K. Irvine, A. Bergmann, and M. Dominguez, for providing flies, antibodies, and molecular reagents. We thank B. Ronsin (Toulouse RIO Imaging) for help with microscopy, A. Alsawadi, A. Dib, and M. Soulard for experimental support. We also thank all members of the Payre lab for critical reading of the manuscript. This work was supported by ANR (Chrononet), Fondation pour le Recherche Médicale (Equipe FRM DEQ20170336739), Agence Universitaire de la Francophonie (PCSI AUF-BMO) for D.O. and F.P., PRES IDEX Université de Toulouse for C.P., and by MEXT KAKENHI (JP26113006) for Y.K. J.B. was supported by fellowships from "Ministère de l'Enseignement et de la Recherche" and "La Ligue contre le Cancer". S.A.H. and A.M.-F. were supported by fellowships from "FRM" and "Ligue contre le Cancer", respectively.

ACKNOWLEDGMENTS

We would like to thank the members of the FP lab and G. Kolahgar for invaluable help and comments on the manuscript. We are grateful to M. Bou Sleiman and Z. Zhai for discussions and critical reading of the manuscript. We thank P. Valenti for excellent assistance in the early stages of this work, O. Bohner, J. Favier and A. Destenabes for help in generating transgenic lines, S. Bosch and other members of the LITC platform (https://www-litc.biotoul.fr/) for imaging. We thank A. Bardin, H. Bellen, J.P. Couso, A. Debec, M. Dominguez, B. Edgar, N. Frankel, D. Stern, D. St Johnston and N. Tapon for providing fly stocks and reagents, and the Bloomington Drosophila Stock Center, Kyoto Drosophila Genomics and Genetic Resources, Vienna Drosophila Resource Center, Flybase and Developmental Studies Hybridoma Bank for key data and materials. SAH was supported by Fondation pour la Recherche Médicale and Communauté des Communes de Dannieh-Université Libanaise. This work was supported by Agence Universitaire de la Francophonie (PCSI AUF-BMO), École Doctorale des Sciences et de Technologie-Université Libanaise, the Federation of European Biochemical Societies, American University of Beirut URB, Agence Nationale de la Recherche (ANR, ChronoNet) and Fondation pour la Recherche Médicale (DEQ20170336739).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

spl)-C : Enhancer of split  FZD : frizzled  GSK3 : Glycogen Synthase Kinase 3  GOF : gain of function  Hes : Hairy Enhancer of Split  Hh : Hedgehog  Hpo : Hippo  20E : 20 hydroxy-ecdysone 11  IIS : Insulin / IGF signaling pathway  ISCs : Intestinal Stem Cells  JAK/STAT : Janus Kinase/signal transducer and activator of transcription  Kr : Krüppel  Lgr5 : Leucin-rich G protein-coupled receptor 5  LOF : loss of function Renal Nephric Stem Cell  Robot2/leak : Roundabout receptor / leak receptor  Sav : Salvador  SC : Stellate Cell  scrib : scribble  Sd : Scalopped  SMAD : Supressor of Mothers Against Decapentaplegic  SmORF : small open reading frame  SoxN : SoxNeuro  Su(H) : Supressor of Hairless

Data availability

The ChIP-seq datasets analyzed during the current study are available in the EMBL-EBI repository: https://www.ebi.ac.uk/biosamples/samples/ SAMEA2439952, https://www.ebi.ac.uk/biosamples/samples/SAMN01041482, https://www.ebi.ac.uk/biosamples/samples/SAMN02231205, https://www.ebi.ac. uk/biosamples/samples/SAMN02231213, https://www.ebi.ac.uk/biosamples/ samples/SAMN02231206. The data that support the findings of this study are given in Supplementary Table 1. Detailed scripts and computer code used for ChIP-seq analysis are available from the corresponding author on request.

Abbreviations

Results

Summary of paper 1

At the beginning of my thesis, Jérôme Bohère a former PhD student in our laboratory was working on the characterization of Svb function within Renal Nephric Stem Cells (RNSC). This work and the one in the gut have emerged following initial observations made by Dani , who originally found the expression of Svb in both intestinal and renal adult stem cells.

Jérôme has demonstrated that the mature SvbACT, resulting from Ubr3-Pri-proteasomal processing, is required for protecting RNSC from apoptosis.

Svb physically interacts with Yorkie (Yki), the nuclear factor of the Hippo pathway well known to control the proliferation/apoptosis balance. The SvbACT/Yki complex directly binds to a regulatory region of Drosophila Inhibitor of Apoptosis, DIAP1 and induces its expression in stem cells. Jérôme's work has therefore revealed a new function of Svb in the Hippo signaling pathway and a way of protecting somatic stem cells to cope with apoptosis.

