Morphogenesis of the paraxial mesoderm in mice

描述(由申请人提供)：活体成像与小鼠遗传学的力量相结合，代表着朝着解开哺乳动物胚胎发育调控机制的关键的下一步。我们的研究项目致力于利用哺乳动物系统中的成像方法。我们正在进行的和拟议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新的、更高水平的组织，而且还讨论了这一过程如何由特定的基因或基因网络机械地调节。我们的方法结合了细胞、发育和计算生物学领域。该项目的长期目标是利用小鼠作为实验上易于处理的模型，阐明哺乳动物的细胞行为、谱系关系和调节原肠形成的分子机制。一个直接的目标是提供原始条纹和新兴中胚层内运行的动态事件的详细图像。具体地说，我们专注于一种中胚层亚型，即旁轴中胚层，它通过一个易于遗传解剖的反复进化保守的过程产生形态上不同的体节。尽管进行了广泛的遗传分析，但轴旁中胚层的形态发生背后的细胞动力学是复杂的，也不是很清楚。我们假设，旁轴中胚层的规范、增殖和图案化涉及精心安排的细胞行为的刻板序列。利用活体成像结合遗传标记和对不同程度干扰这一过程的突变体的分析，我们已经开始研究推动移植后早期小鼠胚胎近轴中胚层规范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，并挑战了既定的血统关系。将在构成这项建议的三个具体目标中进一步探讨这一研究方向。在特定的目标1中，我们研究了从小鼠原始条纹中出现的细胞的命运。利用遗传诱导和光可调节的命运定位方法，我们将确定原始条纹细胞的命运，调查双潜能中胚层祖细胞群体的存在，确认并确定自我更新的近轴中胚层前体细胞的位置。在具体目标2中，我们将定义导致中胚层出现的原始条纹上的细胞行为。实时成像和一组新的报告菌株代表了我们实验室开发的一个独特的平台，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中胚层出现所必需的细胞行为(例如，运动和分裂)。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控的。在具体目标3中，我们将定义在旁轴中胚层内导致体细胞发生的细胞行为。使用实时成像，我们将确定与体节形成一致的细胞动力学，并测试这种形态发生过程在细胞水平上是如何在突变体中被错误调控的。

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.