Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis)

Abstract

Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.

Figure 1. Microinjection method and transient expression of GFP in C. semilaevis.

(A) Schematic drawing of microinjection. (1) A micropipette is passed through the chorion at an approximately 45-degree angle; (2) a droplet which is 1/10~1/5 of the cell volume is injected into the cell. (B) Transient expression of GFP in 2 dah C. semilaevis. (a) Control fish; (b) large magnification of frame area in (a); (c) transient expression of GFP in the muscles; (d) large magnification of frame area in (c) muscle fibres strongly expressing of GFP are indicated by arrows. Scale bar, 100 μm.

Figure 2. Genomic structure of the C. semilaevis dmrt1 gene, design of dmrt1-TALENs and mutated sequences induced by TALENs.

(A) dmrt1-TALENs designed to target exon 1 of the gene. (B) Detection of mutations in the injected embryos by T7E I digestion. (C) Mutant dmrt1 sequences from TALEN injected embryos. The T7E I cut bands were recovered after gel electrophoresis and cloned for sequence analysis. “−” represents deletion of bp, “+” represents addition of bp. The numbers at the right side indicate the number of deleted or inserted base pairs.

Figure 3. Effects of dmrt1 disruption on gonad phenotype, sex differentiation.

(A) Gross morphology of gonads from approximately one year old fish. (1) dmrt1-deficient ‘testes’; (2) wild-type ovaries; (3) wild-type testes. (B) histology of gonads from approximately one year old fish. (a) dmrt1-deficient testis. The development of testis is ceased. The shape of the dmrt1-deficient testes in transverse sections is similar to control ovaries, and there are structures resembling ovarian cavity and ovarian lamella in the gonad of the mutant male fish. ovarian cavity-like (OCL), ovarian lamella-like (OLL); (b) large magnification of frame area in a. No secondary spermatocytes, spermatids and sperm are observed. oogonia-like (OGL), spermatogonia (SG) and primary spermatocytes (PSC). (c) Ovary of control female, including ovarian cavity (OC), ovarian lamella (OL); (d) large magnification of frame area in (c). Four stages of oocytes: stage I - IV and oogonia (OG). (e) Testis of control male. seminiferous lobuli (SL), seminiferous cyst (SC); (f) larger magnification of frame area in (e). Secondary spermatocytes (SSC), spermatids (ST) and sperm (SM). Scale bar is shown in the figures.

Figure 4. Gene expressions of sex differentiation markers on dmrt1-deficient gonad.

Dmrt1 and foxl2 are key transcription factors in testicular and ovarian differentiation, respectively. Cyp19a1a encodes for aromatase which is responsible for estrogen production. Sox9a and Amh are male related genes. Relative mRNA expression of dmrt1, foxl2, cyp19a1a, Sox9a and Amh in dmrt1-deficient gonads at one year of age from one dmrt1-deficient and three wild-type gonads. β-actin was used for calibration.

Figure 5. Growth parameters of dmrt1-deficient C. semilaevis.

(A) Weight of dmrt1-deficient male C. semilaevis (DDM) in 2014 (1 year old) was significantly higher than of wild-type males (WTM) and similar to that of wild-type females (WTF). (B) Body length and (C) width of dmrt1-deficient C. semilaevis were larger than of wild-type male and close to the values of females. (D) Weight of dmrt1-deficient male C. semilaevis (DDM) in 2015 (8 months old) was also higher than of wild-type males (WTM) and similar to that of wild-type females (WTF). (E) Body length and (F) width of dmrt1-deficient C. semilaevis were larger than of wild-type male and close to the values of females.

Figure 6. Effect of dmrt1 disruption on intersex gonad phenotype, sex differentiation and gene expression.

(A) Phenotype of gonads in the intersex. (a) Testis shaped up-side gonad of the intersex; (b) ovary shaped down-side mutant gonad of the intersex; (c) testes of a pseudomale. (B) Histology of the gonads. (a) Testis of the intersex showing normal male structures; (b) dmrt1 mutant gonad (ovary) of the intersex; (c) testis of a pseudomale. Scale bar, 100 μm. (C) Determination of the genetic sex of the intersex by SSR PCR yielding different sized products for the Z (169 bp) and W (134 bp) chromosomes. (D) Sequences of wild-type and mutated target sites of dmrt1 retrieved from ovary, partial; and only wild-type dmrt1 in testis, partial. (E) Relative mRNA expression of dmrt1, foxl2 and cyp19a1a in the dmrt1-deficient gonad at one year of age. β-actin was used as internal standard.

Figure 7. The Schedule of sampling.

Sexual differentiation happens at approximately 60-dph. The tissues for gonadal histology and gene expression studies were sampled at about approximately 1-yph. (I) 1-dph C. semilaevis. (II) 60-dph C. semilaevis. (III) 1-yph C. semilaevis. (A) Wild-type female C. semilaevis; (B) wild-type male C. semilaevis; (C) dmrt1-deficient male C. semilaevis.