Analysis of spontaneous mutations in rodents and humans has proven invaluable in identifying molecular mechanisms essential to brain development. Most notably, proteins essential to patterned migration of cortical neurons have been identified through genetic analyses and positional cloning. In contrast, there have been few spontaneous mutations that have led to the identification of pathways involved in the control of cytokinesis and associated cellular dynamics that occur throughout neurogenesis at the surface of the ventricles. The regulation of cytokinesis plays a central role in neocortical neurogenesis, and may determine the fates of newly generated daughter cells. In the first period of this grant we discovered a novel mutant, flathead, which has now revealed an essential molecular mechanism that operates to regulate cytokinesis and neurogenesis in neuronal progenitors. The CNS-specific phenotype of the flathead mutant is characterized by severe micrencephaly with alterations in the relative numbers of different neuronal cell types, and in abnormal neuronal hypertrophy. A positional cloning strategy was used to identify the flathead mutation as a mutation in the Citron-kinase gene (CitronK or CitK) located on the long arm of rat and human chromosome 12. The flathead mutation is a single base deletion in exon 1 that results in a nonsense codon and absence of CitronK protein expression in proliferative zones. CitronK protein is concentrated at the VZ surface at adherens unctions and cytokinesis furrows. We hypothesize that CitK, which contains multiple protein interaction domains, links fate determining signals with cytoskeletal restructuring at the VZ surface. We propose to use a combination of imaging experiments, in vivo transfection, and biochemical experiments to further elucidate a cellular and molecular pathway necessary for neurogenesis.
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