Neuron-specific gene manipulations in transparent zebrafish embryos

To investigate the molecular basis of neural network formation, we introduced a novel double-cassette vector approach for visualizing and manipulating neuronal development in living zebrafish embryos. Two genes are physically linked in the double-cassette vector system, which ensures co-expression of an effector-protein and an EGFP-reporter in the same neuron. By generating transgenic enhanced green fluorescent protein (EGFP) expressing zebrafish lines, we first established that EGFP under control of either the olfactory marker protein (OMP) gene promoter or the nicotinic acetylcholine receptor β3 (nAChRβ3) gene promoter, directed strong EGFP expression to the olfactory sensory neurons and the retinal ganglion cells (RGCs), respectively. These transgenic lines allowed the visualization of the development of the entire olfactory sensory neurons and RGCs in vivo. By injection of vectors with EGFP under control of either the OMP or the nAChRβ3 gene promoter, we followed the development of individual olfactory sensory neurons and RGCs. The double-cassette expression vector strategy enabled us to clarify the roles of protein kinase A (PKA) and glycogen synthase kinase-3β (GSK-3β) in the development of olfactory sensory neurons and RGCs. The combination of visualization and neuron-specific gene manipulation provides a powerful reverse genetic in vivo approach for the study of genes of interest in neural differentiation, axonal pathfinding, and synaptogenesis.