In this chapter we assess the role of several known mechanosensors in PE cluster formation.

"Causa admiración como trabaja el corazón"

Late el corazón -Amanece que no es poco "I de vegades una tonteria de sobte ens indica que ens en sortim" Captatio Benevolentiae -Manel

List of publications, posters and oral communications

Publications -Peralta, M., Ortiz Lopez, L., Jerabkova, K., Lucchesi, T., Vitre, B., Han, D., Guillemot, L., Dingare, C., Sumara, I., Mercader, N., Lecaudey, V., Delaval, B., Meilhac, S. and Vermot, J., 2020. Intraflagellar To achieve these aims, we defined the following objectives:

-Describe Yap1/Taz-Tead activation in the dorsal pericardium, the PE, and the epicardial layer in vivo.

-Study the role of Yap1/Taz-Tead signalling during PE cluster formation, as well as study models of Yap1/Taz-Tead activity upregulation and dowregulation.

-Find new regulators of PE cluster formation.

-Study possible mechanosensors involved during PE cluster formation.

These objectives are reflected in the structure of this thesis as follows:

-

Plan de la thèse de doctorat

La couche épicardique est essentielle au développement du coeur et à son bon fonctionnement. Sans l'épicarde, le développement embryonnaire échoue en raison de l'absence de formation des vaisseaux coronaires et du myocarde. Il est également un acteur clé lors de la régénération cardiaque. L'épicarde naît d'un groupe de cellules extracardiaques appelé proépicarde (PE). Les cellules proépicardiques émergent du péricarde dorsal, subissant d'importants changements de forme cellulaire. Malgré l'importance du PE et de la couche épicardique, de nombreuses étapes du développement du PE ne sont toujours pas comprises. Dans mon projet de thèse, nous avons cherché à explorer le rôle de l'activité de Yap/Taz-Tead dans la formation du PE, ainsi qu'à identifier de nouveaux régulateurs importants pour ce processus.

Pour atteindre ces buts, nous avons défini les objectifs suivants :

-Décrire l'activation de Yap1/Taz-Tead dans le péricarde dorsal, le PE et la couche épicardique in vivo.

-Étudier le rôle de la signalisation Yap1/Taz-Tead pendant la formation du PE, ainsi qu'étudier les modèles d'upregulation et de dowregulation de l'activité Yap1/Taz-Tead.

-Trouver de nouveaux régulateurs de la formation du PE.

-Étudier les mécanosenseurs possibles impliqués dans la formation du PE.

Ces objectifs sont reflétés dans la structure de cette thèse comme suit:

- -Recherche de mécanorégulateurs possibles de la formation du PE Enfin, l'annexe I contient une fonctionnalité publicitaire que j'ai contribué à créer lorsque je travaillais chez Leica Microsystem. J'ai contribué à la réalisation de ce reportage en réalisant des images d'embryons de poissons et en prenant part au processus de rédaction.

Introduction

The heart is the first organ to acquire its function in the developing embryo, providing the forming organs with oxygen and nutrients. Heart development is a complex, highly regulated process that when dysregulated results in congenital heart disease (CHD). In the human population, 1% of live births present CHD, and heart malformations explain around 30% of fetus loss during gestation [START_REF] Bruneau | The Developmental Genetics of Congenital Heart Disease[END_REF]. Furthermore, cardiovascular disease (CVD) is the leading cause of death worldwide, responsible for 16% of the world's total deaths [START_REF] Who | WHO | Cardiovascular Diseases (CVDs)[END_REF]. Specifically, 4 out of 5 CVD deaths are caused by heart attacks and strokes [START_REF] Who | WHO | Cardiovascular Diseases (CVDs)[END_REF]. For all these reasons, a better understanding of heart development might open new avenues for regenerative medicine after myocardial infarction that are based on enhancing pathways active during embryogenesis to promote cardiac regeneration [START_REF] Xin | Mending Broken Hearts: Cardiac Development as a Basis for Adult Heart Regeneration and Repair[END_REF][START_REF] Tzahor | Cardiac Regeneration Strategies: Staying Young at Heart[END_REF].

Heart development

In adult humans, the heart lies in the anterior part of the body in the middle compartment of the chest. It is surrounded by the pericardium, which contains a small amount of fluid, and it is nourished by the coronary vessels. The heart is divided into four chambers: right atrium, right ventricle, left atrium and left ventricle. Between chambers, we find the heart valves: these structures can be closed or opened, maintaining the flow in a single direction and preventing backflow (Figure 1A). The right atrium receives deoxygenated blood from the vena cava and it passes to the right ventricle. Blood is then pumped to the pulmonary circulation. It is oxygenated in the lungs and it returns to the left atrium, passing through the left ventricle first and then the left atrium, where it is pumped to the systemic circulation to provide the whole body with oxygen and nutrients.

The heart is composed of three layers: the epicardium, the myocardium, and the endocardium. The endocardium is an endothelial layer in direct contact with the blood. The myocardium is the cardiac muscle, responsible for heart contraction. The epicardium is the outermost layer covering the heart. Oxygen and nutrient supply, as well as the removal of metabolic waste, is performed in the heart by the coronary vasculature.

Heart development in model organisms

In order to improve our knowledge and understanding of heart development and cardiovascular diseases, several animal models are currently used. It is important to define which question we would like to address before choosing a model. In chickens and mice, as in humans, the heart is composed of 4 chambers (Figure 1B). In zebrafish, the heart has only 2 chambers: a single atrium and a single ventricle (Figure 1C). In all of these animal models the heart comprises the 3 same layers as the human heart (Figure 1D). Although mice are more similar to humans, the zebrafish offers several advantages for studying heart development [START_REF] Stainier | Cardiovascular Development in the Zebrafish. I. Myocardial Fate Map and Heart Tube Formation -PubMed[END_REF].

Zebrafish embryos are not completely dependent on a functional cardiovascular system in the early steps of heart development, since embryos without blood circulation can still obtain oxygen by passive diffusion and survive until 7 days post fertilization (dpf) [START_REF] Sehnert | Cardiac Troponin T Is Essential in Sarcomere Assembly and Cardiac Contractility[END_REF]. This allows the analysis of embryos with severe cardiovascular defects that is not possible with other animal models. Moreover, the optical transparency of zebrafish embryos, as well as their external development, facilitates in vivo imaging of heart development.

Combined with new transgenic technologies, zebrafish is an animal model widely used for the study of heart development [START_REF] Bakkers | Zebrafish as a Model to Study Cardiac Development and Human Cardiac Disease[END_REF].

Heart development in the mouse

Heart development in mice starts shortly after gastrulation [START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF][START_REF] Vincent | How to Make a Heart. The Origin and Regulation of Cardiac Progenitor Cells[END_REF]. Fate mapping showed that the first cardiac progenitors are located in the anterior region of the primitive streak. After gastrulation, cardiac precursors in the mesoderm differentiate into cardiomyocytes and form a bilateral structure called cardiac crescent (cc). By a complex morphogenetic process, the cc transforms into a primitive heart tube (HT) which rapidly begins to pump blood [START_REF] Vincent | How to Make a Heart. The Origin and Regulation of Cardiac Progenitor Cells[END_REF]. The mature heart is formed by 2 main populations of progenitor cells: the first heart field (FHF) and the second heart field (SHF). The primitive HT (FHF) will give rise to the left ventricle. The SHF contributes to the elongation of the arterial and the venous pole of the heart [START_REF] Buckingham | Building the Mammalian Heart from Two Sources of Myocardial Cells[END_REF],

giving rise to the right ventricle, the left and right atrium, and the outflow tract (Figure 2) [START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF][START_REF] Ivanovitch | Live Imaging of Heart Tube Development in Mouse Reveals Alternating Phases of Cardiac Differentiation and Morphogenesis[END_REF].

Progenitor FHF cells have not any specific gene marker, as it is hard to distinguish genes expressed in the cardiac crescent that are beginning to differentiate into cardiomyocytes [START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF].

Cells from the SHF, on the other hand, express characteristic markers. Fgf10

and Fgf8, for instance, are required for the correct formation of the right ventricle and outflow region of the heart. Another known marker is Isl1. Isl1mutant mice show defects at the venous pole in the atria, as well as at the arterial pole in right ventricular and outflow tract development [START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF].

As the cardiac tube elongates, it undergoes rightward looping and the poles of the heart tube converge and become juxtaposed. The cardiac chamber then grows out from the looping tube in a process called ballooning, leading to the formation of the right and left ventricles and the atria [START_REF] Vincent | How to Make a Heart. The Origin and Regulation of Cardiac Progenitor Cells[END_REF]. Adapted from [START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF] While the endocardium develops in close association with the myocardium, the epicardium emerges from the proepicardium, a transitory mesenchymal structure that forms at the posterior end of the heart tube at E9.5 [START_REF] Vincent | How to Make a Heart. The Origin and Regulation of Cardiac Progenitor Cells[END_REF].

Endocardial cells come from a subset of cardiac progenitor cells mapped in the primitive streak stage. It is not yet known whether endocardial cells derive from cells in close proximity to the cardiomyocytes in the primitive streak or if there is a common progenitor [START_REF] Nakano | The Developmental Origins and Lineage Contributions of Endocardial Endothelium[END_REF][START_REF] Meilhac | The Deployment of Cell Lineages That Form the Mammalian Heart[END_REF].

Heart development in the zebrafish

Although the zebrafish heart comprises 2 chambers, it recapitulates the main development steps during embryonic heart formation in mouse [START_REF] Liu | Zebrafish in the Study of Early Cardiac Development[END_REF]. First, cardiac progenitors can be found at the blastula stage. The ventricular pool resides more dorsally and closer to the margin than the atrial pool, while endocardial progenitors are located across the lateral margin without any specific spatial organization (Figure 3). During gastrulation, these progenitors migrate to reside in the posterior half of the anterior lateral plate mesoderm (LPM) by 15 hpf (Figure 3).

As they differentiate, the bilateral populations of myocardial precursors start to migrate toward the embryonic midline. After making contact, the two populations merge to form the cardiac cone (Figure 3) [START_REF] Stainier | Cardiovascular Development in the Zebrafish. I. Myocardial Fate Map and Heart Tube Formation -PubMed[END_REF]. The embryonic heart starts to beat at 22 hpf. By 24 hpf, the cardiac cone has evolved into a linear HT, where the endocardial precursor stream medially and posteriorly, coating the HT. [START_REF] Sainier | Cardiovascular Development in the Zebrafish I. Myocardial Fate Map and Heart Tube Formation[END_REF]. The HT gradually grows and loops rightwards. Next, the V and At form by ballooning out. At 48 hpf, the ventricle and the atrium are separated by the constriction of the atrio-ventricular canal (AVC) and the heart starts to grow through the addition of cardiac progenitors from the arterial and venous pole [START_REF] Bakkers | Zebrafish as a Model to Study Cardiac Development and Human Cardiac Disease[END_REF]. Like in mammals and birds, the SHF population contributes to the arterial pole myocardium after the initial heart tube formation [START_REF] Hami | Zebrafish Cardiac Development Requires a Conserved Secondary Heart Field[END_REF].

Several signalling pathways are important for SHF cell fate determination. Fgf8, for instance, is required for cardiomyocyte differentiation at the arterial pole (de [START_REF] Pater | Distinct Phases of Cardiomyocyte Differentiation Regulate Growth of the Zebrafish Heart[END_REF]). Hippo pathway is also involved in SHF cell fate determination. An increase in activity of Hippo pathway effectors Yap1/Wwtr1 promotes the size of the SHF while inhibiting Yap1/Wwrt1 restricts it. This regulation happens through the BMP pathway. Yap1/Wwrt1 promotes Bmp2 expression and this promotes venous pole identity and increases atrial CM number [START_REF] Fukui | Hippo Signaling Determines the Number of Venous Pole Cells That Originate from the Anterior Lateral Plate Mesoderm in Zebrafish[END_REF].

At 72 hpf, differentiated cardiomyocytes in the myocardium start to protrude towards the lumen, generating small clusters of cells. These clusters of cells will form the trabeculae, highly organized sheet-like muscular structures that form as a result of the extrusion and expansion of differentiated cardiomyocytes into the lumen of the ventricular chambers. Cardiac trabecular formation is essential for proper heart development and failures in trabeculae formation can result in embryonic lethality or cardiomyopathies (Liu et al. 2010). During heart development, physical forces generated by cardiac contractility and blood flow play an essential role. From the onset of heartbeat, blood flows through the cardiac lumen and this influences heart development at several steps, such as chamber formation (Granados-Riveron and Brook 2012), the progression of trabeculation, and chamber differentiation [START_REF] Rasouli | Regulation of Cardiomyocyte Behavior in Zebrafish Trabeculation by Neuregulin 2a Signaling[END_REF][START_REF] Peshkovsky | Dependence of Cardiac Trabeculation on Neuregulin Signaling and Blood Flow in Zebrafish[END_REF][START_REF] Staudt | High-Resolution Imaging of Cardiomyocyte Behavior Reveals Two Distinct Steps in Ventricular Trabeculation[END_REF]. In particular, mechanical forces are needed for proper valve development.

Several mechanosensors are important in this process. For instance, the opening of mechanosensitive channels like TRP or Piezo families is associated with calcium fluxes, which initiate intracellular signalling cascades [START_REF] Steed | Hemodynamics Driven Cardiac Valve Morphogenesis[END_REF]. Two TRP channels, Pkd2 and Trpv4, regulate the expression of the transcription factor Krüppel like factor 2a (Klf2a). Klf2a

regulates the expression of flow-resposible genes in endothelial cells, which promote the formation of cardiac valves [START_REF] Heckel | Oscillatory Flow Modulates Mechanosensitive Klf2a Expression through Trpv4 and Trpp2 during Heart Valve Development[END_REF][START_REF] Vermot | Reversing Blood Flows Act through Klf2a to Ensure Normal Valvulogenesis in the Developing Heart[END_REF]). Piezo channels, on the other hand, are necessary for proper outflow tract valves formation [START_REF] Duchemin | Mechanically Activated Piezo Channels Modulate Outflow Tract Valve Development through the Yap1 and KLF2-Notch Signaling Axis[END_REF].

The Proepicardium

The epicardium is a single layer of flat mesothelium covering the myocardium. It starts to form at the time of heart looping. It is important for myocardial maturation [START_REF] Männer | Experimental Study on the Formation of the Epicardium in Chick Embryos[END_REF][START_REF] Pennisi | Epicardium Is Required for the Full Rate of Myocyte Proliferation and Levels of Expression of Myocyte Mitogenic Factors FGF2 and Its Receptor, FGFR-1, but Not for Transmural Myocardial Patterning in the Embryonic Chick Heart[END_REF] and gives rise to the coronary vasculature [START_REF] Mikawa | Retroviral Analysis of Cardiac Morphogenesis: Discontinuous Formation of Coronary Vessels[END_REF] and the intracardiac fibroblasts [START_REF] Wessels | Epicardially Derived Fibroblasts Preferentially Contribute to the Parietal Leaflets of the Atrioventricular Valves in the Murine Heart[END_REF]. Importantly, the epicardium plays a key role in heart regeneration (J. [START_REF] Cao | The Epicardium as a Hub for Heart Regeneration[END_REF].

The epicardium derives from the proepicardium (PE), a cluster of mesothelial cells in a cauliflower-like shape that forms close to the venous pole of the cardiac tube [START_REF] Männer | Does the Subepicardial Mesenchyme Contribute Myocardioblasts to the Myocardium of the Chick Embryo Heart? A Quail-chick Chimera Study Tracing the Fate of the Epicardial Primordium[END_REF]. Interestingly, the PE was already known among embryologists of the 19 th century. In 1909, Kurkiewicz identified the PE as the source for epicardial cells in the chick embryonic heart [START_REF] Kurkiewicz | O Histogenezie Miesna Sercowego Zwierzat Kregowych-Zur Histogenese Des Herzmuskels Der Wirbeltiere[END_REF].

This theory was not taken into account until it was rediscovered in the 1970s [START_REF] Bodmer | Cardiovascular Development[END_REF]. The PE has been described in several vertebrates, from fish to human [START_REF] Hirakow | Epicardial Formation in Staged Human Embryos[END_REF][START_REF] Fransen | Epicardial Development in the Axolotl,Ambystoma Mexicanum[END_REF][START_REF] Komiyama | Origin and Development of the Epicardium in the Mouse Embryo[END_REF][START_REF] Kuhn | The Early Development of the Epicardium in Tupaia Belangeri[END_REF][START_REF] Nesbitt | Epicardial Development in the Rat: A New Perspective[END_REF][START_REF] Icardo | The Development of the Epicardium in the Sturgeon Acipenser Naccarii[END_REF][START_REF] Jahr | Development of the Proepicardium in Xenopus Laevis[END_REF][START_REF] Serluca | Development of the Proepicardial Organ in the Zebrafish[END_REF]. In mice, the PE is a cluster formed by a mixture of cells, including cells derived from epithelial and mesenchymal lineages, located in close proximity to the septum transversum [START_REF] Viragh | The Origin of the Epicardium and the Embryonic Myocardial Circulation in the Mouse[END_REF]. In zebrafish, the PE is composed of two clusters located both in the dorsal pericardium (DP). One cluster, which we will refer to as the avcPE, is located in the atrioventricular channel area, and the other one, which we will refer to as the vpPE, is located close to the venous pole. There is an additional source of cells, the apPE, close to the arterial pole area that can also contribute to the epicardium [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF].

Different molecular markers are used to define epicardial and PE identity, such as Wilms' tumor suppressor 1 (Wt1), T-box transcription factor 18 (Tbx18), and the transcription factor Tcf21 [START_REF] Carmona | Localization of the Wilms' Tumour Protein WT1 in Avian Embryos[END_REF][START_REF] Kraus | Cloning and Expression Analysis of the Mouse T-Box Gene Tbx18[END_REF][START_REF] Tanaka | Tbx18 and Boundary Formation in Chick Somite and Wing Development[END_REF][START_REF] Robb | Epicardin: A Novel Basic Helixloop-helix Transcription Factor Gene Expressed in Epicardium, Branchial Arch Myoblasts, and Mesenchyme of Developing Lung, Gut, Kidney, and Gonads[END_REF].

PE formation

In the zebrafish, PE cluster formation begins around 48 hpf (Figure 4A) [START_REF] Serluca | Development of the Proepicardial Organ in the Zebrafish[END_REF]. Live imaging using a wt1a reporter line showed that mesothelial cells from the DP migrate collectively towards a midline between the venous pole In mice live imaging does not allow yet a detailed description of PE cluster formation. The PE cluster is initially present in E9.0 through E9.75 and appears as an outgrowth of mesothelial cells adjacent to the liver bud and the sinus venosus [START_REF] Cossette | The Identification of Different Endothelial Cell Populations within the Mouse Proepicardium[END_REF]. Starting at E9.5, cells from the PE delaminate and are released into the pericardial coelom. Then, they attach to the myocardium and migrate to cover the surface of the heart. There is also a contribution by migrating proepicardial cells directly over the adjacent myocardium [START_REF] Viragh | The Origin of the Epicardium and the Embryonic Myocardial Circulation in the Mouse[END_REF].

