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Understanding the processes that control the formation of blood (haematopoiesis), and blood vessels (vasculogenesis and angiogenesis) in vivo has huge clinical importance. The complex three-dimensional architecture of blood vessels is dynamic and aberrant regulation of either the growth or function of the vascular system may potentiate the spread of tumours, resulting in failure of physiological processes such as implantation and placental development, leading to a range of angiogenesis associated disorders for example diabetic retinopathy. Both embryonic and adult haematopoiesis are also three-dimensional, dynamic processes in which deregulation may result in blood disorders or leukaemia. The experiments herein describe my contribution to investigations into the molecular mechanisms involved in haematopoiesis and angiogenesis over a period of approximately 15 years, taking advantage of technical advances as they became available and adapting them to specific cell models. For example, microarray technology has facilitated discovery of new pathways and transcripts implicated in normal and pathological angiogenesis; central to this mechanism is the role of vascular endothelial growth factor (VEGF), a mitogen specific to endothelial cells. Chromosome immunoprecipitation (ChIP) technology subsequently revealed pathways of early mesoderm formation and the importance of gastrulation in this process. Transcriptional targets of the T-box transcription factor Brachyury were subsequently determined. Throughout this work, the human female reproductive tract provided a unique resource, as one of the rare sites of physiological angiogenesis with which to investigate endothelial cell biology and haematopoiesis. Embryonic stem cell-derived embryoid bodies subsequently proved to be an excellent model for the study of early blood vessel development in three dimensions (2003-5), and to follow early mesoderm development (2006-2010). Targets of Brachyury revealed the close association between blood vessel development, haematopoiesis and early mesoderm formation via a common haemangioblast precursor for blood and endothelial cell lineages. Data gathered by myself, and colleagues, from gene expression and transcription factor analysis is now being used to create lineage codes or routemaps for differentiation of stem cells to mature cells in-vitro and it is now possible to produce mature megakaryocytes and erythrocytes in vitro. The current challenge is to produce fully functional human platelets and enucleated red blood cells. Combined with the use of autologous induced pluripotent stem cells (iPSCs) this makes patientspecific tailoring of cell-based therapies a real possibility.

Les cellules souches embryonnaires (ES) sont des cellules pluripotentes, capables de s'auto-renouveller indéfiniment dans des conditions de culture appropriées. Cela signifie que ces cellules restent dans un état prolifératif et indifferencié en culture et ont le potentiel de se différencier dans les trois feuillets embryonnaires, à savoir l'ectoderme, le mésoderme et l’endoderme, et leurs dérivés. Cette capacité à se différencier dans tous les types cellulaires, souligne la diffculté à contrôler la différenciation des cellules ES in vitro et à les guider vers un lignage spécifique. Mon projet de thèse porte sur la différenciation des cellules ES murines. Une étape importante du développement embryonnaire est le choix entre l’ectoderme et le mésendoderme. Dans ce but, j'ai développé une lignée ES qui permet de suivre exclusivement l'expression de Brachyury (T) dans le mésendoderme à l'exclusion de la notochorde: la lignée TRepV. Pour cela, jai cloné un fragment de 1 kb du promoteur murin de Brachyuryen amont du rapporteur Venus (YFP). Avant d'utiliser cette lignée, j’ai cherché à la valider. Malheureusement l'expression du rapporteur TRepV ne reproduit pas fidèlement l'expression endogène de T. Une hypothèse est que le fragment de 1kb ne contient pas tous les éléments de régulation de T nécessaires pour expression fidèle in vitro. De manière surprenante, j’ai observé que le rapporteur TRepV est exprimé de façon hétérogène dans les cellules ES non différenciées. Au cours de mon travail de thèse, je me suis intéressée à cette expression hétérogène. J'ai montré que les cellules ES TRepV+ représentent une sous-population distincte des cellules souches, qui peut être maintenue séparément exprimant le rapporteur de manère stable, à la difference d'autres gènes exprimés de manière hétérogène dans les cellules ES. Nous avons trouvé un marqueur d'une population distincte parmi les cellules ES et de nouveaux gènes impliqués dans pluripotence, qui seront abordés dans des études futures. / Embryonic stem cells (ESCs) are a powerful system to investigate developmental processes in vitro, and a promising tool to generate specific cell types for cellular therapies and regenerative medicine. ESCs are self-renewing, pluripotent cells, maintaining a proliferative and undifferentiated state in culture, while retaining the capacity to differentiate into the three embryonic lineages: ectoderm, mesoderm and endoderm, and all their derivatives. Here, I established a primitive streak specific Brachyury/T Reporter ESC line (TRepV) to investigate early ESC differentiation. In contrast to previously published Tknock-in line, we established a transgene T ESCs reporter line, in order to avoid the disruption of the T locus, which may result in a hapoinsuficient phenotype. During the validation process, I observed discrepancies in expression between the TRepV and the endogenous T locus. I followed upon these observations with a more detailed analysis and obtained evidence that T is regulated differently in the ESC system compared to in vivo development. Against expectations, I also observed heterogeneous expression of the TRepV reporter in undifferentiated ESCs. Undifferentiated ESCs were found to be a mix of TRepV+ and TRepV- cells. This finding became the focus of my studies: I found TRepV+ cells represent a distinct population of ESCs with a unique identity. Unlike other heterogeneous ESC populations (such as Stella or Nanog), TRepV+ cells do not interconvert in their fate and represent an explicit, stable subpopulation of ESCs. Finally, I performed a microarray analysis of TRepV+ and TRepV- ESCs and identifed new genes which may be involved in the regulation of self-renewal and pluripotency.

