A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis

Abstract

In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by approximately 3 kb of the 5'-flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tadpoles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis.

Fig. 1.

DM-W is a female genome-specific DM-domain gene in X. laevis. (A and B) Southern blot analysis of DMRT1 and DM-W. EcoRI-digested genomic DNA (20 μg) from X. laevis female and male liver was hybridized with the cDNA sequence corresponding to DMRT1 (amino acids 292–336) or DM-W (amino acids 124–194) as a probe. EF-1α (somatic) was used as a control. Numbers 1–3 correspond to the individual numbers. (C) Chromosomal localization of DM-W and DMRT1. Arrow indicates the fluorescence hybridization signal of DM-W (a) or DMRT1 (d). FISH patterns of DM-W are shown on PI-stained metaphase spreads of female (a) and male (c) X. laevis. Hoechst-stained pattern of the same metaphase spread as a is shown in b. The DM-W-located W chromosome was identified as chromosome 3 (16). FISH pattern of DMRT1 (d) and Hoechst-stained pattern (e) are shown on the same chromosomes, which corresponds to chromosomes 1 and 2 (16). (Scale bars, 10 μm.)

Fig. 2.

Structures of the DM-W gene and its protein. (A) Schematic drawing of DM-W and DMRT1. DM-W and DMRT1 have high identity, except in their C-terminal regions. The P/S domain is proline- and serine-rich. DM, a zinc finger-like DNA-binding motif called the DM domain. (B) Genomic structure of the DM-W gene. DM-W consists of 4 exons, and the sequences corresponding to the initiation and stop codons lie within exons 2 and 4, respectively, as indicated. W3k, the ≈3 kb of the 5′-flanking region (see GenBank/EBI Data Bank accession number AB365520) used to construct the DM-W expression vector, pW3k-DM-W (see Materials and Methods); FISH probe, ≈15 kb of the genomic sequence, including exons 2–4, used for FISH analysis. (C) Genotyping ZW and ZZ individuals by PCR using genomic DNA isolated from liver. Sequence information of primers used is described in Materials and Methods. Numbers 1–3 correspond to the individual numbers in Fig. 1A.

Fig. 3.

Expression profiles of DM-W and DMRT1 in the primordial gonads during the sex-determination period are shown. ZZ or ZW status was initially determined by PCR using genomic DNA from individuals, as described in Materials and Methods. (A) In situ hybridization of DM-W and DMRT1 during sex determination (st. 50 and 52). The arrowhead indicates the primordial gonads. (Scale bars, 0.5 mm.) (B) RT-PCR of DM-W and DMRT1 in primordial gonads at st. 48–59. Primordial gonads along with the mesonephros, from two ZZ and ZW tadpoles each at st. 48–52 were used (Left). The data from the same-stage ZZ and ZW individuals show similar expression levels. Because the primordial gonad without the mesonephros is too small to isolate sufficient RNA for RT-PCR at st. 48, RNA was isolated from five ZZ or ZW gonads at st. 50–59 (Right). (C) A comparison of mRNA levels using a competitive RT-PCR method for DM-W and DMRT1 performed during and after sex determination. Total RNA was extracted from the gonad/mesonephros at st. 48–52 or from gonads at st. 53–59, respectively. RT-PCR was performed for each of two ZZ or ZW tadpoles at each stage, which confirmed that there were few individual differences. Lane C shows a control PCR in which the same amounts of pcDNA-FLAG-DM-W and pcDNA-FLAG-DMRT1 were used.

Fig. 4.

Analysis of ZZ transgenic tadpoles carrying an expression vector for DM-W. (A) RT-PCR of DM-W of st. 50 tadpole gonads, including normal ZW gonads and transgenic gonads carrying pW3k-DM-W. Genomic PCRs for transgene insertion and to determine genetic sex are shown in the upper two panels. (B) Sections of normal developing ovary and testis (Left) and developing gonads with both testicular and ovarian structures (ovotestes) from transgenic ZZ tadpoles carrying pW3k-DM-W or pcDNA3-FLAG-DM-W (Right) at st. 56. OC, ovarian cavity; PO, primary oocyte; Sg, spermatogonium; Og, oogonium. Note that Sg and Og were morphologically indistinguishable. (Scale bars, 20 μm.) The ZZ transgenic tadpole, ZZ#3 or ZZ#8, carrying the DM-W expression vector pW3k-DM-W and pcDNA3-FLAG-DM-W, respectively, showed ovotestes. (C) RT-PCR of DM-W and DMRT1 in normal gonads and transgenic gonads carrying pW3k-DM-W or pcDNA3-FLAG-DM-W, from st. 56 tadpoles. Genomic PCRs for transgene insertion and genetic sex are shown in the upper two panels. ZZ#1–ZZ#8 correspond to individual transgenic ZZ tadpoles described in the text, and ZZ#3 and ZZ#8 correspond to individual animals in B.