Origin of the PE and mechanisms inducing PE formation

Genetic mapping in mice has shown that PE cells express Nkx2.5 and isl1 at some point in their development. These both genes are expressed in the primary and secondary heart fields, respectively, suggesting that the PE arises from the LPM [START_REF] Lisandro | Induction of the Proepicardium[END_REF]. In zebrafish, it has been described that a defect in midline migration of the lateral plate mesoderm results in cardia bifida. Interestingly, each of the hearts has its own PE. The presence of bilaterial PE suggests that the origin of the epicardium in zebrafish also lies in the LPM [START_REF] Serluca | Development of the Proepicardial Organ in the Zebrafish[END_REF]. Genetic mapping in the chick mapped the origin of the PE cells in the LPM, adjacent but not mixed to the cardiogenic mesoderm [START_REF] Bressan | Early Mesodermal Cues Assign Avian Cardiac Pacemaker Fate Potential in a Tertiary Heart Field[END_REF].

It is still largely unknown how PE cell identity is induced. In chick, ectopic implantation of quail liver bud induces expression of wt1, tbx18, and tcf21 in adjacent host tissue. In zebrafish lacking mature hepatocytes (hnf1ba mutants), wt1, tbx18, and tcf21 are expressed at wild-type levels, indicating that the liver is not necessary for PE molecular induction [START_REF] Lisandro | Induction of the Proepicardium[END_REF]). The Tbox transcription factor 18 is strongly expressed in the PE and in the epicardium [START_REF] Kraus | Cloning and Expression Analysis of the Mouse T-Box Gene Tbx18[END_REF][START_REF] Tanaka | Tbx18 and Boundary Formation in Chick Somite and Wing Development[END_REF]. Tbx18 inactivation in the mouse embryo causes defects in the epicardium, coronary vasculature, and the venous pole (Pérez-Pomares and de la Pompa 2011).

Wt1 is a known epicardial marker, already expressed in PE cells, that regulates the expression of important genes for epicardium development [START_REF] Kirschner | The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the Α4integrin Gene[END_REF]. In mutant mice lacking Wt1, the epicardium fails to cover the entire heart [START_REF] Moore | YAC Complementation Shows a Requirement for Wt1 in the Development of Epicardium, Adrenal Gland and throughout Nephrogenesis[END_REF].

Tcf21 is a basic helix-loop-helix transcription factor expressed in the PE, the epicardium, and the pericardium. In Xenopus, when tcf21 is lacking, epicardial cells fail to form a cohesive polarized sheet and are retained in a proepicardial state [START_REF] Tandon | Tcf21 Regulates the Specification and Maturation of Proepicardial Cells[END_REF]. In chick and mice, tcf21 depletion results in epicardial blistering [START_REF] Braitsch | Pod1/Tcf21 Is Regulated by Retinoic Acid Signaling and Inhibits Differentiation of Epicardium-Derived Cells into Smooth Muscle in the Developing Heart[END_REF].

Studies in zebrafish show that bone morphogenetic protein (BMP) plays a role in PE cluster formation (Liu and Stainier 2010). Zebrafish mutants lacking Activin A receptor type 1 (acvr1l), a functional type I BMP receptor, lose tbx18 and tcf21 expression in the heart [START_REF] Liu | Zebrafish in the Study of Early Cardiac Development[END_REF].

The importance of BMP during epicardial development was also described in a paper from 2015 where they differentiated functional epicardial cells from human pluripotent stem cells (HPSC) [START_REF] Iyer | Robust Derivation of Epicardium and Its Differentiated Smooth Muscle Cell Progeny from Human Pluripotent Stem Cells[END_REF]. In this study, HPSC cells were first differentiated to an early mesoderm. After Bmp4 treatment, cells increased the expression of epicardial markers. In addition to Bmp, RA and Wnt signaling was also promoted to obtain differentiated epicardial cells. These HPSC-derived epicardial cells not only resembled human fetal epicardial cells, but they were also able to contribute to the coronary vasculature of a developing chicken [START_REF] Iyer | Robust Derivation of Epicardium and Its Differentiated Smooth Muscle Cell Progeny from Human Pluripotent Stem Cells[END_REF].

In Besides being important for actomyosin network regulation, BMP is an upstream effector of the zinc-finger transcription factor Gata4 in the LPM [START_REF] Rojas | Gata4 Expression in Lateral Mesoderm Is Downstream of BMP4 and Is Activated Directly by Forkhead and GATA Transcription Factors through a Distal Enhancer Element[END_REF]. This factor is also important for PE, since it has been described that Gata4 mutant mice lack PE and have severe heart defects [START_REF] Watt | GATA4 Is Essential for Formation of the Proepicardium and Regulates Cardiogenesis[END_REF]). This phenotype is not fully recovered with the expression of other members of the Gata family, such as Gata6, as the PE is smaller than in wild-type, there are fewer epicardial cells attached, and the expression of tbx18 is decreased [START_REF] Borok | Unique Functions of Gata4 in Mouse Liver Induction and Heart Development[END_REF].

Another known regulator of PE formation is Tbx5. In zebrafish, tbx5a is expressed in the LPM and when lacking, the expression of tbx18 and tcf21 is strongly decreased. It has also been proposed that Tbx5a expression allows LPM cells to have the ability to respond to Bmp signals and initiate PE formation (Liu and Stainier 2010). In chick, epicardial cells in early development express tbx5. When the cluster is formed, proepicardial cells are tbx5 negative until they get close to the myocardium, when some of them reactivate tbx5 expression.

This suggests that factors from the myocardium might be regulating PE cluster formation [START_REF] Bimber | Differential Regulation of Tbx5 Protein Expression and Sub-Cellular Localization during Heart Development[END_REF]. In humans, tbx5 is expressed in the coronary vasculature and in the epicardium [START_REF] Hatcher | A Role for Tbx5 in Proepicardial Cell Migration during Cardiogenesis[END_REF]). Loss of tbx5 expression accounts for Holt-Oram syndrome, a rare autosomal dominant disease which is characterized by upper limb malformations and cardiac septation defects (OMIM 2006).

Hand2 is another transcription factor expressed in the LPM that is critical for the differentiation of LPM derivatives and that seems to be important for PE cluster formation. In hand2 zebrafish mutants, the PE cluster is completely absent, suggesting that it is required for the differentiation of mesodermal cells into PE cells. However, hand2 mutants have severe cardiac malformation and the phenotype could be due to myocardial defects (Liu and Stainier 2010).

Genes involved in left-right assymmetry also influence the development of the proepicardium. In chick, the proepicardium forms from bilaterally symmetric structures that develop asymmetrically, as the proepicardium forms just on the right side of the cavity. When right-sided gene expression determintants, such as fgf8 or snai1 are disrupted, there is a loss of tbx18 expression in the right inflow [START_REF] Svensson | Deciphering the Signals Specifying the Proepicardium[END_REF][START_REF] Schlueter | A Right-Sided Pathway Involving FGF8/Snai1 Controls Asymmetric Development of the Proepicardium in the Chick Embryo[END_REF]. In mice, however, left-right asymmetry genes do not seem to be regulating PE formation, as both right and left proepicardial precursors will develop equally and fuse in the midline to generate the proepicardium [START_REF] Svensson | Deciphering the Signals Specifying the Proepicardium[END_REF] . In zebrafish, both proepicardial primordia develop to form the PE cluster [START_REF] Serluca | Development of the Proepicardial Organ in the Zebrafish[END_REF].

In mice, the Hippo signaling components are expressed during epicardium formation [START_REF] Singh | Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development[END_REF]. Their expression in the PE has not yet been described, but mice where both Yap and Taz have been knocked out specifically in the PE and the epicardium show embryonic lethality, suggesting that the PE cluster does not form properly in these mice. Moreover, Yap and Taz regulate Wt1 and Tbx18 expression [START_REF] Singh | Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development[END_REF]. In zebrafish, epicardial cells also express the Hippo signaling component Taz (or Wwtr1) [START_REF] Lai | The Hippo Pathway Effector Wwtr1 Regulates Cardiac Wall Maturation in Zebrafish[END_REF]).

Transfer of PE cells to the myocardium during epicardium formation

There are two main mechanisms of PE cell transfer to the myocardium: the formation of a transient bridge between the PE and the myocardium (Figure 5A), and the release of PE cells to the pericardial cavity followed by their progressive attachment to the myocardial surface (Figure 5B). Depending on the species, PE cell transfer occurs via one of the two mechanisms or via both mechanisms [START_REF] Rodgers | Differential Growth and Multicellular Villi Direct Proepicardial Translocation to the Developing Mouse Heart[END_REF].

In Xenopus, the PE cluster establishes a firm contact with the developing ventricle and a bridge is established. Once the bridge is established, the PE cells transfer to the heart and spread over the myocardial surface to form a primitive epicardium [START_REF] Jahr | Development of the Proepicardium in Xenopus Laevis[END_REF]. A similar mechanism occurs in chick [START_REF] Schulte | Morphological and Molecular Left-Right Asymmetries in the Development of the Proepicardium: A Comparative Analysis on Mouse and Chick Embryos[END_REF].

In mice, however, a bridge does not seem to form. Instead, cells of the PE cluster are released into the pericardial cavity, after which they adhere to the myocardial surface, forming a primitive epicardium [START_REF] Schulte | Morphological and Molecular Left-Right Asymmetries in the Development of the Proepicardium: A Comparative Analysis on Mouse and Chick Embryos[END_REF].

In zebrafish, both methods have been observed. Cells from the apPE transfer directly to the myocardium, similarly as in birds and Xenopus. Cells from the other avcPE and vpPE cluster, however, are released into the pericardial cavity and are advected with a periodical motion coupled to the heartbeat. Epicardial cells preferentially adhere first to the distal half of the ventricle and later colonize the proximal half. The distal half of the ventricle is the area where the cells are advected at the lowest speed in the pericardial cavity, which suggests that PE cell attachment is a process resulting from the balance between PE cell adhesiveness to the myocardium and the differential flow forces. Finally, the myocardium is fully covered at around 6 dpf [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF].

A proposed hypothesis is that differences in PE cell attachment to the myocardium are due to the differences in coelomic cavities. In chicken and

Xenopus, at the time of PE formation, the cavity is not closed and the bridge ensures that cells are not lost. In mammals and zebrafish, the pericardial cavity is lost and PE cell release is predominant [START_REF] Peralta | The Epicardium in the Embryonic and Adult Zebrafish Europe PMC Funders Group[END_REF] .

YAP and TAZ, effectors of the Hippo pathway

The Hippo signalling pathway was discovered in Drosophila and has been shown to be conserved in vertebrates [START_REF] Huang | The Hippo Signaling Pathway Coordinately Regulates Cell Proliferation and Apoptosis by Inactivating Yorkie, the Drosophila Homolog of YAP[END_REF][START_REF] Dong | Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals[END_REF]).

The Hippo signalling pathway was first described as a regulator of organ size [START_REF] Dong | Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals[END_REF]), but since then more roles, such as being a regulator of cell proliferation, differentiation, migration, or EMT, have been described [START_REF] Cheng | YAP Promotes Epithelial Mesenchymal Transition by Upregulating Slug Expression in Human Colorectal Cancer Cells[END_REF][START_REF] Varelas | The Hippo Pathway Effectors TAZ and YAP in Development, Homeostasis and Disease[END_REF]) [START_REF] Cheng | YAP Promotes Epithelial Mesenchymal Transition by Upregulating Slug Expression in Human Colorectal Cancer Cells[END_REF]. The main effectors of the Hippo pathway in mammals are YAP and TAZ.

Traditionally, nuclear localization of YAP is linked to the activation of pro-growth transcriptional programs through association with the TEAD family of transcription factors [START_REF] Dupont | Role of YAP/TAZ in Mechanotransduction[END_REF][START_REF] Zhao | Inactivation of YAP Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control[END_REF].

Several mechanisms regulate YAP localization, therefore controlling Yapdependent gene transcription. The Hippo pathway is the main YAP regulator [START_REF] Piccolo | The Biology of YAP/TAZ: Hippo Signaling and Beyond[END_REF], but other regulators such as Amot proteins are also important [START_REF] Ragni | Amotl1 Mediates Sequestration of the Hippo Effector Yap1 Downstream of Fat4 to Restrict Heart Growth[END_REF]. Importantly, these different mechanisms can coexist in a cell and it is not possible to exclude interplay between them.

Hippo pathway

The Hippo pathway is dysregulated in a broad range of cancers, and it is frequently correlated with cancer progression, but at the same time, it is key for The Hippo pathway is conserved throughout the eukaryotic kingdom. Since the Hippo pathway is essential for correct organ growth, it has been proposed that the emergence of the Hippo pathway is associated and conserved within animals that have an organized body plan that incorporates distinct organs and tissues [START_REF] Hilman | The Evolutionary History of YAP and the Hippo/YAP Pathway[END_REF]. In mammalian model systems, YAP is a functional homolog of the transcriptional regulator Yorkie [START_REF] Dong | Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals[END_REF].

TAZ or Wwrt1 has also been characterized as a paralog of YAP. LATS1 and LATS2 kinases are homologs of Warts. These kinases can phosphorylate YAP and TAZ, resulting in nuclear exclusion, cytoplasmatic sequestration and/or proteasomal degradation [START_REF] Piccolo | The Biology of YAP/TAZ: Hippo Signaling and Beyond[END_REF]. Most upstream components of the canonical Hippo pathway are also conserved in mammals, such as MST1 and MST2 kinases (homologs of Hippo), SAV1

(homolog of Salvador), and MOB1A and MOB1B (homologs of Mob). When YAP or TAZ are active, they can translocate into the nucleus and bind to transcription factors of the TEAD transcription factor family (homolog of Scalloped) and induce the expression of a wide range of genes, such as ctgf [START_REF] Meng | Mechanisms of Hippo Pathway Regulation[END_REF]. Both YAP and TAZ have a shared domain in the N-terminal region that mediates the binding to TEAD transcription factors.

Although most of the domain is conserved between the two proteins, in YAP there is a PxxΦP motif that is lacking in TAZ. This suggests that there are some differences in TEAD activation between the two proteins, but the in vivo implications are not known yet [START_REF] Varelas | The Hippo Pathway Effectors TAZ and YAP in Development, Homeostasis and Disease[END_REF].

Other YAP and TAZ regulators

Angiomotins (Amot) are a family of proteins known to bind to tight-junction proteins and F-actin that regulate YAP and TAZ nuclear localization (Zhao et al. 2011). Amot proteins have a Yap-binding motif. In mice, it has been shown that Amot depletion compromises development, as there is an increased percentage of embryo loss compared to wild-type embryos [START_REF] Leung | Angiomotin Prevents Pluripotent Lineage Differentiation in Mouse Embryos via Hippo Pathway-Dependent and-Independent Mechanisms[END_REF]. In embryos lacking LATS1 and LATS2, there is an increase of proteins expressing Cdx2 (transcription factor downstream of TEAD) at the morula stage that is recovered by the expression of Amot [START_REF] Leung | Angiomotin Prevents Pluripotent Lineage Differentiation in Mouse Embryos via Hippo Pathway-Dependent and-Independent Mechanisms[END_REF].

In zebrafish, Amotl2a is essential to restrict the number of cells in the posterior lateral line by inhibiting proliferation. Amotl2a physically interacts with Yap and Taz, sequestering them in the cytoplasm and inhibiting their proliferationpromoting transcriptional activity [START_REF] Agarwala | Amotl2a Interacts with the Hippo Effector Yap1 and the Wnt/β-Catenin Effector Lef1 to Control Tissue Size in Zebrafish[END_REF].

Mechanical and cytoskeletal inputs also regulate YAP and TAZ activity [START_REF] Dupont | Role of YAP/TAZ in Mechanotransduction[END_REF]. YAP and TAZ have a PDZ-binding motif, which mediates interactions with proteins that are usually transmembrane or cytoskeletonassociated [START_REF] Varelas | The Hippo Pathway Effectors TAZ and YAP in Development, Homeostasis and Disease[END_REF]. The cytoskeletal adaptation of spread cells (including the formation of F-actin stress fibers) causes YAP and TAZ nuclear accumulation. When cell shape is rounded and compact, YAP and TAZ are excluded from the nucleus. This is the case of contact inhibition of proliferation, a process by which cultured cells stop dividing then they become confluent.

YAP and TAZ are nuclear in cells growing in low density and relocate to the cytoplasm when cells are in confluence [START_REF] Zhao | Inactivation of YAP Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control[END_REF][START_REF] Elosegui-Artola | Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores[END_REF]). YAP and TAZ also respond to changes in extracellular matrix (ECM) stiffness:

rigid ECM keeps more active YAP/TAZ to the nucleus than a softer ECM (Figure 7) [START_REF] Piccolo | The Biology of YAP/TAZ: Hippo Signaling and Beyond[END_REF]. YAP activation due to ECM stiffness can also induce EMT [START_REF] Park | Switch-like Enhancement of Epithelial-Mesenchymal Transition by YAP through Feedback Regulation of WT1 and Rho-Family GTPases[END_REF].

Mechanical cues are regulators of Rho-GTPases, a family of GTPases that influence actin cytoskeleton dynamics and ROCK activity (a downstream Rho-GTPase effector). At the same time, YAP and TAZ activity can be regulated by them. Inhibition of Rho-GTPase function inhibits YAP and TAZ activity [START_REF] Piccolo | The Biology of YAP/TAZ: Hippo Signaling and Beyond[END_REF] and activation of Rho-kinase (downstream of Rho-GTPase) promotes nuclear YAP/TAZ activity [START_REF] Varelas | The Hippo Pathway Effectors TAZ and YAP in Development, Homeostasis and Disease[END_REF].

Alterations in F-actin also affect YAP and TAZ localization. F-actin capping proteins inhibit YAP and TAZ nuclear localization, as when these proteins are lacking there is an increase in YAP and TAZ activity [START_REF] Aragona | A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors[END_REF].

The liver kinase B1 (LKB1) also seems to have an important role in regulating YAP and TAZ nuclear translocation, since it is a known tumour suppressor and in vitro studies with several types of cells have shown that Lkb1 expression increases Yap phosphorylation and consequent nuclear exclusion [START_REF] Qiu | EMT Induced by Loss of LKB1 Promotes Migration and Invasion of Liver Cancer Cells through ZEB1-Induced YAP Signaling[END_REF]) [START_REF] Nguyen | LKB1 Tumor Suppressor Regulates AMP Kinase/MTOR-Independent Cell Growth and Proliferation via the Phosphorylation of Yap[END_REF]) [START_REF] Mohseni | A Genetic Screen Identifies an LKB1-MARK Signalling Axis Controlling the Hippo-YAP Pathway[END_REF].

