During mouse embryo gastrulation, the pluripotent epiblast gives rise to the three embryonic germ layers, the ectoderm, mesoderm and endoderm. After somitogenesis begins and pluripotency disappears from the epiblast, bipotent neuromesodermal progenitors (NMPs) drive axis elongation, contributing to the formation of the posterior nervous system, as well as the axial and paraxial mesoderm. Early NMPs arise in the E8.5 mouse embryo, in and near the primitive streak, while late NMPs are found in the tail bud (E9.5 - E13.5). NMP regions are characterized by coexpression of Tbra (Brachyury) and Sox2. Sox1, another neural related transcription factor, has also been detected in NMP regions. Importantly, it has been shown that Sox1 expression increases as NMPs transit from the primitive streak to the tail bud stages. Mouse epiblast derived stem cells (EpiSCs) recapitulate the properties of the post-implantation epiblast and therefore serve as a good in vitro system for the study of early lineage specification events. EpiSCs express pluripotency factors and early differentiation markers, including Sox2, Sox1 and Tbra. Based on studies reporting that EpiSC cultures contain distinct subpopulations that have progressed further into lineage specification, I analyzed the properties of the Tbra expressing EpiSCs and by dissecting their expression profile, I assess whether these cells are pluripotent or they have progressed further into lineage specification, possibly into an NM fate. I show that EpiSC cultures include a large fraction of Tbra/Sox2 double positive cells; however, Nanog expression was detected in the vast majority of Tbra+/Sox2+ EpiSCs suggesting that most of the Tbra+ cells are pluripotent rather than bipotent NMPs. Using a previously published Tbra-GFP reporter cell line, I present that Tbra-GFP+ cells constitute a dynamic fraction of the culture that has not exited pluripotency (as shown by expression of the pluripotency markers), but have adopted an early primitive streak-like character. Similar to the cells of the posterior epiblast, these EpiSCs are in a reversible state and they retain their ability to undergo neural differentiation. In contrast to the overlap of Tbra and Sox2 positivity in self-renewing EpiSCs, it has been shown that Tbra expression is mutually exclusive with expression of Sox1-GFP, that seems to mark a distinct subpopulation with neural-like characteristics. In vitro NMPs can be generated from EpiSCs upon treatment with Fgf2 and the Gsk- 3 antagonist/Wnt agonist CHIRON99021 (FGF/CHI). In these conditions, 80% of the culture becomes Tbra+/Sox2+. Given that Sox1 is present in NMP regions in vivo, I hypothesized that the NMP cultures could contain Tbra+Sox1+ NM bipotent cells. Most importantly, the upregulation of Sox1 at the tail bud stages drove the hypothesis that Sox1 expression could mark the transition from an early- to a late-like NMP state in vitro. In this study, using a Sox1-GFP reporter cell line, I show that Tbra/Sox2/Sox1-GFP triple positive cells emerge in FGF/CHI treated EpiSCs. Importantly, Sox1-GFP+ cells express NMP markers and are enriched in transcripts of Hox genes. The expression profile of Sox1-GFP+ cells resembles the alteration of Hox gene activation that takes place in the caudal progenitor regions during the transition from early NMPs (E8.5) to late NMPs (E9.5-10.5) and hence supports the hypothesis that Sox1-GFP marks NMPs that correspond to the axial progenitors found at tail bud stages. Although the gene activity observed in the Sox1-GFP+ subpopulation correlates with the NM developmental potential, these cells exhibit strong neurogenic capacity, while evidence for their ability to give rise to mesoderm differentiation products is still lacking. Since Tbra and Sox1/Sox2 are not expressed in NMP regions exclusively, but also in mesoderm and neural fated tissues respectively, double rather than single reporter cell lines would be more suitable tools for tracking and isolating bipotent NM progenitors in vivo and in vitro. Here, I present the CRISPR/Cas9-mediated generation of a reliable Tbra-GFP reporter ES cell line that in contrast to the one published before, contains both endogenous Tbra loci intact. By targeting the Sox2 locus in the Tbra-GFP ES cells, I generated a Tbra-GFP/Sox2-tdTomato double reporter ES cell line, that in the future, could help us to dissect the molecular mechanisms underlying the self-renewal and differentiation of NMPs.

