Immunohistochemical analysis of the INL cells in the Egr1-morphant retinas.

<p>Immunohistochemical analysis of the INL cells in the controls (5misCTLMO) and Egr1 morphants (<i>egr1s</i>MO) was performed with several cell markers at 72 and 120 hpf. These include anti-5E11 (5E11; A-D), anti-parvalbumin (Parv; E-H), anti-GABA (GABA; I-L) and anti-Islet1 (Islet1; M-P) for ACs; anti-PKCβ1 (PKC; Q-T) for BCs; anti-GS (GS; U-X) for MCs; and Islet1 and anti-Prox1 (Prox1; Y-AB) for HCs. In short, the analysis has revealed that Egr1 knockdown specifically compromised the differentiation of Parv+ and GABA+ ACs. See text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-t002" target="_blank">2</a> for further discussion and additional results for the specific effects on HCs differentiation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-g006" target="_blank">Figure 6</a>. For all sections, the lens is on the left and dorsal is up. Scale bar  =  50 µm.</p

PR differentiation was delayed in the Egr1-morphant retinas.

<p>Immunohistochemical analysis of the PRs in the controls (5misCTLMO) and Egr1 morphants (<i>egr1s</i>MO) was performed with zpr1 (red-green double cones) and zpr3 (rods) at 72 hpf (A-D) and 120 (E-H) hpf. The signal of zpr1+ and zpr3+ cells was detected in the whole ONL of the controls at 72 hpf (A & C), while they were substantially reduced and restricted to a small region on the ventral ONL in the Egr1 morphants (B & D). Four staining types were defined as follows: Type 1: ≤ ¼, 2: ≤ ½, 3: ≤ ¾, 4  =  full retina. In these example images, the controls are staining Type 4 while the morphant images are staining Type 1. By 120 hpf, the differentiation of the zpr1+ and zpr3+ cells in the Egr1-morphant retinas (F & H) became more comparable to the controls (E & G). For all sections, the lens is on the left and dorsal is up. Scale bar  =  50 µm.</p

A summary of the immunostaining analysis of cell-type specific makers in the Egr1-morphant retinas.

<p>(-): no obvious change between the Egr1 morphants and controls</p><p>↓: intermediate reduction compared with the controls</p><p>↓↓: severe reduction compared with the controls</p><p>The immunostaining analysis results are summarized according to their cell type and markers used. The extent of the staining at 72 and 120 hpf is presented by the following scheme: (-): no obvious change between the Egr1 morphants and controls; ↓: intermediate reduction compared with the controls; ↓↓: severe reduction compared with the controls. The figure numbers of the corresponding immunostaining pictures are also listed.</p

A statistical summary of the cell marker staining results.

<p>For Islet1+ACs, zn8+ GCs, and Prox1+ & Islet1+ HCs, their numbers were counted and normalized by the corresponding retinal area. The mean (), standard deviation (<i>s</i>) and the number of embryos (N) for each group at each stage are listed, and the corresponding U- and <i>p</i>-values from the Mann-Whitney test computed. The figure numbers of the corresponding immunostaining pictures are also listed.</p

Immunohistochemical analysis of the GCs in the Egr1-morphant retinas.

<p>Immunohistochemical analysis of the GCs in the controls (5misCTLMO) and Egr1 morphants (<i>egr1s</i>MO) was performed by anti-zn8 (zn8; green) at 72 hpf (A & B) and 120 hpf (C & D). Phalloidin (red) was used as a counterstain to highlight the plexiform layers. A whole-eye section is shown at the top for each condition, while the magnified view of a selected region (white box) on the dorsal side of the optic nerve is shown at the bottom. The analysis has indicated that Egr1 knockdown suppressed the early dendritic outgrowth of GCs into the IPL at 72hpf (B), which was irregular at this stage. In addition, the cell number per retinal area was not different between the two groups. This defect was largely resolved by 120 hpf, despite the IPL was still thinner as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-g003" target="_blank">Figure 3</a>. This suggests that there were still defects in differentiation of cells that projected neurites into the IPL. One possible cause of the defect is the differentiation problem of ACs as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-g004" target="_blank">Figure 4</a>. See text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056108#pone-0056108-t002" target="_blank">2</a> for further discussion. For the whole-eye sections, the lens is on the left and dorsal is up. Scale bar  =  50 µm for the whole-eye sections and 25 µm for the selected regions.</p

The expression dynamics of <i>egr1</i> during zebrafish retinogenesis.