Hippo pathway in zebrafish heart development

There are several tools available to study the Hippo pathway during zebrafish development.

It is possible to follow YAP and TAZ activity in vivo using different reporter lines. There are several different ways to modulate Yap activity in zebrafish. It is possible to manipulate Yap functionally using heat-shock transgenic lines, such as the hsp70:RFP-CAyap. Fish from this line have a mutation in the motif in Yap that is recognized by Lats 1/2, preventing its inactivation through phosphorylation and consequently promoting its translocation to the nucleus.

Another line that allows us to modify Yap activity is the hsp70:RFP-DNyap line.

A truncated form of the Yap protein is used, in which the transcriptional activation domain is removed but the Tead binding domain is intact. This allows Yap to bind to its DNA partner but it does not activate its targets [START_REF] Mateus | Control of Tissue Growth by Yap Relies on Cell Density and F-Actin in Zebrafish Fin Regeneration[END_REF].

Yap activity can also be regulated with the use of some drugs. XAV939 is a trankyrase inhibitor that suppresses Yap activity. Trankyrases recognize poly-ADP ribosylation sites and can change the subcellular localization of the substrate or cause its proteasome-dependent degradation. Trankyrases are positive regulators of YAP, as they bind to Amot proteins, resulting in more free

Yap that are able to translocate to the nucleus. When trankyrases are targeted, Amot protein levels are increased and YAP activity is downregulated (W. [START_REF] Wang | Tankyrase Inhibitors Target YAP by Stabilizing Angiomotin Family Proteins[END_REF].

Verteporfin is a second-generation photosensitizer that is also able to suppress Yap activity. Verteporfin decreases nuclear YAP levels and activity as a consequence of increasing 14-3-3σ protein levels. 14-3-3 proteins sequestrate phosphorylated proteins in the cytosol, targeting for degradation in the proteasome. When these proteins are upregulated, YAP is trapped in the cytosol (C. [START_REF] Wang | Verteporfin Inhibits YAP Function through Up-Regulating 14-3-3σ Sequestering YAP in the Cytoplasm[END_REF].

Dexamethasone is a synthetic glucocorticoid that promotes the nuclear activity of Yap in vitro and in vivo in zebrafish [START_REF] Astone | Zebrafish Mutants and TEAD Reporters Reveal Essential Functions for Yap and Taz in Posterior Cardinal Vein Development[END_REF]). It has been described that activation of glucocorticoid receptors activated Yap via actin cytoskeleton remodelling in breast cancer cells [START_REF] Sorrentino | Glucocorticoid Receptor Signalling Activates YAP in Breast Cancer[END_REF]).

Yap1 also seems to be involved in OFT valve formation [START_REF] Duchemin | Mechanically Activated Piezo Channels Modulate Outflow Tract Valve Development through the Yap1 and KLF2-Notch Signaling Axis[END_REF]. Yap1 regulates cell fate determination of cardiac precursor cells into smooth muscle cells via a process that involves elastin b [START_REF] Moriyama | Evolution of the Fish Heart by Sub/Neofunctionalization of an Elastin Gene[END_REF]. Moreover, Yap1 is also localized in the nucleus of endothelial and smooth muscle cells of the OFT and a significant fraction of Yap1 mutants have abnormal or not recognizable outflow tract valves [START_REF] Duchemin | Mechanically Activated Piezo Channels Modulate Outflow Tract Valve Development through the Yap1 and KLF2-Notch Signaling Axis[END_REF].

Wwtr1 also has an important role in cardiomyocyte development and proliferation during trabecular morphogenesis [START_REF] Lai | The Hippo Pathway Effector Wwtr1 Regulates Cardiac Wall Maturation in Zebrafish[END_REF].

Primary Cilia

Primary cilia are membrane protrusions, with an antenna-like shape. They consist of a microtubule-based ciliary axoneme assembled from a basal body and they are surrounded by a lipid bilayered membrane [START_REF] Venkatesh | Primary Cilia[END_REF].

According to the internal arrangement, primary cilia have a 9 +0 axoneme (Hildebrandt, Benzing, and Katsanis 2011) (Figure 8A). Inside the cilium, kinesins and dynein motor proteins transport intraflagellar transport (IFT)

proteins and cargo towards the tip or back to the basal body and the cell, respectively [START_REF] Ishikawa | Ciliogenesis: Building the Cell's Antenna[END_REF]. The IFT machinery is composed of two biochemically distinct subcomplexes, IFT-complex A and IFT-complex B [START_REF] Taschner | Architecture and Function of IFT Complex Proteins in Ciliogenesis[END_REF]. Primary cilia are present in several types of cells, as a single primary cilium is found on the apical surface of the majority of cells in the human body [START_REF] Berbari | The Primary Cilium as a Complex Signaling Center[END_REF]). Primary cilia sense Primary cilia have been reported in the embryonic pericardium of the chick and the mouse [START_REF] Rash | Cilia in Cardiac Differentiation[END_REF]. In zebrafish, in vivo analysis of the pericardial cavity shows that there are primary cilia and that they are heterogeneously distributed in the dorsal pericardium [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. At 48 hpf, the right half of the sinus venosus presents a higher number of cilia than the left half. The PE region in the dorsal pericardium also has differences between the right and left side. The right side, where the PE cluster will form,

shows higher cilia number. Heartbeat triggers motion of these primary cilia and their movement is different depending on the position of each cilium. Overall, there are more cilia in the right side of the avc area, where the PE cluster will form [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]) (Figure 8B).

Zebrafish (ZF) husbandry and embryo treatments:

Animal experiments were approved by the Animal Experimentation Committee of the Institutional Review Board of the IGBMC. ZF lines used in the study were: -Klf2b (Generated in the Vermot Lab)

All animals were incubated at 28.5°C for 24 hours before treatment with 1phenyl-2-thiourea (PTU) (Sigma Aldrich) to prevent pigment formation.

Drug treatments were performed as followed:

-Dexamethasone treatment: embryos were exposed to a solution containing 25 μM of dexamethasone during the time needed for the experiment.

-XAV939 treatment: embryos were exposed to a solution containing 5 μM of XAV939 from 36 to 55 hpf.

-Caffeine treatment: embryos were exposed to a solution containing 100 µM of caffeine from 50 to 55 hpf.

-Isoprenaline treatment: embryos were exposed to a solution containing 300 µM of isoprenaline from 50 to 55 hpf.

In vivo imaging:

Zebrafish embryos were staged and anaesthetised with 0.02% tricaine solution.

Brightfield imaging was performed on a Leica DMIRBE inverted microscope using a Photron SA3 high-speed CMOS camera (Photron, San Diego, CA) and water immersion objective (Leica 20X, NA 0.7). Image sequences were acquired at a frame rate of 125 frames per second.

For confocal imaging, embryos were mounted in 0.7% low melting-point agarose (Sigma Aldrich). Confocal imaging was performed on a Leica SP8 confocal microscope. Images were acquired bidirectionally with a lowmagnification water immersion objective (Leica HCX IRAPO L, 25X, N.A. 0.95).

For time lapse, z-stacks were acquired each 15 or 30 min, depending on the experiment. The optical plane was moved 5 µm between the z-sections.

For lightsheet imaging, two mounting methods were used: 3D printed molds to create a phytagel structure, where the fish stay in direct contact with the medium, and U-shaped glass capillaries, where the fish is embedded in 0.7%low melting-point agarose (Sigma Aldrich). Lightsheet imaging was performed on a Leica TCS SP8 DLS with water immersion detection objective (HC Fluotar L 25 0.95 2.5 Water) with a TwinFlect of 5mm and with an illumination objective HC PL Fluotar 2.5 0.007.

Whole mount immunohistochemistry:

Embryos were fixed overnight with 4% Paraformaldehyde in PBS overnight at 4°C, washed with Tween 0.1% in PBS and permeabilized with 0.5% Triton-X100 in PBS for 20 min at room temperature.

After several steps of washing with Tween 0.1% in PBS, samples were blocked for 2h at RT with 5% goat serum, 5% BSA and 20mM MgCl2 in PBS followed by overnight incubation with the primary antibodies diluted in the blocking solution at 4°C. After several washes, secondary antibodies diluted 1:500 in PBS were incubated overnight at 4°C. Nuclei were counterstained with DAPI 1:1000 in PBS for 15 minutes at 4°C.

The antibodies for immunofluorescence detection were as follows: anti-GFP (Aves Lab) at 1:500, anti-ctgf (Abcam) at 1:100, anti-Yap/Taz (specific for Taz in zebrafish) at 1:100 (Cell signaling), anti-Yap (Lecaudey laboratory). Secondary antibodies and stains were: goat anti-chicken Alexa Fluor 488 IgY (H+L) (Invitrogen), goat anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific), Phalloidin 568 (Thermo Fisher Scientific) and DAPI (Thermo Fisher Scientific).

In some experiments, 4xGTIIC:d2GFP embryos were only labelled with phalloidin and DAPI and endogenous GFP was imaged.

Embryos were mounted and imaged with a Leica SP8 confocal microscope fitted with a dipping immersion objective (Leica HCX IRAPO L, 25X, N.A. 0.95).

Z-stacks were taken every 5 µm. Maximal projections of images were 3D

reconstructed in whole-mount views using IMARIS software (Bitplane Scientific Software).

Microinjection:

MOs from Genetools Inc were injected in 1 to 8 cell stage embryos.

The following MOs were used:

-Taz MO: 5'-CTGGAGAGGATTACCGCTCATGGTC-3' (Voltes et al.

2019

) at 2ng concentration.

-tnnt2a MO: 5'-CATGTTTGCTCTGATCTGACACGCA-3' [START_REF] Sehnert | Cardiac Troponin T Is Essential in Sarcomere Assembly and Cardiac Contractility[END_REF]) at 5.8ng concentration.

Video analysis and statistical analysis:

ImageJ was used to manually quantify cell number, as well as fluorescence intensity.

Statistical tests were performed using GraphPad Prism 7.

RNAscope:

Embryos were fixed overnight at 4ºC in 4% paraformaldehyde in PBS and were dehydrated to 100% methanol gradually. RNAscope was performed using ctgf-C3 and from RNAscope Fluorescent Multiplex kit (Advanced Cell Diagnostics).

TSA Plus fluorescein (PerkinElmer) and were used at 1:600 dilution. Embryos were kept for 3 days in 1:50 phalloidin 647 (Thermo Fisher Scientific) at 4ºC after the RNAscope protocol to stain F-actin. Embryos were imaged using a Leica SP8 confocal microscope with a Leica HCX IRAPO L, × 25, NA0.95 water immersion objective.

In vivo heart imaging with Leica TCS SP8 DLS Preface

In order to perform live imaging, lightsheet imaging is broadly used. Lightsheet microscopy, or selective plane illumination microscopy, is a technique that uses a focused light-sheet to illuminate the specimen from the side. This technique is used in live imaging because it can achieve good resolution at high penetration depths and because it is minimally invasive [START_REF] Huisken | Selective Plane Illumination Microscopy Techniques in Developmental Biology[END_REF].

The basis of lightsheet microscopy is to illuminate a well-defined volume around the focal plane of the detection optics from the side. In the Leica TCS SP8 DLS (called DLS from here), the sheet of light is formed by the fast scanning of a laser beam during the acquisition time of the camera [START_REF] Köster | Light Sheet Microscopy Turned Vertically[END_REF].

Although a traditional lightsheet has a perpendicular setup where the illuminating and the detection objectives are arranged at a 90° angle to each other, the DLS has a vertical setup that uses a mirror device (TwinFlect mirrors)

to deflect the light sheet at a 90º angle. The module is fully integrated into the vertical axis of the inverted microscopy stand [START_REF] Köster | Light Sheet Microscopy Turned Vertically[END_REF]. This For the in vivo imaging of this thesis, larvae were first transferred to fish water containing 0.2 mg/ml tricaine and 0.0033% PTU. Then, three different types of sample mounting have been used:

1) Phytagel molds designed at the IGBMC. Special 3D printed stamps were designed at the IGBMC. Placing the stamps in a bottom glass petri dish and adding phytagel at concentration 1% creates a pocket-like structure between the mirrors, where the fish is kept still and in direct contact with the medium (Figure IB).

2) U-shaped capillary glass. U-shaped glass capillaries, customized for the different mirror sizes, are attached to the bottom of the bottom glass petri dish. The zebrafish embryo is embedded with agarose and placed in the U-shape capillary (Figure IC). Pour l'imagerie in vivo de cette thèse, les larves ont d'abord été transférées dans de l'eau de poisson contenant 0,2 mg/ml de tricaine et 0,0033% de PTU.

Ensuite, trois différents types de montage des échantillons ont été utilisés:

Introduction

For image acquisition, the specimen preparation has two major requirements.

The specimen needs to be placed into the focal plane of the detection objective in between the two mirrors of the cap. In addition, it needs to be slightly elevated from the petri dish bottom for the mirrors/light sheet to reach the specimen's distal part. The advantages of this kind of specimen mounting are:

 easy handling and  possibility to use low concentration of scaffolding medium (e.g., agarose

or methylcellulose) which provides enough specimen confinement under physiological conditions for long-term in vivo imaging.

The disadvantage is the difficulty to align the specimens such that the region of interest is exposed to the side used for fluorescence detection.

To overcome this disadvantage, while maintaining the aforementioned advantages, a rotation device has been designed which allows the angle of the FEP tube to be easily adjusted, so that the specimen can be optimally aligned for imaging. Such set-ups have previously been demonstrated elsewhere [START_REF] Voie | Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens[END_REF][START_REF] Preibisch | Software for beadbased registration of selective plane illumination microscopy data[END_REF][START_REF] Bruns | Sample holder for axial rotation of specimens in 3D microscopy[END_REF].

Materials and Methods

Figure 4: Sample Rotation Device

The sample rotation device (see above, 158007064) for DLS imaging comes with:

 the DLS rotation device;

 microscope glass cover slips (21 x 26 mm); and

 FEP tubes (Camitec, outer diameter 2.00 mm, inner diameter 1.80 mm).

However, there is no silicone or other material for sealing the cover slip included with the rotation device.

Step 1: Cleaning of FEP tubes (recommended procedure)

As described in the report by Kaufmann et al. [4], FEP tubes should be treated in the following way before usage:

 first, all solutions must be degassed and syringe filtered (Millex-HV, PVDF, 0.45 µm);

 rinse with 1 M NaOH (Merck);

 place them in 0.5 M NaOH and ultrasonicate for 10 minutes;

 rinse with double-distilled H2O and 70% ethanol  ultrasonicate in 70% ethanol for 10 minutes;

 rinse with double-distilled H2O; and

 finally, store in double-distilled H2O.

Step 2: Sealing the rotation device with the glass cover slip

Sealing of the cover slip onto the rotation device is done in the following manner:

 apply silicone to the inner edge of the recess at the bottom of the rotation device (see A below);

 add a cover glass and press the glass lightly to ensure that it sits evenly for a good seal at all points (see B below); and

 to check the chamber seal, add some distilled water to the rotation device and see if leaks occur (see C below). As seen below, insert into the rotation device's pin ( 1), the end of the FEP tube with both a black cap and magnet [START_REF] Preibisch | Software for beadbased registration of selective plane illumination microscopy data[END_REF]. Finally, the opposite side of the tube is fixed in place (1 in image below). Be careful: Only put the rotation device onto the z-Galvo stage after initialization. Also, pay attention when changing (illumination) objectives. The wheel for the rotation device protrudes downwards and might touch accidentally an objective (it may be best first to remove bulky objectives from the turret).

Results

Using the LightSheet Wizard, which is included with the LAS X software, you can now acquire images of your specimen(s). Moreover, we show that this modulation is acting through BMP signalling, which tunes Yap1-Tead activity. Finally, we also show that IFT88 and IFT20 interact with Yap1 in the cytoplasm, setting Yap1-Tead activity.

I am second author and I contributed to this paper by:

-Assisting with the imaging of the proepicardial cluster in mice, using the protocol explained in the previous chaper.

-Performing immunohistochemistries against Isl1 and ctgf and helping with the analysis.

-Performing In situ hybridization against Bmp4 in iguana mutants and helping with the analysis.

-Assisting with data curation.

-Helping with the review and editing of the manuscript. Je suis le deuxième auteur et j'ai contribué à cet article en :

Préface en français

Dans

-Aidant à l'imagerie de l'amas proépicardique chez la souris, en utilisant le protocole expliqué dans le chapitre précédent.

-Réalisant des immunohistochimies contre Isl1 et ctgf et en aidant à l'analyse. Several epicardial markers have been described, such as tcf21, wt1, or tbx18.

Study of

Nevertheless, the epicardial layer is very heterogeneous and there are different subsets of cells with different gene expression within it (Y. Cao and Cao 2018;[START_REF] Plavicki | Multiple Modes of Proepicardial Cell Migration Require Heartbeat[END_REF][START_REF] González-Rosa | Pan-Epicardial Lineage Tracing Reveals That Epicardium Derived Cells Give Rise to Myofibroblasts and Perivascular Cells during Zebrafish Heart Regeneration[END_REF][START_REF] Weinberger | Functional Heterogeneity within the Developing Zebrafish Epicardium[END_REF]. Several regulation pathways have been described as important regulators of this process, such as the Bmp pathway (Andrés-Delgado et al.

2019

). However, it is still unknown why some specific cells from the dorsal pericardium will acquire PE-like identity, undergoing major cell shape changes to form the PE cluster.

One crucial regulator of cardiovascular development is the Hippo pathway, a signalling pathway involved in the regulation of organ size and cell proliferation among others [START_REF] Zhao | The Hippo Pathway in Organ Size Control, Tissue Regeneration and Stem Cell Self-Renewal[END_REF]. Yap1 and Wwrt1 (or Taz)

are the main effectors of the Hippo pathway, as activation of the Hippo pathway results in Yap/Taz phosphorylation and cytoplasmatic retention [START_REF] Yu | Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer[END_REF]. However, changes in mechanical cues such as extracellular matrix rigidity, shear stress, or biomechanical forces of blood flow can also regulate Yap/Taz nuclear translocation independently from the Hippo pathway [START_REF] Lundin | YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow[END_REF][START_REF] Elosegui-Artola | Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores[END_REF]. In zebrafish, the Hippo pathway promotes venous pole identity and increases atrial cardiomyocyte number through BMP [START_REF] Fukui | Hippo Signaling Determines the Number of Venous Pole Cells That Originate from the Anterior Lateral Plate Mesoderm in Zebrafish[END_REF]). Moreover, it has been described that intraflagellar transport complex B proteins can regulate the Hippo effector Yap1 during cardiogenesis [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. In mice, Yap1 is colocalizing with Wt1 in epicardial cells.

