Ethics statement
All animal experiments and field work were conducted according to the standard animal guidelines approved by the Animal Care Committee of Shanghai Ocean University (Approval No. SHOU-DW-2021-011).
DNA and RNA sequencing
O. kenojei were caught at Bohai Bay in China, and genomic DNA was extracted from their muscle tissues. A short-read paired-end library with a 350-bp insert size was sequenced on the BGISEQ-500 platform (BGI Qingdao, China), and two 20-kb-long read libraries were constructed and sequenced on the RSII platform (Pacific Biosciences, California, USA) in the CLR mode, according to the official standard protocol. For Hi-C sequencing, libraries were constructed and sequenced on the BGISEQ-500 platform with 100-bp paired-end reads, according to the official standard protocol58. RNAs were extracted from six tissues, including the eye, ovarium, dorsal skin, abdomen skin, liver, and spermary, by using a TRIzol kit (Invitrogen, USA), and the libraries were sequenced on the BGISEQ-500 platform (Supplementary Table 7). For P. glauca, caught from the western Pacific Ocean, we constructed a short-read 350-bp library and sequenced it on the Illumina Novaseq platform. Genomic DNA was broken down into approximately 15-kb-long fragments to construct a long-read library and sequenced on the PacBio Sequel II platform in the CCS mode.
Genome assembly and chromosome anchoring
SMARTdenovo59 was used to assemble the O. kenojei genome by using long reads with default parameters. Then, the contigs were polished using Pilon (version 1.22)60 by using the clean short reads filtered using Soapnuke (version 1.6.5) with these parameters: -M 1 -A 0.4 -d -n 0.02 -l 10 -q 0.1 -Q 2 -G. Finally, the Hi-C data were mapped to the draft genome by using Juicer61 and anchored to the chromosome using the 3D-DNA pipeline62. For the P. glauca genome, accurate CCS data were assembled using hifiasm (version 0.16)63. Next, purge_dups (version 1.2.6)64 was used to reduce the number of heterozygous duplications. The chromosome-linking steps were identical to those used for the O. kenojei genome. Finally, the completeness assessment of assembled genomes was conducted using BUSCO based on the metazoa odb10 dataset and the Core Vertebrate Genes65 in gVolante (https://gvolante.riken.jp/).
Genome annotation
For genome repeat annotation, we first used RepeatModeler (version 1.0.8) and LTR-FINDER (version 1.0.6)66 to construct a custom repeat library and then used RepeatMasker (version 4.0.6)67 to search the genome repeats against both our custom library and Repbase (version 21.01) database by using the following parameters: -nolow -no_is -norna -engine ncbi. RepeatProteinMask (version 4.0.6) was also used for perform homolog-based search at the protein level with the following parameters: -engine ncbi -noLowSimple -p-value 0.0001. In addition, tandem repeats were detected using Tandem Repeats Finder (version 4.07)68 with the following parameters: -Match 2 -Mismatch 7 -Delta 7 -PM 80 -PI 10 -Minscore 50 -MaxPeriod 2000. Next, by using the aforementioned method, we detected repeat contents in other cartilaginous fishes for downstream analysis (Supplementary Table 8).
For gene annotation, protein sequences of Chiloscyllium punctatum, S. torazame, Callorhinchus milii, Rhincodon typus, and Carcharodon carcharias (Supplementary Table 9) were downloaded from public databases and mapped to the genome using BLAT (version 35.1)69. Next, gene models were predicted using GeneWise (version 2.4.1)70. RNA reads were aligned to the genome using HISAT271. Furthermore, transcripts were assembled using Stringtie (version 1.2.2)72, and open reading frames were predicted using TransDecoder (version 5.5.0; http://transdecoder.sourceforge.net/). Finally, GLEAN73 was used to integrate a nonredundant gene set.
For gene function annotation, protein sequences were aligned to sequences from various databases including Swiss-Prot, TrEMBL74, and KEGG (version 105)75 by using BLASTP (version 2.2.26)76. Function-specific motifs and domains were determined by InterProScan (version 5.60-92.0)77 according to several protein databases, including Pfam, SMART, PANTHER, PRINTS, PROSITE profiles, and ProSitePatterns. Gene Ontology annotation results were extracted from the InterProScan results. Finally, protein clustering was performed with 70% similarity by using Cd-hit (version 4.8.1)78.
