The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other ...vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system.
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.
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 GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY 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 sex-determining gene in O. luzonensis.
Sexual selection drives rapid phenotypic diversification of mating traits. However, we know little about the causative genes underlying divergence in sexually selected traits. Here, we investigate ...the genetic basis of male mating trait diversification in the medaka fishes (genus Oryzias) from Sulawesi, Indonesia. Using linkage mapping, transcriptome analysis, and genome editing, we identify csf1 as a causative gene for red pectoral fins that are unique to male Oryzias woworae. A cis-regulatory mutation enables androgen-induced expression of csf1 in male fins. csf1-knockout males have reduced red coloration and require longer for mating, suggesting that coloration can contribute to male reproductive success. Contrary to expectations, non-red males are more attractive to a predatory fish than are red males. Our results demonstrate that integrating genomics with genome editing enables us to identify causative genes underlying sexually selected traits and provides a new avenue for testing theories of sexual selection.
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.
Medaka (
) has become an important vertebrate model widely used in genetics, developmental biology, environmental sciences, and many other fields. A high-quality genome sequence and a variety of ...genetic tools are available for this model organism. However, existing genome annotation is still rudimentary, as it was mainly based on computational prediction and short-read RNA-seq data. Here we report a dynamic transcriptome landscape of medaka embryogenesis profiled by long-read RNA-seq, short-read RNA-seq, and ATAC-seq. By integrating these data sets, we constructed a much-improved gene model set including about 17,000 novel isoforms and identified 1600 transcription factors, 1100 long noncoding RNAs, and 150,000 potential
-regulatory elements as well. Time-series data sets provided another dimension of information. With the expression dynamics of genes and accessibility dynamics of
-regulatory elements, we investigated isoform switching, as well as regulatory logic between accessible elements and genes, during embryogenesis. We built a user-friendly medaka omics data portal to present these data sets. This resource provides the first comprehensive omics data sets of medaka embryogenesis. Ultimately, we term these three assays as the minimum ENCODE toolbox and propose the use of it as the initial and essential profiling genomic assays for model organisms that have limited data available. This work will be of great value for the research community using medaka as the model organism and many others as well.
Sex is pivotal for reproduction, healthcare and evolution. In the fish medaka, the Y-chromosomal dmy (also dmrt1bY) serves the sex determiner, which activates dmrt1 for male sex maintenance. However, ...how dmy makes the male decision via initiating testicular differentiation has remained unknown. Here we report that autosomal gsdf serves a male sex initiator. Gene addition and deletion revealed that gsdf was necessary and sufficient for maleness via initiating testicular differentiation. We show that gsdf transcription is activated directly by dmy. These results establish the autosomal gsdf as the first male sex initiator. We propose that dmy determines maleness through activating gsdf and dmrt1 without its own participation in developmental processes of sex initiation and maintenance. gsdf may easily become a sex determiner or other autosomal genes can be recruited as new sex determiners to initiate gsdf expression. Our findings offer new insights into molecular mechanisms underlying sex development and evolution of sex-controlling genes in vertebrates.
The congenic strain, an inbred strain containing a small genomic region from another strain, is a powerful tool to assess the phenotypic effect of polymorphisms and/or mutations in the substituted ...genomic region. Recent substantial progress in the genetic studies of complex traits increases the necessity of congenic strains and, therefore, a quick breeding system for congenic strains has become increasingly important in model organisms such as mouse and medaka. Traditionally, more than ten generations are necessary to produce a congenic strain. In contrast, a quick method has been reported previously for the mouse, in which the use of genetic markers reduces the required number of backcross generations to about a half that of the traditional method, so that it would take around six generations to obtain a congenic strain. Here, we present an even quicker congenic production system, which takes only about four generations. The system can produce medaka congenic strains having part of the HNI-II (an inbred medaka strain derived from the northern Japanese population, Oryzias sakaizumii) genome in the HdrR-II1 (another inbred strain from the southern Japanese population, O. latipes) background. In this system, the availability of frozen sperm and genotype data of the BC1 male population makes it possible to start marker-assisted congenic production after obtaining the BC2 population. Our evaluation revealed that the system could work well to increase the percentage of recipient genome as expected, so that a congenic strain may be obtained in about one year.
Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic ...domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar V
/K
values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.
Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate ...choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are upregulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.
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