Moreover, epicardial inactivation of Yap/Wwrt1 causes embryonic death [START_REF] Singh | Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development[END_REF]. Although Yap1/Wwrt1-Tead activity is involved in different steps of cardiac development, its impact during the process of PE cluster formation remains elusive.

Here, we used zebrafish to study the role of Yap1/Taz-Tead activation during PE cluster formation. We found that Yap1/Taz is active in DP cells from early timepoints and activity is maintained in PE and epicardial cells. Chemical modulation of Yap1/Taz activity affects PE formation and we describe Lkb1 as a new regulator in PE cluster formation. Graphic showing relative intensity (intensity normalized to pericardial cells far from the cluster) of the PE cells and cells close to the pericardium. PE cells, followed by cells close to the cluster, have higher intensity.

To confirm that 4xGTIIC:d2GFP was specifically labelling Yap1/Taz-Tead activation, we performed RNAscope at 55 hpf to check ctgf expression, a known read-out of Yap1/Taz-Tead activity [START_REF] Totaro | YAP/TAZ Upstream Signals and Downstream Responses[END_REF]. We confirmed that ctgf is specifically expressed in PE cells, as well as in some cells in the ventral pericardium, consistent with the results that we previously obtained (Figure 1C).

Furthermore, we performed IMHC against yap, taz, and ctgf at 55 hpf. We confirmed that the localization of these antibodies is consistent with the GFP expression from the reporter line 4xGTIIC:d2GFP, thereby confirming that GFP expression is specific to Yap1/Taz-Tead activity (Figure 1D).

To further describe Yap-Taz/Tead activation, we measured quantitatively GFP intensity in different areas of the DP. We performed confocal in vivo imaging every 30 minutes starting at 50 hpf. For every time point, we measured the intensity of PE cells and pericardial cells close to the PE cluster, and we normalized it to the intensity of the pericardial cells far from the PE cluster (Figure 1E). As expected, cells from the PE cluster have GFP intensity higher than nearby pericardial cells (Figure 1F), confirming that cells from the PE have increased Yap/Taz-Tead activity compared to other areas of the DP.

To investigate in more detail PE formation we sought to follow it in vivo using lightsheet microscopy. For this purpose, we imaged actb2:lifeAct-RFP [START_REF] Behrndt | Forces Driving Epithelial Spreading in Zebrafish Gastrulation[END_REF]) embryos crossed with Et(-26.5Hsa.WT1-gata2:EGFP)cn1 or epi:GFP [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF]), a reporter line of Wt1a where around 70% of PE cells express GFP. Imaging at 48 hpf every 30 seconds (Figure 2B), we observed that some cells from the pericardium express GFP before they start to round up and the PE cluster is not completely formed. Since the PE expresses different known markers, we next wanted to confirm if tcf21 was also expressed before cells extrude from the DP. We imaged tcf21:nls-GFP embryos starting at 36 hpf every 15 minutes. We observe that similarly to wt1a, tcf21 is present in PE cells before they round up and after in the formed PE (Figure 2C).

Next we image 4xGTIIC:d2GFP embryos (n=2) starting at 36 hpf every 15 minutes (Figure 2D). In earlier time points, GFP expression is weak in all the pericardium. At around 40 hpf, some cells in the atrioventricular channel area in the DP increased their GFP intensity. These cells keep increasing their GFP expression through time and at 48 hpf they start to round up, possibly being the ones that will form the PE cluster.

We then imaged an embryo starting at 48 hpf every 15 minutes (Figure 2E). We observed flat cells in the dorsal pericardium with high GFP expression, that when followed in time, gives rise to the PE cluster. Overall, our results show that Yap1/Taz-Tead is specifically active in the first steps of epicardial development.

Chemical modification of Yap1/Taz nuclear activity has an impact on PE cluster formation

We then sought to test the role of Yap1/Taz-Tead in PE formation upregulating and downregulating its activity. For this purpose, we used dexamethasone and XAV939. Dexamethasone is a glucocorticoid that activates in vivo Yap1/Taz-Tead mediated transcription [START_REF] Sorrentino | Glucocorticoid Receptor Signalling Activates YAP in Breast Cancer[END_REF][START_REF] Astone | Zebrafish Mutants and TEAD Reporters Reveal Essential Functions for Yap and Taz in Posterior Cardinal Vein Development[END_REF]), while XAV939 is a compound that inhibits tankyrase stabilizing AMOT proteins, resulting in a decrease of Yap1 nuclear translocation (W. [START_REF] Wang | Tankyrase Inhibitors Target YAP by Stabilizing Angiomotin Family Proteins[END_REF].

We first treated 4xGTIIC:d2GFP embryos from 50 to 55 hpf with a solution containing 25 μM of dexamethasone and fixed them to image them with a confocal microscope after phalloidin staining. Dexamethasone treated embryos show no differences in terms of PE cell number compared to non-treated embryos. However, there is an increase of fluorescence in PE cells from dexamethasone-treated embryos compared to non-treated embryos (Sup.

figure 3A,B).

Taking into consideration these results, we decided to increase the time of These results contradict our main hypothesis, since we were expecting the PE cluster formation to be somewhat impaired in Yap1 mutants. However, since it is already known that Yap1 and Taz have some overlapping functions, these results could be the result of compensation by Taz. To check this, we performed immunohistochemistry against Taz in Yap1 mutants and controls and saw that Taz protein is present in the zebrafish heart, PE, some DP cells, and in some epicardial cells (Figure Sup 3 E). To avoid any possible compensation, we used Taz MO at 2ng concentration in Yap1 mutants. In Yap1 mutants and Taz morphants, as expected, the number of Taz cells in the myocardium (Fig Sup 3 F) and pericardium (Fig Sup 3 G) is decreased compared to Yap1 mutants and controls. However, the number of PE cluster cells is not significantly different (Fig Sup 3 H). Altogether, we see that although Yap1/Taz-Tead is active specifically in PE cells, when we inhibit this pathway starting at the 1 cell stage the PE cluster formation is not affected. Since the epicardial layer is essential, it is possible that there are compensatory ways to ensure proper epicardial formation that can overcome Yap1 and Taz inhibition.

Lkb1 mutants have bigger PE clusters at 72 hpf

Given that Dexamethasone increases Yap1/Taz-Tead activity and results in a bigger PE cluster, we hypothesized that mutations in Yap1/Taz-Tead inhibitors would result too in a bigger cluster. Since lkb1 has been previously described as a regulator of Yap1 nuclear translocation in vitro [START_REF] Mohseni | A Genetic Screen Identifies an LKB1-MARK Signalling Axis Controlling the Hippo-YAP Pathway[END_REF], we sought to investigate the impact of lkb1 loss-of-function during PE cluster formation. We fixed lkb1 mutants (van der [START_REF] Velden | The Serine-Threonine Kinase LKB1 Is Essential for Survival under Energetic Stress in Zebrafish[END_REF]) crossed to tcf21:nls-GFP at 36, 55, 72 hpf, and 5 dpf and performed immunohistochemistry against GFP and phalloidin. At 36 hpf there are no apparent differences between controls and mutants in terms of GFP positive cells in the pericardium (Figure 4A) or myocardium cell number (Figure Sup 4A). At 55 hpf, there no significant differences between lkb1 mutants and controls in terms of PE cluster cell number (Figure 4B). Thus, lkb1 mutants show differences in terms of PE cell number at 72 hpf.

Discussion

In this chapter we study Yap1/Taz-Tead activity during PE cluster formation. We describe in vivo Yap1/Taz-Tead activity in the heart from 36 hpf to 55 hpf using the reporter line 4xGTIIC:d2GFP. We see GFP expressed specifically in the PE cluster. Moreover, GFP expression is on before PE cells extrude to the proepicardial cavity, suggesting that it is involved somehow in PE cluster formation.

PE cells change their shape from flat to round, breaking their cell adhesions.

Although this process is not a complete epithelial to mesenchymal transition (EMT), it shares many similarities [START_REF] Andrés-Delgado | Actin Dynamics and the Bmp Pathway Drive Apical Extrusion of Proepicardial Cells[END_REF]. Moreover, PE cells express twist1a (Peralta 2014), a bHLH transcription factor involved in EMT and a regulator of lineage specification and differentiation [START_REF] Lamouille | Molecular Mechanisms of Epithelial-Mesenchymal Transition[END_REF]. It is also interesting to mention that Yap has been described as a promoter of EMT in several cancers, such as colorectal cancer [START_REF] Cheng | YAP Promotes Epithelial Mesenchymal Transition by Upregulating Slug Expression in Human Colorectal Cancer Cells[END_REF]. In vitro, it has also been seen that Yap-mediated responses to changes in ECM organization promote EMT [START_REF] Park | Switch-like Enhancement of Epithelial-Mesenchymal Transition by YAP through Feedback Regulation of WT1 and Rho-Family GTPases[END_REF]).

This would be consistent with a model in which Yap1/Taz-Tead activity promotes PE cluster formation: Yap1/Taz-Tead is active in DP cells from early time points and these cells will extrude, in an EMT-like process, to form the PE cluster.

Consistent with this, in our chapter we show that modifying Yap1/Taz-Tead activity using drugs results in dysregulation of the PE formation process: an increase in Yap1/Taz-Tead activity results in more PE cell in the cluster while a decrease results in a decreased PE cell number. All of this suggests that Yap1/Taz-Tead activity promotes PE-like cell identity. This is consistent with what has been previously published in the field. It has been published that IFT complex B proteins inhibit Yap1 activity during PE cluster formation and a lack of IFT88 results in an increase in PE cell cluster [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]). Motin proteins have also been shown to regulate PE cluster formation. In zebrafish, Amot proteins bind to Yap/Taz in the cytoplasm sequestering it [START_REF] Agarwala | Amotl2a Interacts with the Hippo Effector Yap1 and the Wnt/β-Catenin Effector Lef1 to Control Tissue Size in Zebrafish[END_REF]. Amotl2a mutants, which would have increased nuclear Yap1/Taz, have bigger PE clusters [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. However, although when we chemically decrease Yap1/Taz-Tead activity in zebrafish results in a smaller PE cluster, we do not see the same results in Yap1 mutants or Yap1 mutants and Taz morphants.

This could be due to different events. In our model, we did not completely eliminate Taz from the system. Double mutants for Yap1/Taz exhibit severe developmental defects and die by 30 hpf [START_REF] Nakajima | Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance[END_REF]. We tried to overcome this by treating our Yap1 mutants with a low concentration of Taz MO. The resultant fish had decreased, but not absent, levels of Taz protein.

However, we found that the PE cluster is still formed in these fish. One possibility could be that even low amounts of Taz is enough to promote PE cluster formation and compensate for the loss of Yap1. For instance, it has been described in retinal development in mice that in Yap heterozygous mice models, depletion of one Yap allele leads to transient TAZ compensatory mechanisms at postnatal stages, associated with a gain of function-like phenotype [START_REF] Masson | Yap Haploinsufficiency Leads to Müller Cell Dysfunction and Late-Onset Cone Dystrophy[END_REF]). However, this hypothesis is very hard to prove since we reached the limit of our model: we need some levels of Taz in order to have viable fish and so we cannot study the consequences of completely deleting the activity of both.

One way to try to answer our question would be to create conditional mutants of Yap1 or Taz. While traditional mutants have a mutation in all cells at all times, conditional mutants allow inactivation of genes in somatic cells in a temporal or tissue specific manner [START_REF] Maddison | Generating Conditional Mutations in Zebrafish Using Gene-Trap Mutagenesis[END_REF]. It is still challenging to create zebrafish conditional mutants due to the unavailability of gene targeting techniques for zebrafish embryonic stem cells and the low efficiency of homologous recombination in fertelized zebrafish eggs [START_REF] Dai | Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos[END_REF]Robles, Martí, and Belmonte 2011). Although challenging, several labs have created conditional mutants restricting expression in a tissue specific manner, for example, Cre lines specific for cardiomyocytes [START_REF] Jopling | Zebrafish Heart Regeneration Occurs by Cardiomyocyte Dedifferentiation and Proliferation[END_REF]) among others. However, this approach would not be suitable for our study. First of all, there are no specific genes only expressed in the PE or epicardium. We could not completely restrict Yap1 or Taz expression only in the PE, other organs of the zebrafish would be affected. For instance, if we used the promoter of one of the most used epicardical markers, wt1a, and conditionally mutate genes under this promoter, we would also be affecting kidney development [START_REF] Perner | The Wilms Tumor Genes Wt1a and Wt1b Control Different Steps during Formation of the Zebrafish Pronephros[END_REF]. Another issue that we would face is that Yap1 and Taz are necessary for development and double mutants die at 30 hpf [START_REF] Nakajima | Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance[END_REF]). If we tried to restrict Yap1 or Taz expression before the PE cluster is formed, the embryos would most likely not survive. A way to overcome these issues would be to change animal model. In mice conditional mutant are widely available and a conditional line with a double mutation in Yap1 and Taz specifically in epicardial cells already exists [START_REF] Singh | Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development[END_REF]. Although in this paper they already described that this double mutants do not survive, it would be useful for our project to try different conditions with this line and study PE cluster formation.

Another explanation could be that other pathways involved in PE formation are compensating for the loss of Yap1/Taz. Since the heart is essential for survival and the epicardium is key for heart development, it is possible that there exists several signalling pathways to ensure PE cluster formation. These pathways might not be effective when we chemically modify Yap1/Taz-Tead activity because we treat from 36 hpf and it is too late to compensate. For instance, it is known that PE is regulated by BMP signalling [START_REF] Liu | Zebrafish in the Study of Early Cardiac Development[END_REF] and that endothelial Notch signalling activates bmp expression, inducing PE cluster formation [START_REF] Andrés-Delgado | Notch and Bmp Signaling Pathways Act Coordinately during the Formation of the Proepicardium[END_REF]). In the future, it should be studied if

Yap1 mutants show any differences in Bmp or Notch levels that could explain why they do not have any differences in terms of PE cell number.

Another possibility is that, although we do see some change in PE cluster formation after drug treatment targeting Yap1/Taz-Tead activity, this pathway is not essential for PE cluster formation and it would have more importance at later points of epicardial development. For instance, it is known that epicardial cells undergo EMT transition and give rise to non-myocardial cardiac cells. It is possible that PE cells activate Yap1 when they acquire their identity but Yap1 function is not relevant until after the cells are attached to the myocardium.

However, to prove this hypothesis we face the same issues as beforewe cannot completely delete Yap1 and Taz as double mutants die at early time points.

Previously it has been described that cells from the DP collectively move towards the DP midline, where the cluster will be formed [START_REF] Andrés-Delgado | Actin Dynamics and the Bmp Pathway Drive Apical Extrusion of Proepicardial Cells[END_REF]. In this same paper they propose a model in which Bmp promotes actin polymerization leading to PE formation. Our hypothesis that Yap1/Taz-Tead activity is involved in PE cluster formation is compatible with this model.

Yap1/Taz are known mechanosensors that can respond to F-actin polymerization, as it has been seen that triggering F-actin polymerization promoted Yap1/Taz activity [START_REF] Dupont | Role of YAP/TAZ in Mechanotransduction[END_REF]. It is possible that the promotion of actin polymerization, as well as the reduction in cell area that cells in the DP midline experience, increases Yap1/Taz-Tead activity.

Finally, we also describe a new possible regulator for PE cluster formation, lkb1.

In vitro it has been described that it inhibits Yap1 nuclear translocation [START_REF] Mohseni | A Genetic Screen Identifies an LKB1-MARK Signalling Axis Controlling the Hippo-YAP Pathway[END_REF]. We show that lkb1 mutants have bigger clusters at later points of proepicardial formation. At 55 hpf there are no differences between controls and mutants. However, at 72 hpf lkb1 mutants have increased PE cell number compared to controls. Since epicardial cell number is not changed, it could mean that time of cell extrusion from the DP is increased. However, further studies about the role of lkb1 during PE cluster formation should be done.

In summary, our study reports specific Yap1/Taz-Tead activation during PE cells. Further studies are needed to understand the exact function of this pathway during PE formation. mécanosenseurs connus qui peuvent répondre à la polymérisation de la Factine, car il a été vu que le déclenchement de la polymérisation de la F-actine favorisait l'activité de Yap1/Taz [START_REF] Dupont | Role of YAP/TAZ in Mechanotransduction[END_REF]. Il est possible que la promotion de la polymérisation de l'actine, ainsi que la réduction de la surface cellulaire que subissent les cellules de la ligne médiane DP, augmente l'activité de Yap1/Taz-Tead.

Discussion en français

Enfin, nous décrivons également un nouveau régulateur possible de la formation du PE, lkb1. In vitro, il a été décrit qu'il inhibe la translocation nucléaire de Yap1 [START_REF] Mohseni | A Genetic Screen Identifies an LKB1-MARK Signalling Axis Controlling the Hippo-YAP Pathway[END_REF]. Nous montrons que les mutants lkb1 ont de PE plus grands à des points plus tardifs de la formation du proépicarde.

A 55 hpf, il n'y a pas de différences entre les contrôles et les mutants. It is known that mechanical forces influence chamber formation, trabeculation, cardiomyocyte proliferation, and valve formation (Granados-Riveron and Brook 2012). However, the role of fluid forces during PE formation remains elusive.

Pericardial advections, formed by the heartbeat, are necessary for the release of PE cells. Moreover, avcPE and vpPE are located in areas of high flow forces [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF]). However, mutants without heartbeat still form the PE cluster, although it is misplaced [START_REF] Serluca | Development of the Proepicardial Organ in the Zebrafish[END_REF][START_REF] Andrés-Delgado | Actin Dynamics and the Bmp Pathway Drive Apical Extrusion of Proepicardial Cells[END_REF].

Since it has been described that subcellular localization and activity of Yap1/Taz can be regulated by cell substrate rigidity, actin cytoskeleton remodelling, and by specific regimens of cell stretching [START_REF] Low | YAP/TAZ as Mechanosensors and Mechanotransducers in Regulating Organ Size and Tumor Growth[END_REF], we thought that specific Yap1/Taz-Tead activity in the DP could be due to mechanical forces caused by heartbeat and this could, in turn, regulate PE cluster formation.

In this chapter we ought to examine further how changes in fluid forces within the pericardial cavity affect PE cluster formation and Yap1/Taz-Tead activity in the dorsal pericardium.

Results

Modifying heartbeat results in affected PE cluster formation

To assess the role of pericardial fluid forces during PE formation, we modified them by treating 4xGTIIC:d2GFP embryos with drugs that affect the heartbeat.

