Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal mesoderm tissue during gastrulation; a process called convergence and extension. These morphogenetic movements are essential to form the early embryo and are important for later development. There are a number of different proteins involved in regulating the morphogenetic movements during gastrulation. The Planar Cell Polarity Signaling Pathway helps establish individual cell polarity and is activated in dorsal mesoderm cells undergoing convergence and extension. In addition, dorsal mesoderm cells migrate by using integrin receptors and the surrounding extracellular matrix to correctly position the mesoderm in the embryo. I will focus my efforts on analyzing the function of ADAM proteins during Xenopus laevis gastrulation. The ADAM family of metalloproteases is important for a variety of biological processes. ADAM proteins function as ectodomain sheddases by cleaving membrane bound proteins involved in signal transduction, cell-cell adhesion, and cell-extracellular matrix adhesion. I will focus on investigating the roles of two ADAM family members; ADAM13 and ADAM19 during gastrulation. Both ADAM13 and ADAM19 are expressed in the dorsal mesoderm during gastrulation. Throughout early embryonic development, ADAM13 is expressed in the somitic mesoderm and cranial neural crest cells. ADAM19 is expressed in dorsal, neural and mesodermal derived structures such as the neural tube, notochord, the somitic mesoderm, and cranial neural crest cells. Since ADAM13 and ADAM19 are expressed in similar tissues, I investigated if both proteins functionally interacted. I show that a loss of ADAM13 protein in the embryo reduces the level of ADAM19 protein by 50%. In the opposite experiment, a loss of ADAM19 protein in the embryo reduces the level of ADAM13 protein by 50%. This suggests that both ADAM13 and ADAM19 are required to maintain proper protein levels in the embryo. This might be explained through their physical interaction in a cell. The ADAM19 Proform binds to the ADAM13 Proform in cultured cells. Through domain analysis, I show that ADAM19 binds specifically to the cysteine-rich domain of ADAM13. When co-overexpressed in a cell, the level of Mature ADAM13 (compared to the Proform) is reduced suggesting a complex form of regulation. I propose a few hypothetical models that discuss how ADAM19 may function as a chaperone to stabilize and regulate the further processing of ADAM13 protein. Some of the unpublished work discussed in this thesis focuses on the roles of ADAM13 and ADAM19 in the dorsal mesoderm during gastrulation. Specific emphasis is made on investigating the axial mesoderm during notochord formation. I show that ADAM19 affects gene expression important for the A-P polarity of the notochord while ADAM13 does not. The changes in gene expression can be partially rescued by the EGF ligand Neuregulin1β, a known substrate for ADAM19 in the mouse. ADAM13 and ADAM19 are important for convergence and extension movements of the axial mesoderm during gastrulation. Specifically, a loss of ADAM13 or ADAM19 causes a delay in mediolateral cell intercalation resulting in a significantly wider notochord compared to control embryos. These defects occur without affecting dishevelled intracellular localization or the activation of the PCP signaling pathway. However, a loss of ADAM13 or ADAM19 reduces dorsal mesoderm cell spreading on a fibronectin substrate through α5β1 integrin. To conclude, the work presented in this thesis focuses on the similarities and differences of ADAM13 and ADAM19 in the early embryo. Although ADAM13 and ADAM19 are required for normal morphogenetic movements during gastrulation, my data suggests they have different functions. ADAM13 appears to function in regulating cell movements while ADAM19 appears to function in regulating cell signaling. I propose a few hypothetical models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation.>

Un tissu épithélial est composé de cellules polarisées, étroitement liées les unes aux autres par des jonctions adhérentes. La perte de ces jonctions adhérentes est la première étape dans le développement des cancers au niveau des tissus épithéliaux. Il est donc important de comprendre les mécanismes d’attachement inter-cellulaire. Pour étudier ces interactions, nous utilisons comme modèle l’embryon de drosophile, où une fine régulation des jonctions adhérentes est requise pour l’une des étapes précoces de développement. Durant cette étape du développement, les cellules épithéliales changent de voisines le long de l’axe antéro-postérieur sans perdre leur adhérence cellulaire. Ce processus d’intercalation cellulaire est dû au recrutement polarisé du moteur moléculaire Myosine II au niveau des jonctions qui se désassemblent. Il a été mis en évidence qu’au cours de ce processus la perte de fonction de la voie JAK/STAT perturbe la localisation de la Myosine II. Au cours de ma thèse, j’ai réalisé un crible génétique dans un contexte mutant pour le ligand de la voie JAK/STAT pour me permettre d’identifier des interacteurs potentiellement impliqués dans le contrôle spatial de Myosine II. J’ai pu mettre en évidence plusieurs gènes pouvant être impliqués dans cette intercalation. Parmi ces candidats, je me suis focalisée sur celui montrant le plus fort phénotype : le gène CG13992. La caractérisation de ce gène a fut la seconde étape de mon travail de thèse (car seules les séquences nucléotidiques et protéiques étaient connues). Les résultats obtenus ont permis de mettre en évidence l’implication de ce gène dans la localisation de la Myosine II mais ils restent à confirmer. / Epithelial tissue is composed of polarized cells, which are closely attached to each other by adherens junctions. The loss of adherens junctions is often a key step in the development of cancer in epithelial tissues. It is therefore important to understand the mechanisms of attachment between the cells. To study such epithelial plasticity, we use the Drosophila embryo as a model system, where a fine regulation of adherens junctions is required for one of the early processes of development: germ band elongation. During this process, epithelial cells change their neighbors along the anterior-posterior axis (cell cell intercalation) without loss of cell adhesion. Polarized recruitment of the molecular motor Myosin II at the junctions, that disassemble and reassemble, underlies the intercalation process. In part, intercalation relies on the normal activity of the the JAK / STAT pathway that is crucial for the spatial control of Myosin II. During my PhD, I conducted a genetic screen, in a mutant for the ligand of the JAK / STAT pathway, designed to identify second site interactors for Myosin II control. I identified several genes that appear to be involved in the intercalation process. Among these candidates, I focused on one with the strongest phenotype: the gene CG13992. The functional characterization of this gene was the second stage of my thesis (because only the nucleotide and the protein sequences were known). Preliminary results highlight the involvement of this gene in the localization of Myosin II that remain to be confirmed.

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

One of the best-known structures in Drosophila is the sex comb, an arrangement of modified bristles on the tarsal forelegs of males. This complex, sexually-dimorphic trait shows striking variation among closely related species, although most other aspects of the tarsal bristle pattern have been conserved. I studied the development of the sex comb in the model organism Drosophila melanogaster and six related species. I confirmed that the D. melanogaster sex comb, although longitudinal in the adult, originates in a transverse orientation and rotates during development, and showed that this process occurs through male-specific convergent extension. However, in the species that I examined that have longitudinally-oriented sex combs that extend the full length of the tarsus, including D. ficusphila and two species of the montium subgroup, the sex comb does not rotate, and instead forms from two longitudinal rows that converge during development. Another species of the montium subgroup, D. nikananu, has a sex comb that is convergently similar to D. melanogaster, but forms in a manner typical of its subgroup, showing that very similar combs can be formed through different processes. In all species, there is a strong correlation between the position of the sex comb and the transverse bristle row on the foreleg tarsus just proximal to it. To test whether it is possible to violate this apparent constraint on development, I perturbed the expression of the leg patterning gene dachshund to generate ectopic sex combs in D. melanogaster. I found that while most patterns showed the same correlation, a few circumvent the constraint. I also demonstrated that the ectopic combs were formed non-autonomously and that overexpression of dachshund can transform certain aspects of the sex comb phenotype to resemble the transverse bristles to which they are homologous.

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