Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Les muscles sont des structures très organisées qui nous permettent d’effectuer un grand nombre de fonctions. Ils sont constitués de cellules musculaires mais aussi de tissus conjonctifs qui comprennent à la fois des cellules et la matrice extracellulaire. Les interactions entre les cellules musculaires et le tissu conjonctif sont cruciales pour la physiologie du muscle. Le collagène VI (COLVI) est une molécule hétérotrimérique ubiquitaire située dans les tissus conjonctifs, qui est impliquée dans un grand nombre de processus biologiques. Les trimères de COLVI sont composés de 2 chaines dites “courtes” et d’une chaine “longue”. Chez les mammifères, il existe à ce jour, 6 chaines COLVI (deux courtes (α1-2(VI) et 4 chaines longues (α3-6(VI)). Peu de choses sont encore connues à propos de l’assemblage des chaines les plus récemment décrites α4-6(VI) avec les chaines courtes ainsi qu’une la potentielle compensation entre les différentes chaines longues. De plus, chez l’homme, un déficit en α1-3(VI) du fait de mutations dans les gènes correspondants COL6A1-3 conduit à un spectre de maladies neuromusculaires appelées myopathies liées au COLVI. Pendant ma thèse, je me suis intéressée au COLVI durant le développement du poisson-zèbre, un modèle pertinent pour l’étude de maladies neuromusculaires. Dans la première partie de mon travail, j’ai identifié 2 orthologues des chaines α4-6(VI) chez le poisson-zèbre grâce à des études bio-informatiques. Du fait de leur plus grande homologie avec la chaine α4(VI) murine, nous les avons nommés col6a4a et col6a4b. Pour mieux comprendre les rôles des protéines correspondantes, j’ai créé des embryons de poissons-zèbres déficients en COLVI en utilisant l’approche transitoire par oligo morpholino antisens (MOs). Nous avons dessiné des MOs ciblant des sites d’épissage des pré-messagers col6a2, col6a4a et col6a4b, provoquant un saut d’exon et conduisant à un stop prématuré (PTC). J’ai observé une forte diminution des transcrits ciblés. Tous les embryons injectés (morphants) ont présenté des phénotypes morphologiques macroscopiques qui ont conduit à des défauts fonctionnels. Ces phénotypes ont été confirmés au niveau ultra-structural par microscopie électronique. Toutefois, l’analyse de la croissance des motoneurones a permis de mettre en évidence des différences entre ces morphants. Par la suite, j’ai voulu créer deux types de lignées transgéniques, pour pouvoir à la fois étudier le déficit en COLVI à plus long terme (grâce à l’utilisation de Zinc Finger Nucleases) et tester des approches de cribles pharmacologiques (lignée transgénique col6a2 contenant un PTC, fusionné à la GFP). J’ai effectué les clonages nécessaires à l’obtention des différentes constructions, et ces dernières ont été testées in vitro pour validation, lorsque cela était possible. Malheureusement, du fait des forts taux de mortalité in vivo dans les deux cas, nous avons dû nous résoudre à arrêter ces projets. En parallèle, ma connaissance du modèle poisson-zèbre m’a donné l’opportunité, dans le cadre d’une collaboration avec l’équipe de Denis Furling, d’aborder une autre problématique. Ce groupe, qui travaille sur la Dystrophie Myotonique de type 1 (DM1), s’est intéressé à la réexpression d’une isoforme fœtale de la dystrophine retrouvée chez les patients DM1 et à sa possible implication dans la pathologie. L’isoforme fœtale diffère de la forme adulte notamment par l’exclusion de l’exon 78, conduisant à un changement de cadre de lecture et un changement dans la partie 3’ de l’ARN de la dystrophine. Nous avons montré que le maintien de l’isoforme fœtale de la dystrophine était délétère pendant le développement du poisson-zèbre, puisque ces embryons ont présenté un phénotype macroscopique dépendant de la dose de MO injectée ainsi que des troubles de la mobilité. / Muscles are highly organized structures that allow us to perform many functions. They are made from muscular cells but also surrounding tissues that comprise both cells and extracellular matrix. The interactions between them are crucial for the muscle physiology. Collagen VI (COLVI) is a heterotrimeric protein, ubiquitously expressed in connective tissues. It plays multiple biological roles in the maintenance of structural integrity, cellular adhesion, migration and survival. COLVI trimers are formed by the assembly of 2 “short” chains and 1 “long” chain. To date, six COLVI chains are recognized in mammalians with 2 short (α1-2(VI)) and 3 long (α3-6(VI)) chains. Little is known regarding the possible assembly of the newly characterized α4-6(VI) polypeptides with the short chains, and a putative functional compensation between the different long chains. Furthermore, in humans, deficiency in α1-3(VI) due to mutations in the COL6A1-3 genes causes a heterogeneous group of neuromuscular disorders collectively termed COLVI-myopathies. During my Ph.D, I got interested in COLVI during the development of zebrafish, a relevant model of neuromuscular disorders. In the first part of my work, I identified 2 orthologs of the α4-6(VI) chains in zebrafish thanks to bio-informatics studies. In light of their stronger homology with the mammalian α4(VI) chain, we named the genes encoding the novel chains col6a4a and col6a4b. To further unveil the roles of the corresponding proteins, we created COLVI deficient zebrafish embryos using a morpholino antisense oligonucleotides approach (MO) . We chose to design MOs that block splicing of col6a2, col6a4a and col6a4b, thereby creating premature termination codons. As expected, the targeted transcripts levels were drastically reduced, likely due to degradation by the nonsense mediated RNA decay. All morphant embryos presented macroscopic and morphologic phenotypes that overall resulted in functional muscle defects: altered muscle structure detected by birefringence analysis and impaired motility upon touch-evoked escape test. These alterations were confirmed at the ultra-structural level by electron microscopy. Nevertheless, some phenotypical specificities were uncovered between the different col6a2, col6a4a and col6a4b morphants, with the discovery of axon outgrowth defects. In a second part, we wanted to create stable zebrafish lines to study COLVI deficiency at later stages using Zinc Finger Nucleases (ZFN) and to be able to carry out pharmacological screenings with a transgenic line containing col6a2 with a premature codon (PTC) fused to the GFP. I performed clonings to obtain the different constructs. When possible, constructs were tested in vitro. Unfortunately, due to high mortality in vivo in both cases, we had to interrupt these projects. In parallel, my knowledge of the zebrafish model gave me the opportunity to be part of another project, in collaboration with the team of Denis Furling...

Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.