Investigation of Müller Glial Responses to Retinal Injury in Zebrafish

The zebrafish retina has the capacity to regenerate all classes of retinal neurons, providing an excellent system to study the underlying cellular and molecular mechanisms of neuronal regeneration. Exposure to constant intense light kills rod and cone photoreceptors, inducing proliferation in cells in the inner nuclear layer (INL) that give rise to clusters of neuronal progenitor cells. The neuronal progenitor cells migrate radially to the site of damage and ultimately differentiate into new photoreceptors.

Recent studies suggest that the Müller glia, a member of the radial glial cell class that is normally involved in nervous system support and modulation of synaptic activity, may serve as a source of regenerated neurons. To clearly label the Müller glia, I generated transgenic zebrafish lines in which the Müller glia express EGFP under control of the Müller-specific 1.5 kb glial fibrillary acidic protein (gfap) promoter fragment. The albino Tg(gfap:EGFP)^nt11 line was used in both light-damage and ouabain-damage experiments to demonstrate that the MÌÄå_ller glia are the source of neurogenesis in the regenerating retina. Through immunohistochemical labeling of either light-treated or ouabain-damaged albino Tg(gfap:EGFP)^nt11 eyes, I demonstrated that the proliferating cells that initiate retinal regeneration in the INL are Müller glia.

To identify the cellular damage cues that induce the Müller glia to divide, I developed a technique for targeted ablation of specific retinal cell classes. Treatment of transgenic zebrafish lines that expressed bacterial nitroreductase in specific cell types with the antimicrobial prodrug metronidazole permitted temporal and spatial control of cell ablation. Supporting conclusions drawn from regeneration in the ouabain model of inner retinal neuronal cell death, metronidazole-induced ablation of amacrine and horizontal cells suggested that photoreceptor cell death was not necessary to induce regeneration of lost retinal neurons. Further, recent studies suggested that rod cell death alone can not induce the Müller glia to enter a regenerative program. To ablate only the rod photoreceptors, I generated two transgenic lines that expressed nitroreductase specifically in all rods or only a subset of rods. Metronidazole treatment of these transgenic lines specifically ablated the nitroreductase-expressing rods, producing a robust Müller glial proliferation response only when all rods were killed. This demonstrated that rod photoreceptor death alone can induce an INL-derived regeneration response, although a threshold of rod cell loss must be achieved.