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

小鼠早期胚层分化过程中的谱系分离和细胞行为分析

• 批准号: 318646549
• 负责人: Professor Dr. Sebastian J. Arnold
• 金额: --
• 依托单位: Abteilung II
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/318646549?language=en
• 关键词: Analysis; lineage; segregation; cell; behaviour

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.

在原肠形成过程中，第一批胚胎细胞谱系是由多能上胚细胞确定的。初级胚层的形态发生涉及中胚层和最终内胚层(DE)祖细胞通过原始条纹的瞬时结构的迁移。残留在外胚层的细胞形成外胚层。信号和转录程序引导细胞谱系从多能状态到分化的细胞类型之前已经被深入研究。相比之下，生殖层的谱系分离和形态发生的细胞生物学基础较少。然而，对生殖层形成机制的分子理解将是从干细胞群体中产生分化细胞的关键。我们以前的工作表明，早期的心脏中胚层和DE是在转录因子Eomesodermin(EOMES)的控制下在原始纹路的前部形成的。这两种类型的细胞共同迁移到中胚层细胞层，然后在第二步，DE祖细胞以目前未知的机制整合到胚胎的最外层。在这项建议中，我们建议使用早期谱系特异性基因座上的谱系特异性荧光报告等位基因Eome和Mesp1来分析活胚胎中的细胞特异性行为。因此，在将DE细胞分选到它们的细胞层之前，我们可以区分不同的细胞系。我们将分析最早的谱系分离的形态迹象，并旨在确定指导特定命运的细胞行为的机制。对谱系分离的初步分析将扩展到中胚层不同亚型的规范，这至少部分受T-box转录因子(Tbx-因子)、Eome、brachyury和Tbx6的作用所支配。使用胚胎干细胞中的基因组编辑工具，我们将描述中胚层亚型指定的TBX因子的重叠和排他性功能。结合全基因组的基因表达分析和表观基因组足迹，我们将描绘出Tbx因子下游中胚层亚型规范的路线图。