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Caractérisation de la différenciation de l'endoderme primitif : Coopération entre la voie de signalisation RTK-FGF et le facteur de transcription Gata 6

Abstract : At E3.5 days of development (E3.5), mouse embryo consists of a monolayer of external cells corresponding to Trophectoderme (TE) and of an intern cell mass (ICM), heterogeneous, constituted by two subpopulations of precursory cells: epiblastic cells (Epi) and primitive endoderm cells (EPr). NANOG, an Epi marker and GATA6, a PrE marker, are co-expressed at E3,5 in the MCI and then adopt an exclusive expression within their respective lineage. EPr differentiation requires both expression of GATA6 and RTK pathway, activated by FGF ligand, in order to induce late markers Sox17 and Gata4 expression.First, I studied the relation GATA6/RTK during this process to understand the mechanism of induction of these target genes during final EPr differentiation. I used embryonic stem cells ES WT or Gata6 mutants (ES Gata6-/-), in which I transfected various Gata6 mutant constructions on different residues characterized as potentially phosphorylable by the RTK pathway. So, I analyzed protein expression of Sox17 and Gata4 target genes as well as RNA expression of characteristic genes expressed in the EPr in different inhibition conditions of RTK pathway. So, I was able to highlight that the transmission of the signal is made through the FGF receptor (FGFR1) and that there is compensation between RTK-MEK-ERK and RTK-PI3K pathways highlighted by later Gata6 overexpression of certain mutant forms. Finally, residue S34, S37 and T509 seems to cooperate, through a mechanism not detailed for the moment, for the induction of the EPr target genes.Then, I was interested to phenotypically characterize the role of Dickkopf1 (DKK1), an inhibitor of the WNT/β-catenin pathway, and NOGGIN, an inhibitor of the Bone Morphogenic Protein (BMP) pathway during the EPr differentiation in parietal endoderm (EP) and visceral (EV). Using models of mouse KO for Dkk1 and Noggin, met in pure background C57Bl6, I was able to observe that OCT4 expression was maintained within the Dkk1-/-, and Dkk1-/- Noggin-/- embryos. However, the potential compensation or cooperation mechanism of these two markers is not understanding well for the moment and deserves the analysis of a largest mutant embryos number.
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Stephanie Hermitte. Caractérisation de la différenciation de l'endoderme primitif : Coopération entre la voie de signalisation RTK-FGF et le facteur de transcription Gata 6. Sciences agricoles. Université Clermont Auvergne, 2017. Français. ⟨NNT : 2017CLFAS014⟩. ⟨tel-03119405⟩

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