Phenotypes ofDrosophilaBrain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

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Abstract
Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons ofDrosophila melanogastermushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E).In vitro, wild-type γ neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtlein vivoattributes are unmasked and amplifiedin vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male γ neurons. Second, the γ neuron-specific “naked runt” phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, pan-neuronal “filagree” phenotype maps tosinged, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of humanFascin1(OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggestFSCN1as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.