Morphogenesis of the paraxial mesoderm in mice

描述(申请人提供)：该项目的广泛目标是使用分子、胚胎学、遗传学和光学技术的组合来了解指导小鼠胚胎内中胚层形态发生的细胞和分子事件。我们的中心假设是，WNT3a是一个关键的信号分子，对中胚层规格和模式的几个方面都是必不可少的。在分离天然荧光蛋白质方面的最新进展，以及光学显微镜技术的改进，为活体成像直接研究胚胎内的细胞和分子事件提供了前所未有的机会。光学成像的应用与我们培育的一组表达小鼠的新型转基因报告相结合，代表了一个独特的平台，可以获得关于细胞行为和体内细胞命运的定量信息。我们将利用这些试剂来研究野生型胚胎中轴旁中胚层形态发生所必需的动态细胞行为，以及一系列扰乱这一过程的等位基因WNT3a突变体。这项建议的具体目的是：(1)使用活体成像来定义小鼠胚胎原始条纹中中胚层发生所必需的细胞行为。(2)用活体成像来确定卵裂期前中胚层所必需的细胞行为。(3)确定小鼠胚胎原始条纹中是否存在近轴向中胚祖细胞。将通过使用Wnt3a突变体的等位基因系列来测试Wnt3a在这些过程中所介导的信号传递的作用。这项工作将阐明由于中胚层形成的扰动而导致的细胞缺陷，并将影响我们对影响骨骼和肌肉系统的人类出生缺陷的发病机制的理解。此外，我们研究中胚祖细胞的工作将有助于分离中胚层干细胞，确定其内在细胞行为，分子特征，并最终定向分化用于基于细胞的治疗。

DESCRIPTION (provided by applicant): The broad aim of this project is to use a combination of molecular, embryological, genetic and optical techniques to understand the cellular and molecular events that direct the morphogenesis of mesoderm within the mouse embryo. Our central hypothesis is that Wnt3a is a key signaling molecule essential for several aspects of mesoderm specification and patterning. Recent advances in the isolation of proteins that naturally fluoresce, and the refinement of techniques for optical microscopy offer unprecedented opportunities for in vivo imaging to study cellular and molecular events directly within embryos. The application of optical imaging combined with a novel panel of transgenic reporter expressing mice that we have generated represent a unique platform for acquiring quantitative information on cell behavior and cell fate in vivo. We will exploit these reagents to investigate the dynamic cell behaviors integral to the morphogenesis of the paraxial mesoderm in wild type embryos and an allelic series of Wnt3a mutants that disrupt this process. The Specific Aims of this proposal are: (1) To use in vivo imaging to define the cell behaviors integral to the genesis of mesoderm within the primitive streak of the mouse embryo. (2) To use in vivo imaging to define the cell behaviors integral to the presomitic mesoderm. (3) To establish if paraxial mesoderm progenitor cells reside within the primitive streak of the mouse embryo. A role for signaling mediated by Wnt3a in these processes will be tested through use of the allelic series of Wnt3a mutants. This work will shed light on the cellular defects resulting from perturbations in mesoderm formation and will impact our understanding of the pathogenesis of human birth defects affecting the skeleton and musculature. Furthermore our work investigating mesodermal progenitor cells will contribute information towards the isolation of mesodermal stem cells, the determination of their intrinsic cell behaviors, their molecular signature and ultimately their directed differentiation for use in cell-based therapies.