Genetic Analysis of Morphogenetic Mechanism During Mouse Development

脊椎动物胚胎的发育过程至少部分是通过分泌生长因子等分子来调节的。 本课题主要研究TGF-β超家族成员骨形态发生蛋白（BMPs）在小鼠发育过程中的作用。 为了揭示BMP信号传导的功能，我们通过常规基因打靶技术产生了BMP IA型受体（Bmpr或Alk 3）和激活素IA型受体（Alk 2）缺陷的突变小鼠。每个受体的非接合性导致严重的胚胎致死性，突变胚胎在胚胎7.5天（Bmpr）或8.5天（Alk 2）死亡。 对于Bmpr突变体胚胎，我们发现1）没有中胚层形成，2）原肠胚形成前的细胞周期延长。 对于Alk 2突变体胚胎，我们发现1）中胚层形成但未完全分化，2）胚外区域（未来胎盘）中的Alk 2信号传导对于原肠胚形成至关重要，以及3）Alk 2突变体细胞不能贡献心脏或眼睛。 这些结果表明，BMP信号在胚胎发育早期对细胞生长、原肠胚形成和心脏等特定器官的形成具有重要作用，为了在发育后期对这些基因产物进行功能分析，我们引入了一种新发明的称为组织特异性基因打靶的技术。 使用这项技术，我们与贝勒医学院的Gerard Karsenty博士合作，以骨特异性方式（对成熟成骨细胞特异性）突变Bmpr。 骨特异性Bmpr缺陷小鼠存活，表明我们可以避免Bmpr破坏的胚胎致死性与组织特异性基因打靶技术。 突变小鼠比正常同窝出生的小鼠更小，并且显示出不规则的钙化和骨骼中骨基质的较少沉积。 这些结果是BMP信号传导是体内正常骨形成所必需的第一证据。 在另一种方法中，我们与熊本大学的Yasutaka Yamauchi和Ken-ichi Yamamura博士合作，以神经嵴特异性方式突变Bmpr。胚胎在妊娠中期死亡，表现出神经嵴细胞衍生组织的异常。 我们还与Fred哈钦森癌症中心的Philipe Soriano博士合作，在原肠胚形成阶段以神经前体特异性方式突变Bmpr。 胚胎原肠胚形成后神经组织过度生长。 这些结果表明，BMP信号在神经组织发育的各个阶段起着关键作用。

Developmental process of vertebrate embryos is regulated, at least in part, by secreting molecules such as growth factors. We are focusing on the function of Bone Morphogenetic Proteins (BMPs) that are the members of TGF-beta superfamily during mouse development. To reveal the function of BMP signaling, we have generated a mutant mouse that is deficient for BMP type IA receptor (Bmpr or Alk3) and activin type IA receptor (Alk2) by conventional gene targeting technologies. Nullizygosity of each receptor caused severe embryonic lethality and mutant embryos die at embryonic day 7.5 (Bmpr) or 8.5 (Alk2). For Bmpr mutant embryos, we found that 1) no mesoderm was formed, and 2) cell cycles prior to gastrulation was prolonged. For the Alk2 mutant embryos, we found that 1) mesoderm was formed but not fully differentiated, 2) Alk2 signaling in the extraembryonic region (future placenta) was critical for gastrulation, and 3) Alk2 mutant cells were not capable to contribute heart or eye. These results suggest that BMP signaling at the early stage of embryogenesis is important for cell growth, gastrulation and formation of particular organs such as heart.For functional analysis of these gene products in a later stage of development, we introduced a newly invented technology called tissue-specific gene targeting. Using this technology, we mutated Bmpr in a bone-specific manner (specific for mature osteoblasts), in collaboration with Dr. Gerard Karsenty, Baylor College of Medicine. The bone-specific Bmpr deficient mice were viable indicating we can avoid embryonic lethality of Bmpr disruption with tissue-specific gene targeting technology. Mutant mice were smaller than normal littermate and show irregular calcification and less deposition of bone matrix in their bones. These results are the first evidences that BMP signaling is required for normal bone formation in vivo. In another approach, we mutated Bmpr in a neural crest specific manner, in collaboration with Drs. Yasutaka Yamauchi and Ken-ichi Yamamura, Kumamoto University. Embryos die at the mid-gestation stage showing abnormalities in neural crest cell-derived tissues. We also mutated Bmpr in a neural-precursor specific manner at the stage of gastrulation, in collaboration with Dr. Philipe Soriano, Fred Hutchinson Cancer Center. Embryos showed an overgrowth of neural tissues after gastrulation. These results indicate that BMP signaling plays a critical role in various stage of neural tissue development.