Characterization of Müller glia and neuronal progenitors during adult zebrafish retinal regeneration

Abstract

The adult zebrafish retina exhibits a robust regenerative response following light-induced photoreceptor cell death. This response is initiated by the Müller glia proliferating in the inner nuclear layer (INL), which gives rise to neuronal progenitor cells that continue to divide and migrate to the outer nuclear layer (ONL), where they differentiate into rod and cone photoreceptors. We previously conducted a microarray analysis of retinal gene expression at 16, 31, 51, 68, and 96 hours of constant intense-light treatment to identify genes and their corresponding proteins that may be involved in the generation and proliferation of the neuronal progenitor cells. We examined the expression of two candidate transcription factors, Pax6 and Ngn1, and one candidate transgene, olig2:EGFP, in the regenerating light-damaged retina. We compared the temporal and spatial expression patterns of these markers relative to PCNA (proliferating cell nuclear antigen), an established marker for proliferating cells in the zebrafish retina, and the Tg(gfap:EGFP) nt11 transgenic line that specifically labels Müller glial cells. We found that Müller glial cells dedifferentiate during regeneration, based on the loss of cell-specific markers such as GFAP (glial fibrillary acidic protein) and glutamine synthetase following their reentry into the cell cycle to produce neuronal progenitors. Pax6 expression was first detected in the proliferating neuronal progenitors by 51 hours of constant light treatment, which is significantly after the Müller glia first reenter the cell cycle after 31 hours of light. This suggests that Pax6 expression increases in neuronal progenitors, rather than in the proliferating Müller glia. EGFP expression from the olig2 promoter was first detected by 68 hours of constant light treatment in the dedifferentiated Müller glia, with Pax6 expressed in the closely-associated proliferating neuronal progenitors migrating to the ONL. Both Pax6 and olig2 expression persisted until three days post light-treatment, when the neuronal progenitors begin differentiating into new rod and cone photoreceptors. Ngn1 protein expression was initially detected in proliferating neuronal progenitors at 68 hours of light treatment. However, Ngn1 expression persisted in a subset of the INL nuclei until 17 days post-light treatment. Using the Tg(gfap:EGFP) nt11 transgenic line, Ngn1 was localized to the Müller glial nuclei that were reestablished following the regenerative response. These markers, therefore, can be used to identify different cell types at particular stages of retinal regeneration: neuronal progenitor formation, proliferation, and the reestablishment of the Müller glia cells. These markers will be important to further characterize the regeneration response in other

Light-induced degeneration and microglial response in the retina of an epibenthonic pigmented teleost: age-dependent photoreceptor susceptibility to cell death

“…Thus, constant intense light induces intense apoptosis of the photoreceptors in albino zebrafish (Danio rerio) and albino trout (Oncorhynchus mykiss), although the proliferative activity of neural stem cells replaces degenerating cells (Yurco and Cameron, 2005;Allison et al, 2006;Fausett and Goldman, 2006;Bernardos et al, 2007;Fimbel et al, 2007;Kassen et al, 2007;Thummel et al, 2008a;Thummel et al, 2008b). Similar studies on pigmented teleost fish are sparse, and have been conducted in species exposed to high light intensities in their natural habitat such as the goldfish (Marotte et al, 1979;Raymond et al, 1988) or rainbow trout (Allen and Hallows, 1997;Allison et al, 2006).…”

“…Albino teleosts are a better choice than pigmented animals for investigating the mechanisms by which light damages the retina because of their relatively high susceptibility to light damage. Thus, constant intense light exposure of albino zebrafish causes widespread photoreceptor cell death (Vihtelic and Hyde, 2000;Vihtelic et al, 2006;Thummel et al, 2008a;Thummel et al, 2008b;Bailey et al, 2010), and rod outer segment degradation ('solar pruning') and photoreceptor loss in the albino rainbow trout (Allen and Hallows, 1997;Allen et al, 1999;Allison et al, 2006) and albino oscar Astronotus ocellatus (Allen et al, 1999). However, albino teleosts are rare in nature.…”

“…The rod-dominated retinas of albino strains of rodents and the cone-diurnal retina of albino fish (Teleostei) are damaged by forced exposures to moderate light and this has led to their choice as animal models for the study of light damage to the human retina (Remé et al, 1998;Vihtelic and Hyde, 2000;Allison et al, 2006;Vihtelic et al, 2006;Bernardos et al, 2007;Fimbel et al, 2007;Kassen et al, 2007;Thummel et al, 2008a;Thummel et al, 2008b;Santos et al, 2010). Albino teleosts are rare in nature and most of the studies on light-induced photodegeneration in fish have focused on albino mutant zebrafish.…”

“…However, it also generates potentially damaging reactive oxygen species within the eye. Thus, light has been an effective and frequently used environmental method to induce photoreceptor cell degeneration experimentally (Vihtelic and Hyde, 2000;Allison et al, 2006;Vihtelic et al, 2006;Bernardos et al, 2007;Fimbel et al, 2007;Kassen et al, 2007;Thummel et al, 2008a;Thummel et al, 2008b). Several factors affect the susceptibility of the fish retina to light damage, such as eye pigmentation, rod:cone ratio and the age of the animal.…”

Mobilizing Endogenous Stem Cells for Retinal Repair

“…After injury, quiescent Müller cells re-enter the cell cycle and de-differentiate to form multipotent progenitors that subsequently generate all retinal neuron types that repair the retina and restore visual function [10][11][12][13][14][15][16] . Over the past decade, efforts have been placed to investigate whether retinal neuroregeneration can be induced from Müller cells of adult mammals, such as mouse and rat.…”