Buac Kristina, Watkins-Chow Dawn E, Loftus Stacie K, Larson Denise M, Incao Arturo, Gibney Gretchen, Pavan William J
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Genet. 2008 Sep 5;4(9):e1000177. doi: 10.1371/journal.pgen.1000177.
The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10(LacZ/+). Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1-4 (msp1-4), which show altered NC patterning. In homozygous msp1 embryos, Sox10(LacZ) expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3(msp1), that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting.
神经嵴（NC）是一群胚胎干细胞，可分化为多种细胞类型，包括外周和肠神经系统的神经胶质细胞和神经元以及皮肤和毛发的黑素细胞。调节神经嵴发育的基因和遗传途径中的突变会导致一系列人类发育障碍，统称为神经嵴病。为了确定调节神经嵴发育的分子途径，并了解这些过程中的改变如何导致疾病，我们利用对神经嵴缺陷敏感的小鼠模型Sox10（LacZ/+）建立了N-乙基-N-亚硝基脲（ENU）诱变筛选。在分析的71个家系中，我们鉴定并定位了四个可遗传位点，称为Sox10表达模式修饰因子1-4（msp1-4），它们显示出神经嵴模式改变。在纯合msp1胚胎中，颅神经节、颅神经和交感神经链中不存在Sox10（LacZ）表达；然而，其他表达Sox10 的细胞的发育似乎不受该突变影响。连锁分析、测序和互补测试证实，msp1是受体酪氨酸激酶Erbb3的一个新等位基因，即Erbb3（msp1），该蛋白在细胞外区域有一个单氨基酸替换。ENU诱导的突变不会改变蛋白质表达，然而，它足以损害ERBB3信号传导，使得在msp1中观察到的胚胎缺陷类似于Erbb3无效等位基因的缺陷。对突变蛋白的生化分析表明，ERBB3在细胞表面表达，但msp1突变使其配体诱导的磷酸化显著降低。这些发现突出了突变残基对ERBB3受体功能和激活的重要性。这项研究强调了使用ENU诱变来鉴定调节神经嵴发育的遗传途径以及剖析离散蛋白质结构域作用的实用性，这两者都有助于在细胞和发育环境中更好地理解基因功能。
