Protein expression pattern of Fgf receptors.

<p><b>A</b>) Fgfr1a protein is detected in the photoreceptor layer colocalizing with UV cones (green) (white arrow), INL and GCL (white arrowhead). <b>B</b>) Expression of Fgfr3 is detected in the outer part of the INL next to the UV cone synaptic terminals (white arrowhead). <b>C, D</b>) Fgfr3 is colocalized with the synaptic terminals of UV cones and rods (white arrows). Scale bars = 20 µm.</p

Double labeling of Caspase-3 positive cells.

<p><b>A</b>) Casp3 (red) and HuC/D (green), marker for mature neurons such as amacrine and ganglion cells, do not colocalize in the INL (white arrow) and GCL (white arrowhead). <b>B</b>) Glutamine synthetase (GS, green), a marker for MGC, colocalizes with Casp3+ (red) cells in the INL (white arrows). Scale bar = 20 µm.</p

No influence of Fgf inhibition on neurogenesis in the CMZ.

<p>The numbers represent the average of BrdU+ cells in the CMZ (± standard deviation). For this experiment, transgenic and control siblings were heatshocked for one- or seven days, respectively. BrdU-positive cells in the CMZ of both eyes of at least three individuals were counted for each time point.</p

Identification of BrdU+ cells.

<p><b>A</b>) Müller glia cells (green) are proliferating in the INL after seven days of HS (white arrowheads). <b>B</b>) The vast majority of BrdU+ cells in control and transgenic fish in the GCL colocalize with the pan-leukocyte marker L-Plastin (white arrowhead). <b>C</b>) BrdU+ cells in the INL and ONL do not colocalize with L-Plastin in seven days HS control fish (white arrows). <b>D</b>) BrdU+ cells in the INL do not colocalize with L-Plastin in transgenic heat shocked fish (white arrows). Scale bar = 20 µm.</p

Fgf function in proliferation during regeneration.

<p><b>A</b>) Many cells proliferate in the adult retina 3 days after light lesion (white arrowheads). <b>B</b>) When Fgf signaling is blocked, proliferation is strongly reduced. <b>C</b>) The quantifications show a significant reduction of proliferation after light lesion when Fgf signaling is blocked during the regeneration phase. Scale bar = 20 µm.</p

Fgf receptors, ligands and downstream target expression in specific layers of the adult zebrafish retina.

<p><b>A</b>) <i>fgfr1a</i> expression in the INL and GCL. <b>B</b>) <i>fgfr2</i> signal in the INL <b>C</b>) <i>fgfr3</i> expression in the outer part of the INL <b>D</b>) <i>fgfr4</i> expression in the INL next to the CMZ (black arrow). <b>E</b>) <i>fgf8a</i> expression in the INL and weakly in the GCL. <b>F</b>) <i>fgf20a</i> expression in the ONL, INL and GCL. <b>G</b>) <i>fgf24</i> is detectable in the INL and GCL. <b>H–M</b>) Fgf pathway target gene expression. <b>H</b>) <i>spry1</i> expression in the INL and GCL. <b>I</b>) <i>spry2</i> signal in POS, INL and GCL. <b>J</b>) <i>spry4</i> expression in the INL and weakly in the GCL. <b>K</b>) <i>dusp6</i> expression is strong in the POS, and in the INL and GCL. <b>L</b>) Strong <i>etv5a</i> expression is found in the POS, INL and GCL. <b>M</b>) <i>etv5b</i> expression is widespread in the ONL, INL and GCL and enriched in the POS. <b>N</b>) Summary of ISH expression data: + expression, − no detectable expression. GCL, ganglion cell layer: white arrowhead; INL, inner nuclear layer: black arrowhead; ONL, outer nuclear layer; POS, photoreceptor layer: black arrow. Scale bar = 20 µm.</p

Fgf signaling withdrawal dependent photoreceptor death triggers proliferation response in ONL and INL.