Gene family analysis
Gene sequences of Amblyraja radiata, Chiloscyllium plagiosum, Callorhinchus milii, Carcharodon carcharias, Chiloscyllium punctatum, Rhincodon typus, Scyliorhinus torazame, Latimeria chalumnae, Lepisosteus oculatus, Danio rerio, Acipenser ruthenus, Coilia nasus, Gasterosteus aculeatus, Takifugu rubripes, and Scleropages formosus were downloaded from the NCBI database (Supplementary Table 10). Next, SonicParanoid79 was used to identify orthologous groups among the species, and the third base from qualifying codons was then extracted and compiled for all species, resulting in a reduced dataset of four-fold degenerate sites to construct a phylogenetic tree using IQ-TREE80 with the maximum-likelihood method. The divergence times were inferred on MCMCtree81, and the calibrating fossil times were searched on TimeTree22: L. chalumnae and Le. oculatus, 424–440 Mya; L. chalumnae and Cal. milii, 442–515 Mya; A. ruthenus and Le. oculatus, 345–372 Mya; Le. oculatus and D. rerio, 298–342 Mya; Sc. formosus and D. rerio, 244–301 Mya; Coi. nasus and D. rerio,151–246 Mya; G. aculeatus and T. rubripes, 82–174 Mya; Car. carcharias and R. typus, 113–289 Mya; Cal. milii and A. radiata, 338–471 Mya; and C. plagiosum and A. radiata, 245–343 Mya. The expansion and contraction of gene families were then defined using CAFE (version 5.0)82 using the base model, and the clade_and_size_filter.py script from the CAFE package was used to filter out gene families with copy numbers exceeding 100 in one or more species to avoid noninformative parameter estimates. In order to verify identify representative expanded gene families, we used miniprot (version 0.12)83 to align reference proteins from pathway in KEGG database to target genomes, obtaining combined homolog results with the genewise results. Multiple animo acid sequences alignments were generated using MAFFT (v7.407)84 and the gene trees were constructed by FastTree (2.1.10)85 using maximum likelihood method.
Opsin gene identification
Protein sequences of cartilaginous fishes and reference opsin genes of 24 species were downloaded from NCBI and published articles (Supplementary Table 11). Then, the opsin genes from NCBI databases (Supplementary Data 8) were aligned with the whole-protein sequences by using BLASTP (version 2.2.26) with the following parameters: -e 1e-5. For Raja brachyura, M. birostris, and S. acanthias (the annotation information of which was unavailable), we used miniprot (version 0.12)83 to align opsin proteins with their genomes and obtain opsin homologs.
Zebrafish
Here, we used AB WT zebrafish procured from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China; they were maintained under a 14-h light–10-h dark cycle and fed Artemia nauplii twice daily. Embryos were cultivated at a consistent temperature of 27 °C ± 1 °C in egg water, formulated by diluting artificial seawater in regular water at a ratio of 1.5:1000. The zebrafish broodstock was selectively paired at a 1:1 male-to-female ratio86. All zebrafish used in this study were anaesthetized with MS-222 before sampling.
Light exposure
For violet light exposure experiments, 10 sws1−/− zebrafish and 10 WT zebrafish (all 15 days post fertilization, dpf) were grouped into three parallel groups. All fish were placed in a dark room with violet LED lamps on the top and fed twice per day (in the morning and evening). The water was changed daily, and a 14-h light–10-h dark irradiation cycle was used. The sustained light stimulation time was 30 days. LED lamp wavelength (λmax) and intensity were set to 370 nm and 2,000 ± 100 lx, respectively. For blue light exposure experiments, we followed identical steps but with the following exceptions: sws1−/− zebrafish were replaced by sws2−/− zebrafish, and λmax was set to 420 nm.
Moreover, zebrafish sws1-overexpressing HEK293 cells were cultured under 370-nm violet light for 30 min, whereas zebrafish sws2– and human sws-overexpressing HEK293 cells were cultured under 420-nm blue light for 30 min; in both experiments, the light intensity was set to 2000 ± 100 lx.
Tissue section preparation
Whole zebrafish (45 dpf) were euthanized and immediately placed in 4% paraformaldehyde for overnight fixation. Next, the specimens were subjected to wash and dehydration steps in an ethanol series at increasing concentrations (75%, 85%, 95%, and 100%; every 30 min). Subsequently, the samples were rendered transparent using xylene, followed by overnight immersion in paraffin. Finally, the paraffin-embedded tissue sample blocks were sectioned into 5-μm-thick slices by using a blade, mounted on gelatin-coated slides, and air-dried.
