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

点击翻译按钮获取中文摘要

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.