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How the timing of development is linked to organismal size is a longstanding question. Although numerous studies have reported a correlation of temporal and spatial traits, the developmental or selective constraints underlying this link remain largely unexplored. We address this question by studying the periodic process of embryonic axis segmentation in-vivo in Oryzias fish. Interspecies comparisons reveal that the timing of segmentation correlates to segment, tissue and organismal size. Segment size in turn scales according to tissue and organism size. To probe for underlying causes, we genetically hybridised two closely related species. Quantitative analysis in ~600 phenotypically diverse F2 embryos reveals a decoupling of timing from size control, while spatial scaling is preserved. Using developmental quantitative trait loci (dev QTL) mapping we identify distinct genetic loci linked to either the control of segmentation timing or tissue size. This study demonstrates that a developmental constraint mechanism underlies spatial scaling of axis segmentation, while its spatial and temporal control are dissociable modules. Synopsis: The molecular relationships that link developmental timing and organismal size remain underexplored. Here, a cross-species analysis in Oryzias fish measures the timing of embryonic body axis segmentation and overall organismal size, providing evidence that timing and size control are dissociable modules. Real-time imaging of the segmentation clock in F2 embryos from interspecies crosses of Oryzias fish reveals a broad phenotypic spectrum. F2 embryos exhibit a linear relationship between PSM and somite size, indicating that a developmental constraint mechanism underlies spatial scaling. developmental quantitative trait loci (dev QTL) analysis of F2 embryos identifies distinct genomic loci correlated with segmentation timing and tissue size, respectively. Interspecies comparison in rice fish (Orzyias spp.) shows that both the rate and size of embryonic segmentation increase in proportion to fish size but are regulated by independent mechanisms. [ABSTRACT FROM AUTHOR]

Background: The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. Results: We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. Conclusions: RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans. [ABSTRACT FROM AUTHOR]

Because speciation might have been promoted by ancient introgression from an extinct lineage, it is important to detect the existence of 'ghost introgression' in focal taxa and examine its contribution to their diversification. In this study, we examined possible ghost introgression and its contributions to the diversification of ricefishes of the genus Adrianichthys in Lake Poso, an ancient lake on Sulawesi Island, in which some extinctions are known to have occurred. Population‐genomic analysis revealed that two extant Adrianichthys species, A. oophorus and A. poptae are reproductively isolated from each other. Comparisons of demographic models demonstrated that introgression from a ghost population, which diverged from the common ancestor of A. oophorus and A. poptae, is essential for reconstructing the demographic history of Adrianichthys. The best model estimated that the divergence of the ghost population greatly predated the divergence between A. oophorus and A. poptae, and that the ghost population secondarily contacted the two extant species within Lake Poso more recently. Genome scans and simulations detected a greatly divergent locus, which cannot be explained without ghost introgression. This locus was also completely segregated between A. oophorus and A. poptae. These findings suggest that variants that came from a ghost population have contributed to the divergence between A. oophorus and A. poptae, but the large time‐lag between their divergence and ghost introgression indicates that the contribution of introgression may be restricted. [ABSTRACT FROM AUTHOR]

Seasonal changes are more robust and dynamic at higher latitudes than at lower latitudes, and animals sense seasonal changes in the environment and alter their physiology and behavior to better adapt to harsh winter conditions. However, the genetic basis for sensing seasonal changes, including the photoperiod and temperature, remains unclear. Medaka (Oryzias latipes species complex), widely distributed from subtropical to cool-temperate regions throughout the Japanese archipelago, provides an excellent model to tackle this subject. In this study, we examined the critical photoperiods and critical temperatures required for seasonal gonadal development in female medaka from local populations at various latitudes. Intraspecific differences in critical photoperiods and temperatures were detected, demonstrating that these differences were genetically controlled. Most medaka populations could perceive the difference between photoperiods for at least 1 h. Populations in the Northern Japanese group required 14 h of light in a 24 h photoperiod to develop their ovaries, whereas ovaries from the Southern Japanese group developed under 13 h of light. Additionally, Miyazaki and Ginoza populations from lower latitudes were able to spawn under short-day conditions of 11 and 10 h of light, respectively. Investigation of the critical temperature demonstrated that the Higashidori population, the population from the northernmost region of medaka habitats, had a critical temperature of over 18 °C, which was the highest critical temperature among the populations examined. The Miyazaki and the Ginoza populations, in contrast, were found to have critical temperatures under 14 °C. When we conducted a transplant experiment in a high-latitudinal environment using medaka populations with different seasonal responses, the population from higher latitudes, which had a longer critical photoperiod and a higher critical temperature, showed a slower reproductive onset but quickly reached a peak of ovarian size. The current findings show that low latitudinal populations are less responsive to photoperiodic and temperature changes, implying that variations in this responsiveness can alter seasonal timing of reproduction and change fitness to natural environments with varying harshnesses of seasonal changes. Local medaka populations will contribute to elucidating the genetic basis of seasonal time perception and adaptation to environmental changes. [ABSTRACT FROM AUTHOR]