Hematoxylin and eosin (HE) staining
The paraffin-embedded sections were deparaffinized at 65 °C and rehydrated using an ethanol series at decreasing concentrations (100%, 95%, 90%, 80%, and 70%; every 2 min). Subsequently, they were immersed in a hematoxylin dye solution for 5 min, followed by rinsing with running water. To enable differentiation, we immersed the sections in a 0.5% hydrochloric acid–alcohol solution for 10 s, followed by rinsing under running water and immersion in a 1% eosin dye solution for 10 s. After gradient dehydration, the sections were exposed to xylene for 1–2 min to develop transparency. Finally, the sections were mounted with resin. For each zebrafish sample, the paraffin section with the largest cross-section of the lens was selected for staining treatment, and the images of the dorsal and ventral center of the retina were selected for statistical and analysis of cell number and cell length. The staining results were recorded with a pathology slide scanner (SQS12P, TEKSORAY, CN).
Immunohistochemically staining
The tissue sections were placed in 3% hydrogen peroxide, followed by incubation at room temperature for 25 min in the dark and then by three phosphate-buffered saline (PBS) washings with shaking for 5 min. Next, 3% bovine serum albumin (BSA) was added to cover the tissue section evenly and air-dried at room temperature for 30 min. Next, the BSA seal was removed, and primary antibodies (IL6 Rabbit mAb, ABclonal, Catlog NO.:A22222 at 1:200 dilution; Anti-Caspase-3 Rabbit pAb(Servicebio,Catalog NO.:GB11009-1 at 1:100 dilution) suspended in PBS were added to the sections. All sections were then placed flat in a wet box and incubated at 4 °C overnight; this was followed by three PBS washings with shaking for 5 min. After the slices dried slightly, the sections were incubated with corresponding horseradish peroxidase-labeled corresponding secondary antibodies (HRP IgG, Servicebio, Catalog NO.:GB23303 at 1:200 dilution) at room temperature for 50 min, followed by three PBS washings with shaking for 5 min After the sections dried slightly, the freshly prepared a DAB color-developing solution was added to the section. Next, the sections were covered with hematoxylin dye solution for 3 min, followed by rinsing under running water. After another gradient dehydration, the sections were exposed to xylene for 1–2 min to increase their transparency. Finally, the slices were mounted using resin. The staining results were recorded with a pathology slide scanner (DS-Ri2, Nikon, Japan).
Immunofluorescence
All paraffin-embedded tissue sections used here were treated similarly to the sections used for immunohistochemical staining until the secondary antibody addition step. After washing off the secondary antibodies, we incubated the sections with DAPI at room temperature for 10 min in the dark to stain the nuclei. Finally, the sections were sealed with an anti-fluorescence quenching agent. The staining results were recorded with a pathology slide scanner (SQS12P, TEKSORAY, CN).
For cell immunofluorescence, cells grown on coverslips were fixed with 4% ice-cold paraformaldehyde PBS for 20 min, followed by treatment with 0.1% TritonX-100 PBS for 10 min. After washing the sections with PBS twice, we blocked the cells with 5% BSA at 37 °C for 30 min and then incubated them with primary antibodies (CDKN2A/p16INK4a Rabbit mAb, ABclonal, Catalog NO.:A11651 at 1:200 dilution) at 4 °C overnight. Next, the cells were washed with PBS and incubated with the corresponding secondary antibodies (ABflo 488-conjugated Goat Anti-Rabbit IgG(H + L), ABclonal, Catalog NO.:AS053 at 1:500 dilution) at 37 °C for 30 min. Finally, DAPI was used to stain the nuclei, and the sections were sealed with an anti-fluorescence quenching agent. The staining results were recorded with a pathology slide scanner (SP8, Leica, Germany).
TUNEL staining
TUNEL assay kit (E-CK-A331, Elabscience, US) and TMB chromogenic (322550, ACD, US) were performed for TUNEL staining according to the kit instructions. The paraffin sections were routinely dewaxed and rehydrated, followed by washing with PBS. Subsequently, the repair solution containing proteinase K from the kit was added and incubated at 37 °C in a humid chamber for 1 h. After washing with PBS, the balance solution from the kit was applied and equilibrated at 37 °C in a humid chamber for 30 min.
