A reporter-assisted mutagenesis screen using α1-tubulin-GFP transgenic zebrafish uncovers missteps during neuronal development and axonogenesis

摘要

α1-tubulinexpressionoccursinaneural-specific,temporallyregulated,andregeneration-induciblefashioninzebrafish.AGFPreporterdrivenbytheα1-tubulinpromoterintransgeniczebrafishactsasastable,invivomoleculartagthatfollowsneuronaldevelopmentfrombirth/specificationthroughpostmitoticdifferentiationtoaxonaloutgrowthandsynaptogenesis.Weexploitedthistransgenicsysteminareporterexpression-dependent(morphology-independent)mutagenesisscreentoidentifydisruptionsingeneticlociessentialforneuronogenesisandaxonelaboration,whichwouldmanifestasvisuallyappreciableperturbationsinGFPfluorescence.Thirty-twosuchrecessivemutationswereobtained,asubsetofwhichwasscreenedthroughasecondaryRNAquantification-basedassaytoeliminatehousekeepinggenedefects.Threerepresentativeloci,whencharacterizedindetail,werefoundtoexhibitmisstepsindiscrete,sequentialstagesofembryonicneuronaldevelopment.Mutationinemsookshma/empanneurallydiminishestheneuralprecursorpoolbyaffectingcellproliferationinthedevelopingembryowhilepatterningalongtheneuraxisremainsunperturbed.Disruptionofemdrishti/emontheotherhandamelioratesthemitoticneuralpopulationbyaffectingcellcycleexitofprogenitorsandstallingtheirprogressiontothepostmitoticneuronalstage,withoutimpairingsubsequentcellfatedeterminationordifferentiation.Finally,emdhruva/emisrequiredduringneuronaldifferentiationforaxonalbranchingandterminalinnervationinspinalmotoaxonsandtheretinotectalprojection.Molecularidentificationoftheselociandanalysisoftheremainingmutationalrepertoirewillofferuniqueinsightsintothegeneticinputsthatgoontomakeamature,differentiatedneuron./p/div