Medaka Mutant Research Articles

Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

BackgroundMedaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.ResultsWe report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.ConclusionWith its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.

Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements

BackgroundSex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination.ResultsWe now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal.ConclusionsOur work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs) as a model of PTEN deficiency disease.

Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty

Although estrogens have been generally considered to play a critical role in ovarian differentiation in non-mammalian vertebrates, the specific functions of estrogens during ovarian differentiation remain unclear. We isolated two mutants with premature stops in the ovarian aromatase (cyp19a1) gene from an N-ethyl-N-nitrosourea-based gene-driven mutagenesis library of the medaka, Oryzias latipes. In XX mutants, gonads first differentiated into normal ovaries containing many ovarian follicles that failed to accumulate yolk. Subsequently, ovarian tissues underwent extensive degeneration, followed by the appearance of testicular tissues on the dorsal side of ovaries. In the newly formed testicular tissue, strong expression of gsdf was detected in sox9a2-positive somatic cells surrounding germline stem cells suggesting that gsdf plays an important role in testicular differentiation during estrogen-depleted female-to-male sex reversal. We conclude that endogenous estrogens synthesized after fertilization are not essential for early ovarian differentiation but are critical for the maintenance of adult ovaries.

Molecular basis of social competence in medaka fish.

Oryzias latipes (Medaka) is an established vertebrate model for studying developmental genetics, genomics, and evolutionary biology. The physiology, embryology, and genetics of this species have been extensively investigated for centuries. Medaka fish recently attracted attention in the field of social neuroscience. This review introduces recent advances in medaka behavioral studies, focusing on female mating preferences and male mate-guarding behaviors. The medaka female has the ability to discriminate male individuals and prefers to mate with socially familiar males (female mating preference). In triadic relationships (two males and one female), the dominant male remains closer to the female and repels the other male (mate-guarding). Interestingly, mate-guarding blocks female social familiarization of the rival male, which can increase the mating success of the dominant male. Importantly, behavioral analyses using a series of medaka mutants revealed critical roles of neuropeptide neuromodulatory systems in regulating their social behaviors. The extra-hypothalamic gonadotropin-releasing hormone system has a central role in activating female mating preference. The arginine-vasotocin system is required for the emergence of mate-guarding behavior.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

Small teleost fish provide new insights into human skeletal diseases.

Small teleost fish such as zebrafish and medaka are increasingly studied as models for human skeletal diseases. Efficient new genome editing tools combined with advances in the analysis of skeletal phenotypes provide new insights into fundamental processes of skeletal development. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. Several unique features of the skeleton relate to the extremely small size of early fish embryos and the small size of adult fish used as models. A detailed analysis of the plethora of interesting skeletal phenotypes in zebrafish and medaka pushes available skeletal imaging techniques to their respective limits and promotes the development of new imaging techniques. Impressive numbers of zebrafish and medaka mutants with interesting skeletal phenotypes have been characterized, complemented by transgenic zebrafish and medaka lines. The advent of efficient genome editing tools, such as TALEN and CRISPR/Cas9, allows to introduce targeted deficiencies in genes of model teleosts to generate skeletal phenotypes that resemble human skeletal diseases. This review will also discuss other attractive aspects of the teleost skeleton. This includes the capacity for lifelong tooth replacement and for the regeneration of dermal skeletal elements, such as scales and fin rays, which further increases the value of zebrafish and medaka models for skeletal research.

Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation

Purpose: Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24 hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24 h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation.Materials and methods: Sexually mature wild-type, WT (Hd-rR) and p53(−/−) adult female medaka (O. latipes) were exposed to 4 Gy dose of γ-IR and sampling were collected after 4, 8 and 24 h and 14 days.Results: Apoptosis and morphological alterations were observed from 4 h after irradiation and remarkably increased 8 h after irradiation in the wild-type. Apoptotic cell death has been observed 8 h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation.Conclusions: We conclude that apoptosis and malformations caused by 4 Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4–24 h and the initial response until 8 h was p53-dependent.

Disruption of dmc1 Produces Abnormal Sperm in Medaka (Oryzias latipes).

DMC1 is a recombinase that is essential for meiotic synapsis. Experiments in extensive species of eukaryotes have indicated the independent role of DMC1 in repairing double strand breaks (DSBs) produced during meiosis I. Mutation of dmc1 in mice and human often leads to obstacles in spermatogenesis and male sterility. Here, we report on the disruption of dmc1 in male medaka (Oryzias latipes). Synapsis was disturbed in the mutant medaka testis nuclei, as observed in mice and other organisms. Unexpectedly, the mutant medaka could produce a few sperm and, although most of these had multiple tail or multiple head malformations, some of them could swim, and few of them even had insemination ability. Our transcriptome analysis showed that there was not a remarkable change in the expression of most of the genes involved in the pathways associated with the meiotic DNA repair and flagella assembly. Our results provided an indication of the accessory mechanisms that might be involved in the repair of DSBs during meiosis. In a species besides humans, we provided evidence that disorders in meiosis recombination might lead to the malformation of sperm.