Following this, the TdT enzyme and HRP-dUTP mixed working solution were prepared according to the instructions provided in the kit. The buffer on the sections was removed by centrifugation before adding the prepared mixed working solution, which was then incubated at 37 °C in a humid chamber for 1 h. After another wash with PBS, the TMB chromogenic substrate was added and allowed to stand at room temperature for 5 min. Finally, observations were made under a light microscope; a blue-green coloration indicated a positive signal. The staining results were recorded with a pathology slide scanner (SQS12P, TEKSORAY, CN).
Total RNA extraction and cDNA synthesis from zebrafish eyes
Three zebrafish eye tissue samples were collected, and RNA extraction was performed according to the protocol outlined in the RNA isoPlus specification (9108; Takara, Japan). Subsequently, first-strand cDNA was synthesized using a HiSlid cDNA Synthesis Kit (MKG840; MIKX, China), according to the manufacturer’s instructions.
RNA sequencing analysis
We obtained three mixed samples of zebrafish eye tissue from each of the following groups: normally fed WT, blue-light–exposed WT, violet-light–exposed WT, blue-light–exposed sws2−/− mutant, and violet-light–exposed sws1−/− mutant. Sequencing was conducted at Shanghai Ouyi Biology. Differential expression analysis was performed using DESeq2, with Q value < 0.05 and fold change > 2 or fold change <0.5 set as the thresholds for significantly differentially expressed genes.
qRT-PCR
We performed RT-qPCR by using the Pro SYBR qPCR Mix (MKG800; MIKX, China). Specifically, divergent primers annealing at the distal ends of circRNA were used to quantify circRNA abundance. Supplementary Table 12 provides the details of the RT-qPCR primers used. Amplification was performed on a StepOnePlus Real-Time PCR System (CFX; Bio-Rad, USA), and Ct thresholds were determined using the relevant software program.
Zebrafish gene editing based on CRISPR/Cas9 system
Knockout targets for zebrafish sws1 and sws2 were designed in The University of California Santa Cruz Genome Browser (https://genome.ucsc.edu/). The sgRNA and cas9 mRNA synthesized in vitro were microinjected into WT zebrafish embryos. These embryos were cultured to the age of 3 months, and F0 generation heterozygous mutants were screened through Sanger sequencing. Heterozygous and WT zebrafish were then used to obtain F1 zebrafish, and homozygous mutants were screened through Sanger sequencing (Supplementary Fig. 8)87. All sequencing was performed at Shanghai Sangong Biological Company.
Behavioral testing
We performed behavioral testing to evaluate sws1 and sws2 knockout in zebrafish by using the Y-maze experiment (20 cm × 50 cm arm length); the three arms of the Y-maze were set as the starting, light, and dark arms. Equal amounts of feed were provided in the light and dark arms. In total, 30 WT and 30 mutant zebrafish were assessed consecutively. Each fish was tested for 2 min, starting from the starting arm, three times. The final swimming direction was recorded.
Vector construction and transfection
To construct an opsin overexpression plasmid, we cloned human sws, zebrafish sws1, and sws2 cDNAs into the pmcherry-N1 vector using the Homologous Recombinant Kit (C113; Vazyme; Supplementary Fig. 9a). Primers used for amplification are listed in Supplementary Table 12. The plasmid was transfected into HEK293 cells by using Lipo8000 (C0533FT; Beyotime, USA), according to the manufacturer’s instructions. In order to construct a stable cell line, the transfected cells were transferred to 10 cm dish, and 800 ng/μl of G418 was added for screening, and the monoclonal cell line was obtained and cultured into a stable cell line. Fluorescence signals were observed under a fluorescence microscope (IX53; Olympus, Japan). Untransfected HEK293 cells and pmcherry-N1 transfected cells were used as controls.
SA-β-gal staining
For SA-β-gal staining, we used the SA-β-Gal Staining Kit (G1073; Servicebio, China), according to the manufacturer’s instructions. Synovial cells were fixed with senescent cell staining fixative for 15 min and washed three times, followed by incubation in SA-β-gal staining solution at 37 °C for 24 h. Images were captured under an inverted fluorescence microscope, and SA-β-gal-positive synovial cells were randomly divided into three regions.
Statistical analysis
All data in this study are expressed as means ± standard deviations. Analyses were performed on Prism (version 9.1.2; GraphPad Software). The means of the groups were compared using a Student’s t test and analysis of variance. P values were calculated using a log-rank test, and P < 0.05 was considered to indicate statistical significance.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.