Fei Liu

Fish show exceptional diversity in sex-determination mechanisms, making them a powerful system for studying how these pathways evolve. However, no definitive master sex-determining gene has yet been identified in Cyprinidae, a widely distributed and species-rich fish family. Here, we identified a male-specific gene in common carp encoding Pangu, a helix-loop-helix (HLH) domain peptide localized to Sertoli cells. Loss- and gain-of-function analyses demonstrate that pangu is both necessary and sufficient for male sex determination. Expression and reporter assays indicate that pangu represses cyp19a1a , a key regulator of ovarian differentiation, and disruption of cyp19a1a restores testicular development in the feminized phenotype of XY pangu mutants. These findings establish pangu as the master sex-determining gene in common carp and link an HLH domain peptide to sex differentiation.

Testis-associated highly conserved oncogenic long non-coding RNA (THOR) is a highly conserved and testis-enriched long non-coding RNA (lncRNA) across vertebrates that plays diverse roles in various cancers. However, its physiological function and regulatory mechanism in testes remain largely unknown. Here, we investigated the genomic location and expression pattern of THOR in the model organism zebrafish, and generated a homozygous THOR knockout model using clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 technology. Loss of THOR in zebrafish impaired spermatogenesis, leading to oligospermia (38.7% reduction in sperm count), reduced sperm motility, sperm ultrastructural defects, and decreased fertilization rates. RNA sequencing (RNA-seq) analysis of wild type (WT) and THOR knockout testes revealed dysregulation of cell cycle-related genes, including cyclin-dependent kinase inhibitor 1D (cdkn1d), forkhead box O1 a (foxo1a), TSC complex subunit 1a (tsc1a), TSC complex subunit 2 (tsc2), ATRX chromatin remodeler (atrx), and RAD21 cohesin complex component b (rad21b). RNA pulldown assays in zebrafish testes identified 486 potential THOR-interacting proteins primarily involved in ribosome biogenesis, RNA splicing, chromatin architecture, and meiotic progression. Notably, the core synaptonemal complex components synaptonemal complex protein 1 (Sycp1), synaptonemal complex protein 2 (Sycp2), and synaptonemal complex protein 3 (Sycp3) were all captured as THOR-binding partners. We further demonstrated that THOR directly interacts with Sycp3 and positively regulates its protein levels. Immunostaining assays on chromosome spreads revealed a significantly higher frequency of discontinuous Sycp3 signals in THOR−/− testes, suggesting the presence of meiosis defects caused by Sycp3 downregulation. Our findings expand the understanding of lncRNA-mediated control of spermatogenesis and male infertility by providing the first evidence that lncRNA THOR interacts with the synaptonemal complex to regulate meiosis progression.

Alternative splicing of ddx4 (DEAD-box helicase 4), a key germline marker gene, has been reported to generate sex-specific transcripts in zebrafish gonads. The biological functions and regulatory mechanisms of the ddx4 ovary-specific transcript (ddx4-L) during oogenesis remain unclear. In this study, we found that ddx4-L mutants, in which ddx4-L was specifically deleted, had enlarged ovaries but laid fewer eggs, along with having a lower fertilization rate compared to WT controls. RNA-seq analysis was performed to detect the changes in gene expression between WT and ddx4-L mutant ovaries. A total of 524 upregulated and 610 downregulated DEGs were identified. GO and GSEA enrichment analyses showed that genes involved in fertilization and reproduction biological processes were significantly downregulated. More specifically, we observed a remarkable reduction in Sycp1, a core component of synaptonemal complex, in ddx4-L mutant ovaries at both the mRNA and protein levels. In addition, the expressions of transposon elements, as well as the events of alternative splicing, alternative polyadenylation, and RNA editing, were analyzed based on the RNA-seq data. We found that the deletion of ddx4-L resulted in derepression of DNA transposons in zebrafish ovaries, possibly causing genome instability. In conclusion, our work demonstrates that the ovary-specific ddx4 transcript plays important roles in oocyte meiosis and DNA transposon repression, which extends our understanding of the biological functions and regulatory mechanisms of sex-specific alternative splicing in zebrafish oogenesis and reproduction.

