Morphogenesis of the paraxial mesoderm in mice

描述（由申请人提供）：本项目的主要目的是结合分子、胚胎学、遗传学和光学技术来了解指导小鼠胚胎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，对中胚层规范和模式的几个方面至关重要。分离天然荧光蛋白的最新进展，以及光学显微镜技术的改进，为直接研究胚胎内的细胞和分子事件提供了前所未有的体内成像机会。光学成像的应用与我们所产生的一组新型转基因报告基因表达小鼠相结合，为获得体内细胞行为和细胞命运的定量信息提供了一个独特的平台。我们将利用这些试剂来研究野生型胚胎中近轴中胚层形态发生的动态细胞行为，以及破坏这一过程的等位基因系列Wnt3a突变体。本提案的具体目的是：(1)利用体内成像来定义小鼠胚胎原始条纹中胚层发生所必需的细胞行为。(2)利用体内成像技术定义体细胞前中胚层的细胞行为。(3)确定近轴中胚层祖细胞是否存在于小鼠胚胎的原始条纹中。将通过使用Wnt3a突变体的等位基因系列来测试Wnt3a介导的信号在这些过程中的作用。这项工作将阐明由中胚层形成扰动引起的细胞缺陷，并将影响我们对影响骨骼和肌肉组织的人类出生缺陷发病机制的理解。此外，我们对中胚层祖细胞的研究将为中胚层干细胞的分离、确定其内在细胞行为、分子特征以及最终将其定向分化用于基于细胞的治疗提供信息。

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.