RESUME Le rat Dumbo présente un aspect malformatif évoquant certains syndromes crânio-faciaux humains. La compréhension du phénotype Dumbo pourrait expliquer les événements cellulaires et moléculaires à l’origine de ces syndromes. Le données recueillies chez le rat Dumbo et comparées à celles du rat Wistar sont susceptibles de constituer de précieuses informations éventuellement transposables à l’espèce humaine. La première étape de cette étude a consisté en des analyses morphologiques et morphométriques afin de vérifier les perturbations morphologiques communes entre les rats Dumbo et les syndromes malformatifs humains : la brièveté des os zygomatique, maxillaire, mandibulaire et la position basse des oreilles. Ces analyses ont été réalisées sur les squelettes embryonnaires âgés de 16 jours à 21 jours de rats Dumbo et Wistar à l’aide d’une coloration in toto au Bleu Alcian – Alizarine. La deuxième étape de cette étude consistait en une analyse cytogénétique. Pour ce faire, nous avons établi le caryotype du rat Dumbo et nous l’avons comparé avec le caryotype du rat Wistar. L’étape suivante fut de procéder à l’analyse histologique des malformations crânio-faciales chez le rat Dumbo en observant la chondrogenèse pendant la morphogenèse crânio-faciale. Enfin, l’examen de l’expression des gènes Msx1 sens (S) , Msx1 antisens (AS) et Dlx1 dans l’extrémité céphalique des rats Dumbo a été réalisé par les techniques de RT–PCR (Reverse Transcription Polymerase Chain Reaction method). Des estimations semi-quantitatives ont été validées en utilisant des dilutions ADNc du rat Wistar. Des densitométries de la densité d’amplicons fluorescence ont été réalisées à l’aide du logiciel VilberLourmat Bio1D software. Les résultats obtenus ont permis de caractériser de manière précise les malformations crânio-faciales chez le rat Dumbo. 1- l’analyse céphalométrique sur les squelettes embryonnaires met en évidence une réduction des dimensions de certaines structures crânio-faciales chez le rat Dumbo : os mandibulaire, maxillaire, zygomatique, ainsi qu’on l’observe également dans le syndrome de Treacher Collins. 2- l’analyse cytogénétique par les techniques de banding G ne montre pas une différence entre le caryotype de Dumbo et le caryotype de rat Wistar (le contrôle). 3- l’analyse histologique de têtes d’embryons montre un retard dans les processus de la chondrification et de l’ossification pendant la morphogenèse crânio-faciale avec une croissance retardée des ébauches oculaires et des bourgeons dentaires. 4- l’analyse de l’expression génique de certains gènes homéobox (Msx1 S et AS) et Dlx1 montre une réduction de ces expressions pendant la morphogenèse crânio-faciale chez le rat Dumbo en comparaison avec la morphogenèse normale dans la souche Wistar.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

La morphogenèse crâniofaciale chez les vertébrés est un phénomène important, strictement régulé dans l’espace et dans le temps. Elle est basée sur une série complexe d'événements moléculaires et morphogénétiques qui implique un réseau interactionnel de gènes et de facteurs de transcriptions, tels les homéoboîtes. La crête neurale (CN) est au cœur de ce processus. Cette dernière fournit la principale source du mésenchyme crâniofacial. Cette population de cellules embryonnaires transitoires va subir une transition épithélio-mésenchymateuse et migrer en plusieurs vagues vers des sites prédéfinis puis se différencier en divers types cellulaires. La CN est à l’origine de plusieurs structures : une grande partie du squelette facial dont le maxillaire, la mandibule, l’os alvéolaire qui entoure les dents ainsi qu’une partie des tissus conjonctifs crâniofaciaux. Les cellules issues des CN sont pluripotentes et offrent un espoir en régénération osseuse et cartilagineuse. Ces caractéristiques ont généré un intérêt particulier des chercheurs pour les utiliser en thérapie cellulaire afin de réparer les défauts osseux des mâchoires. Parmi les tissus crâniofaciaux, nous avons choisi d’étudier plus avant la gencive et les cellules