Wnt4a Is Indispensable for Genital Duct Elongation but Not for Gonadal Sex Differentiation in the Medaka, Oryzias latipes.

In most vertebrates, the oviducts and sperm ducts are derived from the Müllerian ducts and Wolffian ducts, respectively. However, in teleosts, the genital ducts are formed by the posterior extension of gonads in both sexes. Whether the genital ducts of teleosts are newly evolved organs or variants of Müllerian ducts is an important question for understanding evolutionary mechanisms of morphogenesis. One of the genes essential for Müllerian duct formation in mice is Wnt4, which is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we addressed the above question by examining genital duct development in mutants of two Wnt4 genes in the medaka (wnt4a is orthologous to mouse Wnt4, and wnt4b is paralogous). The wnt4b mutants had a short body but were fertile with normal genital ducts. In contrast, both male and female wnt4a mutants had their posterior elongation of the gonads stopped within or just outside the coelom. The mutants retained the posterior parts of ovarian cavities or sperm duct primordia, which are potential target tissues of Wnt4a. The gonads of female scl mutants (unable to synthesize sex steroids) lacked these tissues and did not develop genital ducts. Medaka wnt4a was expressed in the mesenchyme ventral to the genital ducts in both sexes. Taken together, the data strongly suggest that the mouse Müllerian ducts and the medaka genital ducts share homologous developmental processes. Additionally, the wnt4a or wnt4b single mutants and the double mutants did not show sex-reversal, implying that both genes are dispensable for gonadal sex differentiation in the medaka.

Genetic diversification of the Kanehira bitterling Acheilognathus rhombeus inferred from mitochondrial DNA, with comments on the phylogenetic relationship with its sister species Acheilognathus barbatulus.

The Kanehira bitterling, Acheilognathus rhombeus, is a freshwater fish, discontinuously distributed in western Japan and the Korean Peninsula. Unusually among bitterling it is an autumn-spawning species and shows developmental diapause. Consequently, the characterization of its evolutionary history is significant not only in the context of the fish assemblage of East Asia, but also for understanding life-history evolution. This study aimed to investigate the phylogeography of A. rhombeus and its sister species Acheilognathus barbatulus, distributed in China, using a mitochondrial analysis of the ND1 gene from 311 samples collected from 50 localities in Japan and continental Asia. Phylogenetic analysis revealed that A. barbatulus is included in A. rhombeus and genetically closer to Japanese A. rhombeus than to Korean A. rhombeus. Divergence of Korean A. rhombeus and A. barbatulus from Japanese A. rhombeus was estimated to be from the late Pliocene (3.44 Mya) and the early Pleistocene (1.98 Mya), respectively. Each event closely coincided with the time of the Japan Sea opening. Japanese A. rhombeus comprised seven lineages: three in Honshu and four in Kyushu. One lineage in central Kyushu was genetically closer to the Honshu lineages than to other lineages in northern Kyushu. Divergence of Japanese lineages was estimated to be from the early to middle Pleistocene (0.55-0.93 Mya), during a period of geological and paleoclimatic change, including volcanic activity. Population expansion in the late Pleistocene (<0.10 Ma) was suggested in many of the lineages, which accords with other freshwater fishes. Biogeographically the ancestral A. rhombeus/A. barbatulus was likely to have repeatedly colonized Japan from the continent through land bridges in the late Pliocene and the early Pleistocene. However, the close genetic relationship between Japanese A. rhombeus and A. barbatulus suggests another possibility, with the second colonization occurring in reverse, from Japan to China. The small genetic distance between them indicates that the colonization occurred later than colonization events of other freshwater fishes, including other bitterling species.

Genetic Characteristics of the Japanese Serow Capricornis crispus in the Kii Mountain Range, Central Japan.

