Onecut genetic cascade in the vertebrate retina. Schematic representation of the oc1 and oc2 vertebrate transcription factors, their upstream regulators and their target genes involved in the development of retinal tissues.

Territories of expression of oc genes in the zebrafish eye. On the left, schematic representation of the zebrafish eye section showing the retinal layers at 72 hpf. (A–J) In situ hybridization analysis of oc1, oc2, oc-like, oc3a and oc3b expression on zebrafish transverse eye sections at 48 hpf (A–E) and 72 hpf (F–J). oc1 expression is localized in IPL and in RGC and remains in GCL at 72 hpf (A,F). oc2 expression is observed in retinal IPL and INL at 48 hpf (B) and in GCL and ONL at 72 hpf (G), while oc-like transcript is reported in the INL and GCL at 48 hpf (C) and also in IPL at 72 hpf (H). No signal is observed for oc3a (D,I) and oc3b (E,J) in the zebrafish eye at both analyzed developmental stages. RGC, retinal ganglion cells; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer.

Eye phenotypes of oc1, oc2, oc-like and Triple morphant embryos. (A) Eye of control embryo injected with STD MO and (B–E) of embryos injected with oc1, oc2, oc-like and triple oc-MO. (F) Analysis of eye areas in oc1, oc2, oc-like and triple oc-MO embryos at 24 hpf. Data are expressed as mean ± SEM. Non parametric Kruskal–Wallis test with Dunn’s post hoc correction. *** p < 0.001, **** p < 0.0001.

Cell proliferation and apoptosis assays on zebrafish oc morphant retinal cells. (A–E) Immunostaining for the mitotic marker PH3 in (A) Eye of control embryo injected with STD MO and (B–E) of embryos injected with oc1, oc2, oc-like and triple oc-MO. (F) Graphical analysis of proliferating cells in oc1, oc2, oc-like and triple oc-MO embryos at 24 hpf with respect to STD MO embryos. (G–L) Tunel assay showing no significant differences in the number of apoptotic cells (black circles) between control (G) and oc1, oc2, oc-like and triple oc-MO eyes (H–K). (L) Graphical analysis of the number of apoptotic cells observed in (G–K) retinas. Data are expressed as mean ± SEM. Non parametric Kruskal–Wallis test with Dunn’s post hoc correction. *** p < 0.001.

Effect of oc genes depletion on zebrafish retinal organization. (A–E) Sagittal sections of zebrafish retinas at 72 hpf from the SoFa1 transgenic line. (F–J) Immunostaining for Zn-8 in RGCs, and (K–O) for SV2 in IPL and PRC axon terminus (white arrows) of zebrafish eye at 72 hpf. (A,F,K) STD MO, (B,G,L) oc1, (C,H,M) oc2, (D,I,N) oc-like and (E,J,O) triple oc-MO eye sections. Retinal populations are unaltered following STD MO injection (A,F,K). A reduction of RGC (G) and AC (B) is reported in oc1-MO retina, and milder alterations are visible in oc-like-MO (D,I,N) and oc2-MO (C,H,M) respectively. Triple oc-MO retina (E,J,O) shows severe alterations and reductions in the RGC and a complete absence of PRC axon terminals (white arrow). (P) Schematic representation of the retinal alterations. The reduced number of cells represented in this schematic drawing is just an illustration and is not quantitative. AC, amacrine cells; BP, bipolar cells, HC, horizontal cells; PRC, photoreceptor cells; RGC, retinal ganglion cells; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Alteration of the swimming activity in oc morphants. (A) Schematic representation of swimming activity of morphant larvae during light/dark stimuli. The recording traces show the increase in locomotion in oc1-MO and triple oc-MO larvae compared to oc2, oc-like-MO and STD MO injected larvae. (B,C) The graphs show the total distance moved (B) and the mean velocity (C), obtained by combining the values from 12 larvae per group. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Bars represent mean ± SEM. * p < 0.05, *** p < 0.001.

Altered expression of the zebrafish orthologs to Ciona DEGs. Altered levels of cplx2, cplx2-like, tmtc2a, tmtc2b, diras1a, lhx5, prox1a and prox1b transcripts in oc morphant embryos at 48 hpf by RT-qPCR. Gene expression was normalized to rpl13a expression. Data are expressed as mean ± SEM. (n = 2 for each study group). One-way ANOVA with Dunnett’s post hoc correction. * p < 0.05, ** p < 0.01, *** p< 0.001.