Biology subjects

Nishihara, H.

Publications and source records attributed to Nishihara, H..

13 recordsLinked to original sources

bioRxiv2026

Early origin of sugar sensing in jawed vertebrates

Sweet taste guides animals to consume carbohydrate-rich foods, and many different vertebrate groups, from fish to mammals, rely on sugar-rich fruits or nectar produced by flowering plants (angiosperms). Although the genes encoding T1R2-T1R3, the receptor pair that mammals use to sense sugars, exist in the genomes of many vertebrates, their functions are unclear--and whether sugar sensing arose once early in vertebrate evolution or independently in different lineages after angiosperms evolved is currently unknown. Here, we combined ancestral reconstruction and receptor functional profiling to examine the evolutionary history of T1R taste receptors--including recently-described non-canonical receptors--across all major vertebrate clades. Our results pinpoint the origin of sugar sensing to before the emergence of angiosperms and uncover a myriad of alternative T1R-based sugar-sensing mechanisms, suggesting multiple independent T1R trajectories and revealing uncharted sensory diversity across vertebrates.

evolutionary biology↗

bioRxiv2025

Vitamin D differentially modulates effector and regulatory T-cell migration across the blood-brain barrier

Multiple sclerosis (MS) is an inflammatory disease of the CNS influenced by a combination of genetic predisposition and environmental factors. Vitamin D (VitD) deficiency is considered a major risk factor for MS. While VitD is associated with immunomodulatory roles, the exact mechanisms by which VitD protects from disease are still largely unknown. CD4 T cells play a key role in MS pathogenesis with autoimmune effector T cells (Teff) infiltrating the CNS across the blood-brain barrier (BBB) and regulatory T cells (Treg) displaying impaired functions. Here we show that treatment of human CD4 T cells with the active form of VitD (1,25-Dihydroxyvitamin D3; 1,25(OH)2D3) decreased cell-surface expression of 4{beta}1- and L{beta}2-integrins on Teff but not Treg and reduced Teff adhesion to their endothelial ligands VCAM-1 and ICAM-1. By employing live cell imaging, we observed that VitD treatment reduced arrest of Teff but not Treg to the BBB as well as ICAM-1 and VCAM-1 under physiological flow in vitro and differentially affected post-arrest behaviour of Teff versus Treg on the BBB under physiological flow. Furthermore, VitD treatment favoured the migration of Treg over Teff across the BBB under static and flow conditions in vitro. In vivo live cell imaging showed that VitD reduced T cell arrest on the inflamed BBB during autoimmune neuroinflammation. Finally, VitD also reduced expression of integrins mediating CNS homing on pathogenic CD4 T cells isolated from the CSF of persons with MS (PwMS). As VitD treatment did not alter barrier properties or adhesion molecule profile of our BBB model we propose a beneficial effect of VitD supplementation in PwMS by reducing CNS trafficking of pro-inflammatory T cells while leaving CNS entry of Treg unaffected.

immunology↗

bioRxiv2025

Flamenco plasticity tunes somatic piRNAs, rewiring host isoforms and opening a route to heritable transposon spread

Transposons drive genome innovation, yet how they evade somatic piRNA defenses, reach the germline, and rewire host genes with limited cost remains unclear. Using comparative long-read sequencing in Drosophila ovarian somatic cells (OSCs), we show that the LTR transposon Springer rewires gene expression by inserting into promoter-proximal introns at an AT-rich motif. Its 5' LTR initiates transcripts that splice into host exons, expanding isoform diversity without adding coding sequence. We catalog 72 Springer-driven isoforms, indicating broad mutagenic potential. In parallel, the Flamenco (Flam) uni-strand piRNA cluster undergoes structural remodeling that replaces antisense transposon fragments with forward-oriented copies, reshaping piRNA populations and eroding silencing of specific elements, including Springer. This relaxation of somatic repression creates conditions permissive for germ-cell entry, providing a plausible route to heritable genome change. Thus, Flam plasticity, contrasting with the relative stability of dual-strand clusters, mechanistically links transposon activity to genome rewiring and evolutionary innovation.

genetics↗

bioRxiv2024

Selectivity and evolution of Aqp10 in solute permeability influenced by pore molecular weight