<p><b>A</b>) The control retina shows few BrdU+ cells in the ONL (arrowhead). <b>B</b>) Non-heatshocked control transgenic fish show similar numbers of BrdU+ nuclei in the ONL (arrowhead) as the control. <b>C</b>) After 3 days of heat shock treatment, a strong proliferation response is detectable in the INL (arrowhead). <b>D</b>) After five days, cell clusters and fusiform-shaped cells are found in the INL (arrowhead). <b>E</b>) After seven days of heat shock induction, a large number of BrdU+ nuclei are located in the ONL (arrowhead). <b>F</b>) Quantifications of BrdU+ cells per section. Under control conditions, control siblings and non-treated transgenic fish show hardly any BrdU incorporation. After one day of HS induction, proliferation increases in both groups of experimental fish compared to non-heatshocked controls. There is no significant difference between transgenic and WT siblings (p = 0,11). After three days, many cells proliferate mainly in the INL of transgenic siblings (p = 0,01). At seven days of Fgf signaling inhibition, the number of proliferating cells in the INL and ONL increases even further in dn-fgfr1 fish (p = 4,3E-13). Shown are the mean numbers of BrdU+ nuclei/section. The error bars indicate the SEM. p-values: <b>*</b>≤0.05, **≤0.01, ***≤0.001. <b>G</b>) After 5 d of HS, experimental fish were soaked in BrdU, followed by a one month chase time. In control siblings, BrdU+ nuclei were found after one month of regeneration in the ONL (arrowhead). <b>H</b>) In transgenic fish elongated BrdU+ cell nuclei, which are characteristic for photoreceptor cells, are detected in the photoreceptor layer (arrowhead). <b>I</b>) Zpr1 and BrdU (arrowhead) double positive nuclei were not detectable in control fish. <b>J</b>) In contrast, numerous Zpr1 and BrdU double-labeled cells were found in the photoreceptor layer in retinas of transgenic fish (arrowhead). Scale bars = 20 µm.</p

Recovery of retinal tissue architecture and marker gene expression after photoreceptor ablation.

<p><b>A, B</b>) Hematoxylin-eosin staining of control retina after one month. dn-fgfr1 transgenic, regenerated retina has recovered a similar layered structure as the retina of wild-type control siblings. <b>C, D</b>) Expression of the double cone marker Zpr1 is recovered in the photoreceptor cells of dn-fgfr1 fish (arrowheads) after seven days of regeneration and displays a pattern comparable to control retinas. <b>E, F</b>) <i>rho</i> expression recovers in transgenic fish and is highly comparable to control fish (arrowheads). <b>G, H</b>) UV opsin <i>(opn1sw1)</i> expression shows the same pattern in control and in transgenic retinas (arrowheads). <b>I, J</b>) Expression of the downstream target gene <i>etv5b</i>, indicative of Fgf signaling activity, has recovered in dn-fgfr1 fish after seven days of regeneration and expression is indistinguishable from control fish (arrowheads). Scale bar = 20 µm.</p

Müller glia cells after light lesions, labelled by the gfap:GFP reporter line (green) and DAPI (blue).

<p>Somata and processes of MG are indicated by yellow and red arrows, respectively. <b>A:</b> Untreated control retina. MG processes in the ONL are indicated by red arrowheads. <b>B:</b> 3 days after focused light lesion in the lesion centre. Swollen MG nuclei were found (yellow arrows). MG processes in the ONL collapsed (above dashed red line). <b>C:</b> 3 days after diffuse light lesions. ONL processes of MG collapsed (above dashed red line). <b>D:</b> Control side of focused light treated fish after 28 dpl. <b>E:</b> 28 days after focused light lesion in the lesion centre. Somata of MG are larger and sometimes displaced to the ONL (white arrowhead). Gaps in DAPI channel (dashed red line) are filled with GFP+ MG. <b>F:</b> 28 dpl after diffuse light lesion. Some displaced MG were found in the ONL (white arrowhead).</p

Average number of photoreceptor cell bodies per 300 µm retina length ± SEM after light lesion and respective P-value for control vs. 3dpl comparisons.

<p>Average number of photoreceptor cell bodies per 300 µm retina length ± SEM after light lesion and respective P-value for control vs. 3dpl comparisons.</p