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 Ϸ3 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.T he sexual fate of metazoans is determined genetically or by environmental factors, such as temperature. In the former case, heterogametic sex chromosomes determine the male (XY() or female (ZW&) fate in many species of vertebrates. In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY gene of the teleost fish medaka have been characterized as sex-determining genes that initiate testis formation, leading to male sexual development (1-5). In contrast, the molecular mechanism for the ZZ/ZW sex-determining system remains unclear, because no sexdetermining genes have been isolated.The Drosophila melanogaster doublesex (dsx) and Caenorhabditis elegans male abnormal (mab)-3 genes are known to control sexual development in these animals (6, 7). The two genes encode proteins containing a zinc finger-like DNA-binding motif called the DM domain. In vertebrates, the DM-domain gene DMRT1 is implicated in sexual development. In the mouse, DMRT1 is essential for postnatal testis differentiation (8, 9). In some other vertebrates, such as the chicken and turtle, DMRT1 expression is connected to testis formation in undifferentiated gonads (10-12). As mentioned above, the medaka fish gene DMY/Dmrt1bY, which is a coorthologue of DMRT1, causes testis formation as a sex-determining gene (3-5). In the chicken, which has the ZZ/ZW system, DMRT1 is located on the Z chromosome, suggesting that gene dosage may induce male development (...

The mitogen-activated protein kinase (MAPK) cascades, in which the major components are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK), are conserved eukaryotic signaling pathways (2, 7, 14, 46). The general function of the MAPK cascades is to link a variety of extracellular stimuli to nuclear responses, i.e., the modulation of gene expression (45). MAPK is activated by dual phosphorylation on threonine and tyrosine residues catalyzed by MAPKK, and MAPKK is activated by serine/threonine phosphorylation catalyzed by MAPKKK. In mammals, at least three MAPK cascades have been identified. The MAPKs in each pathway are ERK (extracellular signal-regulated kinase), JNK/SAPK (cJun N-terminal kinase/stress-activated protein kinase), and p38. The ERK cascade is mostly responsive to mitogenic and differentiation stimuli, whereas the JNK and p38 cascades are strongly activated by proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-␣), and extracellular stresses, such as UV irradiation and osmotic shock (4,23,29,36).In the ERK cascade, Raf (Raf-1, A-Raf, and B-Raf), MEK (MEK1 and MEK2), and ERK (ERK1 and ERK2) correspond to MAPKKK, MAPKK, and MAPK, respectively (36). The p38 cascade contains p38␣/CSBP/RK/Mxi2 (12,25,37,56) and p38␤ (18) as MAPKs and MKK3 (9) and MKK6 (6,13,32,35,42) as MAPKKs, while in the JNK cascade the MAPKs are JNK1, JNK2, and JNK3 (also known as SAPK␥, SAPK␣, and SAPK␤, respectively) (8,11,19,22,31), and the MAPKKs are SEK1/MKK4/JNKK1 (9,26, 38) and MKK7/JNKK2 (33,44,48). The specificity of the MAPKKKs involved in the JNK and p38 cascades is less clear. For instance, the TAK1 (52), ASK1 (16), and MLK3 (43) MAPKKKs can activate both the JNK and p38 cascades, while the MEKK1 (28, 50, 53) and MEKK4 (10) MAPKKKs selectively activate the JNK cascade.The identification of numerous components of the MAPK cascades as described above suggests that there are a number of these distinct signaling pathways in cells. Furthermore, studies of JNK3-deficient mice (54) indicate the existence of a JNK3-specific cascade that cannot be complemented by the other JNK family members, even though JNK1, JNK2, and JNK3 exhibit over 80% identity, and these JNKs seem to be

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SUMMARYA 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.

Seasonal hibernation in mammals is under a unique adaptation system that protects organisms from various harmful events, such as lowering of body temperature (Tb), during hibernation. However, the precise factors controlling hibernation remain unknown. We have previously demonstrated a decrease in hibernation-specific protein (HP) complex in the blood of chipmunks during hibernation. Here, HP is identified as a candidate hormone for hibernation. In chipmunks kept in constant cold and darkness, HP is regulated by an individual free-running circannual rhythm that correlates with hibernation. The level of HP complex in the brain increases coincident with the onset of hibernation. Such HP regulation proceeds independently of Tb changes in constant warmth, and Tb decreases only when brain HP is increased in the cold. Blocking brain HP activity using an antibody decreases the duration of hibernation. We suggest that HP, a target of endogenously generated circannual rhythm, carries hormonal signals essential for hibernation to the brain.