Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. Here, we used recombinant breakpoint analysis to restrict the sex-determining region in medaka fish (Oryzias latipes) to a 530-kilobase (kb) stretch of the Y chromosome. Deletion analysis of the Y chromosome of a congenic XY female further shortened the region to 250 kb. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only the DM-related PG17 was Y specific; we thus named it DMY. Two naturally occurring mutations establish DMY's critical role in male development. The first heritable mutant--a single insertion in exon 3 and the subsequent truncation of DMY--resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. During normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for testicular development and is a prime candidate for the medaka sex-determining gene.

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that Gsdf Y (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that Gsdf Y showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included Gsdf Y converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of Gsdf Y contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sexdetermining gene in O. luzonensis. IN most vertebrates, sex is determined genetically. Mammals and birds with cytogenetically well-differentiated sex chromosomes have sex determination systems that differ between the taxonomic classes but not within them (Solari 1994). In mammals, for example, the sex-determining (SD) gene SRY/Sry on the Y chromosome has a universal role in sex determination (Gubbay et al. 1990;Sinclair et al. 1990;Koopman et al. 1991;Foster et al. 1992). By contrast, some fish groups, such as salmonids, sticklebacks, and Oryzias fishes, have sex chromosomes that differ among closely related species (Devlin and Nagahama 2002;Woram et al. 2003;Takehana et al. 2007a;Ross et al. 2009).A DM-domain gene, Dmy, was the first SD gene identified in a nonmammalian vertebrate, the fish medaka Oryzias latipes (Matsuda et al. 2002(Matsuda et al. , 2007. In this species, the term Y chromosome is employed to refer to a recombining chromosome that carries the male-determining gene Dmy, and X is used for the homologous chromosome; these are not a heteromorphic pair. This gene is conserved among all wild populations of O. latipes examined to date . The closely related species O. curvinotus also has Dmy on its Y chromosome, which is orthologous to the O. latipes Y chromosome (Matsuda et al. 2003). However, Dmy has not been detected in any other type of fish, including other Oryzias fishes (Kondo et al. 2003). Analysis of the Y-specific region of the O. latipes sex chromosome has demonstrated that Dmy arose from duplication of the autosomal Dmrt1 gene (Nanda et al. 2002;Kondo et al. 2006). This Dmrt1 duplication is estimated to have occurred within the last 10 million years in a common ancestor of O. latipes, O. curvinotus, and O. luzonensis. In O. luzonensis, however, no functional duplicated copy of Dmrt1 has been detected (Kondo et al. 2003) (Figure 5A).O. luzonensis possesses an XX-XY system, which is homologous to an autosomal linkage group (LG 12) and Uwa 1985). In the d-rR strain, the wild-type alle...

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20 -30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation. Developmental Dynamics 231:518 -526, 2004.