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 Vmax/Km 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. [ABSTRACT FROM AUTHOR]

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. The mutations underlying sexually selected traits like the red fins on a male medaka fish can be hard to pinpoint. Using a new genome, transcriptomics and gene editing, Ansai et al. find that the gene csf1 causes male fins to be red, which attracts females and, surprisingly, is less attractive to predators. [ABSTRACT FROM AUTHOR]

Coelomogenesis in the isocrinid sea lily, Metacrinus rotundus, is described through the swimming larval stages. After the late gastrula stage, the archenteron separates from the ectoderm to form an archenteral sac, which develops into a dumbbell shape consisting of anterior and posterior lobes, and a middle part connecting both lobes. The anterior and posterior lobes, and the middle part, become separated into an axo‐hydrocoel, the left and right somatocoels and an enteric sac, respectively. The hydrocoel forms from the left lower edge of the axo‐hydrocoel and becomes separated from the axocoel by the late dipleurula stage, when chambered organs and coelom X bud off from the anterior tip of the right and left somatocoels, respectively. Coelom X does not occur in comatulid crinoids (feather stars), and its fate is unclear. The pore canal extends from the axocoel. The hydrocoel differentiates into a crescent shape at the overtime semidoliolaria stage, a few days after the semidoliolaria becomes competent to settle. Coelomogenesis in M. rotundus is much simpler than in the comatulids and probably represents the ancestral mode of the crinoids. As each portion of the dumbbell sac differentiates almost in situ into each coelom, presumptive fates in the sac are easily followed in M. rotundus. [ABSTRACT FROM AUTHOR]

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metabolic theory predicts that maintenance rate increases faster with animal body size than food intake rate, such that the critical resource density R* at which ingested energy exactly covers maintenance requirements increases with body size. Small-sized (low R*) juveniles may thus exclude their larger-sized (high R*) parents in resource competition, resulting in apparent semelparous life histories and non-overlapping generations. However, empirical support for such a competition-driven semelparity (CDS) remains scarce. Here, we report a high consistency of cohort dynamics with CDS in wild medaka ( Oryzias latipes). As predicted by the theory, there was a strong juvenile-adult diet overlap, and all individuals died after reproduction as semelparous age-1 adults, synchronous with a rapid somatic growth of age-0 juveniles into the adult stage and with dropping abundances of zooplankton food resources. In addition to the theory, we found evidence for increased reproductive allocation under food stress, translating into immune depression and elevated parasite prevalence. Therefore, CDS in medaka emerges both from intercohort competitive exclusion and from food-dependent energy reallocation from maintenance to reproduction, the later presumably representing an adaptive response to the former. The literature data show that the strengths of both intercohort competition and reproductive allocation increase at higher temperatures in many ectotherms, pointing to climate warming as a potentially powerful magnifier of CDS in the wild. [ABSTRACT FROM AUTHOR]

