Yasuhiro Kamei

Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present in zebrafish

Cryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator whose reactions do not depend on light, but Drosophila CRY functions as a light‐dependent suppressor of transcriptional inhibitor.

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser–evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock–mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Three Distinct Classes of the α-Subunit of the Nuclear Pore-targeting Complex (Importin-α) Are Differentially Expressed in Adult Mouse Tissues

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The α-subunit of the complex (importin-α) binds to the NLS and the β-subunit (importin-β-) carries the α-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse α-subunits have been identified and classified into three subfamilies (α-P, α-Q, and α-S). The expression of these α-subunits and the β-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the α-subunit or the β-subunit. The β-subunit was found to be ubiquitously expressed, whereas each subfamily of the α-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of α-P was not observed, whereas α-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, α-P was expressed predominantly in primary spermatocytes, whereas α-Q was found mainly in Leydig cells. Expression of α-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by α-subunit family proteins.

High temperature causes masculinization of genetically female medaka by elevation of cortisol

In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by high water temperature (HT; 32–34°C) treatment during the sex differentiation period. Here we report that cortisol caused female‐to‐male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT‐induced masculinization of XX medaka. HT treatment caused elevation of whole‐body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33°C treatments inhibited female‐type proliferation of germ cells as well as expression of follicle‐stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression. Mol. Reprod. Dev. 77: 679–686, 2010.

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka

DMRT1, which is found in many vertebrates, exhibits testis-specific expression during the sexual differentiation period, suggesting a conserved function of DMRT1 in the testicular development of vertebrate gonads. However, functional analyses have been reported only in mammals. The current study focused on the Dmrt1 function in the teleost medaka, Oryzias latipes, which has an XX–XY sex determination system. Although medaka sex is determined by the presence or absence of the Y chromosome-specific gene Dmy, we demonstrated that in one Dmrt1 mutant line, which was found by screening a gene-driven mutagenesis library, XY mutants developed into normal females and laid eggs. Histological analyses of this mutant revealed that the XY mutant gonads first developed into the normal testis type. However, the gonads transdifferentiated into the ovary type. The mutant phenotype could be rescued by transgenesis of the Dmrt1 genomic region. These results show that Dmrt1 is essential to maintain testis differentiation after Dmy-triggered male differentiation pathway.

Trunk exoskeleton in teleosts is mesodermal in origin

The vertebrate mineralized skeleton is known to have first emerged as an exoskeleton that extensively covered the fossil jawless fish. The evolutionary origin of this exoskeleton has long been attributed to the emergence of the neural crest, but experimental evaluation for this is still poor. Here we determine the embryonic origin of scales and fin rays of medaka (teleost trunk exoskeletons) by applying long-term cell labelling methods, and demonstrate that both tissues are mesodermal in origin. Neural crest cells, however, fail to contribute to these tissues. This result suggests that the trunk neural crest has no skeletogenic capability in fish, instead highlighting the dominant role of the mesoderm in the evolution of the trunk skeleton. This further implies that the role of the neural crest in skeletogenesis has been predominant in the cephalic region from the early stage of vertebrate evolution.

A Neural Mechanism Underlying Mating Preferences for Familiar Individuals in Medaka Fish

Familiarity Does Not Breed Contempt Female mating preference is influenced by social familiarity in various species from fish to primates. Okuyama et al. (p. 91) showed in Japanese rice fish that females prefer to mate with visually familiarized males over unfamiliar males and that this preference is mediated by specific neuromodulatory neurons in the female brain. A particular class of neurons regulates female fish mating preference based on social familiarity. Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

BackgroundDuring the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.ResultsIn this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.ConclusionsThese results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.