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Tilapia as an economically important fish is also an excellent model for studying pigment cell biology and body color formation. In the present study, we engineered a red tilapia by mutation of hps5 using CRISPR/Cas9 gene editing of a target site in exon 2. Disruption of HPS5 led to a significant decrease in the numbers of melanophores and iridophores, and a significant increase in xanthophores, which led to a yellowish-transparent body color in early stages (5–30 dpf, days post fertilization). Slow recovery of iridophore numbers, and increased numbers of xanthophores with shorter nearest-neighbor distances than in wild-type fish was observed at 150 dpf, which finally led to a red tilapia with reddish pigmentation in fins. The hps5−/− mutants also showed several transparent cracks (absence of melanin, iridophores and xanthophores) in iris development. Besides, hps5 was also found to be fundamental for xanthophore development, and even the distance between each of them. Our hps5 mutants provide an excellent new model for studies of HPS5 function. Additionally, the red tilapia mutants may also have potential to serve as new germplasm for aquaculture, or function as a gene resource for genetic modification and breeding of red tilapia and the other related ornamental and food fish in aquaculture. More importantly, this study may have significant values in the area of development and evolution of pigmentation patterns of fish species. 

Abstract Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to the loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using the skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment, and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia. 

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identifiedamhy,dmrt1,gsdfas male andfoxl2,foxl3,cyp19a1aas female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads ofdmrt1;cyp19a1adouble mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads ofdmrt1;cyp19a1a;cyp19a1btriple mutants still developed as ovaries. The gonads offoxl3;cyp19a1adouble mutants developed as testes, while the gonads ofdmrt1;cyp19a1a;foxl3triple mutants eventually developed as ovaries. In contrast, the gonads ofamhy;cyp19a1a,gsdf;cyp19a1a,amhy;foxl2,gsdf;foxl2double andamhy;cyp19a1a;cyp19a1b,gsdf;cyp19a1a;cyp19a1btriple mutants developed as testes with spermatogenesis via up-regulation ofdmrt1in both somatic and germ cells. The gonads ofamhy;foxl3andgsdf;foxl3double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation ofdmrt1. Taking the respective ovary and underdeveloped testis ofdmrt1;foxl3anddmrt1;foxl2double mutants reported previously into consideration, we demonstrated that oncedmrt1mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other thandmrt1, including its upstreamamhyand downstreamgsdf, could be rescued by mutating female pathway gene. Overall, our data suggested thatdmrt1is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia. 

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1􀀀 /􀀀 fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1􀀀 /􀀀 fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1􀀀 /􀀀 fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species. 

Abstract Premelanosome protein (pmel) is a key gene for melanogenesis. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study, we found that most fishes have 2 pmel genes arising from the teleost-specific whole-genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypopigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela−/− mutants. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela−/− mutants, and also an increase in the number and size of xanthophores, producing a golden body color. The RPE pigmentation of pmela−/−;pmelb−/− was similar to pmela−/− mutants, with much less pigmentation than pmelb−/− mutants and wild-type fish. Taken together, our results indicate that, although both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.