Molecular mechanisms of cell lineage specification in the early mouse embryo – Prepispec

How a cell's identity is established and maintained remains a central question of Biology. The Prepispec project aims to study one of the best characterized example of cell specification in mammals, that of the acquisition of Epiblast (Epi) and Primitive Endoderm (PrE) identities within the Inner Cell Mass (ICM) of the preimplantation embryo at the blastocyst stage. The cells that constitute the foetus are descendants from the Epi, a pluripotent tissue that is the source of the ICM-derived Embryonic Stem (ES) cells, while the PrE is an extraembryonic tissue that later gives rise to constituents of the yolk sac in which the foetus is enclosed.
Epi and PrE share a common origin, as they both derive from the ICM of the blastocyst. Finding out how Epi and PrE cells specify within the ICM is the focus of this proposal. Although recent studies, including ours, have identified key regulatory genes involved in this process, important aspects of the underlying mechanisms are still poorly understood. To study them we will combine complementary in vivo and in vitro approaches, using mouse embryos, embryo-derived stem cells (ES and eXtraembryonic ENdoderm (XEN) cells) as well as induced Pluripotent Stem cells (iPS). To reach this goal, we will employ innovative technologies such as single-cell transcriptomics, and CRISPR/Cas9 genome editing strategy to introduce punctual mutations in endogenous genes.
During preimplantation, Nanog and GATA6, respectively Epi and PrE markers, are initially coexpressed. Their expression in the ICM becomes mutually exclusive during blastocyst formation, finally resulting in a mixture of cells expressing either Epi or PrE markers in a salt and pepper pattern. Nanog and Gata6 are required in this binary cell fate decision, and their individual inactivations lead to homogeneous ICMs of PrE or Epi cells respectively. The FGF4 pathway plays a critical role in the acquisition of a PrE identity and perturbation of its activity directly impinges on the PrE/Epi ratio within the ICM. These factors represent the core regulatory network (CRN) controlling Epi/PrE specification.
The Prepispec project aims at deciphering the mechanisms that are upstream and downstream the CRN. We will identify novel genes through the analysis of Nanog/Gata6 double mutants by performing single cell transcriptomics. Indeed our preliminary results show that double-mutant ICMs are still heterogeneous, providing an excellent tool to find upstream factors potentially inducing the CRN. These compound mutant mice will also enable to establish novel in vitro cell lines recapitulating the in vivo "naïve states" of the ICM and PrE. Moreover, we identified a good candidate in modulating the CRN and we will investigate its implication in this process, notably by looking at the impact of its post-translational modifications. Finally, we will identify and dissect the role of different RTK pathways in the early induction of GATA6 expression as it was shown to be FGF4-independent but RTK-dependent.
The Prepispec project concerns the « Santé et bien-être » major societal challenge and is at the interface of developmental biology and stem cells biology, relevant to sub-axis 1 essentially and also to sub-axis 15. Understanding the mechanisms that drive the lineage specification in early embryos can lead to important applications in the field of stem cell biology. The Prepispec project may also generate important data relevant for human health. Indeed, a better knowledge of preimplantation embryo development may impact on assisted reproduction technologies through implementation of protocols for in vitro fertilization or preimplantation diagnosis. The different markers we propose to study as well as novel ones that will be identified during the course of the Prepispec project, will enrich and improve embryo prognosis and will help to develop new, better adapted, embryo culture media.