Pluripotency is defined as the capacity to differentiate into cells from each of the three primary germ layers, the ectoderm, mesoderm and endoderm. This is a property of cells located in the inner cell mass (ICM) of preimplantation blastocysts and in the epiblast layer of postimplantation, presomite embryos. Preimplantation and postimplantation pluripotency can be captured indefinitely in cultured embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively. Preimplantation pluripotency in ES cells is regulated by a network of genes centred on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a mutually-reinforcing circuit and cooperatively stimulate transcription of downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in the postimplantation epiblast. Functional studies established a role for Oct4 and Nanog in the specification of ICM cell identity, and a role for Oct4 in the maintenance of postimplantation pluripotency. Although the role of Sox2 in preimplantation ICM cells is unclear, it is critical for the establishment of egg cylinder following implantation and indispensable for ES cell pluripotency. However, despite the presence of Sox2 in postimplantation pluripotent cells the role of Sox2 in postimplantation pluripotency is unknown. In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog are expressed in opposing gradients in the gastrulation-stage postimplantation epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly suggests that coexpression of Sox2 and Oct4 is important for establishing postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively correlated with NanogHIGH cells. This reciprocal relationship emerged during the transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively influences Sox2 expression post-transcriptionally. The negative influence of Nanog on Sox2 protein level was confirmed using doxycycline-inducible Nanog overexpressing EpiSCs. This negative relationship indicates that the regulation of Sox2 expression is different in postimplantation pluripotency and that Nanog may negatively regulate Sox2 on the protein level in the posterior epiblast. Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this was further investigated by microarray transcription profiling using cells in which a fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2- tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated cell identity and Sox2-tdTomatoLOW cells are associated with non-neural differentiation. Interestingly the global profile of ES cells and EpiSCs that share similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different roles in different pluripotent states. ES cells with enforced Sox2 expression were unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on the maintenance of EpiSC pluripotency. This is likely due to the presence of redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in ES cells. Taken together, these results suggest the hypothesis that postimplantation pluripotency is maintained by multiple Sox factors, while Nanog negatively regulates Sox2 post-transcriptionally to repress neural specification in the posterior epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible negative feedback loop to balance the proneural and pluripotent properties of Sox2 in postimplantation pluripotency.

Elongation of the mouse anteroposterior axis depends on stem cell-like axial progenitors including a neuromesodermal (NM) bi-fated population existing in the primitive streak and later in the tail bud. Fate mapping experiments have demonstrated these NM progenitors reside in precise locations of the embryo. At E8.5, these cells are found in the node-streak border (NSB) and anterior epiblast on either side of the primitive streak. At tail bud stages (E10.5-E13.5), these progenitors reside in the chordoneural hinge (CNH). The coexpression of the transcription factors T (brachyury) and Sox2 has been proposed as a good marker to identify NM progenitors in vertebrates. However, this cell signature has never been thoroughly assessed during mouse axis elongation. In this thesis, I performed T and Sox2 double immunofluorescent stainings on different stages of mouse embryos and reconstructed their expression domains in the 3D images to investigate the spatiotemporal dynamics of NM progenitors during axis elongation. The results show the transient existence of T+Sox2+ cells in the posterior progenitor zone, from the headfold stage (E8.0) to the end of axis elongation (E13.5, 65somites). Moreover, the number of T+Sox2+ cells increases between E8.5 and E9.5 but gradually declines afterwards. I then investigated the time points for initiation and loss of NM progenitors by performing a series of heterotopic grafting experiments. It has been previously shown that distal epiblast (Sox2+T- cells) at LS-EB stages (E7.5) are fated to become NSB cells in E8.5 embryos. However, when cells from the distal region of LS-EB stage embryos (E7.5) were grafted to E8.5 NSB, these cells contribute extensity to the notochord but not either neural tissues or paraxial mesoderm. This indicates that NM progenitors may be not yet specified before the onset of T and Sox2 coexpression, while the notochord progenitors are already specified at E7.5. The grafting experiments also show the loss of NM progenitors at E14.5 after the end of axis elongation, which coincides with the disappearance of T+Sox2+ cells in the tail. Collectively, these results indicate that T+Sox2+ cells may represent a distinct cell state that defines NM progenitors. Wnt/β-catenin signalling has been shown to play an important role in maintaining the posterior progenitor zone. However, due to the wide expression of β-catenin and the early lethality of β-catenin null embryos, the exact effect of losing β-catenin in NM progenitors is still unknown. In this study, I took advantage of the Cre-ERT2 system and grafting technique to conditionally delete β-catenin specifically in NM progenitors during ex vivo culture. The results show that Wnt/β-catenin signalling is required cell autonomously for initiating mesoderm fate choice in NM progenitors. In its absence, mesoderm fated NM progenitors convert their fate and differentiate to neural derivatives. Moreover, the interchangeability between neural and mesodermal fate only exists in NM progenitors, as the loss of β-catenin in mesoderm committed progenitors does not affect their fate choice. Using image analysis and quantification software, I also show that Wnt/β-catenin signalling is crucial for the expansion of T+Sox2+ NM progenitors during axis elongation. Due to difficult access and a limited number of NM progenitors in vivo, in vitro generated NM progenitors from pluripotent cells, such as epiblast stem cells (EpiSCs), can offer an insight into the maintenance and differentiation of NM progenitors. Since the in vivo potential of EpiSCs had never been successfully demonstrated before, I first grafted EpiSCs into postimplantation embryos and cultured them ex vivo for 24-48 hours to assess their cell integration. The results show that EpiSCs can integrate successfully in streak stage embryos (E6.5-E7.5), but not at early somite stages (E8.5), when the epiblast has lost its pluripotency. I then further investigated the in vivo potential of EpiSC derivatives. The results show that increasing Wnt signalling in EpiSCs inhibits their ability to generate anterior neural tissues in vivo, which is consistent with the previous in vitro data. Recently, it has been demonstrated that NM progenitors can be derived from EpiSCs. These in vitro derived NM progenitors can incorporate into E8.5 embryos and give rise to both neural and mesodermal derivatives. In this thesis, I show that these in vitro derived NM progenitors do not incorporate successfully in E7.5 embryos. Collectively, by combining grafting experiments with a chimeric embryo formation assay, I can identify the in vivo stage of the in vitro counterparts of the embryonic cell types.

