Geometric and mechanical control of developmental Yap signaling

发育 Yap 信号的几何和机械控制

• 批准号: 10342966
• 负责人: Eszter Posfai
• 金额: $ 43.26万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10342966
• 关键词: Address; Affect; Apoptosis; Automobile Driving; Behavior; Biocompatible Materials; Biological Models; Cell Communication; Cell Fate Control; Cell Lineage; Cell Polarity; Cell Shape; Cell physiology; Cells; Color; Complement; Complex; Coupled; Coupling; Cues; Data; Development; Embryo; Embryonic Development; Environment; Epithelial; Event; Extracellular Matrix; Failure; Fluorescence Recovery After Photobleaching; Future; G-Protein-Coupled Receptors; Gene Expression; Gene Expression Regulation; Genetic; Geometry; Image; Individual; Inner Cell Mass; Instruction; Joints; Kinetics; Light; Link; Liquid substance; Maintenance; Malignant Neoplasms; Measurement; Measures; Mechanics; Mediating; Membrane; Microscopy; Modeling; Molecular; Monitor; Morphogenesis; Mus; Nuclear; Nuclear Export; Oocytes; Organ; Pathway interactions; Pattern; Placenta; Play; Positioning Attribute; Pre-implantation Embryo Development; Process; Property; Protein Dynamics; Proteins; Regulation; Reporter; Resolution; Rest; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Surface; System; Testing; Time; Tissues; To specify; Transcription Coactivator; Transducers; Work; cell behavior; cell fate specification; cell type; embryo cell; experience; experimental study; genetic approach; genome editing; insight; mechanical force; mechanical signal; mechanical stimulus; mechanotransduction; mimetics; mouse model; preimplantation; progenitor; programs; regenerative; response; segregation; stem; synergism; tool; transcription factor; transcriptomics

In order to build complex and structured tissues, organs and embryos during development, individual cells need to interpret their environment and acquire appropriate cell fates at the right place and time. Failure to do so can result in various developmental abnormalities or the emergence of cancer when cells become unresponsive to the stops and checks imposed by their environment. The preimplantation mouse embryo develops without any spatially pre-patterned information from the oocyte and without any external input, making it an ideal mammalian developmental system to address how cells interact with their surroundings to specify and maintain cell fates. The first cell fate decision is made in the context of a mere ball of cells. Cells allocated to the surface will acquire the trophectoderm fate, becoming the progenitors of the future placenta, and cells within the embryo will become the inner cell mass, giving rise to the embryo proper and extraembryonic membranes. Interpreting this positional information through the presence or absence of apico-basal polarity, which in turn dictates the activity of the conserved Hippo signaling pathway and the subcellular localization of its effector, Yes-associated protein (YAP), was shown to play a role in driving fate specific gene expression programs. However, YAP has been shown to be a key transducer of various mechanical inputs in other systems, including sensing cell shape, extracellular matrix stiffness or tensile forces transmitted by neighboring cells. Although mechanosensing has been proposed to occur during preimplantation development, it is unclear which mechanical inputs are interpreted and whether these directly influence YAP localization and thereby cell fate. Additionally, as most of our understanding of YAP regulation stems from the analysis of fixed samples, how mechanical and polarity cues regulate YAP localization dynamics is not known. Here we will use cutting-edge long term live imaging of endogenously tagged reporters of YAP and downstream lineage markers to simultaneously measure cell shape and position, YAP localization and cell fate specific transcription factor expression to reveal their joint dynamics. To probe the role of mechanical inputs and cell geometry in directing YAP localization, we use various mechanical perturbations coupled with live or single cell transcriptomic readouts. These perturbations include substituting cell-cell interactions normally experienced in the embryo with cell-mimetic biomaterials, which can be fine-tuned to precisely manipulate geometric and mechanical inputs a single cell receives; or applying different amounts of strain to cells to probe the effects experienced when the blastocoel cavity forms. Finally, by using Fluorescence Recovery after Photobleaching and protein stability measurements in live embryos, we will determine how polarity and mechanics regulate the kinetic behavior of YAP and how YAP activity dynamics is linked to cell fate acquisition and maintenance. This work will shed light on how a conserved signaling pathway operates to integrate multiple inputs coupling morphogenesis with robust acquisition of cell fate in the early mammalian embryo.

为了在开发过程中建立复杂和结构化的组织，器官和胚胎 细胞需要解释其环境并在正确的时间和时间获得适当的细胞命运。无法 这样做会导致各种发育异常或细胞变成癌症的出现 对停止的反应和检查其环境所施加的检查。 植入前小鼠胚胎在没有任何空间预图的信息中发展 卵母细胞，没有任何外部输入，使其成为理想的哺乳动物发育系统，以解决细胞的方式 与周围环境互动以指定和维持细胞命运。第一个细胞命运决定是在上下文中做出的 单纯的细胞球。分配给表面的细胞将获得滋养剂命运，成为祖细胞 未来的胎盘和胚胎内的细胞将成为内部细胞质量，从而产生胚胎 适当的外囊膜。通过存在或不存在解释这种位置信息 Apico-Basal极性，这反过来决定了保守的河马信号通路的活性和 其效应子的亚细胞定位，与YES相关蛋白（YAP）被证明在驱动命运中起作用 特定的基因表达程序。但是，YAP已被证明是各种机械的关键传感器 其他系统中的输入，包括传感细胞形状，细胞外基质刚度或拉伸力传播 通过相邻的细胞。尽管已经提出了在植入前进行机械感应 开发，尚不清楚解释哪些机械输入以及它们是否直接影响YAP 本地化，从而使细胞命运。此外，我们对YAP调节的大多数理解都源于 分析固定样品，机械和极性提示如何调节YAP定位动力学。 在这里，我们将使用内源标记的YAP和下游记者的尖端长期实时成像 谱系标记以同时测量细胞形状和位置，YAP定位和细胞命运特异性 转录因子表达以揭示其关节动力学。探测机械输入和细胞的作用 指导YAP定位时的几何形状，我们使用各种机械扰动，连接现场或单个单元格 转录组读数。这些扰动包括通常在 具有细胞模拟生物材料的胚胎，可以对其进行微调以精确操纵几何和 机械输入单个电池接收；或将不同量的应变应用于细胞以探测效果 当胚泡腔形成时会体验。最后，通过光漂白后使用荧光恢复 和蛋白质稳定性测量在活胚胎中，我们将确定极性和力学如何调节 YAP的动力学行为以及YAP活动动力学如何与细胞命运的获取和维护相关。这 工作将阐明保守信号通路如何运作以整合多个输入耦合 在早期哺乳动物胚胎中稳健地获得细胞命运，形态发生。