The Japanese serow, Capricornis crispus, is an indigenous bovid species exclusively inhabiting mountain regions in the main Japanese islands, excepting Hokkaido. It had decreased in abundance to its lowest level due to overhunting and deforestation, with its distribution severely fragmented from the middle of the 20th century, many populations of C. crispus currently facing the risk of extinction. The Kii Mountain Range (KM) on Honshu is one such location that has seen a drastic population decline of C. crispus. In this study, we examined genetic characteristics of C. crispus in KM and neighboring regions of the Chubu district, using mtDNA and microsatellite markers, in order to devise strategies for its conservation. Results for mtDNA were characterized by low nucleotide diversity with five endemic and two dominant haplotypes shared by individuals in neighboring regions. A Bayesian skyline plot indicated a gradual increase after the last glacial maximum. For microsatellites, the genetic diversity of C. crispus in KM was comparable to Shizuoka and higher than Shikoku. Recent genetic bottlenecks were strongly suggested in C. crispus in KM. Bayesian clustering showed a genetic cline between KM and neighboring regions, where multivariate analysis suggested three local populations. A Mantel test indicated male-biased dispersal. These results indicate that C. crispus in KM and neighboring regions constitute multiple local populations, connected through restricted gene flow. For the conservation of C. crispus, it is important to define small-scale conservation units, among which genetic connectivity should be facilitated to prevent further loss of genetic diversity.

Mogamulizumab for relapsed adult T-cell leukemia-lymphoma: Updated follow-up analysis of phase I and II studies.

The present study sought to elucidate the prognosis of adult T-cell leukemia-lymphoma (ATL) patients receiving mogamulizumab, a defucosylated anti-CCR4 monoclonal antibody. Progression-free survival (PFS) and overall survival (OS) of ATL patients enrolled in two studies are herein updated, namely NCT00355472 (phase I study of mogamulizumab in relapsed patients with ATL and peripheral T-cell lymphoma) and NCT00920790 (phase II study for relapsed ATL). Of 13 patients with relapsed aggressive ATL in the phase I study, four (31%) survived >3 years. For 26 relapsed patients with aggressive ATL in the phase II study, median PFS was 5.2 months and 1-year PFS was 26%, whereas median OS was 14.4 months, and 3-year OS was 23%. For patients without a rash or who developed a grade 1 rash only, median PFS was 0.8 months, and 1-year PFS was zero, with a median OS of 6.0 months, and 3-year OS of 8%. In contrast, for patients who developed a rash ≥grade 2, median PFS was 11.7 months, and 1-year PFS was 50%, with a median OS of 25.6 months, and 3-year OS of 36%. Thus, we conclude that mogamulizumab monotherapy may improve PFS and OS in some patients with relapsed aggressive ATL, especially those who develop a skin rash as a moderate immune-related adverse event. Therefore, further investigation is warranted to validate the present observations and to clarify the mechanisms involved in the activity of mogamulizumab.

Safety and efficacy of mogamulizumab in patients with adult T-cell leukemia-lymphoma in Japan: interim results of postmarketing all-case surveillance.

We present the interim results of a postmarketing all-case surveillance study in patients with C-C chemokine receptor 4 (CCR4)-positive, relapsed or refractory adult T-cell leukemia-lymphoma (ATL) treated with the anti-CCR4 monoclonal antibody mogamulizumab since its 2012 launch in Japan. The safety and efficacy analysis populations comprised 484 and 442 patients, respectively. The ATL subtype was acute in 58.9% and lymphoma in 34.2% of patients. All patients were scheduled to receive intravenous infusions of mogamulizumab (1.0 mg/kg) once weekly for eight weeks, alone or in combination with other modalities. Adverse drug reactions (ADRs) were reported in 74.0% of patients, of which 35.7% were serious and 6.2% were fatal. The priority survey items of infusion-related reaction, skin disorder, infection, immune disorder, and tumor lysis syndrome were reported in 29.3, 34.3, 22.1, 3.5, and 2.5% of patients, respectively. Graft-versus-host disease was reported in 25/42 patients who received mogamulizumab before allogeneic hematopoietic stem cell transplantation. The best overall response rate was 57.7% overall, 57.5% in patients treated with mogamulizumab alone, and 58.2% in patients treated with combination therapy. This surveillance indicates that mogamulizumab shows acceptable tolerability in practice; however, because of the risk of serious/fatal ADRs, patients administered mogamulizumab should be carefully monitored.