<p>A time-series whole-mount <i>in situ</i> hybridization was performed to detect the expression pattern of <i>egr1</i> in the WT retina. The signal of <i>egr1</i> was first detected in the anterior-ventral retina at 40 hpf (A, arrow). Then, <i>egr1</i> expression spread to the dorsal retina at 52 hpf (B, arrows). At 72 hpf (C & E), strong signal was detected in the AC and GC regions. Occasionally, positive staining was observed in the HC and PR regions, but it did not become prominent in the peripheral outer retina until 120 hpf (D & F). At this stage, the signal was relatively intense in the GCL and AC region. GC: ganglion cells; AC: amacrine cells; HC: horizontal cells; PR: photoreceptors. Scale bars  =  50 µm.</p

The expression of <i>ptf1a,</i> a TF that specifies ACs and HCs, was abnormal in the Egr1-morphant retinas.

<p>Whole-mount <i>in situ</i> hybridization of <i>ptf1a</i> was performed with the controls (5misCTLMO) and Egr1 morphants (<i>egr1s</i>MO) collected at 52, 72 and 120 hpf. At 52 hpf, <i>ptf1a</i> was primarily expressed in the differentiating retinal neuroepithelium (A & B, arrows) in both types of samples. By 72 hpf, the expression of <i>ptf1a</i> was restricted to the proliferative MZ in the controls (C, arrows), while its expression was maintained in the developing central retina in the Egr1 morphants (D, arrows). This ectopic expression was transient, as <i>ptf1a</i> was finally expressed in MZ in the Egr1 morphants (F, arrows) in a very comparable manner as the controls (E, arrows). The ventral view of the embryos is shown in all pictures. Scale bar  =  100 µm.</p

Irx7 regulates the expression of TFs that specify retinal cell types in ONL and GCL at 52 and 72 hpf.

<p>Whole-mount <i>in situ</i> hybridization of <i>neurod</i> (A–D), <i>crx</i> (E–H), <i>nr2e3</i> (I–L), <i>nrl</i> (M–P) and <i>atoh7</i> (Q–T) was conducted. The most common staining pattern is shown. Embryos were imaged from the ventral side and anterior is up. See text for further discussions. Scale bar = 50 µm.</p

<i>Irx7</i> mRNA can partially rescue the effects caused by Irx7 knockdown.

<p>(A and B) Immunostaining of anti-zpr1 for cones on the retinal section of an morphant and a rescued embryo at 72 hpf respectively. (C and D) Immunostaining of anti-zpr3 for rods on the retinal section of an <i>irx7</i>MO2 morphant and a rescued embryo at 72 hpf respectively. Lateral is to the left and dorsal is up for all sections. The retinal region in the samples is highlighted by a dotted yellow line. The comparable regions in the retinas are highlighted by white arrowheads. Scale bar = 20 µm.</p

<i>Irx7</i> is specifically expressed in the prospective INL during zebrafish retinal development.

<p>Whole-mount <i>in situ</i> hybridization was conducted to elucidate the expression dynamics of <i>irx7</i> in embryonic retinas. (A–G) Dissected eyes obtained from embryos between 38 to 96 hpf. Anterior is to the left and dorsal is up. The black arrowheads indicate the <i>irx7+</i> cells (blue colour) in the retina, the dashed lines indicate the choroid fissure, while the red arrowheads in (D and E) indicate the posterior dorsal region of the retina, the last region to express <i>irx7</i>. (H) A schematic diagram of <i>irx7</i> expression dynamics in the retina from 38 to 52 hpf. The Roman numerals indicate the order of five retinal regions in which <i>irx7</i> appears sequentially. (I–L) Transverse retinal section of the corresponding whole-mount embryo at 38, 50, 72 and 96 hpf. Lateral is to the left and dorsal is up. The black arrowheads indicate the <i>irx7+</i> cells in the retina. Scale bars = 50 µm.</p