Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

Development of the vertebra and fin skeleton in the lamprey and its implications for the homology of vertebrate vertebrae.

BACKGROUND: Although vertebrae are the defining character of vertebrates, they are found only in rudimentary form in extant agnathans. In addition, the vertebrae of agnathans possess several unique features, such as elastin-like molecules as the main matrix component and late (post-metamorphosis) differentiation of lamprey vertebrae. In this study, by tracing the developmental process of vertebrae in lamprey, we examined the homology of vertebrae between lampreys and gnathostomes. RESULTS: We found that the lamprey somite is first subdivided mediolaterally, with myotome cells differentiating medially and non-myotome cells emerging laterally. Subsequently, collagen-positive non-myotome cells surround the myotome. This pattern of somitogenesis is rather similar to that in amphioxi and sheds doubt on the presence of a sclerotome, in terms of mesenchyme cells induced by a signal from the notochord, in lamprey. Further tracing of non-myotome cell development revealed that fin cartilage develops in ammocoete larvae approximately 35 mm in body length. The development of the fin cartilage occurs much earlier than that of the vertebra whose development proceeds during metamorphosis. CONCLUSION: We propose that the homology of vertebrae between agnathans and gnathostomes should be discussed carefully, because the developmental process of the lamprey vertebra is different from that of gnathostomes.

Modeling transcriptional regulation of model species with deep learning.

To enable large-scale analyses of transcription regulation in model species, we developed DeepArk, a set of deep learning models of the cis-regulatory activities for four widely studied species: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, and Mus musculus DeepArk accurately predicts the presence of thousands of different context-specific regulatory features, including chromatin states, histone marks, and transcription factors. In vivo studies show that DeepArk can predict the regulatory impact of any genomic variant (including rare or not previously observed) and enables the regulatory annotation of understudied model species.

Phylogeographic History of Endangered Hokuriku Salamander, Hynobius takedai (Amphibia: Caudata).

Knowledge of the phylogeographic history of organisms is valuable for understanding their evolutionary processes. To the best of our knowledge, the phylogeographic structure of Hokuriku salamander, Hynobius takedai, an endangered species, remains unclear. This study aimed to elucidate the phylogeographic history of H. takedai, which is expected to be strongly influenced by paleogeographic events. Phylogenetic analysis based on partial sequences of the mitochondrial DNA cytochrome b gene confirmed the genetic independence of H. takedai, and the divergence time with closely related species was estimated to be from the Late Pliocene to the Early Pleistocene. In the phylogenetic tree, two clades were identified within H. takedai, and their haplotypes were found in samples collected from the west and east of the distribution range. These intraspecific divergences were strongly influenced by geohistorical subdivisions of the current major distribution areas in the Middle Pleistocene. One clade was further subdivided and its formation may have been influenced by sea level changes in the Late Pleistocene.

Landscape Genetic Analysis for the Japanese Wild Boar in the Early Expanding Stage in the Hokuriku Region of Japan.

As a contribution to improving management of the Japanese wild boar (Sus scrofa leucomystax), which has recently expanded its range and is having some negative effects on the ecosystem, we conducted a landscape genetic study using individual-based genetic analysis and multiple landscape elements to elucidate its dispersal patterns in the early stage of its expansion. Microsatellite DNA analysis of Japanese wild boars in the Hokuriku region of Japan revealed the existence of two ancestral genetic clusters, that they had migrated via different pathways, and that they were inadequately admixed. We also inferred the most suitable habitats for Japanese wild boar using MaxEnt and concluded that lower elevation and snowfall may favor the occurrence of wild boar individuals. Landscape genetic analysis indicated regional differences in Japanese wild boar dispersal patterns, according to the spatial heterogeneity of genetic features and landscape elements. On the western side of the study area, where individuals with a high frequency of one of two ancestral clusters were more abundant, significant effects of isolation by distance and resistance due to the above two landscape factors were detected, suggesting unidirectional dispersion influenced by the alpine landscape. In contrast, on the eastern side, there was indication of resistance to dispersal of individuals predominantly possessing another ancestral cluster, suggesting the influence of irregularly arranged suitable habitats due to the complexity of the mountainous terrain. Based on our findings, we conclude that Japanese wild boar dispersal patterns may be influenced by landscape elements, such as alpine mountains.

Regulation of spatiotemporal limits of developmental gene expression via enhancer grammar.