A Genetic Map for the Only Self-Fertilizing Vertebrate.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

Phylogenetic relationships of bitterling fishes (Teleostei: Cypriniformes: Acheilognathinae), inferred from mitochondrial cytochrome B sequences.

Bitterling (Teleostei: Acheilognathinae) are small cyprinid fishes with a discrete distribution in East Asia and Europe. We used a complete mitochondrial cytochrome b sequence (1141 bp) from 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus), sampled across the major part of their distribution, to elucidate their phylogeny and biogeography, focusing particularly on their origin and dispersal. Based on high support value, the monophyletic Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In the latter clade, the monophyly of Rhodeus was poorly supported, though it was topologically nested in Tanakia. On the basis of molecular-clock calibration, both clades diverged in the middle Miocene, with Tanakia-Rhodeus diverging slightly earlier than Acheilognathus. The Tanakia-Rhodeus clade expanded its distribution westward from the Far East, eventually reaching Europe, while Acheilognathus dispersed in the temperate regions of East Asia. A feature common to both clades is that most extant species, including Japanese endemics, appeared by the end of the Pliocene, corresponding with the present delineation of the Japanese archipelago. Autumn-spawning species with an embryonic diapause, unique to bitterling among cyprinid fishes, formed two distinct lineages (barbatulusrhombeus and longipinnis-typus) within Acheilognathus. The estimated time of divergence of the two lineages was approximately from the late Pliocene, a period characterized by glaciations. The timing of divergence suggests that the shift of spawning from spring to autumn, coupled with embryonic diapause, convergently emerged twice in the evolution of bitterling, possibly as an adaptation to the climate of the late Pliocene.

Phylogeography of the brackish water clam Corbicula japonica around the Japanese archipelago inferred from mitochondrial COII gene sequences.

We investigated the phylogeography of the Asian brackish water clam, Corbicula japonica, to clarify its demographic history using partial mitochondrial COII gene sequences (990 bp) from 283 individuals collected from around the Japanese archipelago and adjacent areas. Phylogenetic analyses revealed the presence of two major groups within our samples: monophyletic Group I comprising Lineages A-E of C. japonica and paraphyletic Group II consisting of Corbicula sp. Lineages A-C were distributed in Japan and Sakhalin Island, and Lineages D, E, and Corbicula sp. were distributed in the Korean Peninsula. Nested clade analysis (NCA) revealed that Lineage A-the dominant lineage in Japan-consisted of Pacific and Japan Sea lineages, the latter comprising southern and northern Japan Sea groups. Genetic diversity indices of the southern group were higher than those of the northern group, suggesting historical range expansion in the Sea of Japan from southwest to northeast. Geographical distribution of these genetic groups appears to have been influenced by major ocean currents around the Japanese archipelago. Dominant haplotypes in the star-shaped haplotype network of Lineage A were distributed throughout the entire distribution range of each genetic group, implying rapid range expansion of this species. The results of mismatch distribution analysis and molecular clock estimation suggest that expansion of lineage A occurred during the late Middle or Late Pleistocene. In contrast, restricted or past gene flow suggested by NCA and the many unique haplotypes (110/123; 89.4%) present in Lineage A suggest that gene flow among extant populations is rather limited.

28S rDNA haplotypes of males are distinct from those of androgenetic hermaphrodites in the clam Corbicula leana.

