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PDF file - 151K, Schematic representation of XPF disruption in DT40 cells.

Supplementary Figure 6 from RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage

PDF file - 50K, Xpf and Mus81 are compensatory to each other in the completion of HR in human and mouse cell lines.

Supplementary Figure Legends 1-6 from RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage

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PDF file - 244K, Ortholog gene of MUS81 is not registered in the chicken database.

PDF file - 130K, Co-immunoprecipitation of Xpf with Eme1 or Slx4.

PDF file - 311K, Sequence alignment among human, mouse, and chicken Xpf.

Supplementary Figure 5 from RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage

The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80−/− cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80−/−BRCA1−/− cells were more sensitive to etoposide than were BRCA1−/− cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80−/− cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1. Cancer Res; 70(21); 8467–74. ©2010 AACR.

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PDF file - 143K, Identification of the interaction partners for Xpf in DT40 cells.

PDF file - 139K, Schematic representation of EME1 disruption in DT40 cells.

Supplementary Figures 1-3 from RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.

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.

Marine medaka Oryzias melastigma at 4months (young), 8months (middle-aged) and 12months old (senior) were employed to determine age-associated change of sex ratios, sex hormones, telomere length (TL), telomerase activity (TA), telomerase transcription (omTERT) and oxidative damage in the liver. Overall, O. melastigma exhibited gradual senescence, sex differences in longevity (FM), TL (FM) and oxidative damage (FM) during aging. In females, the plasma E2 level was positively correlated with TL (TRF5kb), TA and omTERT expression (p≤0.01), and negatively correlated with liver DNA oxidation (p≤0.05). The results suggest high levels of E2 in female O. melastigma may retard TL shortening by enhancing TA via TERT transcription and/or reducing oxidative DNA damage. The findings support TL shortening as a biomarker of aging and further development of accelerated TL shortening, abnormal suppression of TA and excessive oxidative DNA damage as early molecular endpoints, indicative of advanced/premature aging in marine medaka/fish.

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

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.

The endoplasmic reticulum (ER) membrane-bound transcription factors ATF6α and ATF6β mediate adjustment of chaperone levels to increased demands in the ER, which is essential for development of the notochord; the latter synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra., ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.

Myostatin (MSTN) functions as a negative regulator of skeletal muscle mass. In mammals, MSTN-deficient animals result in an increase of skeletal muscle mass with both hyperplasia and hypertrophy. A MSTN gene is highly conserved within the fish species, allowing speculation that MSTN-deficient fish could exhibit a double-muscled phenotype. Some strategies for blocking or knocking down MSTN in adult fish have been already performed; however, these fish show either only hyperplastic or hypertrophic growth in muscle fiber. Therefore, the role of MSTN in fish myogenesis during post-hatch growth remains unclear. To address this question, we have made MSTN-deficient medaka (mstnC315Y) by using the targeting induced local lesions in a genome method. mstnC315Y can reproduce and have the same survival period as WT medaka. Growth rates of WT and mstnC315Y were measured at juvenile (1–2wk post-hatching), post-juvenile (3–7wk post-hatching) and adult (8–16wk post-hatching) stages. In addition, effects of MSTN on skeletal muscle differentiation were investigated at histological and molecular levels at each developmental stage. As a result, mstnC315Y show a significant increase in body weight from the post-juvenile to adult stage. Hyper-morphogenesis of skeletal muscle in mstnC315Y was accomplished due to hyperplastic growth from post-juvenile to early adult stage, followed by hypertrophic growth in the adult stage. Myf-5 and MyoD were up-regulated in mstnC315Y at the hyperplastic growth phase, while myogenin was highly expressed in mstnC315Y at the hypertrophic growth phase. These indicated that MSTN in medaka plays a dual role for muscle fiber development. In conclusion, MSTN in medaka regulates the number and size of muscle fiber in a temporally-controlled manner during posthatch growth.

The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80−/− cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80−/−BRCA1−/− cells were more sensitive to etoposide than were BRCA1−/− cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80−/− cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1. Cancer Res; 70(21); 8467–74. ©2010 AACR.

Perturbations in protein folding and degradation are key pathological mechanisms in neurodegenerative diseases, including Parkinson's disease (PD). Recent evidence suggests that mishandling of proteins may play an important role in the pathogenesis of PD. We have utilized medaka fish to monitor the effects of injecting neurotoxins into the CSF space. In this study, ammonium chloride, tunicamycin, and lactacystin were tested for their ability to disturb lysosomal proteolysis, N-glycosylation in the endoplasmic reticulum, and proteasomal degradation, respectively. All of the substances tested induced selective loss of dopaminergic neurons, movement disorders and inclusion bodies. Among them, the features of the inclusion bodies that developed after ammonium chloride injection mimicked those of PD: co-localization of ubiquitin and phosphorylated α-synuclein, as well as the presence of LC3 protein in the inclusion bodies. Our study demonstrated that medaka fish are useful for examining the effects of environmental toxins and lysosome inhibition, and lysosome inhibitors may be factors in the development of PD.