We treated embryos with caffeine (concentration100 µM) from 50 to 55 hpf, which leads to a nonhomogeneous contraction. Immunohistochemistry experiments performed on caffeine treated embryos and non-treated embryos (Figure 1A) showed that caffeine treatment does not significantly change the number of GFP positive cells in the myocardium or the DP. In caffeine treated embryos there is an increase of PE cluster cells (Figure 1B), as previously published [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF]). Nevertheless, the number of epicardial cells is not changed between treated embryos and controls, suggesting that caffeine is affecting specifically PE cluster formation.

To further study PE formation when heartbeat is altered, we used isoprenaline, a drug that increases heart rate [START_REF] Kossack | Induction of Cardiac Dysfunction in Developing and Adult Zebrafish by Chronic Isoproterenol Stimulation[END_REF]. We treated 4xGTIIC:d2GFP embryos from 50 to 55 hpf with isoprenaline (concentration 300 µM) and fixed them for imaging (Figure 1F). Isoprenaline treated embryos also have increased PE cluster cell number (Figure 1D) and no differences in terms of epicardial cell number (Figure 1E). When we start the treatment at 48 hpf (Figure 2E), the changes in GFP intensity are not significantly different between treated and non-treated embryos. Both in the PE and in the DP, the intensity decreases through time (Figure 2G-H).

However, when we start the treatment at 52 hpf (figure 2F), the results are very different. Although it is very variable between fish, after BDM treatment intensity in the PE and the DP increases compared to non-treated embryos (Figure 2I-J).

Overall we see that disrupting the actin-myosin network also results in a change in Yap1/Taz-Tead activity but it is highly dependent on the time of the treatment.

Discussion

In this chapter we explore further the role of heartbeat during PE cluster formation related to Yap1/Taz-Tead activity.

We see that affecting the heartbeat, either increasing the heart rate or arresting the heart, results in changes in Yap1/Taz-Tead activity. This is consistent with the fact that Yap1/Taz are known mechanosensors whose subcellular localization can be regulated by changes in cell stretching, such as the ones caused by heartbeat.

After we treat with caffeine and isoprenaline, two drugs that affect heart beating, we obtain similar results: an increase in PE cell number without changes in epicardial cell number. However, after caffeine treatment, we see an increase in GFP positive cells that we do not see after isoprenaline treatment. Although both drugs act similarly, their consequences in the zebrafish embryo are not exactly the same. Overall these results suggest that a change in heart rate results in a bigger PE cluster. However, it has to be taken into account that these drugs might not be specific and they could be promoting cell extrusion in the DP independently of the heartbeat. One possible experiment to be done in the future could be to treat fish lacking heartbeat with caffeine and isoprenaline and analyse the changes in 4xGTIIC:d2GFP fish. If the change in cluster cell size is due to changes in heartbeat, we should not see any difference compared to controls once we eliminate heartbeat. Moreover, although we do not see any change in epicardial cell number, we cannot discard that the cluster increased cell size could be due to a change in cell release.

As it has been previously described, arresting the heart does not affect PE cluster size but it affects PE cluster position. We also describe a general decrease in GFP positive cells in the myocardium and an increase of GFP positive cells in the DP. The decrease in the myocardium could be explained by the fact that cell stretching of myocardial cells due to the heartbeat could be translocating Yap1/Taz to the nuclei. In tnnt2a morphants, we lose this cell stretching and consequently also Yap1/Taz-Tead activity. However, we saw an increased number of GFP positive cells in the DP. This could be related to the displacement of the PE cluster. In order to test this hypothesis, one experiment that could be done would be treating tnnt2a morphants with drugs that reduce Yap1/Taz-Tead activity, such as XAV939 or Verteporfin. If we do see a decrease in the number of GFP positive cells in the DP after treatment, we could study were is the cluster formed in these embryos and assess if the misplacement is due to the increase of Yap1/Taz-Tead activity in DP cells.

We also wanted to address whether Yap1 would be important to regulate PE formation in embryos that lack heartbeat. Although Yap1 mutants and tnnt2a morphants seem to have a stronger phenotype in terms of heart shape as heart morphology is more affected than in tnnt2a morphants alone, the PE cluster still forms and there are no significant differences compared to tnnt2a morphants. In our embryos, an early PE cluster was present already at 48 hpf. We did not see a regression of this initial cluster, but we did see an arrest in cell extrusion: no more cells were incorporated in the cluster after BDM treatment.

Overall, we show that Yap1/Taz-Tead activity is sensitive to changes in heartbeat and that PE cluster formation is affected, either in size or place, when we modify heartbeat. Further studies are needed to deduce how mechanical forces are regulating PE cluster formation. [START_REF] Trubelja | Molecular Mechanisms of Mechanosensing and Mechanotransduction in Living Cells[END_REF]. Some known mechanosensors are cilia, integrin signalling components, cell membrane receptor kinases, stretch-sensitive ion channels, intercellular junction proteins, and membrane lipids [START_REF] Lyon | Mechanotransduction in Cardiac Hypertrophy and Failure[END_REF][START_REF] Andrés-Delgado | Interplay between Cardiac Function and Heart Development[END_REF].

Discussion en français

In this chapter we chose some of these already known mechanosensors and evaluated if their disruption affected PE cluster formation.

Results

Primary cilia during PE cluster formation

It has been previously published that iguana mutants, which lack Dzip protein located in the basal body and subsequently also lack primary cilia, have smaller PE clusters compared to controls [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. Moreover, in the context of epidermis development in mice, cilia presence stimulates canonical Notch

Discussion

In this chapter, we assessed the role of several known mechanosensors in the regulation of PE cluster formation.

Since primary cilia are important mechanosensors involved in different steps of development [START_REF] Ferreira | The Cilium as a Force Sensor-Myth versus Reality[END_REF], we studied further its role in PE cluster formation. Since iguana mutants have reduced PE cluster size and it has been seen than Verteporfin and XAV939 treatment, two drugs that inhibit Yap1/Taz-Tead activity, results in a similar phenotype, we studied Yap1/Taz-Tead activity at early developmental timepoints. However, we do not see a decrease in Yap1/Taz-Tead activity, so we must conclude that the reduced size of PE in these mutants is not due to a decrease in Yap1/Taz-Tead.

We further studied the role of primary cilia in this process by investigating PE cluster size in Kif3a mutants, which lack primary cilia, and Pkd2 mutants. Pkd2 is a known mechanosensor in endothelial cells and endocardial cells during valve morphogenesis [START_REF] Goetz | Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development[END_REF][START_REF] Heckel | Oscillatory Flow Modulates Mechanosensitive Klf2a Expression through Trpv4 and Trpp2 during Heart Valve Development[END_REF]. We did not see any change in terms of PE cluster formation. Although there mechanosensors are not involved in PE cluster formation, since iguana mutants have smaller cluster size, we cannot conclude that primary cilia are not important for PE cluster formation. Iguana encodes for Dzip1, a zinc-finger protein required for the proper regulation of Hedgehog signalling [START_REF] Sekimizu | The Zebrafish Iguana Locus Encodes Dzip1, a Novel Zinc-Finger Protein Required for Proper Regulation of Hedgehog Signaling[END_REF]. Further studies should be made to determine if the iguana mutant phenotype is due to the lack of primary cilia or due to misregulation of the Hedgehog signalling pathway. Furthermore, since epicardial regeneration can be regulated by Hedgehog signalling, it would be interesting to further analyse if Hedgehog signalling has an important role also during earlier steps of epicardial formation when the PE cluster is being formed.

Finally, we also conclude that Klf2a/b are not relevant for PE cluster formation.

Although we did not find any new mechanosensors involved in PE cluster formation, there are still many other mechanosensors that have not been studied and more should be done in the future to understand why PE cluster formation is sensitive to mechanical forces and how DP cells sense these changes and differentiate to form epicardial precursor cells. In this thesis we provide new studies about PE cluster formation, focusing on the role of Yap1/Taz-Tead activity during this process.

Discussion en français

First, we created a protocol for in vivo imaging using the DLS, which allows us to follow zebrafish heart development.

Next, I participated in the paper "Intraflagellar Transport Complex B Proteins

Regulate the Hippo Effector Yap1 during Cardiogenesis", were we prove for the first time that IFTB proteins can regulate Yap1-Tead activity through BMP signalling independently of cilia. This restricts proepicardial and myocardial development. Every day new functions for ciliary proteins are being discovered and we found a novel role forIFTB proteins.

We also described Yap1/Taz-Tead activity through all the process of PE cluster formation. Using in vivo and fixed imaging, we show that Yap1/Taz-Tead is active before PE cells extrude from the DP and how this activity is maintained when cells are already attached to the myocardium. Currently, many details about PE cluster formation remain elusive. In this thesis we propose Yap1/Taz-Tead activity as a new pathway involved in epicardial development and cell identity.

Although we do see a change in PE cluster formation when we modify Yap1/Taz-Tead activity using drugs, we do not obtain the same results when we study Yap1 mutants injected with Taz MO. Consequently, we cannot conclude that Yap1/Taz-Tead activity leads to PE cluster formation. However, it should be noted that this activity is specific to PE cluster cells and that it is not possible to completely eliminate Yap1 and Taz in our model since this would cause embryonic death. More studies should be done to try to decipher Yap1/Taz role in epicardial development.

Following previous studies [START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF], we also investigated further the role of mechanical forces in PE cluster formation and tried to find mechanosensors involved in this process. We do show that changing fluid forces in the pericardial cavity affect PE cluster formation, either changing its size or its position, and Yap1/Taz-Tead signalling. However, we did not find new mechanosensors involved in this process.

The epicardial layer is key for proper heart function and it has been widely ignored until recent studies. In this thesis we present an exhaustive study of PE cluster formation in zebrafish, focusing in Yap1/Taz-Tead activity. We hope that this thesis helps to further our knowledge of PE cluster formation. Although we were unable to pinpoint Yap1/Taz-Tead function or find new regulators involved in PE cluster formation, we hope that our results will help to redirect future studies about PE cluster formation.

Dans cette thèse, nous fournissons de nouvelles études sur la formation du PE, en nous concentrant sur le rôle de l'activité de Yap1/Taz-Tead pendant ce processus. Ces résultats indiquent que l'augmentation de l'activité de Yap1/Taz-Tead à des moments précoces augmente le nombre de cellules du groupe PE et que la diminution de la translocation nucléaire de Yap1 a l'effet inverse.

Lkb1 pourrait être un régulateur de la formation des amas de PE

Comme il a été décrit précédemment que lkb1 peut réguler la translocation nucléaire de Yap1 [START_REF] Mohseni | A Genetic Screen Identifies an LKB1-MARK Signalling Axis Controlling the Hippo-YAP Pathway[END_REF], nous avons cherché à étudier l'impact de la perte de fonction de lkb1 lors de la formation de proepicarde. Nous avons analysé le nombre cellules du proepicarde chez les mutants lkb1 (van der [START_REF] Velden | The Serine-Threonine Kinase LKB1 Is Essential for Survival under Energetic Stress in Zebrafish[END_REF] a différents stades embryonnaires.

À 36 hpf, il n'y a pas de différences apparentes entre les témoins et les mutants en termes du nombre de cellules proepicardiques. Cependant, à 72 hpf, les mutants lkb1 présentent un nombre accru de cellules proepicardiques par rapport aux témoins.

Conclusions

Les principales conclusions qui seront examinées plus en détail dans la thèse sont les suivantes :

-Les cellules péricardiques qui donneront naissance au proepicarde ont une activité Yap1/Taz-Tead avant que le groupe ne soit formé.

-L'augmentation de l'activité Yap1/Taz-Tead entraîne une augmentation du nombre de cellules PE.

-Une diminution de l'activité de Yap1/Taz-Tead entraîne une diminution du nombre de cellules PE.

-Les mutants Lkb1 présentent un proepicarde plus grand à 72 hpf.

Résumé en anglais

The epicardium, a mesothelial cell layer covering the myocardium, is essential for heart development, as it promotes myocardial growth, coronary vasculature formation and intracardiac fibroblast generation. In zebrafish, epicardial progenitors arise from the dorsal pericardium (DP). Some DP cells round up and protrude forming the proepicardial (PE) cluster. Yap1 and Wwrt1, central nodes of the Hippo pathway, are localized in the developing epicardium in mice. Although Yap1/Wwrt1-Tead activity is involved in cardiac development in zebrafish, its impact during PE cluster formation remains elusive. We characterized Yap1/Wwrt1-Tead reporter line during PE cluster formation. Next, we assessed the role of Yap1/Wwrt1-Tead activity in this process modifying it. We further studied PE cluster development and how it is affected by changes in mechanical forces. Finally, we searched as well for new regulators of PE cluster formation. Our work suggests that Yap1/Taz-Tead activity is involved during PE cluster formation although Yap1 mutants do no present any PE phenotype. Our work also suggests that fluid forces are key to modulate PE localisation and formation in the pericardial cavity.

Figure 1 .

 1 Figure 1. The heart in various animal models.

Figure 2 .

 2 Figure 2. Heart development in mouse.

Figure 3 .

 3 Figure 3. Heart development in zebrafish.

Figure 4 .

 4 Figure 4. PE cluster formation in zebrafish.

Figure 5 .

 5 Figure 5. PE cell transfer to the myocardium.

Figure 6 .

 6 Figure 6. Canonical Hippo pathway.

Figure 7 .

 7 Figure 7. Non canonical Hippo pathway.

Figure 8 .Figure 1 .

 81 Figure 8. Primary cilia.

Figure 2 .

 2 Figure 2. DLS objective: Sample positioning by using FEP (fluorinated ethylene propylene) tubes.

Figure 3 .

 3 Figure 3. Positioning of specimens inside an FEP tube.

Figure 4 .Figure 5 .

 45 Figure 4. Sample Rotation Device

Figure 6 .

 6 Figure 6. Drawing the specimen into an FEP tube.

Figure 7 .

 7 Figure 7. Length of FEP tube: 38 mm.

Figure 8 .

 8 Figure 8. Attaching the black caps to the FEP tube.

Figure 9 .

 9 Figure 9. Placing the FEP tube in the rotation device (first step).

Figure 10 .

 10 Figure 10. Placing the FEP tube in the rotation device (final step).

Figure 11 .

 11 Figure 11. TCS SP8 DLS scanning stage with rotation device.

Figure 12 .

 12 Figure 12. Drosophila embryo observed from different sides. Single planes were taken from 3D image stacks.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. IFT Complex B Proteins Regulate Proepicardial Development in Zebrafish and Mice

Figure 4 .

 4 Figure 4. IFT88 and IFT20 Are Physically Associated to YAP1, and IFT88 Modulates YAP1 Activity

Figure S2 .

 S2 Figure S2. Cilia protruding into the pericardial cavity are distributed heterogeneously in zebrafish during proepicardial development. Related to Figures1 and 3 and STAR methods.

Figure S3 .

 S3 Figure S3. Ift20 KO mice proepicardial cells lack cilia. Related to Figures1 and 2.

Figure S4 .

 S4 Figure S4. bmp4 is overexpressed in ift88, elipsa/ift54, and Ift20 mutants. Related to Figure 2.

Figure S5 .

 S5 Figure S5. Yap1 activity is increased in the myocardium of ift88 and elipsa/ift54 zebrafish mutants and AMOTL1 activity is increased in IFT20 KO mice proepicardium. Related to Figure 3.

Figure S6 .

 S6 Figure S6. IFT88-GFP co-localize with YAP1 in the cytoplasm. Related to Figure 4.

Figure S7 . 4 .Figure 1 .

 S741 Figure S7. IFT88 and IFT20 regulate YAP1 activity. Related to Figure 4.

Figure 2 .

 2 Figure 2. Yap1/Taz-TEAD in vivo activation during PE cluster formation.

Figure 3 .Figure Supplementary 3 .

 33 Figure 3. Modifying Yap1/Taz-Tead activity chemically impacts PE cluster formation.

Figure 4 .

 4 Figure 4. Description of lkb1 mutants at key points of PE cluster formation.

Figure Supplementary 4 .Figure 1 .

 41 Figure Supplementary 4. Description of lkb1 mutants at key points of PE cluster formation.

Figure 2 .Figure 1 .

 21 Figure 2. PE cluster in Yap1 injected with tnnt2a MO and changes in 4xGTIIC:d2GFP after BDM treatment.

Figure 2 .

 2 Figure 2. Study of PE cluster in Klf2a/b mutants.

-

  In vivo imaging with Leica TCS SP8 DLS -Intraflagellar Transport Complex B Proteins Regulate the Hippo effector Yap1 during Cardiogenesis -Study of Yap1/Taz-Tead activity during PE cluster formation -How is PE cluster formation affected by changes in fluid forces? -Looking for possible mechanoregulators of PE cluster formation Finally, Annex I contains an advertisement feature that I helped to create while I was working at Leica Microsystem. I contributed to the feature by imaging the fish embryos and by taking part in the writing process.

Introduction-

  Matériel et methods -Imagerie in vivo avec Leica TCS SP8 DLS -Les protéines du complexe de transport intraflagellaire B régulent l'effecteur Hippo Yap1 pendant la cardiogenèse -Étude de l'activité de Yap1/Taz-Tead pendant la formation du PE -Comment la formation du PE est-elle affectée par les changements des forces fluides?

Figure 1 .

 1 Figure 1. The heart in various animal models. (A) Frontal view of a human heart (B) Frontal view of a mouse heart (C) Frontal view of a zebrafish heart. In the mammalian and the avian heart we have 4 chambers. The zebrafish heart is composed of 2 chambers. White arrows indicate the blood flow direction. (D) The walls of the heart consist of three layers: the endocardium, the myocardium and the epicardium. RA: Right Atrium; RV: Right Ventricle; LA: Left Atrium; LV: Left Ventricle; A: Atrium; V: Ventricle.

Figure 2 .

 2 Figure 2. Heart development in mouse. First progenitor cells are found in the primitive streak. Then cardiac precursors differentiate into cardiomyocytes and form the cardiac crescent. A: anterior; P: posterior; R: right; L: left; PhA: pharyngeal arches; OFT: outflow tract; LV: left ventricle; RV: right ventricle; LA: left atrium; RA: right atrium.

Figure 3 .