Mutations in TUB-like protein 1 (TULP1) are associated with severe early-onset retinal degeneration in humans. However, the pathogenesis remains largely unknown. There are two homologous genes of TULP1 in zebrafish, namely tulp1a and tulp1b. Here, we generated the single knockout (tulp1a^−/− and tulp1b^−/−) and double knockout (tulp1-dKO) models in zebrafish. Knockout of tulp1a resulted in the mislocalization of UV cone opsins and the degeneration of UV cones specifically, while knockout of tulp1b resulted in mislocalization of rod opsins and rod-cone degeneration. In the tulp1-dKO zebrafish, mislocalization of opsins was present in all types of photoreceptors, and severe degeneration was observed at a very early age, mimicking the clinical manifestations of TULP1 patients. Photoreceptor cilium length was significantly reduced in the tulp1-dKO retinas. RNA-seq analysis showed that the expression of tektin2 (tekt2), a ciliary and flagellar microtubule structural component, was downregulated in the tulp1-dKO zebrafish. Dual-luciferase reporter assay suggested that Tulp1a and Tulp1b transcriptionally activate the promoter of tekt2. In addition, ferroptosis might be activated in the tulp1-dKO zebrafish, as suggested by the up-regulation of genes related to the ferroptosis pathway, the shrinkage of mitochondria, reduction or disappearance of mitochondria cristae, and the iron and lipid droplet deposition in the retina of tulp1-dKO zebrafish. In conclusion, our study establishes an appropriate zebrafish model for TULP1-associated retinal degeneration and proposes that loss of TULP1 causes defects in cilia structure and opsin trafficking through the downregulation of tekt2, which further increases the death of photoreceptors via ferroptosis. These findings offer insight into the pathogenesis and clinical treatment of early-onset retinal degeneration.

Alternative splicing is an important way of regulating gene functions in eukaryotes. Several key genes involved in sex determination and gonadal differentiation, such as nr5a1 and ddx4, have sex-biased transcripts between males and females, suggesting a potential regulatory role of alternative splicing in gonads. Currently, the sex-specific alternative splicing events and genes have not been comprehensively studied at the genome-wide level in zebrafish. In this study, through global splicing analysis on three independent sets of RNA-seq data from matched zebrafish testes and ovaries, we identified 120 differentially spliced genes shared by the three datasets, most of which haven’t been reported before. Functional enrichment analysis showed that the GO terms of mRNA processing, mRNA metabolism and microtubule-based process were strongly enriched. The testis- and ovary-biased alternative splicing genes were identified, and part of them (tp53bp1, tpx2, mapre1a, kif2c, and ncoa5) were further validated by RT-PCR. Sequence characteristics analysis suggested that the lengths, GC contents, and splice site strengths of the alternative exons or introns may have different influences in different types of alternative splicing events. Interestingly, we identified an unexpected high proportion (over 70%) of non-frameshift exon-skipping events, suggesting that in these cases the two protein isoforms derived from alternative splicing may both have functions. Furthermore, as a representative example, we found that the alternative splicing of ncoa5 causes the loss of a conserved RRM domain in the short transcript predominantly produced in testes. Our study discovers novel sex-specific alternative splicing events and genes with high reliabilities in zebrafish testes and ovaries, which would provide attractive targets for follow-up studies to reveal the biological significances of alternative splicing events and genes in sex determination and gonadal differentiation.

Purpose: Bietti crystalline dystrophy (BCD) is a progressive retinal degenerative disease primarily characterized by numerous crystal-like deposits and degeneration of retinal pigment epithelium (RPE) and photoreceptor cells. CYP4V2 (cytochrome P450 family 4 subfamily V member 2) is currently the only disease-causing gene for BCD. We aimed to generate a zebrafish model to explore the functional role of CYP4V2 in the development of BCD and identify potential therapeutic targets for future studies. Methods: The cyp4v7 and cyp4v8 (homologous genes of CYP4V2) knockout zebrafish lines were generated by CRISPR/Cas9 technology. The morphology of photoreceptor and RPE cells and the accumulation of lipid droplets in RPE cells were investigated at a series of different developmental stages through histological analysis, immunofluorescence, and lipid staining. Transcriptome analysis was performed to investigate the changes in gene expression of RPE cells during the progression of BCD. Results: Progressive retinal degeneration including RPE atrophy and photoreceptor loss was observed in the mutant zebrafish as early as seven months after fertilization. We also observed the excessive accumulation of lipid droplets in RPE cells from three months after fertilization, which preceded the retinal degeneration by several months. Transcriptome analysis suggested that multiple metabolism pathways, especially the lipid metabolism pathways, were significantly changed in RPE cells. The down-regulation of the peroxisome proliferator-activated receptor α (PPARα) pathway was further confirmed in the mutant zebrafish and CYP4V2-knockdown human RPE-1 cells. Conclusions: Our work established an animal model that recapitulates the symptoms of BCD patients and revealed that abnormal lipid metabolism in RPE cells, probably caused by dysregulation of the PPARα pathway, might be the main and direct consequence of CYP4V2 deficiency. These findings will deepen our understanding of the pathogenesis of BCD and provide potential therapeutic approaches.

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.