The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, we measured the contribution of individual binding sites in transcriptional regulation. We show that binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, we show that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. We propose that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.

Effects of paddy irrigation-drainage system on water quality and productivity of small rivers in the Himi region of Toyama, Central Japan.

The aim of this study was to clarify the impact of differences between historical and recently introduced irrigation and drainage management systems on water quality in the rivers around paddy fields. We investigated the seasonal variation in nutrients concentration and dissolved organic carbon (DOC) components in single- (used for intake only) and dual-purpose (used for both intake and drainage) channels during a 4-year period in the Himi region of Toyama, Central Japan. The system of dual-purpose channel has traditionally been used in the region of this study. A total of 197 three-dimensional excitation-emission matrix (3DEEM) fluorescence spectra of DOM in waters were applied for the parallel factor analysis (PARAFAC) modeling. Based on the 3DEEM and PARAFAC, the abundance of terrestrial humic-like components in the dual-purpose channel was significantly higher than that in the single-purpose channel. The even long-chain n-fatty acids derived associated with rice cropping in sediments of the dual-purpose channels were 22-30-fold higher than that of the single-purpose channel. In addition, the turbidity values of the river waters had significantly positive linear correlations with concentrations of K+, DOC, and humic-like components. These observations indicate that the dissolved nutrient concentrations in the river water were higher in the dual-purpose channel compared to those of the single-purpose channel, which may be supplied by leaching from the inflow of soil particles from the paddy fields. During the mid-irrigation period, the quantity of epiphytic chlorophyll a on artificial substrate tiles in the dual-purpose channel were 3.1-4.1-fold higher than that in the single-purpose channel. This study clear that the input of paddy drainage during the irrigation season significantly changes the DOC components in river waters and irrigation management is strongly linked to the primary production in agricultural channels. Therefore, it is important to consider the impact of the introduction of different irrigation and drainage management systems on water quality and productivity in order to maintain the riverine ecosystems around rice paddies, which are based on historical water use systems.

Fine-scale genetic structure of the endangered bitterling in the middle river basin of the Kiso River, Japan.

Recently, anthropogenic alterations have had severe and negative impacts on the terrestrial and aquatic species and environments. To conserve species that have a small and limited habitat, it is necessary to focus on fine-scale population structure and its effects on persistence. The deepbodied bitterling Acheilognathus longipinnis is an endangered freshwater fish that occupies ponds scattered in lateral bars in the Kiso River. In this study, we conducted multi-locus microsatellite DNA analysis to evaluate both fine-scale population structure and genetic diversity, in order to conserve A. longipinnis. The smaller number of loci deviating from the Hardy-Weinberg equilibrium in ponds scattered in individual lateral bars compared to the whole river system suggests that A. longipinnis forms a local breeding population in units of ponds. The population was roughly split between the river banks and the local population located in ponds in the mid-channel bar showed intermediate relationships with the river bank populations. Gene flow between local populations was not always homogeneous and was not influenced by geographical distances between local populations or the direction of river flow. The dispersal of A. longipinnis across both river bank sides may be constrained and is probably affected by the ecological characteristics of A. longipinnis and the hydrological regimes. Consequently, A. longipinnis in the Kiso River is maintained as a complex of multiple local populations with appropriate gene flow among them. To conserve A. longipinnis, both the persistence of the unstable ponds and moderate genetic exchanges by individual migration are required.

Hybridization of Two Species of Japanese Toads, Bufo torrenticola and Bufo japonicus formosus, in the Central Part of Japan.

Interspecific hybridization between two species of Japanese toads, Bufo torrenticola (Bt) and Bufo japonicus formosus (Bjf), was not thought to have occurred due to the differences in their spawning habitats, although their hybridization was reported by previous research in a limited area. Such hybridization could have pivotal effects, such as genetic admixture, on Bt, which has a limited distribution and habitat. In this study, to clarify the details of hybridization, a total of 192 individuals were collected from multiple parapatric regions and an only Bjf allopatric region and analyzed based on morphology, mtDNA sequences, and nuclear DNA microsatellite loci. Each individual was identified based on morphological type and mtDNA lineage, and two mismatched combinations, Bt or an intermediate morphological type and the Bjf mtDNA lineage, were detected. In particular, the combination of Bt type and Bjf lineage was dominant in parapatric regions, and it was considered that asymmetric hybridization in which Bjf became a matriline had occurred. In addition, a Bayesian clustering method based on the microsatellite loci was conducted to detect a genetic admixture of the two toad species. Individuals representing a combination of the Bt type and Bjf lineage in the parapatric regions were largely assigned to the Bt cluster or equally to both clusters. These results indicate that backcross with Bt has been repeated, and therefore the traits of Bt might be maintained in this study.