 3 Figure 3. Heart development in zebrafish. In early timepoints, cardiac progenitors are placed in bilateral pools in the late plate mesoderm. Atrial, ventricular and endocardial progenitors are separated. Cells then migrate to the midline were myocardial fuse and form the cardiac tube, while the endocardial cells start to migrate inside the cone. Thislater forms the heart tube, which, starting at 48 hpf, will change its shape in a heartbeat-dependent process involving both looping of the tube and ballooning of the cardiac chambers. Finally, the heart will be formed by an atrium, ventricle, and the bulbus arteriosus. V: ventricle; A: atrium; BA: bulbus arteriosus. Adapted from[START_REF] Brown | Advances in the Study of Heart Development and Disease Using Zebrafish[END_REF][START_REF] Bakkers | Zebrafish as a Model to Study Cardiac Development and Human Cardiac Disease[END_REF]

  area and the outflow tract area. Some cells of the DP rearrange their actin network, round up and form the PE cluster in a process reminiscent of epithelial apical extrusion (Figure4B-C)[START_REF] Andrés-Delgado | Actin Dynamics and the Bmp Pathway Drive Apical Extrusion of Proepicardial Cells[END_REF]. This process is dependent on actomyosin network, since it has been shown that BDM treatment, which disrupts the network, avoids PE cluster formation. While there is no cell division in cells from the midline, it is thought that the process whereby cells converge to the midline is driven by the proliferation of cells in the DP located far from the midline (Andrés-Delgado et al. 2019). PE cells remain loosely attached to neighbouring DP cells (Andrés-Delgado et al. 2019) (Figure 4C). Then, cells from the vpPE and the avcPE are released and are advected into the flow until they attach to the myocardium, while cells from the apPE migrate directly to the myocardium without being released.

Figure 4 .

 4 Figure 4. PE cluster formation in zebrafish. (A) Brightfield image of a zebrafish at 48 hpf, when PE cluster formation starts. (B) Cells from the dorsal pericardium converge through the midline. (C) 2 PE clusters can be found in later stages in the midline: the avcPE and the vpPE. V:ventricle; A:atrium; OFT: outflow tract. Adapted from (Andrés-Delgado et al. 2019; Peralta et al. 2013).

Figure 5 .

 5 Figure 5. PE cell transfer to the myocardium. (A) An example of transient bridge formation. Electron microsocopy image showing PE cell transfer in Xenopus. The PE cluster is formed in the right sinus horn. In later timepoints, the PE cluster acquires a conical shape and attaches to the myocardium transferring the proepicardial cells. (B) An example of the release of PE cells into the pericardial cavity. Brightfield images of a zebrafish heart. The PE cluster forms in the atrioventricular channel area. Cells from the proepicardium are released and float in the pericardial cavity until they eventually attach to the myocardium. O: outflow tract; A: atrium; LS: left sinus horn; L: pericardial surface of the liver forming the bottom of the pericardial cavity; RA: right atrium; LA: left atrium; V: ventricle. Proepicardial and epicardial cells are marked in blue. Adapted from[START_REF] Jahr | Development of the Proepicardium in Xenopus Laevis[END_REF][START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF]).

  proper embryonic development. Further knowledge of Hippo pathway might offer new insight into mechanisms of cancer progression as well as congenital diseases.The Hippo pathway in Drosophila is based on a conserved kinase cascade that comprises the Hippo kinase, the Warts kinase, and the adaptor proteins Salvador and Mob. Activation of these signals promotes phosphorylation of the transcriptional regulator Yorkie by the Warts kinase, resulting in its exclusion from the nucleus. Nuclear Yorkie associates with Scalloped and promotes proliferation and inhibits apoptosis (Figure6)[START_REF] Huang | The Hippo Signaling Pathway Coordinately Regulates Cell Proliferation and Apoptosis by Inactivating Yorkie, the Drosophila Homolog of YAP[END_REF].

Figure 6 .

 6 Figure 6. Canonical Hippo pathway. (A) Hippo pathway in Drosophila Melanogaster. (B) Hippo pathway in mammals. Adapted from (Taha, Janse van Rensburg, and Yang 2018; Varelas 2014).

Figure 7 .

 7 Figure 7. Non canonical Hippo pathway. Differences in cell mechanical properties affect Yap nuclear translocation. Adapted from (MB INFO 2016).

  The 4xGTIIC:d2GFP line contains the 4xGTIIC promoter driving expression of d2GFP. The 4xGTIIC promoter contains 4 GTIIC sequences which are consensus Tead binding sites responsive to Yap/Taz-Tead activity. Co-staining 4xGTIIC:d2GFP embryos with Yap antibody show a strong correlation between nuclear located Yap and GFP expression. The same results are obtained when staining with ctgf antibody. Overall, these results confirm that this transgenic line faithfully reports endogenous Yap-Tead activity[START_REF] Miesfeld | Establishment of Transgenic Lines to Monitor and Manipulate Yap/Taz-Tead Activity in Zebrafish Reveals Both Evolutionarily Conserved and Divergent Functions of the Hippo Pathway[END_REF].Tg(Hsa.CTGF:nlsmCherry) is another useful line for following Yap/Taz activity in vivo. In this line, they use a fragment of the human CTGF gene promoter, which contains three Yap/Taz/Tead-binding sites conserved in the zebrafish ctgfa promoter[START_REF] Astone | Zebrafish Mutants and TEAD Reporters Reveal Essential Functions for Yap and Taz in Posterior Cardinal Vein Development[END_REF]).

  chemical or mechanical signals and transduce them into cell responses such as proliferation, polarity, or differentiation (Berbari et al. 2009). It is proposed that through bending, cilia can initiate downstream mechanotransduction signal cascades, including ionic fluxes within the cilium (R Ferreira et al. 2019).

Figure 8 .

 8 Figure 8. Primary cilia. (A) Schematic representation of a primary cilia. (B) Cilia quantification in dorsal pericardium of zebrafish at 48 hpf. Dorsal pericardium is divided in 3 areas. Adapted from[START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF][START_REF] Hsiao | Trafficking in and to the Primary Cilium[END_REF][START_REF] Higgins | Primary Cilia and Their Role in Cancer (Review)[END_REF].

-

  4xGTIIC:d2GFP[START_REF] Miesfeld | Establishment of Transgenic Lines to Monitor and Manipulate Yap/Taz-Tead Activity in Zebrafish Reveals Both Evolutionarily Conserved and Divergent Functions of the Hippo Pathway[END_REF] -Et.WT1-6451gata2:EGFP)cn1[START_REF] Peralta | Heartbeat-Driven Pericardiac Fluid Forces Contribute to Epicardium Morphogenesis[END_REF] -lkb1Y261 (van der[START_REF] Velden | The Serine-Threonine Kinase LKB1 Is Essential for Survival under Energetic Stress in Zebrafish[END_REF] -tcf21:nls-GFP (J.[START_REF] Wang | The Regenerative Capacity of Zebrafish Reverses Cardiac Failure Caused by Genetic Cardiomyocyte Depletion[END_REF] -actb2:lifeAct-RFP[START_REF] Behrndt | Forces Driving Epithelial Spreading in Zebrafish Gastrulation[END_REF] -Yap fu48[START_REF] Agarwala | Amotl2a Interacts with the Hippo Effector Yap1 and the Wnt/β-Catenin Effector Lef1 to Control Tissue Size in Zebrafish[END_REF] -Kif3a[START_REF] Pooranachandran | Unexpected Roles for Ciliary Kinesins and Intraflagellar Transport Proteins[END_REF] -Pkd2[START_REF] Schottenfeld | Zebrafish Curly up Encodes a Pkd2 Ortholog That Restricts Left-Side-Specific Expression of Southpaw[END_REF] -Klf2a[START_REF] Heckel | Oscillatory Flow Modulates Mechanosensitive Klf2a Expression through Trpv4 and Trpp2 during Heart Valve Development[END_REF]

  vertical axis allows for horizontal mounting. The sample needs to be placed between the TwinFlect mirrors, as the light sheet has to be reflected into the sample and to be placed in the focal plane of the detection objective (Haas 2017) (Figure I A).

Figure

  Figure I. In vivo imaging with Leica TCS SP8 DLS. A) Vertical setup of the microscope. B) Sample mounting with phytagel molds. C) Sample mounting with U-shaped glass capillaries.

3 )

 3 Rotation device, explained in the tutorial below, published in Leica Microsystems webpage[START_REF] Haas | DLS Sample Preparation: Using U-Shaped Glass Capillaries for Sample Mounting[END_REF]. Leica Microsystems designed and created the rotation design and Dr. Remacha and I prepared the published protocol.Préface en françaisPour réaliser des images en direct, l'imagerie par feuille de lumière est largement utilisée. La microscopie à feuille de lumière, ou microscopie à illumination plane sélective, est une technique qui utilise une feuille de lumière focalisée pour éclairer le spécimen par le côté. Cette technique est utilisée pour l'imagerie vivante car elle permet d'obtenir une bonne résolution à des profondeurs de pénétration élevées et parce qu'elle est peu invasive[START_REF] Huisken | Selective Plane Illumination Microscopy Techniques in Developmental Biology[END_REF].La base de la microscopie à feuille de lumière est d'éclairer un volume bien défini autour du plan focal de l'optique de détection depuis le côté. Dans le Leica TCS SP8 DLS (appelé DLS à partir d'ici), la feuille de lumière est formée par le balayage rapide d'un faisceau laser pendant le temps d'acquisition de la caméra[START_REF] Köster | Light Sheet Microscopy Turned Vertically[END_REF].Bien qu'une feuille de lumière traditionnelle ait une configuration perpendiculaire où les objectifs d'éclairage et de détection sont disposés à un angle de 90° l'un par rapport à l'autre, le DLS a une configuration verticale qui utilise un dispositif de miroirs (miroirs TwinFlect) pour dévier la feuille de lumière à un angle de 90º. Le module est entièrement intégré dans l'axe vertical du statif de microscopie inversé[START_REF] Köster | Light Sheet Microscopy Turned Vertically[END_REF]. Cet axe vertical permet un montage horizontal. L'échantillon doit être placé entre les miroirs TwinFlect, car la feuille de lumière doit être réfléchie dans l'échantillon et être placée dans le plan focal de l'objectif de détection[START_REF] Haas | DLS Sample Preparation: Using U-Shaped Glass Capillaries for Sample Mounting[END_REF] (Figure I A).

1 ) 2 ) 3 )

 123 Les moules Phytagel conçus à l'IGBMC. Des tampons spéciaux imprimés en 3D ont été conçus à l'IGBMC. En plaçant les tampons dans une boîte de Pétri en verre de fond et en ajoutant du phytagel à la concentration de 1%, on crée une structure en forme de poche entre les miroirs, où le poisson est maintenu immobile et en contact direct avec le milieu (Figure IB). Capillaires en verre en forme de U. Des capillaires en verre en forme de U, adaptés aux différentes tailles de miroir, sont fixés au fond de la boîte de Pétri en verre. L'embryon de poisson zèbre est enrobé d'agarose et placé dans le capillaire en forme de U (Figure IC). Dispositif de rotation, expliqué dans le tutoriel ci-dessous, publié sur la page Web de Leica Microsystems (Haas et al. 2019). Leica Microsystems a conçu et créé le dispositif de rotation et le Dr Remacha et moi-même avons préparé le protocole publié.Using a Rotation Device for DLS Sample MountingTCS SP8 DLS Digital LightSheet ProtocolThe TCS SP8 DLS microscope utilizes the innovative concept of integrating Light Sheet Microscopy technology into a confocal microscope. Due to its unique optical architecture, specimens/samples can be mounted on standard glass bottom petri dishes, as for standard confocal imaging, but require only a few adaptations when compared to conventional mounting procedures. Images were acquired in the framework of the 2018 EMBO Lightsheet Course at MPI CBG in Dresden with expert support of Bruno Cossermelli Vellutini (MPI CBG), Pavel Tomancak (MPI CBG), and Emmanuel Reynaud (UCD). Authors: Petra Haas, Dr 1 , Elena Remacha Motta 1 , Laia Ortiz Lopez 2 , Bruno Cossermelli Vellutini, Dr 3 , Pavel Tomancak, Dr 3 , Emmanuel Reynaud, Dr 4

Figure 1 :

 1 Figure 1: DLS objective: Positioning of specimens in the free area of the sample space.

Figure 2 :

 2 Figure 2: DLS objective: Sample positioning by using FEP (fluorinated ethylene propylene) tubes.

Figure 3 :

 3 Figure 3: Positioning of specimens inside an FEP tube.

Figure 5 :Step 3 :

 53 Figure 5: Sealing the DLS rotation device.

Figure 6 :

 6 Figure 6: Drawing the specimen into an FEP tube.

Figure 7 :Step 4 :

 74 Figure 7: Length of FEP tube: 38 mm.

Figure 8 :

 8 Figure 8: Attaching the black caps to the FEP tube.

Figure 9 :

 9 Figure 9: Placing the FEP tube in the rotation device (first step).

Figure 10 :Step 3 :

 103 Figure 10: Placing the FEP tube in the rotation device (final step).

Figure 11 :

 11 Figure 11: TCS SP8 DLS scanning stage with rotation device.

Figure 12 :

 12 Figure 12: Drosophila embryo observed from different sides. Single planes were taken from 3D image stacks. Courtesy of Pavel Tomancak, MPI-CBG, Dresden, Germany.

  ce chapitre, je présente l'article "Intraflagellar Transport Complex B Proteins Regulate the Hippo effector Yap1 during Cardiogenesis". Dans cet article, nous étudions le rôle des protéines IFTB pendant la cardiogenèse. Nous décrivons comment les protéines IFT limitent le développement proépicardique et myocardique indépendamment des cils. De plus, nous montrons que cette modulation agit par le biais de la signalisation BMP, qui règle l'activité de Yap1-Tead. Enfin, nous montrons également que IFT88 et IFT20 interagissent avec Yap1 dans le cytoplasme, ce qui règle l'activité de Yap1-Tead.

  the mesothelial cell layer covering the myocardium. It is essential for proper heart development, as it has several key functions such as promoting myocardial growth, coronary vasculature formation, and intracardiac fibroblast generation (Olivey and Svensson 2010; Ruiz-Villalba and Pérez-Pomares 2012; Schlueter and Brand 2012). Furthermore, the epicardium is critical during heart regeneration. Following a cardiac injury, epicardial cells turn on embryonic genes and contribute to new coronary vascular cells and the maintenance of the survived heart muscle (Simões and Riley 2018). Epicardial cell progenitors arise from the DP, the cell layer surrounding the heart and forming the pericardial cavity. Around the time of heart looping, cells from the dorsal pericardium round up and form the PE cluster (Maya-Ramos et al. 2013). In zebrafish, PE formation depends on DP constriction and extrusion of PE cells to the pericardial cavity. After PE cluster formation, PE cells are released into the pericardial cavity and are advected by the pericardial flow until they attach to the myocardium. Once on the myocardium, cells spread and proliferate until they cover the whole heart by around 6 dpf (Peralta et al. 2013; Andrés-Delgado et al. 2019).

Figure 1 .

 1 Figure 1. Yap1/Taz-Tead in the dorsal pericardium. A. Confocal imaging of 4xGTIIC:d2FP embryos stained with phalloidin at 36, 48, and 55 hpf. Arrowheads mark

Figure 2 .

 2 Figure 2. Yap1/Taz-TEAD in vivo activation during PE cluster formation. A. Schematic representation of PE cluster formation. At approximately 48 hpf cells from the dorsal pericardium protrude in the atrioventricular channel area forming the PE cluster. Cells from the cluster are released to the pericardial cavity until they attach to the myocardium. B. Lightsheet timelapse analysis of an actb2:lifeAct-RFP; epi:GFP

  treatment and treated 4xGTIIC:d2GFP embryos from 36 to 55 hpf(Figure 3A, C). Dexamethasone treated embryos have an increased percentage of GFP cells in the pericardium (Figure3B) and myocardium (Figure3B'), showing a general increase of Yap1/Taz-Tead activity. PE cell number is increased in dexamethasone-treated embryos compared to non-treated siblings (Figure3B''). However, epicardial cell number does not significantly change (Figure3B''').Next, we then treated 4xGTIIC:d2GFP embryos with a solution containing 5 μM of XAV939 and treated them from 36 to 55 hpf to fix them and image them with a confocal microscope after phalloidin staining (Figure3A, D). Although the percentage of GFP positive cells in the pericardium is not significantly different in XAV939 treated embryos (Figure3E), they do have a decreased percentage of GFP cells in the myocardium compared to non-treated embryos (Figure3E'),showing a decrease of Yap1/Taz-Tead activity. In addition, PE cell number is lower in XAV939 treated embryos compared to non-treated embryos (Figure3E''), as well as the epicardial cell number (Figure3E''').Taken together, our results indicate that increasing Yap1/Taz-Tead activity starting at 36 hpf increases the number of PE cluster cells, while decreasing Yap1 nuclear translocation has the opposite effect.

Figure 3 .

 3 Figure 3. Modifying Yap1/Taz-Tead activity chemically impacts PE cluster formation. A. 4xGTIIC:d2GFP embryo treatment with dexamethasone and XAV939. B-B'-B''-B'''. Quantification in non-treated and dexamethasone-treated embryos of: GFP positive cells in the dorsal pericardium and the myocardium, PE cell number, and epicardial cell number. C. Confocal plane of a non-treated and a dexamethasone-treated embryo showing PE cluster. D. Confocal plane of a non-treated and a XAV939 treated embryo showing PE cluster.E-E'-E''-E

Figure 4 .

 4 Figure 4. Description of lkb1 mutants at key points of PE cluster formation. A. Percentage of positive GFP cells in the dorsal pericardium at 36 hpf. B. Quantification of PE cluster cell number at 55 hpf. C. Quantification of PE cluster cell number at 72 hpf. D. Quantification of epicardial cell number at 5dpf. A'-D'. Confocal imaging of Lkb1-/-;tcf21:nls-GFP and control siblings at 36, 55, 72 hpf and 5dpf.

  Mutants did not show either any difference in terms of myocardial cell number or epicardial cell number (FigureSup 4B-C). At 72 hpf mutants have an increased number of PE cells compared to controls (Figure4C). As it happened in earlier time points, there were no differences in terms of myocardial and epicardial cell numbers (FigureSup 4 D-E). Finally, we checked at 5 dpf, and there are no apparent differences between controls and mutants in terms of epicardial (Figure4D) or myocardial cell number (FigureSup 3F).

Figure Supplementary 4 .

 4 Figure Supplementary 4. Description of lkb1 mutants at key points of PE cluster formation. A. Quantification of myocardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 36 hpf. B. Quantification of myocardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 55 hpf. C. Quantification of epicardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 55 hpf. D. Quantification of myocardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 72 hpf. E. Quantification of epicardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 72 hpf. F. Quantification of myocardial cell number in lkb1-/-;tcf21:nls-GFP and controls at 5 dpf.

