Retinal ganglion cell axons exit the eye, enter the optic stalk, cross the ventral midline at the optic chiasm, and terminate in the optic tectum of the zebrafish. While in the optic stalk, they grow immediately adjacent to cells expressing the powerful retinal axon repellent slit2. The chemokine stromal cell-derived factor-1 (SDF1) is expressed within the optic stalk and its receptor CXCR4 is expressed in retinal ganglion cells. SDF1 makes cultured retinal axons less responsive to slit2. Here, we show that reducing SDF1 signalingin vivorescues retinal axon pathfinding errors in zebrafish mutants that have a partial functional loss of the slit receptor robo2. In contrast, reducing SDF1 signaling in animals that completely lack the robo2 receptor does not rescue retinal guidance errors. These results demonstrate that endogenous levels of SDF1 antagonize the repellent effects of slit/robo signalingin vivoand that this antagonism is important during axonal pathfinding.

Growing axons navigate a complex environment as they respond to attractive and repellent guidance cues. Axons can modulate their responses to cues through a G-protein-coupled, cAMP-dependent signaling pathway. To examine the role of G-protein signaling in axon guidance in vivo, we used the GAL4/UAS system to drive expression of dominant-negative heterotrimeric G-proteins (DNG) in retinal ganglion cells (RGCs) of embryonic zebrafish. Retinal axons normally cross at the ventral midline and project to the contralateral tectum. Expression of DNGα_S in RGCs causes retinal axons to misproject to the ipsilateral tectum. These errors resemble misprojections in adcy1, adcy8, nrp1a, sema3D, or sema3E morphant embryos, as well as in sema3D mutant embryos. nrp1a is expressed in RGCs as their axons extend toward and across the midline. sema3D and sema3E are expressed adjacent to the chiasm, suggesting that they facilitate retinal midline crossing. We demonstrate synergistic induction of ipsilateral misprojections between adcy8 knockdown and transgenic DNGα_S expression, adcy8 and nrp1a morphants, or nrp1a morphants and transgenic DNGα_S expression. Using qPCR analysis, we show that either transgenic DNGα_S-expressing embryos or adcy8 morphant embryos have decreased levels of nrp1a and nrp1b mRNA. Ipsilateral misprojections in adcy8 morphants are corrected by the expression of an nrp1a rescue construct expressed in RGCs. These findings are consistent with the idea that elevated cAMP levels promote Neuropilin1a expression in RGCs, increasing the sensitivity of retinal axons to Sema3D, Sema3E, or other neuropilin ligands at the midline, and consequently facilitate retinal axon crossing in the chiasm.

The chemokine SDF1 activates a cAMP-mediated signaling pathway that antagonizes retinal responses to the midline repellent slit. We show that knocking down the calmodulin-activated adenylate cyclase ADCY8 makes retinal axons insensitive to SDF1. Experiments in vivo using male and female zebrafish (Danio rerio) confirm a mutual antagonism between slit signaling and ADCY8-mediated signaling. Unexpectedly, knockdown of ADCY8 or another calmodulin-activated cyclase, ADCY1, induces ipsilateral misprojections of retinal axons that would normally cross the ventral midline. We demonstrate a cell-autonomous requirement for ADCY8 in retinal neurons for normal midline crossing. These findings are the first to show that ADCY8 is required for axonal pathfinding before axons reach their targets. They support a model in which ADCY8 is an essential component of a signaling pathway that opposes repellent signaling. Finally, they demonstrate that ADCY8 helps regulate retinal sensitivity to midline guidance cues.

Purpose: We determined whether sirtuins (SIRT1-SIRT7) are expressed in the zebrafish retina, evaluated the modulatory effect of resveratrol in the normal retina, and examined N-Methyl-D-aspartic acid (NMDA)-induced zebrafish retinal damage associated with mitochondrial sirtuins and mitochondrial fusion and fission mediators, OPA1 and Fis1. Methods: Sirtuins, OPA1, and Fis1 mRNA expression was analyzed by RT-PCR and quantitative real time PCR (qPCR) in adult zebrafish (AB type) retina and liver. qPCR showed an effect of resveratrol on SIRTs (SIRT1, 3, 4, 5) and OPA1 and Fis1 in low and high concentrations (5 and 50 mg/L) at different time points (0, 1, 24, and 48 hours) in the retina. Western blots were performed to examine the expression of SIRTs and OPA1 proteins under high concentrations of resveratrol for 24 hours. Hematoxylin and eosin staining, qPCR and mitochondrial copy number, and DNA damage assays then were used to confirm the protective effects of resveratrol on NMDA-induced retinal damage. Results: The seven sirtuins and OPA1 were highly expressed in zebrafish retina compared to the liver. Treatment with resveratrol promoted SIRT1, mitochondrial sirtuins, and OPA1 gene and protein expression, and improved mitochondrial DNA repair in adult zebrafish retina. Interestingly, the effect of resveratrol on SIRT4 gene and protein expression was significantly higher in the zebrafish retina. Importantly, resveratrol offered protection against NMDA-induced retinal damage by activating the SIRT1 gene and subsequent protein expression. Mitochondrial sirtuins and OPA1 genes likely had a role in regulating mitochondrial dynamics. Conclusions: To our knowledge, our study is the first composite analysis of sirtuins in adult zebrafish retina and provides sufficient evidence that resveratrol, as an activator of SIRT1, protects NMDA-induced zebrafish retinal damage by potentially mediating mitochondrial sirtuins and OPA1 genes.