High levels of plasma cortisol and impaired hypoosmoregulation in a mutant medaka deficient in P450c17I

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na+/K+/2Cl− cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na+/K+/2Cl− cotransporter, in seawater.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

Early life exposure to a rodent carcinogen propiconazole fungicide induces oxidative stress and hepatocarcinogenesis in medaka fish

Conazole pollution is an emerging concern to human health and environmental safety because of the broad use of conazole fungicides in agriculture and medicine and their frequent occurrence in aquifers. The agricultural pesticide propiconazole has received much regulatory interest because it is a known rodent carcinogen with evidence of multiple adverse effects in mammals and non-targeted organisms. However, the carcinogenic effect and associated mechanism of propiconazole in fish under microgram-per-liter levels of environmental-relevant exposure remains unclear. To explore whether early life of propiconzaole exposure would induce oxidative stress and latent carcinogenic effects in fish, we continuously exposed larvae of wild type or p53−/− mutant of medaka fish (Oryzias latipes) to propiconazole (2.5–250μg/L) for 3, 7, 14 or 28 days and assessed liver histopathology and/or the oxidative stress response and gene expression during exposure and throughout adulthood. Propiconazole dose-dependently induced reactive oxygen species (ROS) level, altered homeostasis of antioxidant superoxide dismutase, catalase and glutathione S-transferase and caused lipid and protein peroxidation during early life exposure in wild type medaka. Such exposure also significantly upregulated gene expression of the cytochrome P450 CYP1A, but marginally suppressed that of tumor suppressor p53 in adults. Furthermore, histopathology revealed that p53−/− mutant medaka with early life exposure to propiconazole showed increased incidence of hepatocarcionogensis, as compared to the p53−/− control group and wild type strain. We demonstrated that propiconazole can initiate ROS-mediated oxidative stress and induce hepatic tumorigenesis associated with CYP1A- and/or p53 -mediated pathways with the use of wild type and p53−/− mutant of medaka fish. The toxic response of medaka to propiconazole is compatible with that observed in rodents.

Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development

Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene.

Proliferation following tetraploidization regulates the size and number of erythrocytes in the blood flow during medaka development, as revealed by the abnormal karyotype of erythrocytes in the medaka TFDP1 mutant.

For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow. By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant. From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development.

Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

The medaka dhc2 mutant reveals conserved and distinct mechanisms of Hedgehog signaling in teleosts

BackgroundPrimary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction.ResultsTo precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling.ConclusionsCiliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0057-x) contains supplementary material, which is available to authorized users.

Loss of follicle-stimulating hormone receptor function causes masculinization and suppression of ovarian development in genetically female medaka.

FSH, a glycoprotein hormone, is circulated from the pituitary and functions by binding to a specific FSH receptor (FSHR). FSHR is a G protein-coupled, seven-transmembrane receptor linked to the adenylyl cyclase or other pathways and is expressed in gonadal somatic cells. In some nonmammalian species, fshr expression is much higher in the ovary than in the testis during gonadal sex differentiation, suggesting that FSHR is involved in ovarian development in nonmammalian vertebrates. However, little is known of FSHR knockout phenotypes in these species. Here we screened for fshr mutations by a medaka (Oryzias latipes) target-induced local lesion in the genomes and identified one nonsense mutation located in the BXXBB motif, which is involved in G protein activation. Next, we used an in vitro reporter gene assay to demonstrate that this mutation prevents FSHR function. We then analyzed the phenotypes of fshr mutant medaka. The fshr mutant male medaka displayed normal testes and were fertile, whereas the mutant female fish displayed small ovaries and were infertile because vitellogenesis was inhibited. The mutant females also have suppressed expression of ovary-type aromatase (cyp19a1a), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, resulting in decreased 17β-estradiol levels. Moreover, loss of FSHR function caused female-to-male sex reversal in some cases. In addition, the transgenic overexpression of fshr in fshr mutants rescued FSHR function. These findings strongly suggest that in the medaka, FSH regulates the ovarian development and the maintenance mainly by the elevation of estrogen levels. We present the first FSHR knockout phenotype in a nonmammalian species.

Eda/Edar signaling guides fin ray formation with preceding osteoblast differentiation, as revealed by analyses of the medaka all‐fin less mutant afl

Ectodysplasin (Eda) signaling is essential for the morphogenesis of several ectodermal appendages. Here, we report a medaka mutant, all-fin less (afl), which has a nonsense mutation in its eda gene. The adult afl fish displayed various abnormalities of its dermal skeleton, such as short and twisted fin rays, missing and abnormally shaped scales and teeth, and skull deformation. Focusing on the developing fin rays in the caudal region of afl larvae, we found that the fin rays did not elongate; although the initial formation of fin rays proceeded normally. Additionally, eda expression was lost, and the expression pattern of edar, the gene for the receptor of Eda, was different from wild-type one. In vivo imaging of the double-transgenic medaka expressing enhanced green fluorescent protein under control of the edar promoter and DsRed under control of the osterix promoter revealed that edar expression preceded that of osterix and that the edar-expressing cells migrated in the direction of fin ray elongation, indicating that the Eda/Edar signaling event precedes osteoblast differentiation. Our findings provide evidence that Eda signaling accompanied with the binding of Eda to Edar are essential for fin ray formation guided by cell migration.

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.