Regulation by glycogen synthase kinase-3β of the arborization field and maturation of retinotectal projection in zebrafish

The retinotectal projection is one of the best systems to study the molecular basis of synapse formation in the CNS because of the well characterized topographic connections and activity-dependent refinement. Here, we developed a presynaptic neuron-specific gene manipulation system in the zebrafish retinotectal projection in vivo using the nicotinic acetylcholine receptor β3 (nAChRβ3) gene promoter. Enhanced green fluorescent protein (EGFP) expression signals in living transgenic zebrafish lines carrying the nAChRβ3 gene promoter-directed EGFP expression vector visualized the development of entire retinal ganglion cell (RGC) axon projection to the tectum. Microinjection of the nAChRβ3 gene promoter-driven double-cassette vectors directing the expression of both dominant-negative glycogen synthase kinase-3β (dnGSK-3β) and EGFP enabled us to follow the development of individual RGCs and to examine the effect of the molecule on the axonal arborization and maturation of the same neurons in living zebrafish. We found that the expression of the dominant-negative form of zebrafish GSK-3β suppressed the arborization field of RGC axon terminals in the tectum as estimated by the reduction of arbor branch length and arbor areas. Furthermore, the suppression of GSK-3β activity increased the size of vesicle-associated membrane protein 2-EGFP puncta in RGC axon terminals at the early stage of innervation to the tectum. These results suggest that GSK-3β regulates the arborization field and maturation of RGC axon terminals in vivo.