The clam Corbicula leana exists in two forms, hermaphrodites and males. Our previous study on mitochondrial DNA suggested that the male nuclear DNA might have derived from hermaphrodite C. leana relatively recently. To clarify the origin of males in the clam, sequences of the nuclear 28S rDNA divergent domain (which is 441-444 bp long) in androgenetic hermaphrodites and males and dioecious (bisexual) species were analyzed. Unexpectedly, the nuclear 28S rDNA haplotypes of males and hermaphrodites were distinct. Haplotype network analysis indicated that males and hermaphrodites are reproductively isolated from each other without sharing the same nuclear haplotype. These results support a hypothesis that the egg nuclear genome of androgenetic hermaphrodites is replaced by the male sperm genome, and only males develop after fertilization by a male spermatozoon.

The role of propagule pressure in the invasion success of bluegill sunfish, Lepomis macrochirus, in Japan.

The bluegill sunfish, Lepomis macrochirus, is a widespread exotic species in Japan that is considered to have originated from 15 fish introduced from Guttenberg, Iowa, in 1960. Here, the genetic and phenotypic traits of Japanese populations were examined, together with 11 native populations of the USA using 10 microsatellite markers and six meristic traits. Phylogenetic analysis reconfirmed a single origin of Japanese populations, among which populations established in the 1960s were genetically close to Guttenberg population, keeping high genetic diversity comparable to the ancestral population. In contrast, genetic diversity of later-established populations significantly declined with genetic divergence from the ancestral population. Among the 1960s established populations, that from Lake Biwa showed a significant isolation-by-distance pattern with surrounding populations in which genetic bottlenecks increased with geographical distance from Lake Biwa. Although phenotypic divergence among populations was recognized in both neutral and adaptive traits, P(ST)-F(ST) comparisons showed that it is independent of neutral genetic divergence. Divergent selection was suggested in some populations from reservoirs with unstable habitats, while stabilizing selection was dominant. Accordingly, many Japanese populations of L. macrochirus appear to have derived from Lake Biwa population, expanding their distribution with population bottlenecks. Despite low propagule pressure, the invasion success of L. macrochirus is probably because of its drastic population growth in Lake Biwa shortly after its introduction, together with artificial transplantations. It not only enabled the avoidance of a loss in genetic diversity but also formed a major gene pool that supported local adaptation with high phenotypic plasticity.

Phylogeography of the bluegill sunfish, Lepomis macrochirus, in the Mississippi River Basin.

The Mississippi River Basin supports the richest fish fauna in eastern North America and has played a key role in the maintenance of fish biodiversity, especially as a refuge for freshwater fishes during glaciations. In this study, we investigated the phylogeography of the bluegill sunfish, Lepomis macrochirus, in eastern North America, using complete sequence of the mitochondrial ND1 gene from 369 samples collected at 15 sites. Phylogenetic analysis revealed two major lineages (northern and southern clades) in a parsimony network. A sympatric distribution of the lineages was widely observed in the Mississippi Basin. Sequence diversity in the two lineages was significantly lower in glaciated regions around the Great Lakes than in unglaciated regions. The two lineages were estimated to have diverged in the Kansan glaciation, and refugia for both existed around the Ouachita Highlands. The southern clade dispersed during the Yasmouth Interglacial, prior to the dispersal of the northern clade during the Sangamon Interglacial. In the northern clade, low genetic diversity and population fragmentation inferred by nested clade analysis (NCA) were considered due to bottleneck events in the Wisconsin glaciation, while the southern clade showed isolation by distance in a Mantel test. A difference in demographic fluctuation suggests that sympatry of the two lineages has resulted from recent secondary admixture through the range expansion of the northern clade in the post-Pleistocene. Large-scale admixture of multiple mtDNA lineages in L. macrochirus, which has not been recorded in other fishes in the Mississippi River Basin, may result from their high vagility.

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis.

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.

Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis.

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type-specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.

Origin and dispersal of bluegill sunfish, Lepomis macrochirus, in Japan and Korea.

