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Research Article

A Expression Screen Identifies an Amino Acid Essential for Function



Abstract

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, Sox10LacZ/+. 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, Sox10LacZ 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, Erbb3msp1, 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. Author Summary Genome-wide mutagenesis screens provide a valuable tool to identify genes and genetic pathways regulating complex developmental processes. The neural crest is a population of multipotent cells that gives rise to many different tissue and organ systems. Alterations in the pathways coordinating neural crest formation lead to human developmental disorders. To identify genetic components involved in neural crest development, we combined a whole-genome chemical mutagenesis approach with a mouse strain, Sox10LacZ/+, that marks neural crest progenitors during early embryogenesis. We identified and determined the chromosomal location of four mutant lines that display impaired neural crest patterning. One of the mutant lines identified carries a single amino acid change that is sufficient to alter neural crest development and cause embryonic lethality without impeding upon protein expression, highlighting the importance of the mutated residue for gene function. This study demonstrates the feasibility of mutagenesis screens to identify the molecular players required for neural crest development as well as to dissect protein domain functions.


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