The Transcription Factor Jun Is Necessary for Regeneration-associated Gene Expression During Optic Nerve Regeneration in Larval Zebrafish

Abstract

The mammalian central nervous system (CNS) is incapable of regenerating damaged axons; as a result, trauma or neurodegenerative diseases such as glaucoma cause a permanent loss of function. Mature CNS neurons fail to revert to a growth-competent state to develop new axons that successfully navigate through an inhibitory environment and reestablish connections with their targets. Additionally, most cells do not survive more than two weeks after injury. Zebrafish, however, retain the capacity for regeneration into adulthood and facilitate functional recovery by expressing regeneration-associated genes (RAGs). A well-characterized approach to study CNS regeneration in mammals and zebrafish is injury to the retinal ganglion cell (RGC) axons, which form the optic nerve. Although great progress in understanding the mechanisms for CNS regeneration has been made with these models, the key RAGs remain unknown, and full, functional recovery has yet to be achieved in adult mammals. Therefore, zebrafish are a robust model for understanding RAG expression that contributes to RGC survival, axon regrowth, guidance, and target reconnection for optic nerve regeneration. Here, the necessity for jun, an essential RAG for peripheral nervous system (PNS) regeneration, was assessed during optic nerve regeneration in larval zebrafish. To impair endogenous jun function, a heat shock inducible dominant-negative Jun (DN-Jun) line of zebrafish was created. DN-Jun fish were crossed with the Tg(isl2b:GFP) strain that expresses green fluorescent protein (GFP) in RGCs, to visualize the optic nerve in the transparent larvae. Characterization of the DN-Jun zebrafish strain found that early induction of DN-Jun consistently resulted in cranial defects in regions with embryonic jun expression. In the developing retina, jun expression was present in differentiating RGCs and downregulated as they reached their targets in the brain. Optic nerve injury induced and sustained jun expression during axon regrowth, consistent with its expression pattern during adult zebrafish optic nerve regeneration. After optic nerve injury, fish that expressed the DN-Jun protein were more likely to be left with an optic nerve stump, as they failed to regrow RGC axons. Finally, expression of Jun putative target genes (atf3, ascl1a, e2f8, insm1a, klf7b, nfli3, sox11a, and stat5a) that were differentially expressed in adult zebrafish optic nerve regeneration was measured in both standard and DN-Jun conditions. Interestingly, expression patterns differed for many target genes. DN-Jun induction resulted in decreased atf3 and ascl1a expression and increased insm1a, sox11, and stat5a expression, indicating differential regulation downstream of Jun. These data demonstrate the discrete expression patterns of jun between developing and regenerating RGCs and illustrate the importance of jun for successful RGC axon regeneration. Furthermore, these results advance the field in identifying targets for future therapies to treat optic nerve damage.