Neuromesodermal progenitors (NMPs) are bipotent progenitors, located at the caudal end of the embryo and are essential for axis formation. These stem cell-like progenitors possess the ability to self-renew and differentiate to both mesodermal and neural lineages, such as skeletal muscle and spinal cord derivatives. These progenitors arise at E8.5 and are localised in the caudal lateral epiblast (CLE), a posterior region of the embryo near the primitive streak. Later in development, they reside in the tail bud until cessation of axial elongation at E13.5. Throughout these stages NMPs are characteristically marked by co-expression of T(Bra) (Brachyury) and Sox2. This characteristic is also present in in vitro NMPs, which can be derived from Epiblast Stem Cells (EpiSCs) through treatment with Wnt/β-catenin signalling agonists and Fgf2, which simulates their in vivo environment. Protein and mRNA profiling of NMPs and mutant phenotypes in vivo supports the hypothesis that a non-canonical Wnt pathway, the Wnt/Planar Cell Polarity pathway (PCP) could be involved in NMP fate decision and/or maintenance. This thesis focuses on understanding more about the role of PCP by aiming to identify the spatio-temporal profile of Wnt/PCP pathway components in NMP regions during axial elongation, as well as determining its role in NMP behaviour through manipulation of this pathway via in vivo and in vitro assays Employing in situ hybridisation and immunohistochemistry techniques, key Wnt/PCP components, including Pk1, Vangl2 and Ptk7, were confirmed to be present in in vivo and in vitro NMPs, thus, providing strong evidence that Wnt/PCP may be involved regulating NMP behaviour. Disruption of Wnt/PCP signalling through overexpression of Wnt/PCP components was tested in refined in vivo and in vitro assays. Overexpression of Vangl2 and Ptk7, but not Pk1 in NMPs regions in vivo resulted in loss of contribution to neural lineages, as well as lower contribution to NMP regions themselves. Similarly, Wnt/PCP components were disrupted in vitro through generation of dox-inducible overexpression cells lines for Wnt/PCP components. These lines were used to generate NMPs from an optimised novel alternative source Epiblast-Like Cells (EpiLCs), however no clear affect to lineage was observed. Overall this work has successfully advanced our knowledge of Wnt/PCP mediated control of NMP differentiation and maintenance, and provided a finer grained description of the relationships between them.

