A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis Shin Yoshimoto*, Ema Okada*, Hirohito Umemoto*, Kei Tamura*, Yoshinobu Uno†, Chizuko Nishida-Umehara†, Yoichi Matsuda†, Nobuhiko Takamatsu*, Tadayoshi Shiba*, and Michihiko Ito*‡ *Department of Bioscience, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan; and †Division of Genome Dynamics, Creative Research Initiative ‘‘Sousei,’’ Hokkaido University, North10 West8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan Communicated by Satoshi Omura,¯ The Kitasato Institute, Tokyo, Japan, December 27, 2007 (received for review September 11, 2007) In the XX/XY sex-determining system, the Y-linked SRY genes of In some species of amphibians, sex determination is controlled most mammals and the DMY/Dmrt1bY genes of the teleost fish genetically (13), even though the animals’ sex chromosomes are medaka have been characterized as sex-determining genes that morphologically indistinguishable from the autosomes. The trigger formation of the testis. However, the molecular mechanism South African clawed frog Xenopus laevis uses the ZZ/ZW of the ZZ/ZW-type system in vertebrates, including the clawed frog system, which was demonstrated by backcrosses between sex- Xenopus laevis, is unknown. Here, we isolated an X. laevis female reversed and normal individuals (14), but its sex chromosomes genome-specific DM-domain gene, DM-W, and obtained molecular have not yet been identified. Moreover, as with other animals evidence of a W-chromosome in this species. The DNA-binding that use the ZZ/ZW system, no sex-determining gene(s) has domain of DM-W showed a strikingly high identity (89%) with that been identified. We recently showed that X. laevis DMRT1 is of DMRT1, but it had no significant sequence similarity with the expressed during embryogenesis and is then restricted to the transactivation domain of DMRT1. In nonmammalian vertebrates, primordial gonads. Furthermore, our in vitro experiments DMRT1 expression is connected to testis formation. We found showed that the C-terminal region of DMRT1 is a transactiva- DMRT1 or DM-W to be expressed exclusively in the primordial tion domain (15). Here, we report the isolation of a W-linked gonads of both ZZ and ZW or ZW tadpoles, respectively. Although paralogue of DMRT1, DM-W,inX. laevis. Although the DNA- DMRT1 showed continued expression after sex determination, binding domains of DM-W and DMRT1 shared high sequence DM-W was expressed transiently during sex determination. Inter- identity (89%), their C-terminal regions had no significant estingly, DM-W mRNA was more abundant than DMRT1 mRNA in sequence similarity. A comparative analysis of the DM-W and the primordial gonads of ZW tadpoles early in sex determination. DMRT1 mRNA expression patterns showed that DM-W was To assess the role of DM-W, we produced transgenic tadpoles expressed predominantly in the primordial ZW gonads during carrying a DM-W expression vector driven by Ϸ3kbofthe5؅- early sex determination. These findings and phenotypic analyses flanking sequence of DM-W or by the cytomegalovirus promoter. of transgenic animals carrying DM-W expression vectors indi- DM W Importantly, some developing gonads of ZZ transgenic tadpoles cated that the W-linked gene, - , is a probable sex (female)- determining gene in X. laevis. showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Results Taken together, these results suggest that DM-W is a likely sex Isolation of X. laevis Female Genome-Specific Gene DM-W. To clarify (ovary)-determining gene in X. laevis. the role of DM-domain genes in the sex-determining system of X. laevis DMRT1 ͉ ͉ ͉ ͉ , we previously isolated the cDNA (15). Because FISH sex determination transgenic W-chromosome ZZ/ZW medaka DMY/Dmrt1bY is located on the Y chromosome and chicken DMRT1 is located on the Z chromosome (3, 4, 10, 11), he sexual fate of metazoans is determined genetically or by we next examined whether DMRT1 or its putative homologues Tenvironmental factors, such as temperature. In the former were linked to the X. laevis sex chromosomes by performing case, heterogametic sex chromosomes determine the male Southern blot analyses of the genomic DNAs from adult females (XY() or female (ZW&) fate in many species of vertebrates. In and males. Using the sequence encoding amino acids 292–336 of the XX/XY sex-determining system, the Y-linked SRY