Overlapping expression pattern of the IrxAa clustered genes (irx1a, irx2a, and irx4a) in zebrafish retina development. irx1a (A–D), irx2a (E–H), irx4a (I–L), and ath5 (M–P) expression in the GCL (ganglion cell layer) of the retina were revealed by the in situ hybridization of 24-, 30-, 36-, and 48-hpf embryos. The clustered irx genes are all expressed in differentiated GCL. irx1a expression is initiated in the retinal cells at 24 hpf (A) while irx2a expression is observed at 30 hpf (F and K, respectively). At 48 hpf, ath5, irx1a, irx2a, and irx4a are strongly expressed in the GCL. The asterisk (*) marks the choroid fissure of each embryo.

Ocular defects induced by irx2a morpholinos. A: Schematic diagram showing the morpholino MO1 and morpholino MO2 targeted region in the irx2a pre-mRNA. MO1 blocked the translation of Irx2a by targeting the 52 UTR region of irx2a mRNA, whereas MO2 blocked the splicing of irx2a pre-mRNA by targeting the intron1-exon2 splice junction. B: RT-PCR analysis using irx1a- and irx2a-specific primers demonstrated a reduction of irx1a and irx2a transcripts in irx1a MO2- and irx2a MO2-injected embryos, respectively, at 48 and 96 hpf. Beta-actin was used as an internal control. irx2a transcript levels were reduced in irx1a MO2 morphants, suggesting that irx2a is a downstream target of irx1a. No difference in the abundance of irx1a transcripts was found in irx2a MO2 morphants, indicating that the irx2a MO2 was specific to irx2a. C–F: Embryos were raised in a bright environment until 96 hpf. As a result of visual impairment, irx2a MO2 morphants showed hyperpigmentation and smaller eye than the control morphants (E and F). The area of the eye was digitally measured (marked by the white dotted line in E) for each embryo.

irx2a is required for neurogenesis of the retina ganglion cells. A–L: Immunostaining of 72-hpf retina. A–I: Lamination defects of irx2a MO2 morphant retinas were revealed by H&E staining. In the control morphant (A), the retina was segregated into three distinct nuclear layers (outer nuclear layer [ONL], inner nuclear layer [INL, and ganglion cell layer [GCL]). In contrast, the laminar structure was disrupted in the irx2a MO2 morphant retina (E). HuC/D antibody was used to label differentiated RGCs in the GCL (B). However, HuC/D staining was barely detectable in the morphant retina (F). Zn8 immunoreactivity was observed in the surface of RGC and axons in the optic nerve (C). Zn8 staining was completely absent in the morphant (G). Zn12 immunostaining labeled RGCs and amacrine cells in the INL of the control retina (D), but only a small patch of staining was detected in the morphant retina (H). Neurogenesis defects in the GCL and INL of the morphant retina were rescued by injection of irx2a mRNA (I–L). M: Co-injection of irx2a mRNA, but not irx1a, irx4a, rescued the irx2a morphant phenotype.

irx2a is not required for the neurogenesis of photoreceptors cells in ONL. A–F: Section immunostaining of Zpr1 and Zpr3 in 96-hpf retina. Zpr1 immunostaining of control retina (A) labeled a subset of cone photoreceptors in the ONL. Only a small patch of Zpr1-labeled cells could be detected in the irx1a MO2 morphant retina (B), whereas irx2a MO2 morphants (C) showed similar staining of Zpr1 in the ONL as the control. Zpr3 immunostaining of a control retina (D) labeled rod photoreceptors in the ONL. Similar to Zpr1 staining, a small patch of Zpr3-expressing cells could be detected in irx1a MO2 morphant retina (E) whereas the Zpr3-expressing cells could be observed as normal in irx2a MO2 morphant retina (F).

irx2a expression signal is disrupted in irx1a morphant and ath5 morphant retinas. Expression pattern of irx2a and irx4a in irx1a MO2 morphant retinas (G–I and J–L) and ath5 morphant retinas (M–O and P–R) were revealed by in situ hybridization of 30-, 48-, and 72-hpf embryos. In irx1a morphant retina, irx2a expression was completely absent at all the examined developmental stages (G–I), while the expression of irx4a was slightly weaker than the control morphants (J–L). Similar results were observed in ath5 morphant retina; while irx2a expression was disrupted (M–O), similar irx4a expression was detected in the control morphant retina (P–R).