The governance of cell fate decisions during development is a fundamental biological problem. An important aspect of this is how cells exit a multipotent state and choose their fates in a correct manner and proportion. To tackle an aspect of this problem, I have focused on 2 multipotent models: one infinite self-renewal pluripotency in an artificial environment, and the other, bipotent progenitors in the context of the mouse embryo. The first model aimed to explore the effects of chromatin-associated factors on the ability of pluripotent mouse Embryonic Stem Cells (ESCs) to self-renew, via monitoring gene expression heterogeneity of key genes. The second model focused on Neural Mesodermal Progenitors (NMPs), a bipotent cell population found in the Caudal Lateral Epiblast (CLE) of mammalian embryos, which contributes to the spinal cord and paraxial mesoderm. The aim here was to derive NMPs in vitro which exhibit similar gene expression patterns and function like their mouse embryo counterpart and study their renewal and differentiation in detail. The first multipotent model explores the effects of chromatin remodelling on cell fate decisions, specifically investigating the consequences of inhibiting the histone acetyltransferase Kat2a on the ESCs fate. I found first, that the effect of Kat2a inhibition depends on the pluripotent state of the cells; cells in a ground state exhibit a resistance to Kat2a inhibition and maintain their pluripotency, whereas cells in a naïve state experience destabilization of their pluripotency gene regulatory network and shift towards differentiation. Second, that Kat2a inhibition in the naïve state results in a decline in the gene expression noise strength contributed by the promoter activation operation, which suggests that when ESCs become lineage-primed their transcriptional noise is constrained. In the bipotent model, the NMPs are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from ESCs in vitro. Comparison between the different NMPs protocols stresses that Epiblast Stem Cells (EpiSCs) are an effective source for deriving a multipotent population resembling the embryo Caudal Epiblast (CE), that generates NMPs. Furthermore, self-organization of this CE-like population, resulted in axially organized aggregates. Exploiting the mouse embryo CLE as a reference shows that EpiSCs derived NMPs, monolayers and aggregates, consist of a high proportion of cells with the embryo's NMP signature. Importantly, studying this system in vitro sheds light on the sequence of events which lead to NMP emergence in vivo. On this basis, I conclude that understanding the initial state of cells at a crossroads is important to reveal the limitations it imposes on the cells fate exploration, hence makes it possible to mimic more precisely the fate decision process in vitro.

The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.

Lors de la formation du blastocyste, l'embryon de souris est constitué d'un épithélium externe, le trophectoderme (TE), et d'une masse cellulaire interne (MCI). L’épiblaste (EPI) et l’endoderme primitif (EPr) se spécifient au sein de la MCI sous un patron de « sel et poivre » caractérisé par l’expression complémentaire de NANOG, marqueur de l’EPI et de GATA6, marqueur de l’EPr. Nanog est nécessaire pour l’acquisition d’une identité EPI et Gata6 induit le devenir en EPr. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr et la perturbation de son activité impacte directement sur le ratio EPr/EPI dans la MCI. Je recherche des facteurs qui serait exprimés de manière hétérogène avant la spécification des cellules internes et pourraient faire pencher la balance vers un destin ou l’autre. Pour cela, j’ai disséqué l’évolution des cellules de la MCI au sein des embryons Nanog-/- et Gata6-/-. Ces embryons forment correctement le TE et la MCI qui ne se spécifie ni en EPI ni en EPr. En effet, les cellules internes des embryons Nanog-/- ; Gata6-/- restent bloquées autour du stade E3.25. De manière étonnante, dans les cellules de la MCI, le facteur de transcription SOX2 est présent et ce, de manière hétérogène. De plus, grâce à des traitements inhibiteurs de la voie FGF/MAPK, je montre que cette voie n’est pas responsable de l’hétérogénéité d’expression de SOX2. Ainsi, l’expression hétérogène de SOX2 dans les cellules internes des embryons est donc indépendante de Nanog, de Gata6 et de la voie FGF/MAPK. / During mouse blastocyst formation, the embryo consists of an outer epithelium, the trophectoderm (TE), and the inner cell mass (ICM). The epiblast (EPI) and the primitive endoderm (PrE) are specified within the MCI in a "salt and pepper" pattern characterized by the complementary expression of NANOG, marker of EPI and gata6, marker of PrE. Nanog is mandatory to acquire an EPI identity and Gata6 induces the PrE identity. FGF /MAPK pathway plays a critical role in the acquisition of a PrE identity and disruption of its activity directly impacts the PrE/Epi ratio within the ICM. I’m looking for factors that would be expressed heterogeneously before the specification of internal cells and might tilt the balance towards one fate or the other. For this, I dissected the evolution of ICM cells within Nanog-/- ; Gata6-/- embryos. These embryos form properly the TE and MCI that specifies neither EPI nor PrE. Indeed, the internal cells of Nanog-/- ; Gata6-/- embryos remain stuck around the stage of E3.25. Surprisingly, in the MCI cells, the transcription factor SOX2 is present and this, heterogeneously. Moreover, using inhibitors treatments of the FGF/MAPK pathway, I show that this pathway is not responsible for the heterogeneity of expression of SOX2. Thus, the heterogeneous expression of SOX2 in the inner cells of the embryos is therefore independent of Nanog, Gata6 and the FGF/MAPK pathway.

Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.

Au cours de la préimplantation, l'embryon de souris produit deux lignages cellulaires, le trophectoderme (TE), et la masse cellulaire interne (MCI) qui elle-même se différencie en épiblaste (Epi) et en endoderme primitif (EPr), caractérisés respectivement par l'expression exclusive de Nanog et de Gata6. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr. J’ai examiné l’expression de pERK, DUSP4 et ETV5 qui permettent de visualiser l'activité des MAPK. Ces analyses ont été effectuées en activant ou inhibant la voie FGF/MAPK, ainsi que dans des embryons mutants pour Nanog et/ou Gata6. Ceci a permis d’observer l’activation de la voie FGF/MAPK dès E3,25. Un autre volet de mon travail a été d'analyser la voie de l’IGF dans les embryons préimplantatoires afin de comprendre l’influence de cette voie dans les différents lignages. J’ai montré que le récepteur activé pIGF1R est exprimé de manière différentielle dans le TE, l’EPr et l’Epi au cours du développement. Une supplémentation d’IGF1 induit une augmentation du nombre de cellules en deux phases, d'abord de l’Epi puis de l’EPr. A l’inverse, une perte de fonction d’IGF1R induit une diminution du nombre de cellules entre E3,75 et E4,25. / During preimplantation, mouse embryo produces two cellular lineages, the trophectoderm (TE), and the inner cell mass (ICM), which differentiates in epiblast (Epi) and primitive endoderm (PrE), characterized respectively by the complementary expression of Nanog and Gata6. FGF/MAPK pathway plays a critical role in the acquisition of a PrE identity. I examined the expression of the markers of MAPK activity pERK, DUSP4 and ETV5. The analyze was performed with activation or inhibition of FGF/MAPK pathway and in mutant embryos for Nanog or Gata6. This showed that FGF/MAPK pathway is activated as soon as E3,25. I have also analyzed the IGF pathway in preimplantation embryos in order to understand the role of this pathway in embryonic lineages. I showed that active receptor pIGF1R is differentially expressed in TE, PrE and Epi during embryonic development. Supplementation with IGF1 induces an increase in cell number in two phases, first in Epi then in PrE. Conversely, loss of function of IGF1R induces a decrease in cell number between E3,75 and E4,25.

Les cellules souches pluripotentes sont capables de donner naissance à tous les types cellulaires constituant un organisme, ce qui leur confère un fort intérêt thérapeutique. A partir de l’embryon de souris on peut en dériver deux types : les cellules souches embryonnaires (ES) au stade blastocyste et les cellules souches d’épiblaste (EpiSC) au stade œuf cylindre. Ces deux types de cellules partagent leurs propriétés pluripotentes mais se distinguent par de nombreux aspects comme leurs conditions de culture et les gènes qu’elles expriment. Nous avons montré que la reprogrammation par clonage par transfert de noyau permet d’obtenir des EpiSC présentant un méthylome et un transcriptome similaires à ceux des EpiSC issues d’embryons après fécondation. Nous avons également caractérisé le profil de méthylation de l’ADN des EpiSC, et montré une tendance à l’hyperméthylation des promoteurs des EpiSC par-rapport aux cellules ES et à l’épiblaste. De plus, l’absence de méthylation empêche la conversion des cellules ES en EpiSC. Les EpiSC semblent donc dépendre fortement de la méthylation de l’ADN pour réguler l’expression de leurs gènes, ce qui les distingue des cellules ES. / Pluripotent stem cells are of great therapeutic interest because of their capability to give rise to all the cells composing an organism. We can derive two types of these stem cells from the mouse embryo: embryonic stem cells (ESCs) from the blastocyst and epiblast stem cells (EpiSCs) from the egg cylinder stage. These two cell types share their pluripotent properties but are distinct on several features, like their culture conditions and gene expression. We showed that reprogramming using cloning by nuclear transfer allows the obtention of EpiSCs with a methylome and a transcriptome similar to those of EpiSCs derived from embryo after fertilisation. We also characterised the DNA methylation pattern of EpiSCs and showed their tendency to present a hypermethylation at their promoters compared to ESCs and epiblast. We also observed that the absence of DNA methylation blocks the conversion of ESCs into EpiSCs. As a conclusion, it seems that EpiSCs are strongly dependant of DNA methylation to regulate gene expression, which distinguishes them from ESCs.