Figure 3

Injury-induced proliferating retinal progenitors express rx1, vsx2 , and pax6a. A) – D) Histology of the heat-lesioned retina at 1 and 3 dpl; the boxed area in A) is magnified in B). Cell loss is mainly confined to the retinal pigmented epithelium (RPE) and photoreceptors in the outer nuclear layer, ONL. The CMZ (arrow in A) is at the junction between the neural retina and the ciliary epithelium (CE), which is continuous anteriorly with the iris epithelium (IE). Scale bar (A) = 150 μm. C) At 3 dpl double cones immunoreactive with zpr1 (red) are missing from the lesioned area and elongated nuclei appear in the inner nuclear layer (white arrows). Counterstained with DAPI (blue). D) By 3 dpl, radial fibers of Müller glia in the inner nuclear layer, INL, are visible in the region of the lesion (black arrows), indicative of reactive gliosis. Scale bar = 50 μm. D) Proliferating cells in the inner and outer nuclear layers of the retina in the lesioned area (asterisks) at 5 dpl incorporated BrdU (green) injected at 4 dpl. E) Injury-induced proliferating cells are also immunoreactive for proliferating cell nuclear antigen, PCNA (green) and are associated with radial fibers of Müller glia (magenta, anti-human GFAP). Note: the commercial polyclonal GFAP antibody used here is not selective for GFAP in zebrafish but labels other intermediate filaments (data not shown). In contrast, the monoclonal zrf1, which was generated against zebrafish proteins, selectively labels zebrafish GFAP [97]. Scale bar = 50 μm. F) – L) A 4 h or 24 h pulse of BrdU at 4 or 5 dpl labels clusters of nuclei (magenta) that extend between the INL and ONL and express rx1 (F) and vsx2 (J) as visualized by in situ hybridization (red). DAPI (blue); zrf1 (green, anti-zebrafish GFAP). Scale bar = 25 μm. Higher magnification views of rx1 (G,H, I) and vsx2 (K, L) in BrdU⁺ progenitors enclosed in a basket of zrf1⁺ Müller glial fibers.