irx2a acts as downstream of irx1a and ath5 in controlling retinogenesis. The rescue activity of irx2a mRNA in irx1a MO2 morphants was indicated by retinal cell markers of 72 hpf embryos. Differentiation of the GCL (A–C), INL (C), and ONL (D) was disrupted in irx1a morphant retinas. By co-injecting the irx2a mRNA with irx1a MO2, the retinogenesis defects in GCL and INL were rescued. Immunostaining of eye makers was observed in the co-injected embryos (E–G). However, the differentiation defect in the ONL of irx1a morphant could not be rescued by irx2a mRNA (H). Immunostaining revealed retinal defects in irx1a morphants co-injected with irx4a mRNA (I–L) similar to those observed in irx1a morphants (A–D). M: The rescue activity of irx2a in irx1a morphants was dose-dependent whereas the ath5 morphant phenotype could not be rescued by irx2a and/or irx1a. N: The sub-lethal dose of irx1a or irx2a morpholino MO2 could not induce eye defects whereas co-injection of the same dose of irx1a and irx2a morpholinos induced a significant eye phenotype. Similar results were obtained in experiments with co-injection of a sub-lethal dose of irx2a and ath5 morpholinos.

Spread of the waves of shh-GFP expression is rescued by overexpression of irx2a in irx1a morphants. In control shh-GFP transgenic embryos (A–D), shh-GFP is expressed in the ventronasal side of the control retina (A). This expression spreads to the central retina at about 38 hpf (B), and then to the entire GCL and INL from about 42 to 48 hpf (C and D). The expression of shh-GFP is initiated in the irx1a MO2 morphant retina (E) but with a delay of about 8 hr (E–H). Furthermore, shh-GFP is not expressed in the central and dorsal regions of the irx1a morphant retina (G,H). Similar to control embryos, the shh-GFP waves propagated normally in the retina of irx1a morphants co-injected with irx2a mRNA (I–L).

Expression of irx3a, irx5a, and irx6a in retinal ganglion cells of 48-hpf embryos. irx3a (A–D), irx5a (E–H), and irx6a (I–L) expression in the GCL (ganglion cell layer) of the retina was revealed by the in situ hybridization of 24-, 30-, 36-, and 48-hpf embryos. irx3a, irx5a, and irx6a are all initiated at 48 hpf in retinal ganglion cells (D, H, and L respectively). M: The schematic temporal profile for the irx genes and ath5 expression.

Section in situ hybridization of the clustered IrxAa genes in retina. irx1a (A–C), irx2a (D–F), and irx4a (G–I) expression in GCL of the retina were shown by the section in situ hybridization at 30-, 48-, and 72-hpf embryos. irx1a and irx2a are expressed at GCL at 30 hpf and all the irx1a clustered genes are strongly expressed in GCL at 48 and 72 hpf.

The off-target effects of irx1a and irx2a morpholinos are mediated through p53-induced apoptosis. Injection of p53 knockdown morpholino rescued the off-target effects, including reduced body size and curvature of the tail, induced by irx1a morpholino MO2 (D, E) and irx2a morpholino MO2 (B, C). Embryos with co-injection of p53 MO and irx2a MO2 (C, F) or irx1a MO2 (E) still displayed a small eye phenotype (F). Thus, the eye defect was a specific loss of function phenotype of irx1a or irx2a knockdown embryos.

ath5 is required for neurogenesis of the retina ganglion cells. A: HuC/D immunoreactivity was observed in the surface of RGC at 72 hpf. D: However, HuC/D staining was dramatically decreased in the ath5 morphant retina. The Zn12 antibody labeled RGCs and amacrine cells in the INL of the control retina at 72 hpf (B), but only the immunostaining in the INL, not the GCL, was detected in the ath5 morphant retina (E). C: Zpr1 immunostaining of control retina labeled a subset of cone photoreceptors in the ONL at 96 hpf. F: Similar to control, ath5 morphants showed a normal Zpr1 immunostaining pattern in the ONL.

irx1a expression signal is disrupted in ath5 morphant retinas. Expression pattern of irx1a in control retina (A–C) and ath5 morphant retina (D–F) were revealed by in situ hybridization of 30-, 48-, and 72-hpf embryos. In ath5 morphant retina, irx1a expression was absent at 30 hpf (D), while only weak expression signal was detected at 48 hpf (E) and 72 hpf (F) in ath5 morphant retinas.