Immunodetection of Smarce1 in retinas at 30 hpf and 5 dpf. (A) Confocal sections of a 30 hpf retina stained with a Smarce1 antibody (green) (upper panel); the signal is detected in all RPCs nuclei. Cryostat histological sections of 5 dpf retinas showing DAPI stained nuclei (gray) and Smarce1 signal (green) in amacrine cells (Am), retinal ganglion cells (RGC), Photoreceptor layer (PL), and outer nuclear layer (ONL) (lower panel). The high magnification images on the right show Smarce1 signal in most photoreceptor nuclei, except for a few basally-localized ones, which appear negative (arrowheads). Signal in outer segments (PL) is due to autofluorescence. (B) Bright-field photographs of wild type (Ctrl) and mutant (smarce1-/-) heads showing the eye size reduction (upper panel). H&E sections of 5 dpf wild type (Ctrl) and mutant (smarce1-/-) retinas (lower panel). Scale bars in A: 20 μm; in B: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mitosis of RPCs in the CMZ of 5 dpf retinas. (A) Immunostaining with phospho-histone H3 (PH3) reveals mitotic cells in central retinal cryosections from wild-type (Ctrl) and mutant (smarce1-/-) samples at 5 dpf. DAPI staining labels nuclei. A reduced number of PH3-positive cells is observed in smarce1-/- retinas compared to Ctrl. Scale bar: 50 μm. (B) Quantification of PH3 positive cells in WT and smarce1-/- retinas. The upper graph shows the average number of PH3 cells per section (7–10 sections); each dot represents one retina. For both control and smarce1-/- mutants, n = 10. The lower graph shows the total number of PH3-positive cells per retina. A significant reduction in PH3-positive cells was observed in smarce-/- samples compared to Ctrl (cells per section: t18 = 4.14, ***p = 0.0006; in total cells U = 9 **p = 0.002).

Transcriptomic analysis of smarce1-mutant retinas confirms downregulation of cell cycle and mitosis genes. (A) Heatmap showing a list of the top downregulated genes of the Cell Cycle Term. The color bar shows the average FPKM value of three replicates. Yellow color indicates a high expression level, and black color indicates a low expression level. Genes are organized according to their Padj value from small to large (range 1.53e-57 to 3.7e-08). (B) GO enrichment analysis. The most significant 30 Terms were selected for display; the x-axis indicates GO Term, and the y-axis displays GO Term's level of significance of enrichment, expressed as −log10 (padj). Different colors represent different functional categories; BP: biological processes, CC: cell components, MF: molecular function. (C) Confocal images showing immunodetection of Smarce1 in embryos at the oblong stage (3.7 hpf). DAPI staining (blue) labels nuclei in S-phase and chromosomes at mitosis. Phalloidin (green) marks the cell cortex. Smarce1 signal (red) is localized to nuclei but is absent from mitotic chromosomes. Late anaphase/early telophase chromosomes in a dividing cell are indicated (white arrow heads). Scale bar: 20 μm. (D) KEGG enrichment results showing that the top downregulated pathways are related to the cell cycle (red characters); the most significant 20 KEGG downregulated pathways are displayed. The x-axis represents the ratio of the number of differential genes linked with the KEGG pathway to the total number of differential genes; the y-axis lists the KEGG pathways. The size of a point represents the number of genes annotated to a specific KEGG pathway. The color from red to purple represents the significant level of enrichment. (E) Heatmap showing the top downregulated genes of the Mitosis Term. The bar shows the average FPKM value of three replicates. Yellow color indicates a high expression level, and black color indicates a low expression level. Genes are organized according to their Padj value from small to large (range 1.32e-36 to 3.3e-18). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Cell death of retinal neurons in the smarce1-mutants. (A) Confocal sections of 72 hpf and 5 dpf retinas stained with methyl green. Pyknotic nuclei representing apoptotic cells exhibit enhanced fluorescence (examples marked with an asterisk). Scale bar: 40 μm. (B and D). Quantification of the total number of pyknotic nuclei in WT (Ctrl) and smarce1-/- (Mut) retinas at 72 hpf (B) and 5 dpf (D). At 72 hpf, n = 8 for both, control and smarce1-/- mutants; at 5 dpf, n = 8 for control and n = 11 for mutant retinas. A Welch́s t-test was performed showing a significant increase in pyknotic nuclei in smarce-/- samples compared to Ctrl. At 72 hpf (t6 = 4.11, p** = 0.0058); at 5 dpf (U = 0,*** p = 0.0004). (C and E). Quantification of the number of pyknotic nuclei per layer in the smarce1-/- retinas at 72 hpf (C) and 5 dpf (E). PR: photoreceptors, INL: inner nuclear layer, GC, ganglion cell layer. (F) KEGG enrichment results show the upregulation of the apoptosis pathway (red), the most significant 20 KEGG upregulated pathways were selected for display. The x-axis represents the ratio of the number of differential genes linked with the KEGG pathway to the total number of differential genes; the y-axis lists the KEGG pathway. The size of a point represents the number of genes annotated to a specific KEGG pathway. The color from red to purple represents the significant level of enrichment. (G) Heatmap showing the top upregulated genes of the Apoptosis Term. The bar shows the average FPKM value of three replicates. Yellow color indicates a high expression level, and black color indicates a low expression level. Genes are organized according to their Padj value from small to large (range 1.72e-93 to 0.021). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Defective organization of ganglion, amacrine, and horizontal neurons. Confocal sections of 5 dpf Ctrl and smarce1-/- retinas expressing the ganglion cell reporter atoh7:gapGFP (upper panel) and ptf1a:cytGFP (lower panel). GC, ganglion cells; PR, photoreceptors; Hrz, horizontal cells; Am, amacrine cells; IPL, inner plexiform layer. Scale bars 10 μm.

Terminal differentiation of PRs is defective. (A) Heatmap showing the top downregulated genes of the Phototransduction Term. The bar shows the average FPKM value of three replicates. Yellow color indicates a high expression level, and black color indicates a low expression level. Genes are organized according to their Padj value from small to large (range 3.8–64 to 0.00749). (B) Confocal sections of 5 dpf Ctrl and smarce1-/- retinas stained with the Zpr1 antibody (green). Ectopic cones are visible in inner locations (yellow arrows) in the mutant images. Right lower panel shows a closer caption of the PRs where disorganization can be appreciated. Scale bars, upper panel:40 μm; left lower panel: 20 μm; right lower panel: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)