Recent findings suggest that a defect in the ubiquitin-proteasome system plays an important role in the pathogenesis of Parkinson's disease (PD). A previous report (McNaught et al. 2004) demonstrated that rats systemically injected with proteasome inhibitors exhibited PD-like clinical symptoms and pathology. However, because these findings have not been consistently replicated, this model is not commonly used to study PD. We used medaka fish to test the effect of systemic administration of proteasome inhibitors because of the high level of accessibility of the cerebrospinal fluid in fish. We injected lactacystin or epoxomicin into the CSF of medaka. With proteasome inhibition in the medaka brain, selective dopaminergic and noradrenergic cell loss was observed. Furthermore, treated fish exhibited reduced spontaneous movement. Treatment with proteasome inhibitors also induced the formation of inclusion bodies resembling Lewy bodies, which are characteristic of PD. Treatment with 6-OHDA also induced dopaminergic cell loss but did not produce inclusion bodies. These findings in medaka are consistent with previous results reporting that non-selective proteasome inhibition replicates the cardinal features of PD: locomotor dysfunction, selective dopaminergic cell loss, and inclusion body formation.

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., Author summary How individual cell fates become specified from multipotent progenitors is a fundamental question in developmental and stem cell biology. Body pigment cells derive from a multipotent progenitor, but while in zebrafish there are three types of pigment cells (melanocytes, iridophores and xanthophores), in medaka these progenitors form four (as zebrafish, plus leucophores). Here, we address whether mechanisms generating each cell-type are conserved between the two species. We focus on two key regulatory proteins, Sox5 and Sox10, which we previously showed were involved in pigment cell development in medaka and zebrafish, respectively. We compare experimentally how the two proteins interact in regulating development of each of the pigment cell lineages in these fish. We show that development of all pigment cells, except leucophores, is dependent on Sox10, and that Sox5 modulates Sox10 activity antagonistically in all pigment cells in zebrafish, and melanocytes and iridophores in medaka. Surprisingly, in medaka, Sox5 acts co-operatively with Sox10 to promote xanthophore fate and to repress leucophore fate. Our findings reveal surprising diversity how Sox5 and Sox10 interact to govern pigment cell development in medaka and zebrafish, and suggest that this likely relates to the evolution of the novel leucophore pigment cell type in medaka.

Parkinson's disease is a neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. The PTEN-induced kinase 1 gene (PINK1) is responsible for recessive inherited familial Parkinson's disease (PARK6). Neither the function of PINK1 nor its role in the prevention of Parkinson's disease is fully understood. Gene disruption of PINK1 causes remarkably different phenotypes in animal models such as Drosophila melanogaster, zebrafish, and mouse, none of which recapitulate Parkinson's-disease-like symptoms. We established PINK1-gene-disrupted medaka fish. These mutant fish grew normally at first, then developed significant decrease in the frequency of spontaneous swimming movements in the late-adult stage. Although the mutants did not show any dopaminergic cell loss, the amount of 3,4-dihydroxyphenylacetic acid, a major metabolite of dopamine, decreased. Thus, PINK1 contributes to the maintenance of dopamine metabolism, even before the selective death of dopaminergic neurons. Our animal model is therefore a valuable tool to detect pathogenesis in Parkinson's patients in the early stages.

Background Many bacterial or mammalian cell-based test systems, such as the Ames test, chromosomal aberration assays, or gene mutation assays, are commonly used in developed countries to detect the genotoxicity of industrial chemicals. However, the specificity is generally limited and the sensitivity is not sufficiently high. In addition, most assays cannot provide information on mechanisms of genotoxicity of a given chemical. Objectives We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity. Methods We developed a novel bioassay employing gene-disrupted clones of the chicken DT40 B-lymphocyte line, which are designed to be deficient in several specific DNA repair pathways. Genotoxic chemicals can delay cellular proliferation in DNA-repair–deficient clones more significantly than in wild-type cells by interfering with DNA replication, thereby inducing DNA damage. In addition, we verified the validity of this assay by analyzing the genotoxicity of γ-rays, ultraviolet (UV) light, and sodium metaarsenite (NaAsO2). We also characterized DNA lesions induced by NaAsO2. Results Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair–deficient mutants in 48 hr. We also found that NaAsO2 apparently induces at least two types of damage: chromosomal breaks and UV photoproduct-like DNA lesions. Conclusion This bioassay is a reliable and sensitive screening tool for environmental mutagens as well as for further characterizing the nature of detected genotoxicity.

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM(-/-) cells. In addition, BLM(-/-) cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM(-/-) cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.

ASCIZ (ATMIN) was recently identified as a novel DNA damage response protein. Here we report that ASCIZ-deficient chicken DT40 B lymphocyte lines displayed markedly increased Ig gene conversion rates, whereas overexpression of human ASCIZ reduced Ig gene conversion below wild-type levels. However, neither the efficiency of double-strand break repair nor hypermutation was affected by ASCIZ levels, indicating that ASCIZ does not directly control homologous recombination or formation of abasic sites. Loss of ASCIZ led to mild sensitivity to the base damaging agent methylmethane sulfonate (MMS), yet remarkably, suppressed the dramatic MMS hypersensitivity of polβ-deficient cells. These data suggest that ASCIZ may affect the choice between competing base repair pathways in a manner that reduces the amount of substrates available for Ig gene conversion.

