Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use ...positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.
Sex chromosomes and the sex-determining (SD) gene are variable in vertebrates. In particular, medaka fishes in the genus Oryzias show an extremely large diversity in sex chromosomes and the SD gene, ...providing a good model to study the evolutionary process by which they turnover. Here, we investigated the sex determination system and sex chromosomes in six celebensis group species. Our sex-linkage analysis demonstrated that all species had an XX-XY sex determination system, and that the Oryzias marmoratus and O. profundicola sex chromosomes were homologous to O. latipes linkage group (LG) 10, while those of the other four species, O. celebensis, O. matanensis, O. wolasi, and O. woworae, were homologous to O. latipes LG 24. The phylogenetic relationship suggested a turnover of the sex chromosomes from O. latipes LG 24 to LG 10 within this group. Six sex-linkage maps showed that the former two and the latter four species shared a common SD locus, respectively, suggesting that the LG 24 acquired the SD function in a common ancestor of the celebensis group, and that the LG 10 SD function appeared in a common ancestor of O. marmoratus and O. profundicola after the divergence of O. matanensis. Additionally, fine mapping and association analysis in the former two species revealed that Sox3 on the Y chromosome is a prime candidate for the SD gene, and that the Y-specific 430-bp insertion might be involved in its SD function.
Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among ...lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene
in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of
in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in
copies, indicating its key role in freshwater colonization.
Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine ...model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.
•The cyp1a gene of Oryzias javanicus was knocked out using CRISPR/Cas 9.•An environmental DNA (eDNA) extraction method was employed for genotyping.•The cyp1a-knockout strain (KO) is more sensitive to pyrene than wild-type fish (WT).•The KO strain is less sensitive to phenanthrene than WT.•Combined use of WT and KO fish reveals different modes of pollutant toxicity.
To clarify the basal aspects of sexual development in Javafish medaka, Oryzias javanicus (ZZ/ZW), a model marine species for ecotoxicity testing, we examined the details of gonadal sex ...differentiation and exogenous sex hormone-dependent sex reversals using genetic sex-linked DNA markers. Sex differences in germ cell numbers were observed at 5 days post hatching (dph), in which there was a significant increase in the germ cells of ZW. In ZW, diplotene oocytes and the ovarian cavity appeared at approximately 10, and 30 dph, respectively. In ZZ, spermatogonial proliferation was observed at approximately 20 dph. A ZZ-dominant expression of Gonadal soma-derived factor (Gsdf) mRNA was detected before hatching. The exposure of embryos to 17α-ethinylestradiol (EE2; 0.1, 1, 10 ng/mL) did not cause sex reversals in most cases. However, EE2 exposures led to significant Choriogenin-H (ChgH) mRNA expression, an estrogen up-regulated gene, in all fry; these exposures did not suppress Gsdf expression in ZZ fry. The exposure of embryos to 17α-methyltestosterone (MT; 0.1, 1, 10 ng/mL) caused sex reversals but only at low frequencies in ZW and ZZ fish. Although the 10 ng/mL MT exposure was accompanied by induction of significant Gsdf expression in ZW fry, induction of ChgH expression was also observed in several fry. Together, the present study indicates for the first time that male-dominant sexual dimorphic expression of Gsdf precedes the first morphological sex difference, i.e., the sex difference in germ cell number, and results strongly suggest that exogenous sex hormone-dependent sex reversal is not induced easily in O. javanicus.
•Gonadal differentiation was examined in Oryzias javanicus (ZZ/ZW).•ZZ dominant expression of Gsdf precedes the first morphological sex differences.•ChgH/Gsdf mRNA expression was used to assess the impacts of sex hormones on sexuality.•Exogenous sex hormones do not cause sex reversals easily.
Cytochrome P450 1A (Cyp1a) catabolizes polycyclic organic compounds. In this study, we sequenced the
cyp1a
gene from two Javanese medaka
Oryzias javanicus
strains and examined gene expression in ...response to two organic pollutants, phenanthrene and pyrene. Sequence comparisons among the two strains identified several single-nucleotide polymorphisms (SNPs), two of which resulted in amino acid substitutions. Reverse transcription PCR analysis of major organs revealed that the
cyp1a
mRNA level is highest in the liver. After determining LC
50
s against phenanthrene and pyrene for 96 h (3.9 and 2.5 µM, respectively),
cyp1a
mRNA expression in the liver against sublethal concentrations of phenanthrene and pyrene was examined. Using quantitative real-time PCR, a positive correlation was discovered between the
cyp1a
mRNA levels and pyrene concentration, and similar tendency was observed for phenanthrene.
cyp1a
may serve as a quantitative indicator of a broad range of pollutants.
Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of ...tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.
•CRISPR/Cas9 effectively introduced site-directed mutations in the firebrat Thermobia domestica.•CRISPR/Cas9-induced mutations are successfully transferred to following generations.•This study reports the first successful genome editing in an apterygote insect.
See-through medaka lines are suitable for observing internal organs throughout life. They were bred by crossing multiple color mutants. However, some of the causal genes for these mutants have not ...been identified. The medaka has four pigment cell types: black melanophores, yellow xanthophores, white leucophores, and silvery iridophores. The causal genes of melanophore, xanthophore, and leucophore mutants have been elucidated, but the causal gene for the iridophore mutant remains unknown. Here, we describe the iridophore mutant,
(
), which exhibits a strong reduction in visible iridophores throughout its larval to adult stages. The
locus was previously mapped to chromosome 5, but was located near the telomeric region, making it difficult to integrate into the chromosome. We sought the causal gene of
using synteny analysis with the zebrafish genome and found a strong candidate,
(
). Gene targeting and complementation testing showed that
is the causal gene of
This result will allow the establishment of inbred medaka strains or other useful strains with see-through phenotypes without major disruption in the genetic background of each strain.
The medaka Oryzias latipes and its two sister species, O. curvinotus and O. luzonensis, possess an XX-XY sex-determination system. The medaka sex-determining gene DMY has been identified on the ...orthologous Y chromosome O. latipes linkage group 1 (LG1) of O. curvinotus. However, DMY has not been discovered in other Oryzias species. These results and molecular phylogeny suggest that DMY was generated recently approximately 10 million years ago (MYA) by gene duplication of DMRT1 in a common ancestor of O. latipes and O. curvinotus. We identified seven sex-linked markers from O. luzonensis (sister species of O. curvinotus) and constructed a sex-linkage map. Surprisingly, all seven sex-linked markers were located on an autosomal linkage group (LG12) of O. latipes. As suggested by the phylogenetic tree, the sex chromosomes of O. luzonensis should be "younger" than those of O. latipes. In the lineage leading to O. luzonensis after separation from O. curvinotus approximately 5 MYA, a novel sex-determining gene may have arisen and substituted for DMY. Oryzias species should provide a useful model for evolution of the master sex-determining gene and differentiation of sex chromosomes from autosomes.
Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 ...causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.
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