genes of DMRT1, which does not include the DM domain, as a probe, we most mammals and the DMY/Dmrt1bY gene of the teleost fish detected bands of Ϸ4.0 kb in both males and females. However, medaka have been characterized as sex-determining genes that no specific bands in the female genome or doubly dense bands initiate testis formation, leading to male sexual development in the male genome were observed, suggesting that DMRT1 was BIOLOGY (1–5). In contrast, the molecular mechanism for the ZZ/ZW autosomal (Fig. 1A). Interestingly, the full-length cDNA probe, DEVELOPMENTAL sex-determining system remains unclear, because no sex- containing the DM-domain sequence, hybridized with an 8.0-kb determining genes have been isolated. DNA fragment only in samples from females, as well as with the The Drosophila melanogaster doublesex (dsx) and Caenorhabditis 4.0-kb bands in both sexes (data not shown). elegans male abnormal (mab)-3 genes are known to control sexual We cloned the 8.0-kb fragment by screening an X. laevis development in these animals (6, 7). The two genes encode proteins female genomic library with the full-length DMRT1 cDNA. This containing a zinc finger-like DNA-binding motif called the DM domain. In vertebrates, the DM-domain gene DMRT1 is implicated Author contributions: S.Y., Y.M., N.T., T.S., and M.I. designed research; S.Y., E.O., H.U., Y.U., in sexual development. In the mouse, DMRT1 is essential for C.N.-U., and M.I. performed research; K.T. contributed new reagents/analytic tools; S.Y. and postnatal testis differentiation (8, 9). In some other vertebrates, M.I. analyzed data; and S.Y. and M.I. wrote the paper. such as the chicken and turtle, DMRT1 expression is connected to The authors declare no conflict of interest. testis formation in undifferentiated gonads (10–12). As mentioned Data deposition: The sequences reported in this paper have been deposited in the GenBank above, the medaka fish gene DMY/Dmrt1bY, which is a coortho- database (accession nos. AB259777 and AB365520). logue of DMRT1, causes testis formation as a sex-determining gene ‡To whom correspondence should be addressed. E-mail: [email protected]. (3–5). In the chicken, which has the ZZ/ZW system, DMRT1 is This article contains supporting information online at www.pnas.org/cgi/content/full/ located on the Z chromosome, suggesting that gene dosage may 0712244105/DC1. induce male development (10, 11). © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712244105 PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2469–2474 Downloaded by guest on September 29, 2021 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 Ϸ3kbofthe5Ј-flanking region (see GenBank/EBI Data Fig. 1. DM-W is a female genome-specific DM-domain gene in X. laevis.(A and Bank accession number AB365520) used to construct the DM-W expression B) Southern blot analysis of DMRT1 and DM-W. EcoRI-digested genomic DNA (20 vector, pW3k-DM-W (see Materials and Methods); FISH probe, Ϸ15 kb of the ␮ g) from X. laevis female and male liver was hybridized with the cDNA sequence genomic sequence, including exons 2–4, used for FISH analysis. (C) Genotyping corresponding to DMRT1 (amino acids 292–336) or DM-W (amino acids 124–194) ZW and ZZ individuals by PCR using genomic DNA isolated from liver. Se- ␣ as a probe. EF-1 (somatic) was used as a control. Numbers 1–3 correspond to the quence information of primers used is described in Materials and Methods. individual numbers. (C) Chromosomal localization of DM-W and DMRT1. Arrow Numbers 1–3 correspond to the individual numbers in Fig. 1A. indicates the fluorescence hybridization signal of DM-W (a)orDMRT1 (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 somes, chromosomes 1 and 2 (Fig. 1Cd and Ce). The duplicate shown in b. The DM-W-located W chromosome was identified as chromosome 3 signals most likely reflect the duplication of a chromosomal pair (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, during tetraploidization (16). 10 ␮m.) DM-W Is a Paralogue of DMRT1. DM-W consisted of 194 aa residues, and its DM domain (amino acids 20–86) had a strik- screen revealed another DM domain-encoding sequence, cor- ingly high identity (89%) with that of DMRT1 [Fig. 2A and responding to amino acids 75–129 of DMRT1 and with high supporting information (SI) Fig. 5]. In contrast, the DM domains identity to the DM domain of DMRT1.