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。骨形态发生蛋白(BMPs)是转化生长因子-β超家族成员之一，在小鼠发育过程中的功能是我们关注的焦点。为了揭示BMP信号转导的功能，我们用传统的基因打靶技术获得了BMP IA受体(Bmpr或Alk3)和激活素IA受体(Alk2)缺失的突变小鼠。每个受体的缺失会导致严重的胚胎死亡，突变的胚胎在胚胎7.5天(Bmpr)或8.5天(Alk2)死亡。对于Bmpr突变胚胎，我们发现1)没有形成中胚层，2)原肠形成前的细胞周期延长。对于Alk2突变的胚胎，我们发现1)中胚层已形成，但尚未完全分化，2)胚外区域(未来的胎盘)中的Alk2信号对原肠形成至关重要，3)Alk2突变的细胞不能贡献心脏或眼睛。这些结果表明，胚胎发育早期的BMP信号对细胞生长、原肠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技术，称为组织特异性基因打靶。利用这项技术，我们与贝勒医学院的杰拉德·卡森蒂博士合作，以骨骼特有的方式(针对成熟的成骨细胞)突变了Bmpr。骨特异性Bmpr缺陷小鼠是存活的，这表明我们可以通过组织特异性基因打靶技术避免Bmpr中断造成的胚胎死亡。突变小鼠比正常小鼠个头小，表现出不规则的钙化，骨骼中的骨基质沉积较少。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，我们与熊本大学的Yasutaka Yamuchi博士和Ken-ichi Yamamura博士合作，以特定的神经峰方式突变Bmpr。胚胎在妊娠中期死亡，在神经脊细胞来源的组织中表现出异常。我们还与弗雷德·哈钦森癌症中心的菲利普·索里亚诺博士合作，在原肠形成阶段以神经前体特有的方式突变了Bmpr。胚胎原肠发育后表现为神经组织过度生长。这些结果表明，BMP信号在神经组织发育的不同阶段起着关键作用。

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.