(A–D) Hematoxylin and eosin (H&E) staining. (A,B) Histological longitudinal sections of retinas from 3 dpf wild-type and cdh23^−/− embryos showed no significant changes in overall retinal morphology. (C,D) Histological transverse sections of retinas from 3 dpf wild-type and cdh23^−/− embryos showed no significant changes in overall retinal morphology. (A,B) Scale bar = 200 μm; (C,D) scale bar = 400 μm.

Transcriptome analyses of cdh23^−/−. (A) Differential gene clustering heatmap: the x-axis represents the sample names and hierarchical clustering results, while the y-axis represents the hierarchical clustering results of differential genes. The red color represents up-regulated expression genes, and the blue color represents down-regulated expression genes. (B) Volcano plot of differentially expressed genes: the x-axis represents |log₂ (foldchange)|, and the y-axis represents −log₁₀ (p value).

Pathways and differential gene expression associated with cdh23 knockout in relation to photoreceptor degeneration. (A) Gene Ontology (GO) enrichment analysis of differentially expressed genes was conducted across three categories: biological process (BP), cellular component (CC), and molecular function (MF). The y-axis represents enrichment of each GO term, with redder color indicating higher significance, and size of circle representing number of genes enriched in pathway, with larger circles indicating higher number of enriched genes. (B) KEGG pathway enrichment analysis of differentially expressed genes was also performed, with p-adjust value used to assess the statistical significance of each pathway. Smaller p-adjust values indicate greater significance of enrichment. (C–E) Heatmap illustrating differential expression of genes involved in phototransduction, retinol metabolism, and purine metabolism pathways is presented. Vertical axis represents genes, and horizontal axis represents sample groups and control groups, with red indicating up-regulated genes and blue indicating down-regulated genes.

GO and GSEA. (A) Heatmap of differentially expressed genes in photoreceptor cilium GO enrichment pathway. (B) GSEA results of retinol metabolism pathway. Abnormal cGMP metabolism in cdh23^−/− zebrafish retinas generates action potential in ganglion cells.

Visualization of network construction analysis of specific pathways. (A) PPI network representing cdh23 gene shows proteins as nodes in graph, with their interactions depicted by lines connecting them. Various colored lines indicate different types of interactions, with thicker lines representing stronger interactions. (B,C) Key genes involved in optical conduction and retinol metabolism, as well as their interactions. (D,E) Gene expression analysis of light sensor degeneration-related genes using RT-qPCR. Additionally, statistical significance was determined with * representing a p value of <0.05, ** representing a p value of <0.01, and *** representing a p value of <0.001. (F) RT-qPCR expression analysis of genes related to retinol metabolism pathway was conducted. The results showed significant differences in gene expression levels (*: p value < 0.05; **: p value < 0.01; ***: p value < 0.001), indicating potential regulatory roles of these pathways in studied biological processes. ns: not significant.

Enrichment analysis of differentially expressed genes in cdh23^−/− reveals expression of Ca²⁺ pathway and cell apoptosis-related pathways. (A) GO enrichment analysis reveals differential expressions. Abscissa represents Gene Ratio, which is ratio of actual enriched genes in gene set for each GO term to total number of genes. Ordinate represents various enriched GO terms. p value measures significance of enrichment, with smaller values indicating greater statistical significance. (B) Differential expression genes with KEGG enrichment. Volcano plot shows enrichment of various KEGG terms and genes enriched in each pathway. Count value represents number of genes with different enrichment results. -log10 (p value) value measures the significance of enrichment, with a higher value indicating greater statistical significance. (C,D) Increased TUNEL staining in cdh23^−/− retinas. TUNEL staining (green) on cryosections of 3 dpf zebrafish retinas revealed apoptotic cells. Zebrafish larvae were embedded for transverse sectioning. Scale bar = 400 μm.

Identification of differentially expressed genes. (A–F): GSEA for calcium signaling pathway, MAPK signaling pathway, apoptosis, glycolysis/gluconeogenesis, oxidative phosphorylation, and TCA cycle.

Effect of cdh23 on calcium signaling pathway and MAPK signaling pathway. (A) Cluster heatmap of differentially expressed genes in calcium signaling pathway. (B) Cluster heatmap of differentially expressed genes in calcium ion transmembrane transport. (C) Cluster heatmap of differentially expressed genes related to MAPK signaling pathway. (D,E) RT-qPCR expression analysis of genes related to calcium signaling pathway and MAPK signaling pathway was conducted. Results showed significant differences in gene expression levels (*: p value < 0.05; ***: p value < 0.001), indicating potential regulatory roles of these pathways in studied biological processes.

Schematic diagram of cdh23 regulating rod cell calcium ion transport (image created by Figdraw). (A) In WT rod photoreceptor cells, genes such as Rho, Rhol, Grk1, Grk7a, Gnat1, Pde6a, Pde6b, and Guca1b play essential roles in phototransduction. Additionally, Atp2b1b is responsible for transport of calcium ions (Ca²⁺). Rhodopsin is synthesized in endoplasmic reticulum of inner segment (IS) and transported to outer segment (OS) through vesicular transport from Golgi apparatus, with IS and OS being connected by connecting cilium. Purine metabolism generates sufficient ATP to properly position rhodopsin in outer segment. Upon light stimulation, rhodopsin undergoes conformational change, activating Gt transducin protein, which subsequently activates phosphodiesterase (PDE). PDE hydrolyzes cGMP, leading to decrease in its concentration, which causes closure of CNG channels. This reduces inward flow of Ca²⁺ and release of neurotransmitter glutamate, completing phototransduction process. (B) In cdh23^−/− embryos, disrupted purine metabolism leads to insufficient ATP production, impairing vesicle transport from Golgi apparatus and movement of cilium. As a result, rhodopsin fails to localize properly to outer segment, preventing cGMP hydrolysis by PDE. Increased cGMP concentration activates CNG channels, causing continuous inward flux of Ca²⁺, which activates Ca²⁺-dependent MAPK signaling pathway and triggers apoptosis in rod photoreceptor cells.