Investigating the role of nuclear mechanics in the regulation of chromatin structure and embryonic cell fate

总结 内细胞团（ICM）和滋养外胚层（TE）是哺乳动物发育过程中最先确定的两种细胞类型。 发展TE细胞支持着床并产生胎盘，而ICM细胞形成胚胎 和一些胚外组织因此，它们的分化对于成功怀孕至关重要。的 机械调节的Hippo信号通路在ICM和TE细胞中被差异激活，驱动基因 定义这些细胞状态的表达程序。这些程序也依赖于细胞类型特异性染色质 的风景.机械力如何调节染色质结构和胚胎细胞的命运， 然而，植入并没有被完全理解。我假设机械力通过 细胞骨架，通过调节Hippo信号传导和染色质结构来调节TE转录程序。 在这个提议中，我将通过定义核张力如何调节Hippo信号来测试这个假设， 早期胚胎分化过程中的染色质组织。我的最终目标是定义 将机械和调节途径连接到细胞和染色质状态。这项工作将提高我们的 了解细胞命运规范，与早期胚胎发生和植入的关系，等等 大致上我在Giraldez实验室的博士后工作表明，核纤层蛋白A/C在TE中转录上调， 与ICM相比，它调节TE识别; LMNA/C缺失导致ICM样转录状态 这让人联想到河马通路的激活。在目标1（K99）中，我将研究核纤层蛋白A/C对Hippo的调节， 确定细胞骨架网络在调节这种信号传导中的机械传感作用。在目标2中 (K99/R00），为了确定机械力如何调节染色质，我将应用先进的电子显微镜 方法可视化核小体分辨率染色质结构在体内。在培训期间， Giraldez实验室，我将应用一种新的标记策略，结合冷冻电镜来表征板层异染色质 交互.在R00期间及以后，我将应用这些方法来确定 胚胎和细胞核上产生的机械力影响染色质结构和ICM/TE 命运在目标3（R00）中，我将使用嵌合体胚胎和其他发育试验来研究 细胞核的机械性质影响分化潜能。我还将量化核硬度， 发育中的小鼠胚胎的染色质结构。这项工作为更深入地了解 机械力在调节基因表达和细胞特性中的作用。这项建议将培训 我在整个教育过程中获得的概念和新方法（RNA测序和冷冻EM）， 我将在我的导师（Giraldez）的实验室学习，从耶鲁大学的其他科学家和专家那里学习，并在我的合作伙伴的实验室学习。 导师伊丽莎白·维拉和伯纳·索曾这个建议将完成我的博士后培训，并为我做好准备。 我的最终目标是运行一个有竞争力的独立研究项目。

SUMMARY The inner cell mass (ICM) and trophectoderm (TE) are the first two cell types specified during mammalian development. TE cells support implantation and give rise to the placenta, whereas ICM cells forms the embryo and some extraembryonic tissues. Their differentiation is therefore critical for successful pregnancy. The mechanically-regulated Hippo signaling pathway is differentially activated in ICM and TE cells, driving gene expression programs that define these cell states. These programs also depend on cell type-specific chromatin landscapes. How mechanical forces regulate chromatin structure and embryonic cell fates during pre- implantation is however not fully understood. I hypothesize that mechanical forces transmitted though the cytoskeleton, regulate TE transcriptional programs by modulating both Hippo signaling and chromatin structure. In this proposal, I will test this hypothesis by defining how nuclear tension regulates Hippo signaling and chromatin organization during early embryonic differentiations. My ultimate goal is to define the mechanistic links connecting mechanical and regulatory pathways to cell and chromatin states. This work will enhance our understanding of cell fate specification, both in relationship to early embryogenesis and implantation, and more broadly. My postdoctoral work in the Giraldez lab showed that Lamin A/C is transcriptionally up-regulated in TE, compared to ICM, and that it regulates TE identify; LMNA/C depletion leads to an ICM-like transcriptional state reminiscent of Hippo pathway activation. In Aim 1 (K99), I will investigate regulation of Hippo by Lamin A/C and determine the role of mechanical sensing by cytoskeletal networks in the regulation of this signaling. In Aim 2 (K99/R00), to determine how mechanical forces regulate chromatin, I will apply advanced electron microscopy approaches to visualize nucleosome resolution chromatin structure in vivo. During the training period in the Giraldez lab, I will apply a novel labeling strategy, combined with cryo EM to characterize lamina-heterochromatin interactions. During the R00 period and beyond, I will apply these approaches to determine how compaction of the embryo and the generation of mechanical forces on the nucleus impact chromatin structure and ICM/TE fates. In Aim 3 (R00), I will use chimeric embryos and other developmental assays to examine how changes in the mechanical properties of the nucleus affects differentiation potential. I will also quantify nuclear stiffness and chromatin structure in developing mouse embryos. This work paves the way for a deeper understanding of the role of mechanical forces in regulating gene expression and cell identity. This proposal brings together training and concepts that I have acquired throughout my education and new approaches (RNA-seq and cryo-EM) that I will learn in my mentor’s (Giraldez) lab, from other scientists and specialists at Yale, and at the lab of my co- mentors, Elizabeth Villa and Berna Sozen. This proposal will complete my postdoctoral training and prepare me for my ultimate goal of running a competitive independent research program.