gingivales car leur accès est le plus facile et leurs capacités de différenciation autorisent l’observation d’autres phénotypes cellulaires.La gencive est un tissu kératinisé qui entoure les dents et recouvre l’os alvéolaire. Ce tissu est composé principalement de fibroblastes gingivaux (GFs). Parmi ces cellules, se trouvent des cellules souches gingivales (GSCs) caractérisées par leur auto-renouvellement et leur multipotence. Les GSCs sont facilement recueillies chez les patients adultes, elles montrent une plasticité importante et une activité immunomodulatrice qui en font un outil de choix pour la thérapie cellulaire. De plus, la biopsie se fait sans douleur et n’entraîne ni cicatrice ni problème fonctionnel.La première partie de mon travail de doctorat avait pour objectif d’évaluer le rôle de Msx1 dans la morphogenèse crâniofaciale et par la suite d’analyser l’os alvéolaire après une extraction dentaire afin d’analyser les mécanismes associés à ce processus et l’impact de Msx1 sur la cicatrisation osseuse.La deuxième partie de mon travail est axé sur la gencive et avait pour objectif de mettre en évidence l’origine embryologique des cellules souches orales, dont les GSCs, et de déterminer si elles proviennent des crêtes neurales, du mésoderme ou d’une mosaïque des deux. Enfin, pour appliquer nos connaissances sur l’origine embryologique des cellules souches gingivales, nous avons étudié le profil immunitaire des cellules dérivées des CN. Pour cela, nous avons déterminé la capacité phagocytaire des cellules souches gingivales murines dérivées des CN et comparé à des cellules de CN d’autres espèces vertébrées. / Craniofacial morphogenesis in vertebrates is an important phenomenon, strictly regulated in space and in time. It is based on a complex series of molecular and morphogenetic events involving an interactional network of genes and transcription factors, such as the homeobox. Neural crest (NC) is at the heart of this process. The latter provides the main source of craniofacial mesenchyme. This transient population of embryonic cells will undergo epithelial-mesenchymal transition and migrate in waves to predefined sites and to differentiate into various cell types. NC is the source of several structures: a large part of the facial skeleton including the maxillary, mandibular alveolar bone around the teeth as well as connective tissue in craniofacial portion. Cells from NC are pluripotent and offer hope for bone and cartilage regeneration. These characteristics have generated particular interest to researchers for use in cell therapy to repair bone defects of the jaw. Among the craniofacial tissues, we decided to further investigate the gums and gingival cells because their access is easier and differentiation capabilities allow observation of other cellular phenotypes.The gum is a keratinized tissue around the teeth and covers the alveolar bone. This tissue is composed mainly of populations of gingival fibroblasts (GFs). Among these populations, there are gingival stem cells (GSCs) characterized by their self-renewal and pluripotency. The GSCs are easily collected in adult patients, they show significant plasticity and immunomodulatory activity that make it a tool of choice for cell therapy. In addition, the biopsy is painless and involves neither scar nor functional problem.The first part of my PhD work was to evaluate the Msx1 role in craniofacial morphogenesis and subsequently analyse the alveolar bone after tooth extraction to analyse the mechanisms involved in this process and the impact of Msx1 on bone healing.The second part of my work focuses on the gingiva and was intended to highlight the embryological origin of oral stem cells, including GSCs and determine if they come from the neural crest, mesodermal or mosaic two. Finally, to apply our knowledge of the embryological origin of gum stem cells, we studied the immune profile derived NC cells. For this, we determined the phagocytic capacity gingival murine stem cells derived from CN and compared to cells of CN other vertebrate species.