The bluegill sunfish, Lepomis macrochirus, is a notorious exotic species in many freshwater ecosystems, currently expanding its distribution worldwide. In 1960, a small group of bluegills captured in the Mississippi River at Guttenberg in Iowa were imported to Japan as a gift from the mayor of Chicago to the Japanese government. The offspring of these fish were released into the wild in Japan and also in Korea. Over 40 years after this first introduction, L. macrochirus now occupies all the freshwater ecosystems of both countries. We compared invading populations of L. macrochirus in Japan and Korea with native populations in the USA, using PCR-RFLP (polymerase chain reaction-restricted fragment length polymorphism) analyses of mitochondrial DNA, to estimate the origin and dispersal of L. macrochirus in Japan and Korea. Five haplotypes of mitochondrial DNA detected in Japanese and Korean populations completely coincided with the haplotypes of the Guttenberg population. Haplotype diversity of invading populations was shown to be highest in populations established in the 1960s, while genetic variability was lower in more recently established populations. Our results suggest that all L. macrochirus in Japan and Korea have originated from the 15 fish first introduced in 1960. Low haplotype diversity in newly established populations is probably due to genetic drift arising from repeated population bottlenecks, while the high similarity of haplotypes among neighbouring populations is considered to reflect the history of transplantation by humans.

Low genetic variation and inbreeding depression in small isolated populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus.

The Japanese rosy bitterling, Rhodeus ocellatus kurumeus, has been affected not only by the invasion of another subspecies, R. o. ocellatus, from China, but also by habitat fragmentation. In this study, the effects of habitat fragmentation on the fitness of R. o. kurumeus were investigated. Owing to exclusion by R. o. ocellatus, R. o. kurumeus in Honshu and Shikoku has disappeared entirely, except for small populations in isolated man-made ponds in Osaka and Kagawa. In Kyushu it still occupies open water systems, into which R. o. ocellatus has only recently invaded. Meristic and genetic data show that the diversity of R. o. kurumeus is significantly lower in the isolated Osaka and Kagawa populations than the non-isolated Fukuoka population. The Osaka population is inferior to the Fukuoka population in terms of viability and growth. The viability of reciprocal inter-population hybrids between the Osaka and Fukuoka populations was, however, as high as that of the Fukuoka population. In addition to the high scores of band sharing index (BSI) in RAPD-PCR analysis, acceptance of transplanted scales among individuals, irrespective of natal pond, indicates that the Osaka population forms a highly inbred line. These results suggest that low genetic variation is associated with inbreeding depression in the small isolated Osaka populations. Consequently, the management of ponds, including the free movement of individuals, in addition to measures to prevent the invasion of R. o. ocellatus, is necessary for the conservation of R. o. kurumeus.

Normalization of high-flow or removal of flow cannot stop high-flow induced endothelial proliferation.

Endothelial cells (ECs) are activated in response to high-flow. Our previous studies using arteriovenous fistula (AVF) model have demonstrated that high-flow in blood vessels induces an early and rapid proliferation of ECs before arterial dilatation. Here, we investigated the proliferation of ECs, which had once been stimulated by high-flow loading, in a situation without the influence of high-flow. First, we induced high-flow in the rabbit common carotid artery by using AVF. Then, we removed the influence of high-flow by normalization of high-flow with the closure of AVF or by removal of flow itself with tissue isolation and organ culture or with cell culture of ECs, at the timing considered that ECs began to proliferate. Kinetics of ECs was investigated by a laser scanning confocal microscopy, phase-contrast microscopy and light microscopy using bromodeoxyuridine labeling method. We found that ECs, which had once been stimulated by high-flow, transiently proliferated even after normalization of high-flow or removal of flow. We assume that proliferation of ECs is promised when these cells start to proliferate after high-flow loading.

Androgenetic reproduction in a freshwater diploid clam Corbicula fluminea (Bivalvia: Corbiculidae).

Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.