Morphogenesis of the paraxial mesoderm in mice

描述（由申请人提供）：实时成像与小鼠遗传学的力量相结合，代表了揭示哺乳动物胚胎发育调节机制的重要下一步。我们的研究项目致力于开发哺乳动物系统的成像方法。我们正在进行的和提出的实验不仅解决了细胞与其近邻之间的局部相互作用如何产生紧急的、更高水平的组织，而且还解决了这一过程如何由特定基因或基因网络进行机制调节。我们的方法协同细胞，发育和计算生物学领域。本项目的长期目标是利用小鼠作为实验模型，阐明哺乳动物原肠胚形成的细胞行为、谱系关系和分子机制。一个直接的目标是提供在原始条纹和新兴中胚层内运作的动态事件的详细图像。具体来说，我们关注的是一种中胚层亚型，即旁轴中胚层，它通过重复的进化保守过程产生了形态不同的体，可以进行遗传解剖。尽管进行了广泛的遗传分析，但产生身体轴向肌肉组织的旁轴中胚层的形态发生背后的细胞动力学是复杂的，而且尚未得到很好的理解。我们假设，近轴中胚层的规格、增殖和模式涉及精心安排的细胞行为的刻板序列。利用实时成像技术，结合基因标记和对不同程度破坏这一过程的突变体的分析，我们已经开始研究驱动着床后早期小鼠胚胎近轴中胚层形成和形态发生的细胞动力学。我们的观察已经揭示了意想不到的细胞行为，并挑战了既定的谱系关系。这方面的研究将在构成本提案的三个具体目标中进一步探讨。在特异性目标1中，我们研究了从小鼠原始条纹中出现的细胞的命运。利用遗传诱导和光调节的命运定位方法，我们将确定原始条纹细胞的命运，研究双电位中胚层祖细胞群体的存在，确认自我更新的旁轴中胚层祖细胞的存在并确定其位置。在特异性目标2中，我们将定义导致中胚层出现的原始条纹的细胞行为。实时成像和一组新的报告菌株代表了我们实验室开发的独特平台，用于获取小鼠胚胎细胞动力学的定量信息。我们将使用这些工具来定义细胞行为（例如，运动和分裂）与中胚层的出现是不可分割的。然后，我们将测试这些在影响中胚层形成的突变体中是如何被错误调节的。在特异性目标3中，我们将定义在近轴中胚层内导致体细胞发生的细胞行为。使用实时成像，我们将确定与体体形成相一致的细胞动力学，并测试这种形态发生过程在突变体的细胞水平上是如何被错误调节的。

DESCRIPTION (provided by applicant): Live imaging combined with the power of mouse genetics represents the essential next step forward towards unraveling the mechanisms regulating mammalian embryonic development. Our research program is committed to exploiting imaging methods in mammalian systems. Our ongoing and proposed experiments address not only how local interactions between cells and its immediate neighbors give rise to an emergent, higher-level of organization, but also how this process is regulated mechanistically by specific genes or gene networks. Our approach synergizes the fields of cell, developmental and computational biology. The long-term goal of this project is to elucidate the cell behaviors, lineage relationships and molecular mechanisms regulating gastrulation in mammals, using the mouse as an experimentaly tractable model. An immediate goal is to provide a detailed picture of the dynamic events operating within the primitive streak and emergent mesoderm. Specifically, we are focusing on one mesoderm subtype, the paraxial mesoderm, which gives rise to morphologically-distinct somites through a reiterative evolutionarily-conserved process amenable to genetic dissection. Despite extensive genetic analysis, the cellular dynamics underlying the morphogenesis of paraxial mesoderm, the tissue that gives rise to axial musculature of the body, are complex and not well understood. We hypothesize that the specification, proliferation and patterning of the paraxial mesoderm involves a carefully orchestrated stereotypical sequence of cell behaviors. Using live imaging combined with genetic labeling and the analysis of mutants which disrupt this process to varying degrees, we have begun to investigate the cell dynamics driving paraxial mesoderm specification and morphogenesis in the early postimplantation mouse embryo. Our observations have already revealed unexpected cell behaviors and challenged established lineage relationships. This line of research will be further explored in the three Specific Aims that constitute this proposal. In Specific Aim 1 we investigate the fate of cells emerging from the mouse primitive streak. Using genetic inducible and photomodulatable fate mapping approaches, we will determine the fate of cells of the primitive streak, investigate the existence of a bipotential mesendoderm progenitor population, confirm the presence and identify the location of self-renewing paraxial mesoderm progenitors. In Specific Aim 2 we will define the cell behaviors at the primitive streak leading to emergence of mesoderm. Live imaging and a panel of novel reporter strains represent a unique platform developed by our laboratory for acquiring quantitative information on cellular dynamics in mouse embryos. We will use these tools to define the cell behaviors (for example, movement and division) integral to the emergence of mesoderm. Then, we will test how these are misregulated in mutants affecting mesoderm formation. In Specific Aim 3 we will define the cell behaviors operating within the paraxial mesoderm leading to somitogenesis. Using live imaging, we will determine the cell dynamics coincident with somite formation, and test how this morphogenetic process is misregulated at a cellular level in mutants.