Optic axon guidance during development and regeneration in the zebrafish
Directed regeneration of axons in the CNS has potential for the treatment of CNS disorders and injuries. In contrast to mammals, following optic nerve lesion zebrafish regenerate axons that navigate to their correct targets and form new synapses leading to functional recovery. Correct pathfinding is thought to rely on a range of molecular cues in the CNS which the growing axon expresses receptors for. However, the specific guidance cues are not well elucidated. It is likely that a proportion of them will be the same as during development, while some may be specific to regeneration. Alternatively, axons may simply retrace former trajectories guided by the molecular environment or mechanical constraints of degenerating tracts, as demonstrated in the mammalian PNS. To elucidate this, we investigated regeneration in the astray/robo2 knockout mutant which exhibits misprojection of optic axons during development leading to the establishment of ectopic tracts. We show that degenerating tracts do not provide a strong guidance cue for regenerating axons in the CNS as ectopic tracts in the astray mutant are not repopulated following lesion despite presenting a similar environment to entopic degenerating tracts. We also find that as astray mutant (knockout) and robo2 morphant (transient knockdown) projection and termination errors persist in the adult, it is clear that there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development. In addition, we find a reduced importance of the axon guidance receptor Robo2 and its repellent ligand Slit2 for pathfinding during regeneration as specific developmental pathfinding errors of optic axons in astray mutants are corrected during adult optic nerve regeneration and global overexpression of Slit2 elicits pathfinding defects during development but not regeneration. To address regeneration-associated gene regulation in axotomised retinal ganglion cells, we carried out a microarray analysis. We found that many genes detected as a gradient in the adult retina during regeneration are not differentially expressed in the embryonic eye, despite having distinct expression patterns in other embryonic tissues. Of the genes which exhibit strong differential expression in the retina of both regenerating adults and developing embryos, foxI1 is one of the most interesting candidates as other fox genes have been implicated in axon guidance and due to its highly restricted retinal expression pattern. Surprisingly, further investigation has revealed that foxI1 knockout mutant embryos have retinotectal projections which appear normal in terms of axon pathfinding and mapping. Another family of genes indicated by the array, which are cytosolic phosphoproteins known to be involved in the signal transduction cascade of multiple inhibitory guidance cues during axon growth, are the crmps. Knocking down crmp2 with morpholinos during development resulted in a sparser innervation of the tectum with individual axons which trend towards having less complex arbors with shorter branches and reduced overall axon length. As a whole this work adds to our current knowledge of optic axon guidance during development and regeneration and the relative importance and effect of selected potential guidance cues, which may help toward informing future mammalian CNS regeneration research.
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