Supplementary Information

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis Jérôme Bohère 1 , Alexandra Mancheno-Ferris 1 , Sandy Al Hayek 1,4,5 , Jennifer Zanet 1 , Philippe Valenti 1 , Kohsuke Akino 2 , Yuya Yamabe 2 , Sachi Inagaki 3 , Hélène Chanut-Delalande 1 , Serge Plaza 1 , Yuji Kageyama 2,3 , Dani Osman 4,5 , Cédric Polesello 1* & François Payre 1 * * Correspondence and requests for materials should be addressed to C.P. or F.P.

(email: cedric.polesello@univ-tlse3.fr, francois.payre@univ-tlse3.fr ) Disrupting renal homeostasis in the adult through expression of tsh-RNAi in stellate cells (c724-Gal4 driver) reduces their number and favour mitotic figures, as followed by PH3 staining. Flies were kept at 18°C before hatching and dissected after 4 days at 29°C. Values are presented as average +/- standard error of the mean (SEM) and boxes with whiskers (10-90 percentile). ). P-values from Mann-Whitney test are <0.0001 (***). (c) G-TRACE lineage-tracing experiments performed with esg-Gal4 and dome-MESO-Gal4, two drivers specific of RNSCs, or Aph4-Gal4 that drives specific expression in neighbouring principal cells located in lower tubules. In G-TRACE experiments, the nuclear DsRed protein (Red-Stinger, red) that is expressed under the control of UAS sequences accumulates in cells in which the Gal4 driver is active. The nuclear GFP protein (Stinger, green) -controlled by a flp/FRT cassette- is expressed both in progenitors (Gal4-positive cells) and in their progeny (Gal4-negative cells). Many G-TRACE progeny cells are visible in the lower tubule region (white arrows) for both RNSC drivers, while we never observed progeny from Aph-Gal4. Scale bar is 50 µm (d) Closeup of esg G-TRACE experiments, focusing on the region of lower tubules. Progeny cells (GFP positive, RFP negative) are highlighted by white arrowheads. On the right panel, tubules were processed for anti-Cut staining (blue), revealing that RNSC progeny differentiate into large polyploid cells principal cells, characterized by high levels of Cut. Flies were raised at 18°C until hatching and kept 21 days at 25°C before dissection. Scale bar is 10 µm. Supplementary Methods

This file includes:

Fly stocks

The following Drosophila melanogaster stocks were used in this study: tsh-LacZ (BL#11370), esg-lacZ (BL#10359) Aph4-LacZ (BL#12285), esg-Gal4, UAS-GFP; tubulin-Gal80 ts / SM6-TM6B 1 (B. Edgar), y, w, hsFLP, tubulin-Gal80 FR19A; UAS-mcd8::GFP/Cyo; tubulin-Gal4/TM6B,Tb (N. Tapon), esg-Gal4,UAS-mcd8::GFP/Cyo; UAS-H2B::RFP, tubulin-Gal80 ts /TM2 2 (M. Dominguez), col-Gal4, UAS-mcd8::GFP/Cyo and dome-MESO-Gal4 (M. Crozatier), GMR-Gal4/Cyo (BL#9146), tal-Gal4/TM3, Sb (J.P. Couso), c507-Gal4

Supplementary References

Summary of paper 2

Here we show that the OvoL/Shavenbaby (Svb) transcription factor is required for adult gut homeostasis in flies. We find that Svb is specifically expressed and processed into the activator isoform in adult gut progenitors, i.e. intestinal stem cells and enteroblats (ISC/EB). Svb-ACT is required to protect stem cells from apoptosis, and sufficient to promote their proliferation.

Indeed, genetic assays reveal that two main mitogenic pathways, EGFR and Wnt, activate svb expression in ISCs. We identified an enhancer driving svb expression in gut progenitors and demonstrate that its activity relies on the direct binding of nuclear effectors of the EGFR and Wnt pathways. In addition, we delineated a second svb enhancer driving expression in differentiated enterocytes (ECs). Strikingly, we find that this is the Svb-REP isoform that is expressed and required within ECs. Svb-REP induces the differentiation of EBs to ECs and is required to maintain proper differentiation and survival of ECs.

Finally, we show that Svb-ACT is required for the growth of ISC-derived tumors and that Svb-REP is sufficient to override deregulated signaling pathways, blocking tumorous behavior and leading to enterocyte differentiation.

Taken together, our data in flies therefore demonstrate that controlled expression and maturation of the OvoL/Svb transcription factor plays a key role in the balance between stem cell maintenance/proliferation and enterocyte differentiation in the adult gut.

SUMMARY

AUTHOR CONTRIBUTIONS

The project was initially setup by DO, SP and FP.

