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

Gene expression analysis in the adult zebrafish pallium

<p>Dataset 1 Expression of eomesb in the embryonic brain and the adult pallium in zebrafish.<br>Raw data of Figure S2 and additional image files of eomesb expression in the embryo and the adult pallium.</p> <p>Dataset 2 Images of negative control.<br>No signal was detected in the absence of the riboprobe, demonstrating that the antibody reacts specifically with the synthetic RNA.</p> <p>Dataset 3 Expression of eomesa in the zebrafish pallium.<br>Raw data of Figure 1 and additional image files of eomesa expression in the adult pallium.</p> <p>Dataset 4 Expression of emx1, emx2 and emx3 in the zebrafish larval brain. Raw data of Figure S3 and additional image files of emx gene expression in the zebrafish larvae.</p> <p>Dataset 5 Expression of emx1, emx2 and emx3 in the zebrafish pallium.<br>Raw data of Figure 2 and additional image files of emx gene expression in the adult pallium.</p> <p>Dataset 6 Expression of Prox1 in the zebrafish pallium.<br>Raw data of Figure 3 and additional image files of Prox1 expression in the adult pallium.</p> <p>Dataset 7 Expression of ascl1a in the zebrafish pallium.<br>Raw data of Figure 4 and additional image files of ascl1a expression in the adult pallium.</p> <p> </p

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

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

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

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