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Development
2010 Aug 01;13715:2519-26. doi: 10.1242/dev.048751.
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Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system.
Yoshimoto S
,
Ikeda N
,
Izutsu Y
,
Shiba T
,
Takamatsu N
,
Ito M
.
Abstract
A Y-linked gene, DMY/dmrt1bY, in teleost fish medka and a Z-linked gene, DMRT1, in chicken are both required for male sex determination. We recently isolated a W-linked gene, DM-W, as a paralogue of DMRT1 in Xenopus laevis, which has a ZZ/ZW-type sex-determining system. The DNA-binding domain of DM-W shows high sequence identity with that of DMRT1, but DM-W has no significant sequence similarity with the transactivation domain of DMRT1. Here, we first show colocalization of DM-W and DMRT1 in the somatic cells surrounding primordial germ cells in ZW gonad during sex determination. We next examined characteristics of DM-W and DMRT1 as a transcription factor in vitro. DM-W and DMRT1 shared a DNA-binding sequence. Importantly, DM-W dose-dependently antagonized the transcriptional activity of DMRT1 on a DMRT1-driven luciferase reporter system in 293 cells. We also examined roles of DM-W or DMRT1 in gonadal formation. Some transgenic ZW tadpoles bearing a DM-W knockdown vector had gonads with a testicular structure, and two developed into frogs with testicular gonads. Ectopic DMRT1 induced primary testicular development in some ZW individuals. These observations indicated that DM-W and DMRT1 could have opposite functions in the sex determination. Our findings support a novel model for a ZZ/ZW-type system in which DM-W directs female sex as a sex-determining gene, by antagonizing DMRT1. Additionally, they suggest that DM-W diverged from DMRT1 as a dominant-negative type gene, i.e. as a ;neofunctionalization' gene for the ZZ/ZW-type system. Finally, we discuss a conserved role of DMRT1 in testis formation during vertebrate evolution.
Fig. 1. Colocalization of the DM-W and DMRT1 proteins in somatic cells surrounding the PGCs of the ZW gonad at stage 50. (A) In situ hybridization analyses of DM-W, DMRT1 and XVLG-1 in cross-sections of primordial ZW (upper) or ZZ (lower) gonads at stage 50. Asterisks indicate primordial germ cells (PGCs). Scale bars: 5 μm. (B) Western blot analysis using the anti-DM-W and anti-DMRT1 antibodies. 293T cells were transiently transfected with pcDNA3-FLAG-DM-W or pcDNA3-FLAG-DMRT1. The extracts of the 293 cells (left) and primordial ZZ and ZW gonads (stages 50-52) (right) were examined by immunoblotting with each antibody followed by an HRP-conjugated anti-rabbit antibody. DM-W was only detected in the ZW samples. (C) Immunofluorescence analysis using anti-DM-W and anti-DMRT1 antibodies on cross-sections of primordial ZW gonads at stage 50. Scale bars: 2 μm. Asterisk indicates a PGC.
Fig. 3. Analyses of transgenic X. laevis individuals carrying the knockdown vector for DM-W. (A) Sections of ovarian gonads from two non-transgenic and two transgenic (knockdown) ZW tadpoles carrying psh-DM-W1 and -W2, at stage 56. GC, germ cell; OC, ovarian cavity. Scale bars: 20 μm. (B) Non-transgenic normal gonads (left), and underdeveloped ovaries (middle) and testicular gonads (right) of transgenic ZW frogs six months after metamorphosis. The gonad is within the dotted line. MS, mature sperm; OC, ovarian cavity; Oo, oocyte; Sc, spermatocyte; Sg, spermatogonium. Scale bars: 0.5 mm (upper); 20 μm (middle and lower). (C,D) RT-PCR of gonadal marker genes in normal ZW ovary, ZZ testis and transgenic ZW (#3-#5) gonads. ZW#3-#5 correspond to the individual animals in B. Genomic PCRs for the transgene insertion and genetic sex are shown in the upper two panels. The data in C and D are from two or one non-transgenic ZZ and ZW individuals, and three or one transgenic ZW individuals. The marker genes were as follows. VLG-1, germ cells; EF-1O, oocytes; Sox3 and P450cArom, ovary-specific cells; DMRT1 and Sox9, testis-specific cells; megs and SP4, spermatogenic cells; ODC-1 and EF-1S, control.
Fig. 4. Analysis of transgenic ZW frogs carrying an expression vector for DMRT1. Non-transgenic normal ZZ and ZW gonads (left), and transgenic ovotestis (middle and right) carrying pcDNA3-FLAG-DMRT1, 1 month after metamorphosis, are shown. Genomic PCRs for transgene insertion and genetic sex are shown in the upper two panels. The gonad is inside the dotted line. The asterisks indicate germ cells. Scale bars: 0.5 mm (upper); 20 μm (lower).