DECLARATION OF INTEREST

The authors of this manuscript declare no competing interests. In all panels GFP+ cells are in green, anti-Prospero staining is in red and DAPI in blue. Scale bar is 20µm. with EMT-TF to maintain stemness. Hence, the Drosophila gut is an attractive model system to study the distinct role/mode of action of the two isoforms of Svb.

KEY RESOURCES

We have searched for Svb target genes within the ISC lineage. We started by profiling the activity of svb downstream target enhancers already identified in the embryonic epidermis (Chanut-Delalande et al., 2006;Menoret et al., 2013). Among this set of 22 validated enhancers, we observed a specific activity of three enhancers within small diploid duplet cells, thus very likely ISCS, ie: SnE1, an enhancer of singed encoding an actin binding protein, and two enhancers from uncharacterized genes CG4914 and CG31022. Thanks to tools available in the lab, we observed that mutations of Svb binding sites within the SnE1 enhancer reduce its activity within ISC/EBs compared to control. Thus, we have putative candidate target genes for Svb in the ISCs and further investigations of their putative role will require loss of function assays. So far, we only find few potential target genes of Svb in ISCs, and not in ECs. In order to find additional target genes, we are performing an unbiased approach using Dam-ID approach. This method consists of coupling the protein of interest, Svb in our case, to a DNA adeninemethylase (Dam) [START_REF] Van Steensel | Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase[END_REF]. The resulting chimeric protein binds to Svb target enhancers and methylates the adenine present in GATC Dam recognition sites; methylated sites are next quantitatively mapped to the Drosophila genome by next-gen.

sequencing. This approach should unravel the different subset of genes activated by Svb-Rep vs Svb-Act forms in ISCs/EBs and ECs, respectively. This should also help us to delineate the function of Svb in the balance between stemness and differentiation.

Hormonal control of stem cells behavior

Adapted stem cell comportment is regulated by a myriad of signals emanating from the surrounding environment. Both local and systemic signals can regulate stem cells behavior directly or through their niche. Accumulating evidences point to an important role of endocrine hormones in regulating stem cell establishment, maintenance, proliferation and differentiation [START_REF] Gancz | Hormonal control of stem cell systems[END_REF]. In Drosophila, both insulin and the ecdysone hormone are required for primordial germ cells accumulation at embryonic stages and for their differentiation at larval stages. In adults, ecdysone is required for germinal stem cells (GSC) maintenance [START_REF] Gancz | Coordinated regulation of niche and stem cell precursors by hormonal signaling[END_REF]. In addition, ecdysone is known to be required for the proliferation of progenitors (AMPs), responsible for adult midgut generation during metamorphosis [START_REF] Micchelli | Identification of adult midgut precursors in Drosophila[END_REF].

Although likely present at low levels, the ecdysone hormone is also detected in adults and its receptors EcR is expressed in various adult organs, within the central nervous system, gut, male and female reproductive apparatus and fat body among others [START_REF] Schwedes | Ecdysone signaling in adult Drosophila melanogaster[END_REF].

Interestingly, ecdysone inhibition in the adult can affect physiological functions including sexual behavior, sleep, nutrient, stress response as well as life span.

In mice, the thyroid hormone T3 is crucial for intestinal crypt establishment and ISC proliferation by enhancing Wnt signaling [START_REF] Flamant | Congenital hypothyroid Pax8(-/-) mutant mice can be rescued by inactivating the TRalpha gene[END_REF]. In flies, the insulin hormone is required for ISC proliferation, symmetric stem cell division, as well as for their proper differentiation [START_REF] Biteau | Lifespan extension by preserving proliferative homeostasis in Drosophila[END_REF][START_REF] Choi | Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway[END_REF][START_REF] Mcleod | Stem cell dynamics in response to nutrient availability[END_REF][START_REF] O'brien | Altered modes of stem cell division drive adaptive intestinal growth[END_REF].

In addition to ISCs, insulin and growth factors regulate hematopoietic stem cells self-renewal and expansion [START_REF] Benmimoun | Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila[END_REF]. Evidence from our work supports that ecdysone is required to maintain ISC/EBs in the midgut and RNSCs in Malpighian tubules. Indeed, lack of Ecdysone signaling leads to a reduction of stem cell numbers, a phenotype that can be rescued by the re-expression of either Pri or Svb-Act. As a detailed study of the role of ecdysone in ISCs is ongoing in the lab of Bruce Edgar, we decided to restricted our study on the possible effect of this signaling on Pri expression.

Work from our lab has demonstrated that the ecdysone hormone activates the expression of Pri throughout development (Chanut-Delalande et al., 2014). The active form of the hormone, 20 hydroxyedsysone (20E) is synthesized from dietary cholesterol and well known to control the timing of developmental transitions, such as larval molting and metamorphosis. Chip-seq