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.

为了在发育过程中建立复杂和结构化的组织，器官和胚胎， 细胞需要解释它们的环境，并在正确的地点和时间获得适当的细胞命运。未能 这样做会导致各种发育异常或癌症的出现，当细胞变得 对周围环境所施加的拦截和检查反应迟钝。 植入前小鼠胚胎的发育没有来自胚胎的任何空间预图案化信息。 卵母细胞和没有任何外部输入，使其成为一个理想的哺乳动物发育系统，以解决细胞如何 与周围环境相互作用以指定和维持细胞命运。第一个细胞命运决定是在 一个细胞球。分配到表面的细胞将获得滋养外胚层的命运，成为祖细胞 胚胎内的细胞将成为内细胞团，从而产生胚胎。 固有膜和胚外膜。通过存在或不存在来解释这个位置信息 顶端-基底极性，这反过来又决定了保守的Hippo信号通路的活性， 其效应子--Yes相关蛋白（雅普）的亚细胞定位被证明在驱动命运中起作用 特定的基因表达程序。然而，雅普已被证明是各种机械的关键换能器。 其他系统中的输入，包括感测细胞形状、细胞外基质硬度或传递的张力 邻近的细胞。虽然机械感知已经被提出发生在植入前 发展，目前还不清楚哪些机械输入的解释，以及这些是否直接影响雅普 定位和细胞命运。此外，由于我们对雅普调节的大多数理解源于 固定样品的分析，机械和极性线索如何调节雅普定位动力学是未知的。 在这里，我们将使用最先进的长期活成像的内源性标记的记者的雅普和下游 同时测量细胞形状和位置、雅普定位和细胞命运特异性的谱系标志物 转录因子表达，以揭示其联合动力学。为了探索机械输入和细胞 几何在指导雅普定位，我们使用各种机械扰动耦合活细胞或单细胞 转录组读数这些干扰包括替代细胞间的相互作用， 胚胎与细胞模拟生物材料，可以微调，以精确地操纵几何和 单个细胞接受的机械输入;或对细胞施加不同的应变量以探测效应 在囊胚腔形成时所经历的。最后，通过使用光漂白后的荧光恢复， 和蛋白质稳定性测量活胚胎，我们将确定极性和力学如何调节 雅普的动力学行为以及雅普活性动力学如何与细胞命运获得和维持相关联。这 这项工作将揭示保守的信号通路如何整合多个输入耦合 在早期哺乳动物胚胎中具有细胞命运的稳健获得的形态发生。