A Fissura Oral (FO) é uma malformação craniofacial comum na espécie humana e sua etiologia é complexa com aspectos genéticos e ambientais envolvidos na sua formação. Por ser uma malformação de prevalência variável, estudos de associação em populações distintas são necessários, principalmente em populações heterogêneas como no Brasil. A suplementação com ácido fólico está envolvida na redução do risco de recorrência para algumas malformações, mas a natureza da reação entre a ingestão do ácido fólico, a interação entre os genes da rota metabólica e o seu efeito nas concentrações de folato é pouco caracterizada. Além disso, existem poucos estudos envolvendo um grande número de genes e a suplementação com ácido fólico a longo prazo. O objetivo desse trabalho foi estudar o papel dos genes MSX1 e IRF6 e região 8q24 em indivíduos com fissuras orais não sindrômicas de diferentes regiões do Brasil e analisar o efeito da suplementação com ácido fólico e dos polimorfismos nos genes da rota metabólica do folato nos níveis de folato séricos e eritrocitários. Nossos resultados mostram associação positiva entre o alelo 4 do polimorfismo de repetição CA (MSX1) e FO, alelo A da variante rs987525 (8q24) foi associado com FL/P e o haplótipo G/A (rs2235371/rs642961) do gene IRF6 associado com o aumento do risco para FL/P. Dos 23 genes da rota metabólica do ácido fólico estudados, 5 (FPGS, FOLR1, FOLR2, SHMTI e MTHFR) foram relacionados com os níveis de folato sérico e eritrocitário. As variantes rs7033913 (FPGS), rs11235462 (FOLR1) e rs2276048 (FOLR2) foram associadas com os níveis de folato sérico após suplementação. Os polimorfismos rs2168781 e rs2461837 (SHMT1) foram relacionados com os níveis de folato eritrocitário basal e o rs1801131(MTHFR) com os níveis de folato eritrocitário durante a suplementação. Conhecer a etiologia das fissuras orais e entender os efeitos da suplementação e de variantes dos genes da rota do folato nos níveis basais de folato é essencial tanto para auxiliar no manejo clínico através de uma medicina personalizada quanto para aconselhamento genético. / Oral cleft (OC) is a common craniofacial malformation. The etiology is complex and involves genetic and environmental factors. OC have a variable prevalence and association studies are needed in different populations, especially in heterogeneous populations as the Brazilian. Folic acid supplementation reduce the recurrence risk for some malformation, but the reaction between folic acid intake, the interaction between genes of metabolic pathway and effect on folate concentrations is poorly characterized. Furthermore, there are few studies with a large number of genes and long-term folic acid supplementation. The aim was to analyze the role of MSX1 and IRF6 gene and 8q24 region in individuals with non-syndromic oral clefts in different regions of Brazil and to analyze the effect of folic acid supplementation in folate pathway genes and correlate to levels of serum and red blood cell (RBC) folate. Our results have shown a positive association between the CA repeat polymorphism 4 allele (MSX1) and OC, between rs987525 A allele (8q24) and CL/P and the G/A haplotype (rs2235371 / rs642961) of IRF6 associated with increased risk of CL/P. The 23 folate pathway genes studied, 5 (FPGS, FOLR1, FOLR2, SHMTI and MTHFR) were correlated to serum and red blood cell (RBC) folate levels. The variants rs7033913 (FPGS), rs11235462 (FOLR1) and rs2276048 (FOLR2) were associated to serum folate levels after supplementation. Polymorphisms in SHMT1 (rs2168781 and rs2461837) were associated with basal RBC folate while MTHFR (rs1801131) were associated with RBC folate levels during supplementation. Understanding of oral cleft etiology and folate gene pathway will assist clinic management and genetic counseling since folate is involved in important biologic processes.

Made available in DSpace on 2014-12-17T14:13:45Z (GMT). No. of bitstreams: 1 MariaLC_TESE.pdf: 1193515 bytes, checksum: 3a2d36973fa8df734305dbfc5a101581 (MD5) Previous issue date: 2012-12-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / O objetivo deste estudo foi investigar a contribui??o de 6 polimorfismos do gene MSX1 em pacientes com fenda l?bio e/ou palatina n?o-sindr?mica. Avaliou-se tamb?m a contribui??o materna, de forma qualitativa, do etilismo e tabagismo durante a gesta??o como fatores etiol?gicos, bem como iniciou-se a pesquisa de novas variantes no gene MSX1. Este estudo foi realizado com a colabora??o de uma equipe multidisciplinar que atende os pacientes fissurados. Foram estudados 358 indiv?duos dos quais 158 s?o pacientes com fendas orais e 200 s?o crian?as saud?veis constituindo o grupo controle. Todos os indiv?duos e/ou respons?veis legais responderam ao question?rio com informa??es sobre h?bitos de vida e hist?rico familiar. Para a pesquisa de polimorfismos foram utilizados ensaios pr?