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Analysis of the mechanisms of mammalian palate and limb morphogenesis

哺乳动物上颚和肢体形态发生机制分析

基本信息

批准号：
12670016
负责人：
TAKIGAWA Toshiya
金额：
$ 2.5万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Cleft palate, the most frequent congenital craniofacial birth defect in humans, is caused by genetic and/or environmental perturbations in the multi-step process of palate development. Mutations in the TGF-β3 gene are associated with non-syndromic cleft palate in humans. However, little is known about why cleft palate is variable in its phenotype and penetrance and why mutation analysis using specific gene-deficient mice does not necessarily predict phenotypic consequences.In this study, we developed a novel suspension culture method of fetal mouse palatal shelves. By using the 'single palatal shelf in suspension culture' , we evaluated the differentiation of palatal medial edge epithelial (MEE) cells and its roles in palatal fusion. In addition, we established TGF-β3-defficient mice in various strains (C57BL/6J, 129/Sv, FVB/N, and ICR) as model systems for analyzing phenotypic variability of genetic cleft palate and the synergism of the master and modifier genes that regulate mammalian palatogenesis. In vivo analysis indicated that TGF-β3-null mutation causes severe cleft palate in inbred strains but partial cleft palate in outbred strains. In vitro analysis suggested that phenotypic variation of cleft palate in TGFβ3-defficient mice is closely associated with the difference in MEE cell differentiation between inbred and outbred mouse strains. However, such severe cleft palate in TGF-β3-null mice in inbred strains was partially rescued with a DNA methyltransferase inhibitor and produced milder phenotypes similar to those observed in outbred strains. Our results strongly suggest that TGFβ3-associated or -interacting imprinting genes may act as modifier genes and bring about phenotypic variation of mammalian non-syndromic cleft palate.

腭裂是人类最常见的先天性颅面部出生缺陷，是由腭部发育的多步骤过程中的遗传和/或环境扰动引起的。转化生长因子-β3基因突变与人类非综合征性腭裂有关。然而，为什么腭裂的表型和外显率是不同的，以及为什么使用特定基因缺陷小鼠的突变分析不一定能预测表型后果，人们还知之甚少。在本研究中，我们开发了一种新的胎鼠腭架悬浮培养方法。采用单层腭架悬浮培养的方法，研究了腭侧缘上皮(MEE)细胞的分化及其在腭突融合中的作用。此外，我们在不同品系(C57BL/6J、129/Sv、FVB/N和ICR)中建立了转化生长因子-β3缺陷小鼠作为模型系统，以分析遗传性腭裂的表型变异以及调控哺乳动物腭裂发生的主基因和修饰基因的协同作用。体内分析表明，转化生长因子-β3零突变在近交系中会导致严重的腭裂，而在近交系中会导致部分腭裂。体外分析表明，转化生长因子β3缺陷小鼠腭裂的表型变异与近交系和近交系小鼠MEE细胞分化的差异密切相关。然而，在近交系的转化生长因子-β3缺失小鼠中，这种严重的腭裂可以用DNA甲基转移酶抑制剂部分挽救，并产生与近交系中观察到的类似的较温和的表型。我们的结果强烈提示转化生长因子β3相关或相互作用的印记基因可能作为修饰基因，导致哺乳动物非综合征性腭裂的表型变异。