  Dans ce chapitre, nous étudions l'activité de Yap1/Taz-Tead pendant la formation du PE. Nous décrivons l'activité in vivo de Yap1/Taz-Tead dans le coeur de 36 hpf à 55 hpf en utilisant la lignée rapporteur 4xGTIIC:d2GFP. Nous constatons que la GFP est exprimée spécifiquement dans le PE. De plus, l'expression de la GFP est présente avant l'extrusion des cellules PE vers la cavité proépicardique, ce qui suggère qu'elle est impliquée d'une manière ou d'une autre dans la formation du PE.Les cellules PE changent de forme, passant d'une forme plate à ronde, en rompant leurs adhésions cellulaires. Bien que ce processus ne soit pas une transition épithéliale à mésenchymateuse (EMT) complète, il présente de nombreuses similitudes[START_REF] Andrés-Delgado | Actin Dynamics and the Bmp Pathway Drive Apical Extrusion of Proepicardial Cells[END_REF]. De plus, les cellules PE expriment twist1a[START_REF] Peralta | Dissecting the Role of Heartbeat-Driven Pericardiac Fluid Forces on Epicardium Morphogenesis Through[END_REF], un facteur de transcription bHLH impliqué dans l'EMT et un régulateur de la spécification et de la différenciation des lignées[START_REF] Lamouille | Molecular Mechanisms of Epithelial-Mesenchymal Transition[END_REF]. Il est également intéressant de mentionner que Yap a été décrit comme un promoteur de l'EMT dans plusieurs cancers, comme le cancer colorectal[START_REF] Cheng | YAP Promotes Epithelial Mesenchymal Transition by Upregulating Slug Expression in Human Colorectal Cancer Cells[END_REF]. In vitro, il a également été observé que les réponses médiées par Yap aux changements dans l'organisation de l'ECM favorisent l'EMT[START_REF] Park | Switch-like Enhancement of Epithelial-Mesenchymal Transition by YAP through Feedback Regulation of WT1 and Rho-Family GTPases[END_REF]. Cela serait cohérent avec un modèle dans lequel l'activité de Yap1/ Taz-Tead favorise la formation du PE: Yap1/Taz-Tead est actif dans les cellules DP dès les premiers stades et ces cellules vont s'extruder, dans un processus de type EMT, pour former le PE.Dans notre chapitre, nous montrons que la modification de l'activité de Yap1/Taz-Tead à l'aide de médicaments entraîne une dérégulation du processus de formation du PE: une augmentation de l'activité de Yap1/Taz-Tead entraîne une augmentation du nombre de cellules PE, tandis qu'une diminution entraîne une diminution du nombre de cellules PE. Tout ceci suggère que l'activité de Yap1/Taz-Tead favorise l'aquisition de l'identité des cellules de type PE. Ceci est cohérent avec ce qui a été publié précédemment dans le domaine. Il a été publié que les protéines du complexe B de l'IFT inhibent l'activité de Yap1 pendant la formation des PE et que l'absence d'IFT88 entraîne une augmentation des cellules du PE[START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. Il a également été démontré que les protéines Motin régulent la formation du PE. Chez le poisson zèbre, les protéines Amot se lient à Yap/Taz dans le cytoplasme en le séquestrant[START_REF] Agarwala | Amotl2a Interacts with the Hippo Effector Yap1 and the Wnt/β-Catenin Effector Lef1 to Control Tissue Size in Zebrafish[END_REF].Les mutants Amotl2a, qui auraient une augmentation de Yap1/Taz au niveau nucléaire, ont de PE plus grands[START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF]. Cependant, bien que lorsque nous diminuons chimiquement l'activité de Yap1/Taz-Tead chez le poisson zèbre, nous obtenons un PE plus petit, nous ne voyons pas les mêmes résultats chez les mutants Yap1 ou les mutants Yap1 et les morphants Taz. Cela pourrait être dû à différents événements. Dans notre experience, nous n'avons pas complètement éliminé Taz du système. Les mutants doubles pour Yap1/Taz présentent de graves défauts de développement et meurent avant 30 hpf (Nakajima et al. 2017). Nous avons essayé de surmonter ce problème en traitant nos mutants Yap1 avec une faible concentration de Taz MO. Les poissons résultants présentaient des niveaux diminués, mais pas absents, de protéine Taz. Cependant, nous avons constaté que le PE est toujours formé chez ces poissons. Une possibilité pourrait être que même de faibles quantités de Taz sont suffisantes pour promouvoir la formation du PE et compenser la perte de Yap1. Par exemple, il a été décrit dans le développement de la rétine chez la souris que dans les modèles de souris hétérozygotes Yap, la déplétion d'un allèle Yap entraîne des mécanismes compensatoires TAZ transitoires aux stades postnatals, associés à un phénotype de type gain de fonction (Masson et al. 2020). Cependant, cette hypothèse est très difficile à prouver car nous avons atteint la límite de notre modèle: nous avons besoin de certains niveaux de Taz pour avoir des poissons viables et nous ne pouvons donc pas étudier les conséquences de la suppression totale de l'activité des deux. Une façon d'essayer de répondre à notre question serait de créer des mutants conditionnels de Yap1 ou de Taz. Alors que les mutants traditionnels présentent une mutation dans toutes les cellules à tout moment, les mutants conditionnels permettent l'inactivation de gènes dans les cellules somatiques d'une manière temporelle ou tissulaire spécifique (Maddison, Lu et Chen 2012). Il est encore difficile de créer des mutants conditionnels de poisson zèbre en raison de l'indisponibilité de techniques de ciblage de gènes pour les cellules souches embryonnaires de poisson zèbre et de la faible efficacité de la recombinaison homologue dans les oeufs de poisson zè bre fertilisés (Dai et al. 20 10 ; Robles, Martí et Belmonte 2011). Bien qu'il s'agisse d'un défi, plusieurs laboratoires ont créé des mutants conditionnels limitant l'expression d'une manière spécifique aux tissus, par exemple, des lignées Cre spécifiques aux cardiomyocytes (Jopling et al. 2010), entre autres. Cependant, cette approche ne serait pas adaptée à notre étude. Tout d'abord, il n'existe pas de gènes spécifiques expresées uniquement dans le PE ou l'épicarde. Nous ne pourrions pas restreindre complètement l'expression de Yap1 ou Taz uniquement dans le PE, d'autres organes du poisson zèbre seraient affectés. Par exemple, si nous utilisons le promoteur de l'un des marqueurs épicardiques les plus utilisés, wt1a, et que nous mutons conditionnellement les gènes sous ce promoteur, nous affecterions également le développement des reins (Perner, Englert et Bollig 2007). Un autre problème auquel nous serions confrontés est que Yap1 et Taz sont nécessaires au développement et que les doubles mutants meurent à 30 hpf (Nakajima et al. 2017). Si nous essayions de restreindre l'expression de Yap1 ou de Taz avant la formation du PE, les embryons ne survivraient probablement pas. Un moyen de surmonter ces problèmes serait de changer de modèle animal. Chez la souris, les mutants conditionnels sont largement disponibles et une lignée conditionnelle avec une double mutation de Yap1 et Taz spécifiquement dans les cellules épicardiques existe déjà (Singh et al. 2016). Bien que dans cet article ils décrivent déjà que ces doubles mutants ne survivent pas, il serait utile pour notre projet d'essayer différentes conditions avec cette lignée et d'étudier la formation du PE. Une autre explication pourrait être que d'autres voies impliquées dans la formation du PE compensent la perte de Yap1/Taz. Comme le coeur est essentiel à la survie et que l'épicarde est la clé du développement du coeur, il est possible qu'il existe plusieurs voies de signalisation pour assurer la formation du PE. Ces voies pourraient ne pas être efficaces lorsque nous modifions chimiquement l'activité de Yap1/Taz-Tead car nous traitons à partir de 36 hpf et il est trop tard pour compenser. Par exemple, il est connu que le PE est régulé par la signalisation BMP (Liu et Stainier 2012) et que la signalisation Notch endothéliale active l'expression de bmp, induisant la formation du PE (Andrés-Delgado et al. 2020). À l'avenir, il faudrait étudier si les mutants Yap1 présentent des différences dans les niveaux de Bmp ou de Notch qui pourraient expliquer pourquoi ils ne présentent pas de différences en termes de nombre de cellules PE. Une autre possibilité est que, bien que nous observions un certain changement dans la formation du PE après un traitement médicamenteux ciblant l'activité de Yap1/Taz-Tead, cette voie n'est pas essentielle pour la formation du PE et elle aurait plus d'importance à des moments ultérieurs du développement épicardique. Par exemple, il est connu que les cellules épicardiques subissent une transition EMT et donnent naissance à des cellules cardiaques non myocardiques. Il est possible que les cellules PE activent Yap1 lorsqu'elles acquièrent leur identité mais que la fonction de Yap1 ne soit pertinente qu'après la fixation des cellules au myocarde. Cependant, pour prouver cette hypothèse, nous sommes confrontés aux mêmes problèmes que précédemment -nous ne pouvons pas supprimer complètement Yap1 et Taz car les mutants doubles meurent à des moments précoces. Auparavant, il a été décrit que les cellules du DP se déplacent collectivement vers la ligne médiane du DP, où le cluster sera formé (Andrés-Delgado et al. 2019). Dans ce même article, ils proposent un modèle dans lequel Bmp favorise la polymérisation de l'actine conduisant à la formation du PE. Notre hypothèse selon laquelle l'activité de Yap1/Taz-Tead est impliquée dans la formation du PE est compatible avec ce modèle. Yap1/Taz sont des

  Cependant, à 72 hpf, les mutants lkb1 ont un nombre accru de cellules PE par rapport aux témoins. Comme le nombre de cellules épicardiques n'est pas modifié, cela pourrait signifier que le temps d'extrusion des cellules du DP est augmenté. Cependant, d'autres études sur le rôle de lkb1 pendant la formation des grappes PE devraient être menées. En résumé, notre étude rapporte une activation spécifique de Yap1/Taz-Tead au cours des cellules PE. Des études supplémentaires sont nécessaires pour comprendre la fonction exacte de cette voie pendant la formation des PE.How is PE cluster formation affected by changes in fluid forces? IntroductionThe heart is the first functional organ to form in the embryo, ensuring a supply of oxygen and nutrients to de developing tissues[START_REF] Lindsey | Mechanical Regulation of Cardiac Development[END_REF]. The cardiovascular system is constantly subjected to the mechanical forces generated by the beating heart, and changes form considerably during development and disease[START_REF] Garoffolo | Mechanotransduction in the Cardiovascular System: From Developmental Origins to Homeostasis and Pathology[END_REF][START_REF] Andrés-Delgado | Interplay between Cardiac Function and Heart Development[END_REF].

Figure 1 .

 1 Figure 1. Modifying heartbeat affects PE cluster formation. A. Confocal plane of nontreated and caffeine treated 4xGTIIC:d2GFP embryos at 55hpf. B. Quantification of PE cell number at 55hpf of caffeine treated embryos and non-treated. C. Quantification of epicardial cell number at 55hpf of caffeine treated embryos and non-treated. D.

Figure 2 .

 2 Figure 2. PE cluster in Yap1 injected with tnnt2a MO and changes in 4xGTIIC:d2GFP after BDM treatment. A. Confocal imaging of Yap1 mutants with tnnt2a MO at 55hpf.Maximum intensity projection. B. Quantification of PE cell number in Yap1 mutants, Yap1 injected with tnnt2a MO and controls in tcf21:nls-GFP background at

Finally, we performed

  in vivo imaging of 4xGTIIC:d2GFP embryos for 4 hours to monitor changes in GFP expression in the heart after BDM treatment. Our results show that changes in Yap1/Taz-Tead activity after BDM treatment are very dependent on the stage of the treatment. Treating embryos starting at 48 hpf does not change significantly Yap1/Taz -Tead activation. However, treating after 52 hpf gives similar results to embryos injected with Tnnt2a MO: we see a general increase of GFP intensity in the DP, as well as in the PE cells. It was previously published that BDM treatment, due to the disruption of the actinmyosin network (Andrés-Delgado et al. 2019), avoids PE cluster formation.

Figure 1 .

 1 Figure 1. Study of PE cluster in mutants with primary cilia defects. A. Schematic representation of a primary cilia showing the localization of Pkd2, Kif3a, and Iguana. B. Percentage of GFP positive cells in the pericardium in iguana mutants and controls in 4xGTIIC:d2GFP background at 36 hpf. C. Percentage of GFP positive cells in the myocardium in iguana mutants and controls in 4xGTIIC:d2GFP background at 36 hpf. D. Quantification of PE cell number in Kif3a mutants and controls at 55 hpf. E.

2 .

 2 Tout d'abord, nous avons créé un protocole d'imagerie in vivo utilisant le DLS, qui nous permet de suivre le développement du coeur du poisson zèbre. Ensuite, j'ai participé à l'article "Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis", où nous prouvons pour la première fois que les protéines IFTB peuvent réguler l'activité de Yap1-Tead par la signalisation BMP indépendamment des cils. Cela limite le développement proépicardique et myocardique. Chaque jour, de nouvelles fonctions pour les protéines ciliaires sont découvertes et nous avons trouvé un nouveau rôle pour les protéines IFTB. Nous avons également décrit l'activité de Yap1/Taz-Tead tout au long du processus de formation du PE. En utilisant l'imagerie in vivo et fixe, nous montrons que Yap1/Taz-Tead est actif avant que les cellules PE n'extrudent du DP et comment cette activité est maintenue lorsque les cellules sont déjà attachées au myocarde. Actuellement, de nombreux détails sur la formation du PE restent insaisissables. Dans cette thèse, nous proposons l'activité de Yap1/Taz-Tead comme une nouvelle voie impliquée dans le développement épicardique et l'identité cellulaire. Bien que nous observions un changement dans la formation du PE lorsque nous modifions l'activité Yap1/Taz-Tead à l'aide de médicaments, nous n'obtenons pas les mêmes résultats lorsque nous étudions des mutants Yap1 injectés avec la MO Taz. Par conséquent, nous ne pouvons pas conclure que l'activité Yap1/Taz-Tead entraîne la formation du PE. Cependant, il faut noter que cette activité est spécifique aux cellules du PE et qu'il n'est pas possible d'éliminer complètement Yap1 et Taz dans notre modèle car cela provoquerait la mort embryonnaire. D'autres études devraient être menées pour essayer de déchiffrer le rôle de Yap1/Taz dans le développement épicardique. Suite à des études précédentes (Peralta et al. 2013), nous avons également étudié le rôle des forces mécaniques dans la formation du PE et essayé de trouver des mécanosenseurs impliqués dans ce processus. Nous montrons que le changement des forces fluides dans la cavité péricardique affecte la formation du PE, soit en changeant leur taille ou leur position, et la signalisation Yap1/Taz-Tead. Cependant, nous n'avons pas trouvé de nouveaux mécanosenseurs impliqués dans ce processus. La couche épicardique est essentielle au bon fonctionnement du coeur et elle a été largement ignorée jusqu'à des études récentes. Dans cette thèse, nous présentons une étude exhaustive de la formation du PE chez le poisson zèbre, en nous concentrant sur l'activité de Yap1/Taz-Tead. Nous espérons que cette thèse contribuera à approfondir nos connaissances sur la formation du PE. Bien que nous n'ayons pas été en mesure d'identifier la fonction de Yap1/Taz-Tead ou de trouver de nouveaux régulateurs impliqués dans la formation du PE, nous espérons que nos résultats aideront à réorienter les études futures sur la formation du PE. Bibliography Introduction Dans la population humaine, 1% des naissances présentent des maladies cardiaques congénitales. Le coeur est le premier organe à acquérir sa fonction dans l'embryon en développement, fournissant aux organes en formation l'oxygène et les nutriments nécessaire a la croissance et la survie. Le développement du coeur est un processus complexe, hautement régulé, qui n'a pas encore été entièrement caractérisé. Il est fondamental de mieux comprendre comment le coeur se développe pour améliorer notre connaissance des maladies cardiovasculaires. L'épicarde est la couche de cellules mésothéliales qui recouvre le myocarde. Il est essentiel au bon développement du coeur en favorisant la croissance du myocarde, la formation de la vascularisation coronaire et la génération de fibroblastes intracardiaques. L'épicarde joue un rôle majeur dans la régénération. Plusieurs marqueurs épicardiques ont été décrits, tels que Wt1 ou tcf21 ont été identifié dans plusieurs espèces de vertébrés. Notre projet est basé sur l'étude du poisson zèbre, qui est un modèle animal largement utilisé pour étudier le développement et la régénération. Le poisson zèbre a la capacité de régénérer un muscle cardiaque blessé à l'âge adulte. Il est particulièrement utile en raison de ses propriétés optiques optimales pour l'imagerie in vivo, car les embryons peuvent être rendu transparents jusqu'à 5 jours après la fécondation (dpf). Chez le poisson-zèbre, les progéniteurs des cellules épicardiques proviennent du péricarde, la couche cellulaire qui entoure le coeur et forme la cavité péricardique. Les cellules péricardiques dorsales convergent en une ligne entre le pôle veineux et la zone du pôle artériel, où les cellules péricardiques s'arrondissent et forment une saillie vers la cavité péricardique. Ce groupe de cellule se regroupe et forment le proépicarde (PE) environ 48 heures après la fécondation (hpf). Les cellules de l'EP sont libérées dans la cavité péricardique et se déplacent en suivant le flux péricardique provoqué par les battements du coeur, jusqu'à ce qu'elles se fixent au myocarde. Chez d'autres espèces de vertébrés, l'epicarde présente un développement similaire, bien que les 4xGTIIC:d2GFP (Miesfeld et Link, 2014) pour suivre l'activation de Yap1/Taz-TEAD dans l'épicarde avant et pendant la formation des amas de PE. Nous avons donc imagé des embryons à partir de 36 heures après fertilisation (hours post fertilization, hpf). Avant ce stade, l'expression de la GFP est faible dans tout le péricarde. À environ 40 hpf, certaines cellules de la zone proche du canal atrio-ventriculaire du Coeur dans la partie du péricarde dorsal augmentent leur intensité de GFP. Ces cellules continuent d'augmenter leur expression de la GFP au fil du temps et à 48 hpf, elles commencent à s'arrondir pour former le proepicarde. Nous avons ensuite analyse l'expression du rapporteur à partir de 48hpf. Nous avons observé des cellules plates dans le péricarde dorsal avec une expression élevée de la GFP, qui donneront naissance au proepicarde. Dans l'ensemble, nos résultats suggèrent que les cellules qui formeront l'amas PE ont une activité Yap1/Taz-Tead avant de commencer à changer de forme et que l'activité Yap1-Taz/Tead est plus élevée dans les cellules PE. La modification de l'activité nucléaire de Yap1/Taz a un impact sur la formation des grappes de PE Nous avons ensuite cherché à tester le rôle de Yap1/Taz-Tead dans la formation des PE en régulant à la hausse et à la baisse son activité. Pour ce faire, nous avons utilisé la dexaméthasone et le XAV939. La dexaméthasone est un glucocorticoïde qui active in vivo la transcription médiée par Yap1/Taz-Tead (Sorrentino et al., 2017 ; Astone et al., 2018) et XAV939 est un composé qui inhibe les protéines AMOT stabilisatrices de la tankyrase, entraînant une diminution de la translocation nucléaire de Yap1 (Wang et al., 2015). Nous avons traité des embryons 4xGTIIC:d2GFP de 36 à 55 hpf avec de la dexaméthasone et les avons fixés pour les imager avec un microscope confocal après coloration à la phalloïdine. Les embryons traités à la dexaméthasone ont un pourcentage accru de cellules GFP dans le péricarde et le myocarde, ce qui montre une augmentation générale de l'activité Yap1/Taz-Tead. Le nombre de cellules PE est plus élevé chez les embryons traités à la dexaméthasone que chez les contrôles non traités. Ensuite, nous avons quantifie l'effet du traitement au XAV939 sur le nombre de cellules du proepicarde. Bien que le pourcentage de cellules GFP positives dans le péricarde ne soit pas significativement différent chez les embryons traités avec XAV939, ils ont un pourcentage de cellules GFP dans le myocarde inférieur à celui des embryons non traités, ce qui montre une diminution de l'activité Yap1/Taz-Tead. En outre, le nombre de cellules PE est plus faible chez les embryons traités par le XAV939 que chez les embryons non traités, de même que le nombre de cellules épicardiques.

  de la voie Hippo, Yap1 et Wwrt1, pendant la formation du proépicarde Résumé en français L'épicarde est une couche de cellules mésothéliales recouvrant le myocarde. L' épicarde est essentiel pour le développement cardiaque, car il favorise la croissance myocardique, la formation de la vascularisation coronaire et la génération de fibroblastes intracardiaques. Chez le poisson zèbre, les progéniteurs épicardiques proviennent du péricarde dorsal. Lors du développement des progéniteurs épicardiques certaines cellules du péricarde dorsal s'arrondissent et font saillie pour former le cluster proépicardique (PE). Yap1 et Wwrt1 sont des protéines essentielles de la voie Hippo qui sont exprimées dans l'épicarde en développement chez la souris. Bien que l'activité Yap1 / Wwrt1 soit impliquée dans le développement cardiaque chez le poisson zèbre, son impact pendant la formation du PE reste inexploré. Nous avons caractérisé une lignée rapportant l'activité transcriptionelle de Yap1 / Wwrt1-Tead lors de la formation du PE. Ensuite, nous avons évalué le rôle de Yap1 / Wwr1-Tead lors de la formation du PE en modifiant l'activité Yap1 / Wwrt1-Tead. Nous avons également étudié le développement de PE en réponse aux forces mécaniques. Enfin, nous avons également recherché un nouveau régulateur de la formation de cluster PE. Nos travaux suggèrent que Yap1 / Wwrt1 est nécessaire pour la formation du cluster PE bien que les mutants yap1 ne présentent aucun phénotype PE. Nos travaux suggèrent également que les forces mécaniques sont essentielles pour moduler la localisation PE lors de sa formation dans la cavité péricardique.