-desenhados (TaqMan?) empregando t?cnica de discrimina??o al?lica e para novas variantes foi aplicada a t?cnica High Resolution Melt Curve (HRM), ambos pela PCR em Tempo Real (ABI Prism 7500 Fast Real Time). O sequenciamento foi realizado em parceria com o Centro de Estudos do Genoma Humano (CEGH USP; ABI 3730 DNA Analyser - Applied Biosystems - Foster City, CA, USA) para confirma??o das variantes. Observou-se que, em rela??o ao g?nero, 60% dos pacientes eram do g?nero masculino e 40% do g?nero feminino. O etilismo materno durante a gravidez aumentou o risco de fendas orais em crian?as 3,37 vezes (IC95%: 1,56-7,28, p=0,002). Os resultados demonstraram que nenhum dos 6 polimorfismos estudados demonstraram associa??es significantes com a fenda l?bio e/ou palatina n?o-sindr?mica (p>0,05). Na pesquisa de novas variantes foi aplicada a t?cnica (HRM) para as regi?es 3 UTR e 5? pr?xima do gene MSX-1, dois polimorfimos foram identificados e caracterizados para a regi?o 3`UTR (rs1095, rs2229262) e um polimorfismo para regi?o 5? (rs3821949). N?o foram identificadas novas muta??es. Os resultados obtidos pelo HRM n?o mostraram relev?ncia estat?stica dos polimorfismos encontrados com a etiologia das fendas orais. Conclui-se que, o presente estudo n?o mostra associa??o significativa dos polimorfismos estudados do gene MSX1 com o desenvolvimento das fendas l?bio e/ou palatina n?o-sindr?micas, mas a an?lise de fatores ambientais sugere que o etilismo materno durante a gesta??o ? um importante fator de risco na etiologia das fendas orais. Al?m disso, este estudo possui uma abordagem in?dita do gene MSX-1 em pacientes com fendas orais no Rio Grande do Norte contribuindo para o esclarecimento da etiologia das fendas orais n?o-sindr?micas

A Fissura Oral (FO) é uma malformação craniofacial comum na espécie humana e sua etiologia é complexa com aspectos genéticos e ambientais envolvidos na sua formação. Por ser uma malformação de prevalência variável, estudos de associação em populações distintas são necessários, principalmente em populações heterogêneas como no Brasil. A suplementação com ácido fólico está envolvida na redução do risco de recorrência para algumas malformações, mas a natureza da reação entre a ingestão do ácido fólico, a interação entre os genes da rota metabólica e o seu efeito nas concentrações de folato é pouco caracterizada. Além disso, existem poucos estudos envolvendo um grande número de genes e a suplementação com ácido fólico a longo prazo. O objetivo desse trabalho foi estudar o papel dos genes MSX1 e IRF6 e região 8q24 em indivíduos com fissuras orais não sindrômicas de diferentes regiões do Brasil e analisar o efeito da suplementação com ácido fólico e dos polimorfismos nos genes da rota metabólica do folato nos níveis de folato séricos e eritrocitários. Nossos resultados mostram associação positiva entre o alelo 4 do polimorfismo de repetição CA (MSX1) e FO, alelo A da variante rs987525 (8q24) foi associado com FL/P e o haplótipo G/A (rs2235371/rs642961) do gene IRF6 associado com o aumento do risco para FL/P. Dos 23 genes da rota metabólica do ácido fólico estudados, 5 (FPGS, FOLR1, FOLR2, SHMTI e MTHFR) foram relacionados com os níveis de folato sérico e eritrocitário. As variantes rs7033913 (FPGS), rs11235462 (FOLR1) e rs2276048 (FOLR2) foram associadas com os níveis de folato sérico após suplementação. Os polimorfismos rs2168781 e rs2461837 (SHMT1) foram relacionados com os níveis de folato eritrocitário basal e o rs1801131(MTHFR) com os níveis de folato eritrocitário durante a suplementação. Conhecer a etiologia das fissuras orais e entender os efeitos da suplementação e de variantes dos genes da rota do folato nos níveis basais de folato é essencial tanto para auxiliar no manejo clínico através de uma medicina personalizada quanto para aconselhamento genético. / Oral cleft (OC) is a common craniofacial malformation. The etiology is complex and involves genetic and environmental factors. OC have a variable prevalence and association studies are needed in different populations, especially in heterogeneous populations as the Brazilian. Folic acid supplementation reduce the recurrence risk for some malformation, but the reaction between folic acid intake, the interaction between genes of metabolic pathway and effect on folate concentrations is poorly characterized. Furthermore, there are few studies with a large number of genes and long-term folic acid supplementation. The aim was to analyze the role of MSX1 and IRF6 gene and 8q24 region in individuals with non-syndromic oral clefts in different regions of Brazil and to analyze the effect of folic acid supplementation in folate pathway genes and correlate to levels of serum and red blood cell (RBC) folate. Our results have shown a positive association between the CA repeat polymorphism 4 allele (MSX1) and OC, between rs987525 A allele (8q24) and CL/P and the G/A haplotype (rs2235371 / rs642961) of IRF6 associated with increased risk of CL/P. The 23 folate pathway genes studied, 5 (FPGS, FOLR1, FOLR2, SHMTI and MTHFR) were correlated to serum and red blood cell (RBC) folate levels. The variants rs7033913 (FPGS), rs11235462 (FOLR1) and rs2276048 (FOLR2) were associated to serum folate levels after supplementation. Polymorphisms in SHMT1 (rs2168781 and rs2461837) were associated with basal RBC folate while MTHFR (rs1801131) were associated with RBC folate levels during supplementation. Understanding of oral cleft etiology and folate gene pathway will assist clinic management and genetic counseling since folate is involved in important biologic processes.

