Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.

The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.

P-Smad3 is activated in proliferating cells.

Top: The co-localization of P-Smad3 and proliferating cell nuclear antigen (PCNA) indicates that Smad3 is activated in proliferating cells. Bottom: P-Smad3-positive cells in the inner nuclear layer (INL) co-localized with glutamine synthetase (GS), suggesting that these cells are Müller glia. Representative immunohistochemical staining at day 3 is depicted. Cell nuclei are stained with DAPI (blue). The scale bar indicates 25 μm. GC: ganglion cells, ONL: outer nuclear layer.

In situ hybridization with activin A and B as well as tgfβ1a, 2 and 3 antisense probes in zebrafish after the induction of retinal degeneration by MNU.

Expression of these genes was detected beginning at day 1 and peaking at day 5. The highest staining intensity was observed for tgfβ3 and activins A and B, whereas only modest staining was observed for tgfβ1a and 2. These ligands were primarily detected in the inner nuclear layer (INL). The scale bar indicates 50 μm. GC: ganglion cells, ONL: outer nuclear layer.

H&E staining of zebrafish retinas before (uninjured) and after induction of retina degeneration with MNU.

In the non-inhibited (0.1% dimethyl sulfide, DMSO) and inhibited group (small molecule inhibitor SB431542), a reduction of rod cells was observed starting at day 3. In the non-inhibited group the reduction of rod photoreceptors persisted until day 8, whereas in the group with the inhibited TGFβ pathway (small molecule inhibitor SB431542) a rapid recovery was observed already at day 5. Scale bar indicates 50 μm. GC: ganglion cells, INL: inner nuclear layer, ONL: outer nuclear layer, SB: SB431542

Cell proliferation in the zebrafish retina exposed to 150 mg/l MNU.

Proliferating cell nuclear antigen (PCNA) positive cells (red) indicate proliferation. Cell proliferation in the inner nuclear layer (INL) was highest at day 3 and 5, with no relevant difference between the non-inhibited (0.1% dimethyl sulfide, DMSO) and inhibited group (small molecule inhibitor SB431542). In contrast, proliferation in the outer nuclear layer (ONL) was higher in the inhibited group between 3 and 8. Cell nuclei are stained with DAPI (blue). Scale bar indicates 50 μm. GC: ganglion cells.

TUNEL positive cells in the zebrafish retina after exposure to MNU.

In uninjured zebrafish retina there are merely no TUNEL positive cells. Three days after exposure to 150 mg/l MNU, both the non-inhibited (0.1% dimethyl sulfide, DMSO) and the inhibited group (small molecule inhibitor SB431542) show a considerable amount of TUNEL positive cells (green) in the outer nuclear layer (ONL) and to a lesser degree in the inner nuclear layer (INL). Cell nuclei are stained with DAPI (blue). Scale bar indicates 50 μm. GC: ganglion cells.