  Dans ce chapitre, nous explorons plus avant le rôle du rythme cardiaque pendant la formation du PE en relation avec l'activité de Yap1/Taz-Tead. Si le changement de la taille du PE est dû aux changements du rythme cardiaque, nous ne devrions pas voir de différence par rapport aux contrôles une fois que nous avons éliminé le rythme cardiaque. De plus, bien que nous n'observions aucun changement dans le nombre de cellules épicardiques, nous ne pouvons pas exclure que l'augmentation de les cellules du PE puisse être due à un changement dans la libération des cellules. Comme cela a été décrit précédemment, l'arrêt du coeur n'affecte pas le nombre des cellules du PE mais il affecte la position du PE. Nous décrivons également une diminution générale des cellules positives pour la GFP dans le myocarde et une augmentation des cellules positives pour la GFP dans le DP. après le traitement par BDM. Nos résultats montrent que les changements de l'activité de Yap1/Taz-Tead après le traitement BDM sont très dépendants du stade du traitement. Le traitement des embryons à partir de 48 hpf ne change pas significativement l'activation de Yap1/Taz-Tead. Cependant,

	l'isoprénaline et d'analyser les changements chez les poissons
	le traitement après 52 hpf donne des résultats similaires à ceux des embryons injectés avec la MO Tnnt2a: on observe une augmentation générale de l'intensité de la GFP dans les cellules DP, ainsi que dans les cellules PE. Il a été publié précédemment que le traitement par BDM, en raison de la perturbation du réseau actine-myosine (Andrés-Delgado et al. 2019), évite la formation du PE. Chez nos embryons, un PE précoce était déjà présent à 48 hpf. Nous n'avons pas observé de régression de ce cluster initial, mais nous avons constaté un arrêt de l'extrusion cellulaire: plus aucune cellule n'était 4xGTIIC:d2GFP. La diminution dans le myocarde pourrait être expliquée par le fait que l'étirement des cellules du myocarde dû aux battements du coeur pourrait incorporée dans le cluster après le traitement par BDM.
	Nous constatons que le fait d'affecter le rythme cardiaque, que ce soit en transloquer Yap1/Taz vers les noyaux. Chez les morphants tnnt2a, nous
	augmentant la fréquence cardiaque ou en arrêtant le coeur, entraîne des perdons cet étirement cellulaire et par conséquent aussi l'activité Yap1/Taz-
	changements dans l'activité de Yap1/Taz-Tead. Ceci est cohérent avec le fait Tead. Cependant, nous avons observé un nombre accru de cellules positives
	que Yap1/Taz sont des mécanosenseurs connus dont la localisation pour la GFP dans le DP. Ceci pourrait être lié au déplacement du PE. Afin de
	subcellulaire peut être régulée par des changements dans l'étirement des tester cette hypothèse, une expérience qui pourrait être réalisée serait de traiter
	cellules, tels que ceux provoqués par les battements cardiaques. les morphants tnnt2a avec des médicaments qui réduisent l'activité de
	Après avoir traité avec de la caféine et de l'isoprénaline, deux médicaments qui Yap1/Taz-Tead, tels que XAV939 ou Verteporfin. Si nous constatons une
	affectent les battements du coeur, nous obtenons des résultats similaires: une diminution du nombre de cellules positives pour la GFP dans le DP après le
	augmentation du nombre de cellules PE sans changement du nombre de traitement, nous pourrions étudier où se forme le PE dans ces embryons et
	cellules épicardiques. Cependant, après le traitement à la caféine, nous évaluer si le mauvais placement est dû à l'augmentation de l'activité Yap1/Taz-
	constatons une augmentation des cellules positives pour la GFP que nous ne Tead dans les cellules du DP.
	voyons pas après le traitement à l'isoprénaline. Bien que les deux médicaments Nous avons également voulu savoir si Yap1 serait important pour réguler la
	agissent de manière similaire, leurs conséquences dans l'embryon de poisson formation du PE dans les embryons dépourvus de battements cardiaques. Bien
	zèbre ne sont pas exactement les mêmes. Dans l'ensemble, ces résultats que les mutants Yap1 et les morphants tnnt2a semblent avoir un phénotype
	suggèrent qu'une modification de la fréquence cardiaque entraîne une plus fort en termes de forme du coeur, la morphologie du coeur étant plus
	augmentation du nombre de cellules PE. Cependant, il faut tenir compte du fait affectée que chez les morphants tnnt2a seuls, le groupe PE se forme toujours
	que ces médicaments ne sont peut-être pas spécifiques et qu'ils pourraient et il n'y a pas de différences significatives par rapport aux morphants tnnt2a.
	favoriser l'extrusion des cellules dans le PE indépendamment du rythme
	cardiaque. Une expérience possible à réaliser dans le futur pourrait être de
	traiter des poissons sans rythme cardiaque avec de la caféine et de

Enfin, nous avons réalisé une imagerie in vivo d'embryons 4xGTIIC:d2GFP pendant 4 heures pour suivre les changements de l'expression de la GFP dans le coeur Dans l'ensemble, nous montrons que l'activité de Yap1/Taz-Tead est sensible aux changements du rythme cardiaque et que la formation du PE est affectée, que ce soit en taille ou en lieu, lorsque nous modifions le rythme cardiaque. D'autres études sont nécessaires pour déduire comment les forces mécaniques régulent la formation du PE. Introduction Mechanical forces play a big role during heart development: the heartbeat, as well as the blood flow, regulate cell behaviour in the cardiac system (Andrés-Delgado and Mercader 2016). As we showed in the last chapter, changes in mechanical forces in the heart can also affect PE cluster formation. Mechanical forces are sensed by cells via mechanosensors, which induce downstream signalling cascades. It is likely that different types of cells use different mechanisms and that multiple mechanisms are working together to sense and respond to mechanical forces

  Dans ce chapitre, nous avons évalué le rôle de plusieurs mécanosenseurs connus dans la régulation de la formation du PE.

	Les cils primaires étant d'importants mécanosenseurs impliqués dans
	différentes étapes du développement (R. Ferreira et al. 2019), nous avons
	étudié plus avant leur rôle dans la formation du PE. Puisque les mutants
	d'iguane ont une taille réduite du PE et qu'il a été vu que le traitement par
	Verteporfin et XAV939, deux médicaments qui inhibent l'activité de Yap1/Taz-
	Tead, entraîne un phénotype similaire, nous avons étudié l'activité de
	Yap1/Taz-Tead à des points de temps précoces du développement.
	Cependant, nous ne constatons pas de diminution de l'activité de Yap1/Taz-
	Tead, nous devons donc conclure que la taille réduite du PE chez ces mutants
	n'est pas due à une diminution de Yap1/Taz-Tead.
	Iguana code pour Dzip1, une protéine à doigts de zinc nécessaire à la
	régulation correcte de la signalisation Hedgehog (Sekimizu et al. 2004).
	D'autres études devraient être menées pour déterminer si le phénotype mutant
	d'iguana est dû à l'absence de cils primaires ou à une mauvaise régulation de la
	voie de signalisation Hedgehog. De plus, étant donné que la régénération
	épicardique peut être régulée par la signalisation Hedgehog, il serait intéressant
	d'analyser plus en détail si la signalisation Hedgehog joue un rôle important
	également au cours des premières étapes de la formation épicardique, lorsque
	le PE est en cours de formation.

Nous avons étudié plus avant le rôle des cils primaires dans ce processus en examinant la taille du PE chez les mutants Kif3a, qui sont dépourvus de cils primaires, et chez les mutants Pkd2. Pkd2 est un mécanosenseur connu dans les cellules endothéliales et les cellules endocardiques pendant la morphogenèse des valves

[START_REF] Goetz | Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development[END_REF][START_REF] Heckel | Oscillatory Flow Modulates Mechanosensitive Klf2a Expression through Trpv4 and Trpp2 during Heart Valve Development[END_REF]

. Nous n'avons pas observé de changement en termes de formation du PE. Bien que les mécanosenseurs ne soient pas impliqués dans la formation du PE, puisque les mutants d'iguana ont une taille de PE plus petite, nous ne pouvons pas conclure que les cils primaires ne sont pas importants pour la formation du PE.

PhD thesis outline

Acknowledgements

First, I would like to thank the members of my jury Dr. Juliette Godin, Dr. Miguel Torres and Dr. Chris Jopling for accepting to evaluate my work. Next, I would like to thank my advisors Dr. Julien Vermot and Dr. Florian Fahrbach for the continuous support of my PhD study and related research and for their patience, motivation and knowledge. I thank my labmates for the discussions and constant support: Elena, Helene, Renee, Pedro, Rita, Nathalie and Hajime. Especially I would like to thank Marina for all her support, knowledge, help, guidance and friendship during my PhD.

List of figures Intraflagellar Transport Complex B Proteins Regulate the Hippo effector

Yap1 during Cardiogenesis

-Réalisant les hybridations in situ contre Bmp4 chez les mutants iguanes et aide à l'analyse.

-Aidant à la conservation des données.

-Aidant à la révision et à l'édition du manuscrit.

Preface

In this chapter I present my main work during my PhD, the study of Yap1/Taz-Tead activity during PE cluster formation. The conceptualization of the project was done by me, Marina Peralta and Julien Vermot. Investigation, data curation and writing of the original draft were done by me.

Results

Yap/Taz-Tead activity during PE cluster formation

To investigate in detail PE formation, represented in Figure 2A, we sought to describe Yap1/Taz-Tead activity during this process. Since wt1 is an epicardial marker and its promoter is downstream of TEAD [START_REF] Singh | Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development[END_REF], we used the Yap1/Taz-TEAD reporter line 4xGTIIC:d2GFP [START_REF] Miesfeld | Establishment of Transgenic Lines to Monitor and Manipulate Yap/Taz-Tead Activity in Zebrafish Reveals Both Evolutionarily Conserved and Divergent Functions of the Hippo Pathway[END_REF] to follow Yap1/Taz-TEAD activation in the epicardium before and during PE cluster formation.

For this purpose, we fixed 4xGTIIC:d2GFP embryos at 36, 48, and 55 hpf and stained them with phalloidin (cell shape readout) to image them with confocal microscopy (Figure 1A). At 36 hpf, cells from the pericardium are flat and expression in the DP is low. At 48 hpf, before the PE is formed, pericardial cells are flat but we see some cells in the atrioventricular channel area with high GFP expression. At 55 hpf, cells from the PE are rounded and have high GFP expression and there are some high GFP flat cells next to the PE (Figure 1B).

We believe that these are the cells that will be incorporated into the PE cluster.

Preface

In this chapter I present a study on how fluid forces are affecting PE cluster formation. The conceptualization of the project was done by me, Marina Peralta and Julien Vermot. Investigation, data curation and writing of the original draft were done by me. We next sought to study PE formation and Yap1/Taz-Tead activity in models without heartbeat. We injected tnnt2a-morpholino (5.8ng concentration) to 4xGTIIC:d2GFP embryos at the 1 cell stage and fixed them either at 36 or at 55 hpf. At 36 hpf, when there already are GFP positive cells in the DP and the myocardium, we observe that the percentage of GFP positive cells is increased in the DP of the tnnt2a morphant (Figure 1H), while it is decreased in the myocardium (Figure 1I). Consistently with what has been previously published, we also see that while the number of PE cluster cells is not significantly different, its position is shifted towards the VP.

Préface en français

Dans

Yap1 mutants without heartbeat do not have impaired PE cluster formation

To further study the role of Yap1/Taz-Tead activity in PE cluster formation related to the sensing of mechanical forces, we injected tnnt2a-morpholino (5.8ng concentration) to Yap1-/-:tcf21:nls-GFP and Yap1+/+:tcf21:nls-GFP embryos at the 1 cell stage and fixed them at 55 hpf (Figure 2A). Although heart morphology in Yap1-/-and tnnt2a morphants seems more affected than in the tnnt2a morphants alone, there are no differences in terms of PE cluster number (Figure 2B). As expected, since there are no pericardial advections to transfer PE cells to the myocardium, the tnnt2a MO have significantly less epicardial cells than the controls (Figure 2C).

We conclude that arresting the heartbeat affects Yap1/Taz-Tead activity.

Looking for possible mechanoregulators of PE cluster formation

signalling [START_REF] Ezratty | A Role for the Primary Cilium in Notch Signaling and Epidermal Differentiation during Skin Development[END_REF]). Since Notch is also involved in PE cluster formation [START_REF] Andrés-Delgado | Notch and Bmp Signaling Pathways Act Coordinately during the Formation of the Proepicardium[END_REF]) and cilia number in the DP where the cluster is formed is higher than in other areas [START_REF] Peralta | Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis[END_REF], we wanted to further study which role could primary cilia have during this process.

First of all we sought to investigate the role of Yap1/Taz-Tead signalling in early steps of development in iguana mutants to investigate if the reduced cluster size is due to changes in Yap1/Taz-Tead activity. We crossed iguana mutants with the reporter line 4xGTIIC:d2GFP and analysed them at 36 hpf. We did not see any change in terms of the percentage of GFP positive cells in the pericardium (Figure 1B) or the pericardium (Figure 1C).

Next, we studied two different mutants to investigate further the possible role of primary cilia in PE cluster formation: Kif3a and Pkd2 mutants (its position in the primary cilia is shown in figure 1A).

Kinesin family member 3A (Kif3a) is the ciliary kinase part of the anterograde IFT kinesin motor, required for ciliogenesis [START_REF] Sipe | Kif3a Regulates Planar Polarization of Auditory Hair Cells through Both Ciliary and Non-Ciliary Mechanisms[END_REF]. Mutations in Kif3a cause embryonic lethality [START_REF] Marszalek | Situs Inversus and Embryonic Ciliary Morphogenesis Defects in Mouse Mutants Lacking the KIF3A Subunit of Kinesin-II[END_REF]) and typical phenotypes of ciliogenesis defects, such as curved body axis. Consistently, mutant embryos lack cilia in the nasal pit, lateral line, spinal canal, and the kidney. In other organs of the embryos, cilia were still present but in a decreased number and some of these cilia were shorter than in wild type [START_REF] Pooranachandran | Unexpected Roles for Ciliary Kinesins and Intraflagellar Transport Proteins[END_REF].

We studied Kif3a-/-;epi:GFP and controls at 55 hpf. We performed immunohistochemistry analysis by counting the number of cells in the PE and in the epicardium (Figure 1B-C). We did not detect any significant differences between controls and mutants.

Pkd2 is a calcium-sensitive cation channel located in the cilia. Calcium signalling depends on Pkd2 in several ciliated structures, such as the left-right organizer or kidney epithelial cells [START_REF] Schottenfeld | Zebrafish Curly up Encodes a Pkd2 Ortholog That Restricts Left-Side-Specific Expression of Southpaw[END_REF]. It has been suggested that Pkd2 is mechanosensor in endothelial cells [START_REF] Goetz | Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development[END_REF]. In zebrafish, Pkd2 mutants are also known as Curly up or Cup mutants [START_REF] Schottenfeld | Zebrafish Curly up Encodes a Pkd2 Ortholog That Restricts Left-Side-Specific Expression of Southpaw[END_REF]. In order to assess the possible role of Pkd2 during PE cluster formation, we used the same protocol as with the Kif3a mutants and counted to the number of PE and epicardial cells in Cup-/-;epi:GFP and controls. We found again that there is no significant difference between mutants and controls (Figure 1D-E).

Klf2a/b during PE cluster formation

Klf2a/b are flow-responsive transcription factors that become expressed in the zebrafish heart at 36 hpf [START_REF] Vermot | Reversing Blood Flows Act through Klf2a to Ensure Normal Valvulogenesis in the Developing Heart[END_REF]). Endothelial klf2a expression has been shown to be flow-dependent and knockdown by antisense morpholino affects cardiac valve development (Steed et al. 2016). Since pericardial cells are exposed to flow within the pericardial cavity, we investigated the role of Klf2a/b during PE formation. We used klf2a and klf2b double mutants and performed live imaging to determine if these mutants have PE clusters (Figure 2A). klf2a and klf2b double mutants presented no differences in terms of PE cluster presence (Figure 2B). We conclude that klf2a and klf2b have not a major role in the regulation of the PE cluster formation.