A Fissura Oral (FO) é uma malformação craniofacial comum na espécie humana e sua etiologia é complexa com aspectos genéticos e ambientais envolvidos na sua formação. Por ser uma malformação de prevalência variável, estudos de associação em populações distintas são necessários, principalmente em populações heterogêneas como no Brasil. A suplementação com ácido fólico está envolvida na redução do risco de recorrência para algumas malformações, mas a natureza da reação entre a ingestão do ácido fólico, a interação entre os genes da rota metabólica e o seu efeito nas concentrações de folato é pouco caracterizada. Além disso, existem poucos estudos envolvendo um grande número de genes e a suplementação com ácido fólico a longo prazo. O objetivo desse trabalho foi estudar o papel dos genes MSX1 e IRF6 e região 8q24 em indivíduos com fissuras orais não sindrômicas de diferentes regiões do Brasil e analisar o efeito da suplementação com ácido fólico e dos polimorfismos nos genes da rota metabólica do folato nos níveis de folato séricos e eritrocitários. Nossos resultados mostram associação positiva entre o alelo 4 do polimorfismo de repetição CA (MSX1) e FO, alelo A da variante rs987525 (8q24) foi associado com FL/P e o haplótipo G/A (rs2235371/rs642961) do gene IRF6 associado com o aumento do risco para FL/P. Dos 23 genes da rota metabólica do ácido fólico estudados, 5 (FPGS, FOLR1, FOLR2, SHMTI e MTHFR) foram relacionados com os níveis de folato sérico e eritrocitário. As variantes rs7033913 (FPGS), rs11235462 (FOLR1) e rs2276048 (FOLR2) foram associadas com os níveis de folato sérico após suplementação. Os polimorfismos rs2168781 e rs2461837 (SHMT1) foram relacionados com os níveis de folato eritrocitário basal e o rs1801131(MTHFR) com os níveis de folato eritrocitário durante a suplementação. Conhecer a etiologia das fissuras orais e entender os efeitos da suplementação e de variantes dos genes da rota do folato nos níveis basais de folato é essencial tanto para auxiliar no manejo clínico através de uma medicina personalizada quanto para aconselhamento genético. / Oral cleft (OC) is a common craniofacial malformation. The etiology is complex and involves genetic and environmental factors. OC have a variable prevalence and association studies are needed in different populations, especially in heterogeneous populations as the Brazilian. Folic acid supplementation reduce the recurrence risk for some malformation, but the reaction between folic acid intake, the interaction between genes of metabolic pathway and effect on folate concentrations is poorly characterized. Furthermore, there are few studies with a large number of genes and long-term folic acid supplementation. The aim was to analyze the role of MSX1 and IRF6 gene and 8q24 region in individuals with non-syndromic oral clefts in different regions of Brazil and to analyze the effect of folic acid supplementation in folate pathway genes and correlate to levels of serum and red blood cell (RBC) folate. Our results have shown a positive association between the CA repeat polymorphism 4 allele (MSX1) and OC, between rs987525 A allele (8q24) and CL/P and the G/A haplotype (rs2235371 / rs642961) of IRF6 associated with increased risk of CL/P. The 23 folate pathway genes studied, 5 (FPGS, FOLR1, FOLR2, SHMTI and MTHFR) were correlated to serum and red blood cell (RBC) folate levels. The variants rs7033913 (FPGS), rs11235462 (FOLR1) and rs2276048 (FOLR2) were associated to serum folate levels after supplementation. Polymorphisms in SHMT1 (rs2168781 and rs2461837) were associated with basal RBC folate while MTHFR (rs1801131) were associated with RBC folate levels during supplementation. Understanding of oral cleft etiology and folate gene pathway will assist clinic management and genetic counseling since folate is involved in important biologic processes.

