title: A reporter-assisted mutagenesis screen using alpha 1-tubulin-GFP transgenic zebrafish uncovers missteps during neuronal development and axonogenesis.
abstract: Alpha1-tubulin expression occurs in a neural-specific, temporally regulated, and regeneration-inducible fashion in zebrafish. A GFP reporter driven by the alpha1-tubulin promoter in transgenic zebrafish acts as a stable, in vivo molecular tag that follows neuronal development from birth/specification through postmitotic differentiation to axonal outgrowth and synaptogenesis. We exploited this transgenic system in a reporter expression-dependent (morphology-independent) mutagenesis screen to identify disruptions in genetic loci essential for neuronogenesis and axon elaboration, which would manifest as visually appreciable perturbations in GFP fluorescence. Thirty-two such recessive mutations were obtained, a subset of which was screened through a secondary RNA quantification-based assay to eliminate housekeeping gene defects. Three representative loci, when characterized in detail, were found to exhibit missteps in discrete, sequential stages of embryonic neuronal development. Mutation in sookshma panneurally diminishes the neural precursor pool by affecting cell proliferation in the developing embryo while patterning along the neuraxis remains unperturbed. Disruption of drishti on the other hand ameliorates the mitotic neural population by affecting cell cycle exit of progenitors and stalling their progression to the postmitotic neuronal stage, without impairing subsequent cell fate determination or differentiation. Finally, dhruva is required during neuronal differentiation for axonal branching and terminal innervation in spinal motoaxons and the retinotectal projection. Molecular identification of these loci and analysis of the remaining mutational repertoire will offer unique insights into the genetic inputs that go on to make a mature, differentiated neuron.
authors.affiliation: Molecular and Behavioral Neuroscience Institute, Department of Biological Chemistry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109-070, USA.
authors.forename: Abhilasha
authors.initials: A
authors.lastname: Gulati-Leekha
authors.forename: Daniel
authors.initials: D
authors.lastname: Goldman
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001369:Axons / Q000502:physiology*; D002454:Cell Differentiation / Q000235:genetics*; D017930:Genes, Reporter; D006054:Goldfish / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008297:Male; D016296:Mutagenesis; D009154:Mutation; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D019076:Transgenes; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D014404:Tubulin; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins