Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish

Abstract

In a microarray-based gene profiling analysis of Müller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Müller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Müller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.

Fig. 1.

Cone photoreceptor regeneration in adult zebrafish. Flat-mounted zebrafish retinas are immunolabeled with cone-specific zpr-1 (red). Retinas are oriented dorsal up, ventral down, nasal left, and temporal right. (A, B) Intact retina. Asterisk, attached retinal pigment epithelium. (D, E) At 3 days after exposure to intense light, cones are missing in a horizontal band across the retina. (G, H) By 14 days, cones have regenerated within the lesioned region (dashed lines). (C, F, and I) Magnified images of the boxes in B, E, and H, respectively. (Scale bars: 300 μm in A, B, D, E, G, and H; 20 μm in C, F, and I.)

Fig. 2.

hspd1 and mps1 are up-regulated in injury-activated Müller glia during zebrafish photoreceptor regeneration. Expression patterns of hspd1 (A–C) and mps1 (D–F). (A and D) Fluorescent in situ hybridization of hspd1 and mps1 on retinal sections of Tg(gfap:GFP)mi2002 zebrafish. Autofluorescence in cones (arrow), rods (solid arrowhead), and red blood cells (empty arrowhead). Asterisks, lesioned area (note the disrupted retinal pigment epithelium). DIC, differential interference contrast. (B and E) Expression fold changes of hspd1 and mps1 in isolated GFP⁺ cells detected by qRT-PCR (gray) and microarray (red). Error bars represent SEM for 3 independent biological replicates. (C and F) Within the lesioned region at 48 hpl: in situ hybridization with hspd1 and mps1, respectively (magenta), produces discrete fluorescent dots associated with GFP⁺ neurogenic Müller glia (green) and anti-PCNA (red). Arrows indicate triple-labeled cells; onl, outer nuclear layer; inl, inner nuclear layer. These are not microglia, which are confined to the onl in the lesioned region (23). (Scale bars: 50 μm in A and D; 10 μm in C and F.)

Fig. 3.

Cones fail to regenerate in nbl and ncp mutants at the restrictive temperature. Flat-mounted retinas at 7 dpl immunolabeled with zpr-1 (red). (A and C) Regenerated cones between dashed lines in WT. (B and D) Few or no cones are seen in the lesioned central area in nbl and ncp, respectively. The occasional zpr-1⁺ profile in the region of the lesion might represent a spared cone photoreceptor. (Scale bars: 300 μm.)

Fig. 4.

Retinal regeneration defects of nbl and ncp. (A) Neurogenic clusters at 2 dpl in the inner nuclear layer (inl) immunolabeled with anti-PCNA (magenta) and weakly labeled with anti-Pax6 (green) in WT and nbl. Note that Pax6 is also expressed at high levels in amacrine cells at the inner boundary of the inl. (B) PNCA⁺ photoreceptor progenitors at 3 dpl in the outer nuclear layer (onl) of WT and ncp. (C) Number of PNCA⁺ cells in the inl or onl per 100 μm of linear length retina at 2 or 3 dpl, respectively. Error bars represent SEM for 3 individuals. *, P < 0.05; **, P < 0.0001. (D) Transmission electron micrographs of injury-activated Müller glia in WT and nbl. See text for description of temperature shift paradigm. Müller glia (M) are shown by the magenta wash. Mitochondria (arrows) in Müller glia of WT appear normal after 8 h at 33°C, whereas in nbl mutants, Müller glia contain swollen mitochondria. (Scale bars: 10 μm in A and B; 100 μm in D.)

Fig. 5.

Model for Müller glia-based photoreceptor regeneration in adult zebrafish retina. Four steps in the regeneration of photoreceptors in the light-damaged retina. In nbl mutants, regeneration is blocked at step 3, and in ncp mutants, regeneration is blocked at step 4. See text for further description.