Population Structure, Admixture, and Migration Patterns of Japanese Sika Deer (Cervus nippon) Inhabiting Toyama Prefecture in Japan.

Rapid expansion of sika deer, in both number and distribution, in the Japanese Archipelago has resulted in serious ecological disturbance. In the present study, the population structure and migration patterns of sika deer (Cervus nippon) among Toyama and adjacent Prefectures were investigated using 11 polymorphic microsatellite loci. Deviation from Hardy-Weinberg equilibrium was detected in both total and individual regional sika deer samples from Toyama Prefecture. Results of pairwise FST results, factorial correspondence analysis, and STRUCTURE analysis indicated that sika deer in Toyama are not genetically distinct from those in adjacent Prefectures. Bayesian STRUCTURE results suggested the existence of two distinct clusters. However, multiple lines of genetic structure and high admixture were detected across the populations located in the central region of Toyama Prefecture. Both contemporary and historical migration analyses showed that dispersal into Toyama Prefecture from neighboring prefectures was high, especially migration from the prefecture on the east into Toyama Prefecture, and bidirectional dispersion between Toyama Prefecture and the prefecture to the south. Knowledge of such genetic structures and population dynamics is required for appropriate management and conservation of sika deer populations in the Japanese Archipelago.

Dose-dependent role of claudin-1 in vivo in orchestrating features of atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. It was recently noted that the characteristics of epidermal barrier functions critically influence the pathological features of AD. Evidence suggests that claudin-1 (CLDN1), a major component of tight junctions (TJs) in the epidermis, plays a key role in human AD, but the mechanism underlying this role is poorly understood. One of the main challenges in studying CLDN1's effects is that Cldn1 knock-out mice cannot survive beyond 1 d after birth, due to lethal dehydration. Here, we established a series of mouse lines that express Cldn1 at various levels and used these mice to study Cldn1's effects in vivo. Notably, we discovered a dose-dependent effect of Cldn1's expression in orchestrating features of AD. In our experimental model, epithelial barrier functions and morphological changes in the skin varied exponentially with the decrease in Cldn1 expression level. At low Cldn1 expression levels, mice exhibited morphological features of AD and an innate immune response that included neutrophil and macrophage recruitment to the skin. These phenotypes were especially apparent in the infant stages and lessened as the mice became adults, depending on the expression level of Cldn1 Still, these adult mice with improved phenotypes showed an enhanced hapten-induced contact hypersensitivity response compared with WT mice. Furthermore, we revealed a relationship between macrophage recruitment and CLDN1 levels in human AD patients. Our findings collectively suggest that CLDN1 regulates the pathogenesis, severity, and natural course of human AD.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.

BACKGROUND & AIMS: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. METHODS: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. RESULTS: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. CONCLUSION: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. LAY SUMMARY: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Immune responses against Lewis Y tumor-associated carbohydrate antigen displayed densely on self-assembling nanocarriers.

Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were studied. The high surface density of LY was obtained by the A2B-type amphiphilic polypeptides having LY at the two terminals [LY-poly(sarcosine)2-b-(l- or d-Leu-Aib)6]. The equimolar mixture of these two amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single administration. However, the amount of anti-LY IgM produced was moderate and significantly less than that induced by two administrations of the other molecular assembly (G1) with the average surface density of LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the avidity more than after one administration.

Genetic Population Structure of Sika Deer, Cervus nippon, Derived from Multiple Origins, Around Toyama Prefecture of Japan.

Recently, expansion of the number and distribution of sika deer, Cervus nippon, in the Japanese Archipelago has resulted in the disturbance of indigenous gene pools and ecosystems. There are also concerns that the artificial introduction of sika deer to certain areas may aggravate this situation. In order to contribute to the conservation of ecosystems, I examined the current state of genetic disturbance and dispersal routes in the sika deer populations around Toyama Prefecture, one of the main areas of expanding sika deer distribution. Of 12 haplotypes detected by mitochondrial DNA D-loop sequence analysis, 10 were found to belong to a previously detected sika deer group in northern Japan, although the remaining two haplotypes corresponded to the southern Japanese sika deer group. The latter two haplotypes were detected at especially high frequencies in the southern area of Toyama Prefecture, suggesting that these haplotypes may derive from artificially introduced individuals. Occurrence patterns of indigenous haplotypes around Toyama Prefecture revealed immigration into Toyama Prefecture through different routes, mainly in the east and south-west. The genetic results presented here may have application in predicting future dispersal routes, as well as aid in the establishment of effective measures for management of sika deer.