The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.

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., Author Summary Males of various species, including humans, remain near a (potential) mating partner and repel their rival males (mate-guarding). Mate-guarding is mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. Here we show that the arginine-vasotocin (AVT), a non-mammalian homolog of arginine-vasopressin (AVP), system mediates the molecular mechanisms underlying how mate presence (intersexual relationship) affects male competitive motivation (intrasexual relationship) in mate-guarding (a triadic relationship). We first established a novel behavioral paradigm in which medaka robustly exhibit mate-guarding behavior, that allowed us to study the genetic/molecular mechanisms underlying mate-guarding. Behavioral analysis of courtship behaviors, aggressive behaviors and mate-guarding using medaka mutants in this paradigm suggested that the AVT system is involved in the process in which mate (female) presence drives sexual motivation of males, which may facilitate mate guarding in the triadic relationship. Our study provides genetic evidence that the AVT/AVP system regulates mate-guarding behaviors, suggesting that the role of this system in mate-guarding is evolutionarily conserved from teleosts to mammals.

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.

Notch receptors undergo three distinct proteolytic cleavages during maturation and activation. The third cleavage occurs within the plasma membrane and results in the release and translocation of the intracellular domain into the nucleus to execute Notch signaling. This so-called γ-secretase cleavage is under the control of presenilins, but it is not known whether presenilins themselves carry out the cleavage or whether they act by means of yet-unidentified γ-secretase(s). In this article, we show that Notch intracellular cleavage in intact cells completely depends on presenilins. In contrast, partial purification of the Notch cleavage activity reveals an activity, which is present only in protein extracts from presenilin-containing cells, and which does not comigrate with presenilin. This finding provides evidence for the existence of a specific Notch-processing activity, which is physically distinct from presenilins. We conclude from these experiments that presenilins are critically required for Notch intracellular cleavage but are not themselves directly mediating the cleavage.

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)., Author Summary How individual cell fates are specified from multipotent progenitor cells is a fundamental question in developmental and stem cell biology. Accumulating evidence indicates that stem cells develop into each of their final, diverse cell-types after progression through one or more partially-restricted intermediates, but the molecular mechanisms underlying final fate choice are largely unknown. Neural crest cells (NCCs) give rise to diverse cell-types including multiple pigment cells and thus are a favored model for understanding the mechanism of fate specification. We have investigated how a specific fate choice is made from partially-restricted pigment cell progenitors in medaka. We show that Sry-related transcription factor Sox5 is required for fate determination between yellow xanthophore and white leucophore, and its loss causes excessive formation of leucophores and absence of xanthophores. We demonstrate that Sox5 functions cell-autonomously in the xanthophore lineage in medaka. Furthermore, pax7a is expressed in the partially-restricted progenitor cells shared with xanthophore and leucophore lineages, and Sox5 acts in some of these cells to promote xanthophore lineage. Our work reveals the role of Sox5 as a molecular switch determining xanthophore versus leucophore fate choice from the shared progenitor, and identifies an important mechanism regulating pigment cell fate choice from NCCs.

SummaryThe thymus is a lymphoid organ unique to vertebrates, and it provides a unique microenvironment that facilitates the differentiation of immature hematopoietic precursors into mature T cells. We subjected the evolutionary trajectory of the thymic microenvironment to experimental analysis. A hypothetical primordial form of the thymus was established in mice by replacing FOXN1, the vertebrate-specific master regulator of thymic epithelial cell function, with its metazoan ancestor, FOXN4, thereby resetting the regulatory and coding changes that have occurred since the divergence of these two paralogs. FOXN4 exhibited substantial thymopoietic activity. Unexpectedly, histological changes and a functional imbalance between the lymphopoietic cytokine IL7 and the T cell specification factor DLL4 within the reconstructed thymus resulted in coincident but spatially segregated T and B cell development. Our results identify an evolutionary mechanism underlying the conversion of a general lymphopoietic organ to a site of exclusive T cell generation.

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.

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.

The intracellular region (RAMIC) of the mouse Notch1 receptor interacts with RBP-J/CBF-1, which binds to the DNA sequence CGTGGGAA and suppresses differentiation by transcriptional activation of genes regulated by RBP-J. Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for immortalization of human B cells by the virus. EBNA2 is a pleiotropic activator of viral and cellular genes and is targeted to DNA at least in part by interacting with RBP-J. We found that EBNA2 and the Notch1 RAMIC compete for binding to RBP-J, indicating that their interaction sites on RBP-J overlap at least partially. EBNA2 and Notch1 RAMIC transactivated the same set of viral and host promoters, i.e., the EBNA2 response element of the Epstein-Barr virus TP1 and the HES-1 promoter. Furthermore, EBNA2 functionally replaced the Notch1 RAMIC by suppressing differentiation of C2C12 myoblast progenitor cells.