Vertebrate wnt8a occupies a position at a crossroads linking anteroposterior and dorsoventral axis patterning. While functional aspects of wnt8a are beginning to be understood, the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways are unclear. Three inputs that control wnt8a expression in the zebrafish embryonic margin have been identified: the Brachyury-related T-box transcription factors No tail a (Ntla) and No tail b (Ntlb, previously called Bra) and the maternal zinc-finger transcription factor Zbtb4 (previously called Kzp) are known direct regulators, and Nodal signaling is genetically upstream of wnt8a expression. The transcriptional mechanisms by which the wnt8a locus integrates these diverse temporal inputs are not yet known. We have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Using this transgenic line, we identified two phases of wnt8a transcriptional regulation in zebrafish: phase I comprises Nodal-dependent activation during early gastrulation and phase II comprises No tail (Ntl)-dependent regulation from mid to late gastrula stages onwards. These phases mirror the transition from Nodal-dependent mesoderm induction to Ntl-dependent mesoderm maintenance. To further understand how the wnt8a locus integrates these signals to achieve its transcriptional output, we analyzed upstream cis-regulatory regions through transgenic reporter assays. We identified three promoters in the bicistronic wnt8a locus, two of which drive expression of the upstream coding region (wnt8a.1). We identified two regulatory regions, proximal and distal: the proximal regulatory region contains a mesodermal enhancer with potential binding sites for FoxH1 and Ntl that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal signaling through the mesoderm enhancer in combination with the distal regulatory region, which bears a Zbtb4 consensus binding site. Phase II expression requires Ntl regulation of the mesoderm enhancer in the context of the proximal regulatory region. The distal regulatory region negatively impacts phase II expression driven by the proximal regulatory region, indicating a complex relationship of regulatory elements.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La thérapie cellulaire représente une alternative intéressante aux approchespharmacologiques dans le cadre de certaines pathologies comme les dystrophiesneuromusculaires ou l’ischémie du myocarde. La transplantation de précurseurs adultes deces tissus peut améliorer ces pathologies. Toutefois, le faible nombre de ces précurseursdans l’organisme et la difficulté de leur culture et expansion in vitro sont des facteurslimitants. Grâce à leurs propriétés spécifiques, les cellules souches embryonnaires (ES)constituent une source alternative pour la thérapie cellulaire. Cependant, leur efficacité dedifférenciation dans un lignage donné doit être finement contrôlée avant de pouvoir lesutiliser avec succès.Afin de mieux connaître le potentiel thérapeutique des cellules dérivées de cellulesES, il est essentiel de caractériser les mécanismes moléculaires qui engagent les cellules ESvers différents lignages. Nous nous sommes plus particulièrement intéressés à la voie designalisation p38MAPK, qui est largement impliquée dans la différenciation cellulaire et lasurvie cellulaire. Nous avons plus précisement étudié l’implication de p38MAPK au coursdes différenciations endothéliale, du muscle lisse et du muscle squelettique.Nous avons mis en évidence que les cellules ES p38!-/- ne se différencient plus encellules endothéliales, en cellules du muscle lisse et en cellules du muscle squelettique. Laré-expression de p38MAPK dans ces cellules restaure partiellement les différenciationsdérivées du mésoderme (les différenciations endothéliale, du muscle lisse,cardiomyocytaire et de muscle squelettique). Parallèlement grâce à une inhibitionspécifique de la voie p38MAPK au cours de la différenciation des cellules ES, nous avonsmontré que la voie p38MAPK agit via deux mécanismes moléculaires distincts successifspour réguler la différenciation mésodermique des cellules ES. Le premier mécanisme estcorrèlé à l’expression de Brachyury, un marqueur précoce du mésoderme, alors que lesecond mécanisme est indépendant de Brachyury.Nous avons ensuite poursuivi l’étude de l’implication de p38MAPK dans lamyogénèse des cellules ES et nous avons pu mettre en évidence que p38MAPK estnécessaire à la fois pour l’engagement précoce et la différenciation terminale des cellulesmusculaires.En combinant des approches biochimiques et génétiques, nous avons démontré que lavoie de signalisation p38MAPK est nécessaire très précocement à la différenciation deslignages issus du mésoderme.Ces résultats permettent une meilleure compréhension des mécanismes moléculairesimpliqués dans la différenciation des cellules ES, ce qui constitue une étape préalable ausuccés de futures thérapies cellulaires. / Embryonic stem (ES) cells give rise, in vivo, to all of the three germ layers and, invitro, to differentiate into a broad variety of cell lineages which opens up largeperspectives in regenerative medicine. We previously found that the p38MAPKpathway controls the commitment of ES cells toward either cardiomyogenesis (p38on) or neurogenesis (p38 off ). In this study, we show that p38a knock-out ES cellsdo not differentiate into cardiac, endothelial, smooth muscle, and skeletal musclelineages. Reexpression of p38MAPK in these cells partially rescues theirmesodermal differentiation defects and corrects the high level of spontaneousneurogenesis of knock-out cells. Wild-type ES cells were treated with a p38MAPKspecificinhibitor during the differentiation process. These experiments allowed us toidentify 2 early independent successive p38MAPK functions in the formation ofmesodermal lineages. Further, the first one correlates with the regulation of theexpression of Brachyury, an essential mesodermal-specific transcription factor, byp38MAPK. Moreover, we also showed that p38MAPK is required for the late stageskeletal muscle differentiation. In conclusion, by genetic and biochemicalapproaches, we demonstrate that p38MAPK activity is essential for the commitmentof ES cell into cardiac, endothelial, smooth muscle, and skeletal muscle mesodermallineages.