Time course analysis of gene expression during light‐induced photoreceptor cell death and regeneration in albino zebrafish

Abstract

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.

Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish

“…The down-regulation of genes involved in chromatin assembly and ion homeostasis (cluster III, Fig. S3C) is consistent with the dedifferentiation of injury-activated Müller glia described previously (14,17,18,22,23).…”

“…Our study differs from 3 previously published microarray-based gene expression profile studies of retinal regeneration in adult zebrafish (13-15) in 2 fundamental ways: (i) we used brief exposures to ultra-intense light to induce widespread and rapid photoreceptor death, whereas the earlier studies exposed fish to continuous light at lower intensities for several days (14,15) or surgically removed a small piece of retina (13), and (ii) we isolated the injury-activated Müller glia for RNA extraction and gene profiling analysis, whereas the other studies harvested RNA from the entire retina (13,14) or from laser-captured ONL tissue (15). Retinal injury induces a series of complex cellular responses in many cell types, including neurodegeneration and apoptosis of the damaged cells, stress responses in other retinal cells, and activation of microglia/macrophages (6,14,18).…”

“…Retinal injury induces a series of complex cellular responses in many cell types, including neurodegeneration and apoptosis of the damaged cells, stress responses in other retinal cells, and activation of microglia/macrophages (6,14,18). By purifying the GFP ϩ Müller glia, we increased the sensitivity of our analysis to identify injury-induced changes in gene expression that activate the retinal stem cell population and initiate a neurogenic program.…”

“…A forward mutagenesis screen for temperature-sensitive mutations that interfere with regeneration of amputated caudal fin identified several genes whose functions are critical for specific steps in fin regeneration, including mps1 (monopolar spindle 1, also called ttk, a kinase required for mitotic checkpoint regulation), hspd1 (heat shock 60-kDa protein 1, a mitochondrial chaperone), and fgf20 (fibroblast growth factor 20) (8)(9)(10). In addition, gene profiling analysis of regenerating tissues has provided lists of candidate genes associated with regeneration in fin (11), heart (12), and neural retina (13)(14)(15).…”

Cellular expression of midkine‐a and midkine‐b during retinal development and photoreceptor regeneration in zebrafish

“…Recent studies identified Müller glia as stem cells responsible for both persistent rod genesis (see above) and neuronal regeneration (Bernardos et al,, 2007, Yurco and Cameron, 2005, Kassen et al, 2007, Fausett and Goldman, 2006Fimbel et al, 2007;Thummel et al, 2008). Following the death of extant neurons, Müller glia de-differentiate, reenter the cell cycle and give rise to multipotent progenitors, which continue to proliferate, migrate and differentiate to replace the missing neurons.…”