MYPT1调控小鼠内脏内胚层迁移及原肠形成的作用机制研究

Early embryonic development is a coordinated process of cell proliferation, differentiation and migration. Recent findings show that visceral endoderm plays an important role during early embryonic development. However, it is still largely unknown about the mechanisms that regulating cell movements in visceral endoderm and its impact on anterior-posterior axis determination and gastrulation. Our preliminary data have showed that visceral endoderm specific knockout of the myosin phosphatase target subunit-1 (MYPT1) caused early embryonic developmental defect. Gastrulation was abnormal and three-layer structure was not formed in the mutant embryos. In this study, we will further investigate the molecular mechanisms by which MYPT1 regulating cell migration in the visceral endoderm and its impact on anterior-posterior axis determination and gastrulation. We will further: (1) analyze how MYPT1 deletion affects cell migration in visceral endoderm in a transgenic mouse carrying a fluorescence protein reporter gene; (2) determine the formation of anterior-posterior axis and primitive streak by in situ hybridization; (3) explore the mechanisms by which MYPT1 regulating visceral endoderm migration in cultured visceral endoderm cells. Our study will definitely help us to understand the mechanisms of visceral endoderm development and migration, anterior-posterior axis determination and gastrulation during early embryonic development, and ultimately, improve our understanding on mechanisms of developmental defects in human.

早期胚胎发育过程是一个有序的细胞增殖、分化和迁移的过程，近年来的研究显示内脏内胚层在早期胚胎发育阶段起了重要的作用。目前对内脏内胚层细胞迁移的分子机制及其对胚胎前后轴决定和原肠形成的调控机制尚不清楚。前期研究发现，在内脏内胚层特异性敲除肌球蛋白磷酸酶的调节亚基MYPT1，小鼠早期胚胎发育异常，原肠发生受阻，不能形成三胚层的结构。本研究将进一步研究MYPT1调控内脏内胚层细胞迁移的分子机制及其对前后轴决定、原肠形成的作用。研究内容包括：(1)利用荧光蛋白报告基因小鼠，研究敲除小鼠内脏内胚层细胞的迁移情况；(2)使用原位杂交技术研究胚胎前后轴和原条形成情况；(3)通过体外培养小鼠内脏内胚层细胞，探讨MYPT1调节内脏内胚层细胞迁移的分子机制。本项目的研究成果有助于我们更好地了解内脏内胚层的发育、迁移，前后轴形成及原肠形成的调控机制，也有助于我们探索早期胚胎发育终止的发生机制。

早期胚胎发育过程是一个有序的细胞增殖、分化和迁移的过程，近年来的研究显示内脏内胚层在早期胚胎发育阶段起了重要的作用。目前对内脏内胚层细胞对胚胎前后轴决定和原肠形成的调控机制尚不清楚。我们的研究发现，在内脏内胚层特异性敲除肌球蛋白磷酸酶的调节亚基MYPT1，小鼠早期胚胎发育异常，原肠发生受阻，不能形成三胚层的结构。我们进一步研究发现：(1)内脏内胚层和肠上皮细胞中，MYPT1主要表达在细胞-细胞连接处；(2) MYPT1缺失的内脏内胚层细胞发生了细胞坏死；(3) 培养MYPT1flox/flox的MEF细胞，并在体外感染Cre病毒，可引起MEF细胞坏死；(4) 在L929细胞中敲低MYPT1，细胞坏死增加，并且这种MYPT1敲低引起的细胞坏死可被程序性坏死特异性抑制剂Necrostatin-1所抑制。本项目的研究有助于我们更好地了解MYPT1调控内脏内胚层细胞存活的分子机制及其在早期胚胎发育中的重要作用，也有助于我们探索早期胚胎发育终止的发生机制。