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和有结构的组织、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。未能做到 这样做会导致各种发育异常或当细胞变成 对环境强加的拦截和检查反应迟钝。 植入前的小鼠胚胎发育时没有任何来自 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，以解决细胞如何 与周围环境相互作用，以指定和维持细胞的命运。第一个细胞命运决定是在上下文中做出的 仅仅是一团细胞。分配到表面的细胞将获得滋养外胚层的命运，成为祖细胞 未来的胎盘，胚胎内的细胞将成为内部细胞团，从而产生胚胎 正常的和胚外的膜。通过存在或不存在来解释该位置信息 ，这反过来又决定了保守的河马信号通路的活性和 其效应器的亚细胞定位，是相关蛋白(YAP)，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反应的关键转导因子 其他系统的输入，包括感测细胞形状、细胞外基质硬度或传递的拉力 被相邻的细胞。尽管机械传感被认为发生在植入前 发展，目前还不清楚解释了哪些机械输入，以及这些输入是否直接影响YAP 定位，从而影响细胞的命运。此外，由于我们对YAP监管的大部分理解都源于 对固定样本的分析，机械和极性提示如何调节YAP定位动力学尚不清楚。 在这里，我们将使用YAP和下游的内生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 转录因子的表达，以揭示它们的关节动力学。探讨机械输入和细胞的作用 几何学在指导YAP定位时，我们使用各种机械扰动与活细胞或单个细胞相结合 转录读数。这些扰动包括替换通常在 胚胎采用了模拟细胞的生物材料，可以进行微调，以精确地操纵几何和 单个细胞接受机械输入；或者对细胞施加不同程度的应变以探测其影响 当胚腔形成时所经历的。最后，利用光漂白后的荧光回收 和活胚胎中蛋白质稳定性的测量，我们将确定极性和力学如何调节 YAP的动力学行为以及YAP活动动力学如何与细胞命运的获得和维持联系在一起。这 这项工作将阐明保守的信号通路如何运作来整合多个输入耦合 早期哺乳动物胚胎的形态发生与细胞命运的强健获得。