Temporal program for cell fate specification in the mouse embryo

摘要哺乳动物中的第一个细胞命运决定将包含相同细胞的全能胚胎转化为 两种细胞类型：外部滋养外胚层细胞，将产生胎盘，和内部多能细胞， 会孕育出胎儿该决定取决于细胞极化。尽管我们知道细胞 极化连接到下游信号以指定两种细胞类型，即较早起作用的机制， 确保细胞极化及其特定的发育时间仍然完全未知。以标识此 机制，我们首先需要确定上游监管机构，这一直难以捉摸-直到现在。我们最近 发现两个合子表达的转录因子Tfap 2c和Tead 4，与Rho介导的 肌动球蛋白活性是必需的，足以触发从头细胞极化。这些结果为我们提供了 这是一个前所未有的机会，可以确定触发特定发展时机的机制， 哺乳动物早期发育中的胚胎极化。本提案的目的是揭示 导致从头极化，从而在关键的第一个细胞命运的规范， 哺乳动物胚胎模型，小鼠胚胎。我们的中心假设是合子转录合作 与细胞骨架连接到细胞并驱动第一个细胞命运决定。我们将通过以下方式来验证这一假设： 以下具体目的：目的1：确定什么调节胚胎极化的时间。我们 假设胚胎极化的时间由Tfap 2c和Tead 4控制。我们会改变剂量， Tfap 2c和Tead 4表达的发育阶段，并检查对其下游靶标的影响， 胚胎极化的时间。目的2：确定顶端域是如何建立的。我们 假设Tfap 2c和Tead 4通过调节肌动球蛋白来调节顶端结构域形成 细胞骨架，这反过来又控制Par复合物簇的缀合。我们将使用先进的成像技术 技术和药理学和光遗传学方法来确定肌动球蛋白细胞骨架的作用 在极化过程中调节顶端结构域的形成和Par复合物的动力学。我们还将 研究Tfap 2c和Tead 4如何控制肌动球蛋白细胞骨架的行为和组织的结构。 顶端蛋白成簇形成顶端结构域。目标3：确定极化时间的改变 影响胚胎发育。胚胎极化通常发生在8-细胞期晚期，即第一个细胞期之前。 细胞命运的决定，这表明这种极化的不变时间是至关重要的，为随后的发展 进展我们将确定在胚胎发育前将胚胎极化的时间提前一个细胞周期， 着床阶段影响随后的发育，特别是细胞命运和胚泡形成之前， 着床和着床后胚胎形态发生。我们希望能发现 调节小鼠胚胎细胞极化的精确时间。我们的工作可能会有重大的医疗 这是一项重要的研究，因为它将揭示人类早期发育缺陷和妊娠失败的原因。

SUMMARY. The first cell fate decision in mammals transforms a totipotent embryo comprising identical cells into two cell types: outer trophectoderm cells, which will give rise to the placenta, and inner pluripotent cells, which will give rise to the fetus. This decision depends on cell polarization. Although we understand how cell polarization connects to downstream signaling to specify the two cell types, the mechanisms that act earlier, to ensure cell polarization and its specific developmental timing, remain entirely unknown. To identify this mechanism, we first need to identify the upstream regulators, which have been elusive – until now. We recently found that two zygotically expressed transcription factors, Tfap2c and Tead4, together with Rho-mediated actomyosin activity, are essential and sufficient to trigger de novo cell polarization. These results provide us with an unprecedented opportunity to determine the mechanism that triggers the specific developmental timing of embryo polarization in early mammalian development. The objective of this proposal is to reveal the principles of self-organization that lead to de novo polarization and consequently the first cell fate specification in the key model mammalian embryo, the mouse embryo. Our central hypothesis is that zygotic transcription cooperates with the cytoskeleton to polarize cells and drive the first cell fate determination. We will test this hypothesis via the following Specific Aims: Aim 1: To determine what regulates timing of embryo polarization. We hypothesize that the timing of embryo polarization is controlled by Tfap2c and Tead4. We will alter the dose and developmental stage of Tfap2c and Tead4 expression and examine the effects on their downstream targets and the timing of embryo polarization. Aim 2: To determine how the apical domain becomes established. We hypothesize that Tfap2c and Tead4 regulate apical domain formation by modulating the actomyosin cytoskeleton, which in turn controls the conjugation of Par complex clusters. We will use advanced imaging techniques and pharmacological and optogenetic methods to determine the role of the actomyosin cytoskeleton in regulating apical domain formation and the dynamics of the Par complex during polarization. We will also examine how Tfap2c and Tead4 control the behavior of the actomyosin cytoskeleton and the organization of apical proteins into clusters to form the apical domain. Aim 3: To determine how altered timing of polarization affects embryo development. Embryo polarization always happens at the late 8-cell stage, just before the first cell fate decision, suggesting that the invariant timing of this polarization is critical for subsequent developmental progression. We will determine if accelerating the timing of embryo polarization by one cell cycle at the pre- implantation stage affects subsequent development, specifically cell fate and blastocyst formation before implantation and embryo morphogenesis post-implantation. We expect to discover the triggers and mechanisms that regulate the precise timing of cell polarization in the mouse embryo. Our work will likely have major medical relevance, as it will shed light on the causes of early developmental defects and pregnancy loss in humans.