Le bourgeon de membre est, dès son émergence dans le flanc de l'embryon, divisé en deux domaines distincts, l'un antérieur, l'autre postérieur. Nous analysons le rôle des gènes Msx dans cette polarisation. Chez la souris, les gènes Msx constituent une petite famille multigénique comprenant Msx1 et Msx2: ces gènes codent pour des facteurs de transcriptions à homéodomaine. Les souris mutantes pour Msx1 et Msx2 présentent également une surcroissance de la région antérieure du bourgeon de membre, associée à la perte d'un domaine apoptotique, qui conduit à des polydactylies antérieures. La projet vise à élucider les mécanismes par lesquels les Msx agissent sur la morphogenèse de cette région du membre. Au cours de cette investigation, nous avons fait d'intéressantes observations qui indiquent que les Msx interagissent avec Gli3, et nous avons obtenu d'intéressants résultats expérimentaux par co-immunoprécipitation des Msx et de Gli3 (co-IP) dans des cellules en culture transfectées, et par test de ligation de proximité (PLA) sur le bourgeon de membre in situ. La stratégie repose aussi sur l'analyse différentielle des transcriptome d'embryons doubles mutants pour Msx1 et Msx2 et d'embryons normaux, de façon à identifier les cibles qu'ils peuvent avoir en commun avec Gli3, ces dernières étant déjà décrites. Ces modèles devraient permettre de corréler le niveau d'apoptose avec la surcroissance du bourgeon et la polydactylie qui en résulte, apportant une importante contribution aux mécanismes de la morphogenèse qui sont encore très mal compris. / The vertebrate limb is a paradigmatic model for morphogenesis. The anteroposterior (AP) growth and patterning of the limb bud rely on an intricate regulatory genetic network involving many genetic players such Shh and Gli3. Shh is produced in the posterior region of the limb mesoderm and acts as a morphogen along the AP axis. It regulates both digit number and identity of different AP positions. As the main function of Shh is to prevent proteolysis of Gli3FL into Gli3R, Gli3R concentrates anteriorly which is also where apoptosis takes place. In the mouse, paradoxically, despite a quasi-symmetrical expression profile, Msx1/2 null mutants show systematic anterior defects with an overgrowth in the anterior limb domain, resulting in anterior polydactyly. Both Gli3 and Msx mutant limb defects are concentrated anteriorly in a similar fashion suggesting an interaction between these genes and a possible role of this interaction in AP patterning in the limb bud as well as dysregulation of apoptosis. Indeed, we demonstrate interactions between Msx and Gli3 genes and even proteins. Mutations of Msx and Gli3 result in anterior polydactylous phenotypes and a similar loss of anterior apoptosis. This morphological analysis is associated with a search for common Gli3 and Msx transcriptional targets and partners in an attempt to link gene activity with changes in cell physiology that underlie morphogenesis. We have performed a differential transcriptome (RNA-Seq) on whole limb buds of Msx1-/-Msx2 narrowing down on a number of potential common targets of Gli3 and Msx. We demonstrate that Msx and Gli3 work together in regulating the AP axis of limb development, independent of Shh.

The Wnt signaling pathway represents the principal evolutionarily conserved signaling cascade found in all multicellular organisms. It plays a key role not only in many processes during embryogenesis, but also in maintaining tissue homeostasis and regeneration. By contrast, mutations in genes encoding components of the pathway often result in increased activation of Wnt signaling and underlie onset of many human diseases, particularly cancer. The canonical Wnt signaling pathway is essential for proliferation and maintenance of the pluripotent state of intestinal stem cells and thus for homeostatic renewal of the intestinal epithelium. However, aberrant (hyper)activation of the Wnt signaling pathway is the initial step in development of intestinal neoplasia. Understanding the causes and identifying the consequences of the Wnt signaling hyperactivation is crucial for deciphering mechanisms leading to malignant transformation. Although the canonical Wnt signaling pathway has been the subject of scientific studies for several decades, all regulatory mechanisms and consequences of its hyperactivation have not been completely elucidated yet. During my PhD studies, I focused on understanding function(s) of some components and target genes of this signaling cascade. In this theses, results of my first...