Analysis of lineage segregation and cell behaviour during early germ layer differentiation in mouse

在原肠胚形成过程中，第一个胚胎细胞系是由多能外胚层细胞分化而来的。初级胚层的形态发生涉及中胚层和定形内胚层（DE）祖细胞通过原条的瞬时结构的迁移。保留在上胚层中的细胞形成外胚层。引导细胞谱系从多能状态到分化细胞类型的信号和转录程序先前被深入研究。相反，细胞生物学基础的谱系分离和形态发生的胚层是不太确定的。然而，对胚层形成机制的分子理解将是从干细胞群产生分化细胞的关键。我们以前的工作表明，早期，心脏中胚层和DE形成在前原条的转录因子Eomesodermin（Eomes）的控制下。两种细胞类型共迁移到中胚层细胞层中，然后在第二步中，DE祖细胞通过目前未知的机制整合到胚胎的最外层中。在这个建议中，我们建议使用谱系特异性荧光报告等位基因在早期谱系specifyers，Eomes和Mesp1的基因位点的活胚胎中的细胞特异性行为的分析。因此，我们可以在DE细胞分选到它们的细胞层之前区分不同的细胞谱系。我们将分析谱系分离的最早形态学迹象，并旨在确定指导命运特异性细胞行为的机制。谱系分离的初始分析将扩展到中胚层的不同亚型的说明，中胚层的不同亚型至少部分地由T盒转录因子（Tbx因子）、Eomes、Brachyury和Tbx6的作用控制。使用胚胎干细胞中的基因组编辑工具，我们将描绘Tbx因子在中胚层亚型规范中的重叠和排他性功能。结合基因表达的全基因组分析与表观基因组足迹法，我们将描绘Tbx因子下游中胚层亚型规范的路线图。

During gastrulation the first embryonic cell lineages are specified from cells of the pluripotent epiblast. Morphogenesis of the primary germ layers involves migration of mesoderm and definitive endoderm (DE) progenitors through the transient structure of the primitive streak. Cells remaining in the epiblast layer form the ectoderm. Signals and transcriptional programmes that guide cell lineage specification from pluripotent states to differentiated cell types were previously studied intensively. In contrast, the cell biological basis of lineage segregation and morphogenesis of the germ layers are less defined. However, a molecular understanding of mechanisms of germ layer formation will be key for the generation of differentiated cells from stem cell populations. Our previous work demonstrated that early, cardiac mesoderm and DE are formed in the anterior primitive streak under control of the transcription factor Eomesodermin (Eomes). Both cell types co-migrate into the mesoderm cell layer, before in a second step DE progenitors integrate into the outermost layer of the embryo by currently unknown mechanisms. In this proposal we suggest the analysis of cell-specific behaviour in living embryos using lineage specific fluorescent reporter alleles in the gene loci of early lineage specifyers, Eomes and Mesp1. We thus can distinguish the different cell lineages before the sorting of DE cells into their cell layer. We will analyse earliest morphological signs of lineage segregation and aim for identifying the mechanisms that guide fate-specific cell behaviour. Initial analysis of lineage segregation will be extended to the specification of different subtypes of mesoderm which is, at least partially, governed by the action of T-box transcription factors (Tbx-factors), Eomes, Brachyury and Tbx6. Using genome editing tools in embryonic stem cells, we will delineate overlapping and exclusive functions of Tbx-factors for mesodermal subtype specification. Combining genome wide analysis of gene expression with epigenomic footprinting we will delineate the roadmap of mesoderm subtype specification downstream of Tbx-factors.