Characterization of the gene regulatory network governing the first cell fate decision in mammalian embryonic development

项目总结 尽管维持多能胚胎干细胞自我更新和 胚胎外滋养层干细胞相对了解较多，但对这些细胞如何 转录因子在胚胎发育的早期阶段在体内受到调节。分子遗传学和 胚胎学研究已经确定了引导细胞成为滋养层细胞或 胚胎上皮细胞，但激活多能性表达的转录因子主控调节 每个细胞中的主要调节因子(PMRs)或滋养层主要调节因子(TMRs)在很大程度上仍不清楚。我们的这一重大差距 对早期发育的理解至少有两个因素。首先，技术壁垒阻碍了对 许多分子和基因组学方法在由低细胞数组成的胚胎中。第二，有以下几种 早期胚胎全能卵裂球之间的显著差异(大多数PMR和 TMR开始表达)和全能细胞的细胞培养模型。因此，一种新的方法， 将鉴定PMR和TMR表达调控因子的综合方法与 在体内测试它们的功能的灵敏的新方法是必要的，以填补我们的 了解早期胚胎基因调控网络(GRN)。我们提出了三个目标来解决 这个问题。目标1集中于全面鉴定调节PMRs和PMRs的转录因子 TMR并揭示其监管职能。目标2将生成一份表观遗传学的谱系解析图谱 当细胞选择上胚层或滋养层细胞命运以及确定直接靶点时发生的变化 参与细胞命运指定的转录因子。在目标3中，我们建议剖析 发育转录因子帮助诱导这些表观遗传变化的机制 生成一个全能GRN的模型来协调这一决定。成功完成这些研究 将重塑我们对早期胚胎基因调控的理解，以及外胚层和外胚层的规范 滋养层细胞的命运。

PROJECT SUMMARY Although the “master regulators” that maintain the self-renewal of pluripotent embryonic stem cells and extraembryonic trophoblast stem cells are relatively well understood, little is known about how these transcription factors are regulated in vivo at early stages of embryogenesis. Molecular genetics and embryology studies have identified signaling pathways required to direct cells to become trophoblast or embryonic epiblast, but the transcription factors that activate expression of pluripotency master regulators (PMRs) or trophoblast master regulators (TMRs) in each cell remain largely unknown. This major gap in our understanding of early development is due to at least two factors. First, technical barriers prevent the use of many molecular and genomics approaches in embryos consisting of low cell numbers. Second, there are significant discrepancies between the totipotent blastomeres of early embryos (where most PMRs and TMRs begin to be expressed) and cell culture models of totipotent cells. Therefore, a new approach that combines comprehensive methods for identification of regulators of PMR and TMR expression with sensitive new methods of testing their functions in vivo is necessary to fill this major gap in our understanding of the early embryonic gene regulatory network (GRN). We propose three aims to address this problem. Aim 1 is focused on comprehensive identification of transcription factors regulating PMRs and TMRs and uncovering their regulatory functions. Aim 2 will generate a lineage-resolved atlas of epigenetic changes that occur as cells select either the epiblast or trophoblast fate, as well as identify the direct targets of transcription factors that participate in cell fate specification. In Aim 3, we propose to dissect the mechanisms by which developmental transcription factors help elicit these epigenetic changes and generate a model of the totipotent GRN that mediates this decision. Successful completion of these studies will reshape our understanding of early embryonic gene regulation, as well as specification of epiblast and trophoblast cell fate.