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.

为了在发育过程中构建复杂且结构化的组织、器官和胚胎，个体 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 这样做可能会导致各种发育异常或当细胞变成癌症时出现癌症 对环境所施加的阻止和检查反应迟钝。 植入前小鼠胚胎的发育没有来自胚胎的任何空间预图案信息。 卵母细胞，无需任何外部输入，使其成为解决细胞如何发育的理想哺乳动物发育系统 与周围环境相互作用来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 仅仅是一个细胞球。分配到表面的细胞将获得滋养外胚层的命运，成为祖细胞 未来胎盘的细胞，胚胎内的细胞将成为内细胞团，产生胚胎 固有膜和胚外膜。通过存在或不存在来解释该位置信息 顶端-基底极性，这反过来又决定了保守的 Hippo 信号通路和 其效应子 Yes 相关蛋白 (YAP) 的亚细胞定位被证明在驱动命运中发挥作用 特定的基因表达程序。然而，YAP 已被证明是各种机械的关键传感器 其他系统的输入，包括传感细胞形状、细胞外基质刚度或传输的拉力 由邻近细胞。尽管机械传感已被提议在植入前发生 发展，目前尚不清楚哪些机械输入被解释以及这些是否直接影响 YAP 定位，从而决定细胞命运。此外，由于我们对 YAP 监管的大部分理解源于 通过对固定样本的分析，机械和极性线索如何调节 YAP 定位动态尚不清楚。 在这里，我们将使用 YAP 和下游内源标记记者的尖端长期实时成像 谱系标记可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它们的联合动态。探讨机械输入和细胞的作用 在指导 YAP 定位的几何学中，我们使用各种机械扰动与活细胞或单细胞相结合 转录组读数。这些扰动包括替代通常经历的细胞间相互作用。 具有细胞模拟生物材料的胚胎，可以对其进行微调以精确地操纵几何和 单个细胞接收的机械输入；或对细胞施加不同量的应变以探究效果 当囊胚腔形成时经历。最后，通过光漂白后使用荧光恢复 和活胚胎中的蛋白质稳定性测量，我们将确定极性和力学如何调节 YAP 的动力学行为以及 YAP 活动动态如何与细胞命运的获得和维持相关。这 这项工作将揭示保守信号通路如何整合多个输入耦合 形态发生与早期哺乳动物胚胎中细胞命运的稳健获取。