Immune activation with peptide assemblies carrying Lewis y tumor-associated carbohydrate antigen.

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Ley ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine)m -block-poly(L-lactic acid)30 (m = 15 or 50, Lactosome), poly(sarcosine)m -block-(D/L-Leu-Aib)n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Ley on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Ley and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Ley IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Ley IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Ley on the molecular assembly was required for anti-Ley IgM production. The antigenicity of Ley is therefore dependent on the molecular assemblies on which Ley is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Liver maturation deficiency in p57(Kip2)-/- mice occurs in a hepatocytic p57(Kip2) expression-independent manner.

Fetal hepatic stem/progenitor cells, hepatoblasts, are highly proliferative cells and the source of both hepatocytes and cholangiocytes. In contrast, mature hepatocytes have a low proliferative potency and high metabolic functions. Cell proliferation is regulated by cell cycle-related molecules. However, the correlation between cell cycle regulation and hepatic maturation are still unknown. To address this issue, we revealed that the cell cycle inhibitor p57(Kip2) was expressed in the hepatoblasts and mesenchymal cells of fetal liver in a spatiotemporal manner. In addition, we found that hepatoblasts in p57(Kip2)-/- mice were highly proliferative and had deficient maturation compared with those in wild-type (WT) mice. However, there were no remarkable differences in the expression levels of cell cycle- and bipotency-related genes except for Ccnd2. Furthermore, p57(Kip2)-/- hepatoblasts could differentiate into mature hepatocytes in p57(Kip2)-/- and WT chimeric mice, suggesting that the intrinsic activity of p57(Kip2) does not simply regulate hepatoblast maturation.

A Novel Chemoenzymatic Synthesis of Sulfated Type†2 Tumor-Associated Carbohydrate Antigens by Transglycosylation of Sulfated Lewis X Oxazoline Catalyzed by Keratanase†II.

Sulfated type 2 carbohydrate chains are known tumor-associated carbohydrate antigens (TACAs). Many reports on cancer vaccines employing TACAs as specific antigens have been published, but structurally specified sulfated TACAs have not been used because of the low natural abundance and difficulties in chemical synthesis. We demonstrate for the first time the synthesis of the sulfated type 2 TACAs with an l-fucose branch by keratanase-II-catalyzed transglycosylation of the sulfated Lewis X (Galß(1→4)[Fucα(1→3)]GlcNAc(6-OSO3- ); su-Lex ) oxazoline derivative. Two keratanase IIs (from Bacillus sp. Ks36 and Bacillus circulans KsT202) efficiently catalyzed the transglycosylation reaction of the su-Lex oxazoline derivative, thereby giving the su-Lex dimer as the main product in good yields. Structural analysis of the oligomers confirmed exclusive formation of the ß(1→3) glycosidic bond.

Reduced immune response to polymeric micelles coating sialic acids.

Effects of sialic acid coatings on polymeric micelle consisting of poly(sarcosine)-block-poly(l-lactic acid) (Lactosome) in the aim of prevention of the accelerated blood clearance (ABC) phenomenon are studied. Two kinds of the sialic acid-presenting Lactosomes targeting the immunosuppressive receptors of Siglec-G and CD22 have been successfully prepared. Lactosome presenting 5-N-acetylneuraminic acid or 5-N-acetylneuraminyl-α(2→6)-galactosyl-ß(1→4)-N-acetylglucosamine at the nanocarrier surface diminished the ABC phenomenon due to the reduction of the anti-poly(sarcosine) IgM production. Further, the sialic acid moieties could interact possibly with Siglec-E on immune cell to suppress phagocytosis of the opsonized nanocarriers.

Investigation of bipotent differentiation of hepatoblasts using inducible diphtheria toxin receptor-transgenic mice.

AIM: Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bipotential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bipotency and proliferative ability. METHODS: Hepatic transplantation model using Cre-inducible diphtheria toxin receptor-transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein (AFP) regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and we analyzed their differentiation and proliferation abilities by immunostaining and gene expression profiles. RESULTS: Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes. CONCLUSION: In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo.