Fig. 2

HB-EGF Stimulates MG Dedifferentiation into Multipotent Retinal Progenitors in the Uninjured Retina (A) Diagram of experimental protocol and low-magnification photomicrographs showing that HB-EGF injection into the eye of 1016 tuba1a:gfp fish stimulates GFP expression and BrdU incorporation throughout the INL of the uninjured retina. Scale bar, 150 μm. (B) High-magnification view of (A) showing that GFP⁺/BrdU⁺ cells express the MG marker GS (arrows). Scale bar, 50 μm. (C and D) RT-PCR (C) and in situ hybridization assays (D) showing that HB-EGF stimulates the expression of genes characteristic of dedifferentiated MG. In (D), the PBS plus BSA-treated retinas did not show an in situ hybridization signal above background for any of the probes tested—data only shown for the ascl1a probe. Scale bars, 50 μm. (E) The matrix metalloprotease inhibitor, GM6001, suppresses injury-dependent proliferation of MG-derived progenitors, and this regulation is rescued by soluble HB-EGF. Asterisk (^*) identifies the injury site. (F) Quantification of the effects observed in (E). ^*p < 0.01. (G) BrdU and retinal cell type-specific immunofluorescence shows that HB-EGF-generated progenitors in the uninjured retina at 14 days postinjection are multipotent and generate all major retinal cell types. Scale bar, 50 μm. (H) Quantification of regenerated cell types observed in (G). Error bars represent SD. Scale bars, 50 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. See also Figures S1 and S2.