Ricefishes of the genus Oryzias, including Japanese medaka ( O. latipes), are known as excellent model organisms for studies in various fields of science. Some species of the genus inhabit brackish water, and such species are recognized to be useful to investigate physiological phenomena in seawater. However, only a limited number of species have been recorded from brackish waters. In addition, there is no information about the genetic relationship among populations inhabiting sites with different salinities. Here we report the discovery of Oryzias fish in two locations near Haiphong, northern Vietnam, a brackish mangrove planting area and a freshwater pond. A phylogenetic analysis using mitochondrial 12S and 16S ribosomal RNA (rRNA) gene sequences indicated that the fish from the two localities are the same species, Hainan medaka, O. curvinotus. Population genetic analysis using the mitochondrial 12S and 16S rRNA gene sequences revealed a close genetic relationship between the two populations. These results suggest that O. curvinotus is adaptable to both hyperosmotic and hypoosmotic environments. Due to its osmotic adaptability and ease of rearing in the laboratory, this species is expected to become a model for marine environmental and toxicological studies, as well as for studies of osmotic adaptation mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gene replacement (GR) via homologous recombination is a powerful tool for genome editing. Recently, direct GR is achieved successfully by coinjection of mRNAs for engineered endonucleases such as zinc finger nucleases (ZFNs) and donor DNA in developing embryos of diverse organisms. Here, we report the procedures and efficiency for direct GR by using ZFNs in the fish medaka. Upon zygotic coinjection of mRNAs encoding ZFNs that target the gonad-specifically expressed gsdf locus, linear DNA of GR vector pGRgsdf containing the red fluorescent protein ( rfp) gene flanked by two homology arms of ~1-kb each underwent GR via homologous recombination. Specifically, 15 of 231 adults from manipulated embryos contained a GR allele in the caudal fin, producing an efficiency of ~7 % for somatic GR. Progeny test revealed that two out of nine fertile fish containing the GR allele in the fin were capable of transmitting the GR allele to ~6 % of F generation at adulthood, generating an efficiency of ~22 % for germline transmission. Sequencing and Southern blotting validated precise GR. We show that the GR allele expressed a chimeric gsdf:rfp RNA between gsdf and cointegrated rfp specifically in the gonad, demonstrating recapitulation of endogenous RNA expression as predicted for the defined GR allele. Most importantly, RFP expression coincides faithfully with the gonad-specific gsdf expression in developing embryos and adults. These results demonstrate, for the first time, the feasibility and efficiency of ZFN-mediated precise GR directly in the developing embryo of medaka as a lower vertebrate model. [ABSTRACT FROM AUTHOR]

Genetic polymorphisms are thought to generate intraspecific behavioral diversities, both within and among populations. The mechanisms underlying genetic control of behavioral properties, however, remain unclear in wild-type vertebrates, including humans. To explore this issue, we used diverse inbred strains of medaka fish (Oryzias latipes) established from the same and different local populations. Medaka exhibit a startle response to a visual stimulus (extinction of illumination) by rapidly bending their bodies (C-start) 20-ms after the stimulus presentation. We measured the rates of the response to repeated stimuli (1-s interval, 40 times) among four inbred strains, HNI-I, HNI-II, HO5, and Hd-rR-II1, and quantified two properties of the startle response: sensitivity (response rate to the first stimulus) and attenuation of the response probability with repeated stimulus presentation. Among the four strains, the greatest differences in these properties were detected between HNI-II and Hd-rR-II1. HNI-II exhibited high sensitivity (approximately 80%) and no attenuation, while Hd-rR-II1 exhibited low sensitivity (approximately 50%) and almost complete attenuation after only five stimulus presentations. Our findings suggested behavioral diversity of the startle response within a local population as well as among different populations. Linkage analysis with F2 progeny between HNI-II and Hd-rR-II1 detected quantitative trait loci (QTL) highly related to attenuation, but not to sensitivity, with a maximum logarithm of odds score of 11.82 on linkage group 16. The three genotypes (homozygous for HNI-II and Hd-rR-II1 alleles, and heterozygous) at the marker nearest the QTL correlated with attenuation. Our findings are the first to suggest that a single genomic region might be sufficient to generate individual differences in startle behavior between wild-type strains. Further identification of genetic polymorphisms that define the behavioral trait will contribute to our understanding of the neural mechanisms underlying behavioral diversity, allowing us to investigate the adaptive significance of intraspecific behavioral polymorphisms of the startle response. [ABSTRACT FROM AUTHOR]

Zinc finger nucleases (ZFNs) can generate targeted gene disruption (GD) directly in developing embryos of zebrafish, mouse and human. In the fish medaka, ZFNs have been attempted on a transgene. Here, we developed procedures and parameters for ZFN-mediated direct GD on the gonad-specifically expressed gsdf locus in medaka. A pair of ZFNs was designed to target the first exon of gsdf and their synthetic mRNAs were microinjected into 1-cell stage embryos. We reveal dose-dependent survival rate and GD efficiency. In fry, ZFN mRNA injection at 10 ng/μl led to a GD efficiency of 30 %. This value increased up to nearly 100 % when the dose was enhanced to 40 ng/μl. In a typical series of experiments of ZFN mRNA injection at 10 ng/μl, 420 injected embryos developed into 94 adults, 4 of which had altered gsdf alleles. This leads to a GD efficacy of ∼4 % in the adulthood. Sequencing revealed a wide variety of subtle allelic alterations including additions and deletions of 1∼18 bp in length in ZFN-injected samples. Most importantly, one of the 4 adults examined was capable of germline transmission to 15.2 % of its F1 progeny. Interestingly, ontogenic analyses of the allelic profile revealed that GD commenced early in development, continued during subsequent stages of development and in primordia for different adult organs of the three germ layers. These results demonstrate the feasibility and-for the first time to our knowledge-the efficacy of ZFN-mediated direct GD on a chromosomal gene in medaka embryos. [ABSTRACT FROM AUTHOR]

