Genetic Analysis of Morphogenetic Mechanism During Mouse Development

脊椎动物胚胎的发育过程至少在一定程度上受生长因子等分子的调节。我们关注的是tgf - β超家族成员骨形态发生蛋白（BMPs）在小鼠发育过程中的功能。为了揭示BMP信号的功能，我们利用传统的基因靶向技术培养了缺乏BMP IA型受体（Bmpr或Alk3）和激活素IA型受体（Alk2）的突变小鼠。每个受体的失合性导致严重的胚胎致死性，突变胚胎在胚胎期7.5 （Bmpr）或8.5 （Alk2）死亡。对于Bmpr突变胚，我们发现1)未形成中胚层，2)原肠胚形成前的细胞周期延长。对于Alk2突变胚胎，我们发现1)中胚层形成但未完全分化，2)胚胎外区域（未来的胎盘）的Alk2信号传导对原肠胚形成至关重要，3)Alk2突变细胞不能形成心脏或眼睛。这些结果表明，胚胎发生早期的BMP信号对于细胞生长、原肠胚形成和特定器官（如心脏）的形成非常重要。为了在开发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技术，称为组织特异性基因靶向。利用这项技术，我们与贝勒医学院的Gerard Karsenty博士合作，以骨特异性方式（成熟成骨细胞特异性）突变Bmpr。骨特异性Bmpr缺陷小鼠存活，表明我们可以利用组织特异性基因靶向技术避免Bmpr破坏的胚胎致命性。突变小鼠比正常窝鼠小，骨质钙化不规则，骨基质沉积较少。这些结果首次证明BMP信号是体内正常骨形成所必需的。在另一种方法中，我们以神经嵴特异性的方式突变Bmpr。熊本大学的Yasutaka Yamauchi和kenichi Yamamura。胚胎在妊娠中期死亡，神经嵴细胞衍生组织出现异常。我们还与Fred Hutchinson癌症中心的philippe 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至关重要的作用。

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.