A reporter-assisted mutagenesis screen using α1-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.

Effect of combined exposure to lead and decabromodiphenyl ether on neurodevelopment of zebrafish larvae

“…These genes are only expressed in the central nervous system during early developmental stages, and can be considered as biomarkers for developmental neurotoxicity (Fan et al, 2010). These genes encode proteins that play important roles for myelination of axons (e.g., mbp) (Yoshida and Macklin, 2005), formation of microtubules (e.g., α1-tubulin) (Gulati-Leekha and Goldman, 2006), and regulation of the astrocyte cytoskeleton (e.g., gfap) (Nielsen and Jorgensen, 2003) in the developing central nervous system. Down-regulation of expression of these genes was only observed in the co-exposure groups, suggesting a detrimental impact on cytoskeleton regulation, neuronal myelination, and neuron growth during central nervous system development.…”

Acute exposure to DE‐71: Effects on locomotor behavior and developmental neurotoxicity in zebrafish larvae

“…Western blotting assay further demonstrated that acute PBDE exposure had an effect on protein in the brain. The genes of mbp (a marker of myelination) and a1-tubulin (a neuron-specific microtubule) are expressed exclusively in the nervous system of developing zebrafish larvae [42,43]. Sonic hedgehog a is a morphogen that patterns many organ systems (including the nervous system) and is considered an important signaling molecule in zebrafish embryogenesis [44].…”

Endothelial cell–cell adhesion during zebrafish vascular development

“…Mutants in this broader class displayed brain hemorrhaging with mild additional cardiovascular defects and several of these mutants remain to be characterized. 14,41 Genetic mapping of bubblehead (bbh) and redhead (rhd) revealed that the genetic lesions were positioned in p21-activated kinase (Pak)-interacting exchange factor protein β (βpix) 42 and p21-activated kinase2a (pak2a), 43 respectively. In vitro studies had previously provided evidence of a βPix-Pak interaction acting upstream of the small GTPase Rac to induce cell motility.…”