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor). [ABSTRACT FROM AUTHOR]

Background: Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. Methodology/Principal Findings: To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. Conclusions/Significance: We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages. [ABSTRACT FROM AUTHOR]

Gene duplication is the primary source of novel genes, and is followed by non-, sub-, or neo-functionalization. In this study, we compared the egg envelope digestion mechanism of hatching enzymes between three-spined stickleback and nine-spined stickleback species, and found that the function of the hatching enzymes of nine-spined sticklebacks was uniquely derived by gene duplication, followed by sub-functionalization. The hatching enzyme of euteleosts consists of two metalloproteases, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE). LCE, especially, has an important role in solubilizing egg envelope protein by cleaving two specific sites. Three-spined stickleback had a single copy of the LCE gene, like other euteleosts. However, nine-spined stickleback had two types of LCE genes, α-type and β-type, suggesting that a duplication of the LCE gene occurred during the evolution of sticklebacks. The α-type and β-type each cleaved one of the two sites. Therefore, in the nine-spined stickleback, the function of the ancestral LCE was driven by a single copy gene, which was partitioned into two functions separately driven by two duplicated genes, and egg envelope was solubilized by the cooperative action of the two LCEs, α-type and β-type. Herein, we provide a molecular mechanism for an evolutionary adaptation driven by gene duplication and sub-functionalization. J. Exp. Zool. (Mol. Dev. Evol.) 320B:140-150, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Among the medaka fishes of the genus Oryzias, most species have homomorphic sex chromosomes, while some species, such as Oryzias hubbsi and Oryzias javanicus, have heteromorphic ZW sex chromosomes. In this study, a novel family of repetitive sequence was molecularly cloned from O. hubbsi and characterized by chromosome in situ and filter hybridization, respectively. This repetitive element, which we designated as a BstNI family element, localized at heterochromatin regions on the W chromosome, as well as on two pairs of autosomes. Homologous sequences to this element were found only in O. javanicus, which is a sister species of O. hubbsi, suggesting that this repeated element originated in the common ancestor of these two species. However, the intensity of the hybridization signals was lower in O. javanicus than in O. hubbsi, and the chromosomal location of this element in O. javanicus was confined to heterochromatin regions on one pair of autosomes. Thus, we hypothesize that this repetitive element was extensively amplified in the O. hubbsi lineage, especially on its W chromosome, after the separation of the O. javanicus lineage. In addition, we also found the W chromosomal location of the 18S-28S ribosomal RNA genes in both O. hubbsi and O. javanicus. Our previous studies showed no linkage homology of the sex chromosomes in these species, indicating that the RNA genes were shared between W chromosomes of different origins. This situation may be explained by a translocation of the sex-determining region with the ribosomal RNA genes in either species or an independent accumulation of the RNA genes as a convergent process during W chromosome degeneration. [ABSTRACT FROM AUTHOR]

Tol2 is a member of the hAT (hobo/Activator/Tam3) transposable element family, residing as 10–30 copies per diploid genome in the medaka fish. We previously reported that this element is highly homogeneous in structure at both the restriction map level and the nucleotide sequence level. It was, however, possible that there is variation of such a low frequency as not to have been detected in our previous surveys, in which samples from 12 geographical locations were used. In the present study, we first conducted searches of genome sequence databases of medaka, and found a 119-bp-long internal deletion. We then conducted a survey of samples from 58 locations for this deletion by performing PCR preceded by restriction enzyme digestion to increase the sensitivity to this deletion. We found that copies suffering this deletion have spread, or have been generated by multiple origins, in the northern-to-central part of mainland Japan. Thus, although the high homogeneity in structure is a distinct feature of Tol2, variation does exist at low frequencies in natural populations of medaka. The current status of Tol2 is expected to provide information with which results of future surveys can be compared for clarification of determinants of population dynamics of this DNA-based element. [ABSTRACT FROM AUTHOR]

The proteasome subunit β-type 8 (PSMB8) gene in the jawed vertebrate MHC genomic region encodes a catalytic subunit of the immunoproteasome involved in the generation of peptides to be presented by the MHC class I molecules. A teleost, the medaka (Oryzias latipes), has highly diverged dimorphic allelic lineages of the PSMB8 gene with only about 80% amino acid identity, termed "PSMB8d" and "PSMB8N," which have been retained by most wild populations analyzed. To elucidate the evolutionary origin of these two allelic lineages, seven species of the genus Oryzias were analyzed for their PSMB8 allelic sequences using a large number of individuals from wild populations. All the PSMB8 alleles of these species were classified into one of these two allelic lineages based on their nucleotide sequences of exons and introns, indicating that the Oryzias PSMB8 gene has a truly dichotomous allelic lineage. Retention of both allelic lineages was confirmed except for one species. The PSMB8d lineage showed a higher frequency than the PSMB8N lineage in all seven species. The two allelic lineages showed curious substitutions at the 31st and 53rd residues of the mature peptide, probably involved in formation of the S1 pocket, suggesting that these allelic lineages show a functional difference in cleavage specificity. These results indicate that the PSMB8 dimorphism was established before speciation within the genus Oryzias and has been maintained for more than 30-60 million years under a strict and asymmetric balancing selection through several speciation events. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the β-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae. [ABSTRACT FROM AUTHOR]

Background: In animal groups such as herds, schools, and flocks, a certain distance is maintained between adjacent individuals, allowing them to move as a cohesive unit. Proximate causations of the cohesive and coordinated movement under dynamic conditions, however, have been poorly understood. Methodology/Principal Findings: We established a novel and simple behavioral assay using pairs of small fish (medaka and dwarf pufferfish) by eliciting a simultaneous optomotor response (OMR). We demonstrated that two homospecific fish began to move cohesively and maintained a distance of 2 to 4 cm between them when an OMR was elicited simultaneously in the fish. The coordinated and cohesive movement was not exhibited under a static condition. During the cohesive movement, the relative position of the two fish was not stable. Furthermore, adult medaka exhibited the cohesive movement but larvae did not, despite the fact that an OMR could be elicited in larvae, indicating that this ability to coordinate movement develops during maturation. The cohesive movement was detected in homospecific pairs irrespective of body-color, sex, or albino mutation, but was not detected between heterospecific pairs, suggesting that coordinated movement is based on a conspecific interaction. Conclusions/Significance: Our findings demonstrate that coordinated behavior between a pair of animals was elicited by a simultaneous OMR in two small fish. This is the first report to demonstrate induction of a schooling-like movement in a pair of fish by an OMR and to investigate the effect of age, sex, body color, and species on coordination between animals under a dynamic condition. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients. [ABSTRACT FROM AUTHOR]

The medaka fish ( Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [ABSTRACT FROM AUTHOR]

A DM-domain gene on the Y chromosome was identified as the sex-determining gene in the medaka, Oryzias latipes, and named DMY (also known as dmrt1bY). However, this gene is absent in most Oryzias fishes, suggesting that closely related species have another sex-determining gene. In fact, it has been demonstrated that the Y chromosome in O. dancena is not homologous to that in O. latipes, whereas both species have an XX/XY sex-determination system. Through a progeny test of sex-reversed fish and a linkage analysis of isolated sex-linked DNA markers, we show that O. hubbsi, which is one of the most closely related species to O. dancena, has a ZZ/ZW system. In addition, genetic and fluorescence in situ hybridization mapping of the sex-linked markers revealed that sex chromosomes in O. hubbsi and O. dancena are not homologous, indicating different origins of these ZW and XY sex chromosomes. Furthermore, we found that O. hubbsi has morphologically heteromorphic sex chromosomes, in which the W chromosome has 4,6-diamidino-2-phenylindole (DAPI)-positive heterochromatin blocks and is larger than the Z chromosome, although such differentiated sex chromosomes have not been observed in other Oryzias species. These findings suggest that a variety of sex-determining mechanisms and sex chromosomes have evolved in Oryzias. [ABSTRACT FROM AUTHOR]

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including ∼2,900 new genes, using 5′-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of ∼50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow. [ABSTRACT FROM AUTHOR]

We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon–intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon–intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon–intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns. [ABSTRACT FROM AUTHOR]

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants. [ABSTRACT FROM AUTHOR]