Aqp10 is an aquaglyceroporin that transports not only water but also uncharged low-molecular-weight compounds. We previously demonstrated the evolution of solute permeability in Aqp10 paralogs and showed that the urea and boric acid permeabilities of Aqp10.2 were much weaker than those of Aqp10.1 and plesiomorphic Aqp10s. However, the molecular mechanism responsible for the weak permeability of Aqp10.2 to urea and boric acid remains unclear. Herein, we present a novel hypothesis that explains the solute selectivity of Aqp10. We deduced the ancestral sequences of Aqp10.1 and Aqp10.2 paralogs via molecular phylogenetic analysis. Constructed structural models of these sequences revealed that both the well known amino acid site at position 3 and the sum of molecular weights of the four amino acid sites in the ar/R region were important for the formation of the Aqp10 selectivity filter. Site-directed mutagenesis revealed that a decrease in the sum of the molecular weights of the four amino acid sites enhanced the Aqp10 permeability to urea and boric acid. Based on this, we proposed a model in which the presence of two or more bulky amino acids in the ar/R region, which increases the sum of the molecular weights of amino acids in the ar/R region, was essential for the formation of a filter that limited urea and boric acid transport. Our results outline the molecular mechanism by which Aqp10.2 acquired a selectivity filter during evolution and provide structural insights into the narrowly tuned filter responsible for the solute selectivity of aquaglyceroporins. Significance StatementIn aquaglyceroporins, particularly Aqp10s, the urea and boric acid permeabilities of Aqp10.2 paralogs are much weaker than those of plesiomorphic Aqp10s. Here, we deduced the ancestral sequences of Aqp10.1 and Aqp10.2 via molecular phylogenetic analysis. Structural models of these sequences revealed that the sum of the four amino acid site molecular weights in the ar/R region, if more than one is bulky, contributed to the selectivity filter formation in the pore region. Using site-directed mutagenesis, reduction in the molecular weight of one bulky amino acid residue in the ar/R region restricted urea and boric acid permeability. Therefore, Aqp10.2 acquired a selectivity filter during evolution, and structural differences in this selectivity filter are responsible for the variable solute permeability of aquaglyceroporins.

physiology↗

bioRxiv2024

Establishment of a novel amyotrophic lateral sclerosis patient-derived blood-brain barrier model: Investigating barrier dysfunction and immune cell interaction

Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no effective treatment. The main feature of the disease is the loss of motor neurons in the central nervous system (CNS), but other CNS components and the immune system also appear to be involved in the pathogenesis of the disease. The blood-brain barrier (BBB) is formed by specialized brain microvascular endothelial cells (BMECs) and plays a crucial role in maintaining homeostasis within the central nervous system (CNS). Autopsy studies have reported BBB abnormalities in ALS patients, and studies in animal models suggest that BBB breakdown may precede neurodegeneration. However, the difficulty of obtaining patient samples has hampered further studies. We used the method of differentiating BMEC-like cells from human induced pluripotent stem cells (hiPSCs), which have robust barrier functions and express adhesion molecules necessary for immune cell adhesion, to investigate BBB functions in ALS patients. We also established a method using automated analysis techniques to analyze immune cell adhesion to the BBB in multiple samples simultaneously and with minimal bias. BMEC-like cells derived from ALS patients carrying TARDBP N345K/WT mutations exhibited increased permeability to small molecules due to loss of tight junction, as well as increased expression of cell surface adhesion molecules, leading to enhanced immune cell adhesion. BMEC-like cells derived from hiPSCs with other types of TARDBP gene mutations (TARDBP K263E/K263E and TARDBP G295S/G295S) introduced by genome editing technology did not show such BBB dysfunction compared to the isogenic control. These results indicate that our model can recapitulate BBB abnormalities reported in ALS patients using patient (TARDBP N345K/WT) -derived hiPSCs and that two types of missense mutation, TARDBP K263E/K263E and TARDBP G295S/G295S, do not contribute to the BBB dysfunction. This novel ALS patient-derived BBB model is useful for the challenging analysis of the involvement of BBB dysfunction in the pathogenesis and to promote therapeutic drug discovery.

cell biology↗

bioRxiv2023

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B or BAZ2A could determine the localization of H3K36me2 in different cell types. To explore the physiological role of heterochromatic H3K36me2, we analyzed mouse tissues and embryos. As a result, H3K36me2 was found in heterochromatin at the 2- to 4-cell stages of mouse preimplantation embryos, suggesting its involvement in developmental regulation. SummaryThe authors discovered histone H3K36me2, which is believed to be enriched in potentially active genomic regions, is also located in transcriptionally inactive regions called heterochromatin in some cell types. The detailed molecular mechanism of its heterochromatin targeting is now revealed.

cell biology↗

bioRxiv2023

Morc1 re-establishes heterochromatin on activated transposons and shapes the host transcriptome in gonocytes

Following reprogramming of DNA methylation, numerous transposable elements (TEs) are transiently activated in male prenatal gonocytes. Persistent expression of such TEs to adulthood leads to arrest of germ cell development. However, how these TEs are re-silenced has been unexplored. Here, we found that DNA-binding protein Morc1 re-established H3K9me3-marked heterochromatin on activated TEs, which involved methyltransferase SetDB1. Although Morc1 also triggered DNA methylation, the types of Morc1-targeted TEs for each epigenetic modification were different, suggesting that these two mechanisms were largely independent of each other. Significant overlap between TEs targeted by Morc1 and those by Miwi2, a nuclear PIWI protein, indicated that piRNA-loaded Miwi2 conferred target specificity to Morc1. Activated TEs drove transcription of adjacent genes by acting as ectopic cis-regulatory elements. Such a disrupting effect of TEs on the transcriptome was compensated by Morc1. Thus, Morc1 ensures proper interactions of TEs with host genes through re-establishment of heterochromatin in gonocytes.

bioinformatics↗

bioRxiv2023

Latent taste diversity revealed by a vertebrate-wide catalogue of T1R receptors

Taste is a vital chemical sense for feeding behavior. In mammals, the umami and sweet taste receptors are composed of three members of the taste receptor type 1 (T1R/TAS1R) family: T1R1, T1R2, and T1R3. Because their functional homologs exist in teleosts, only three TAS1R genes generated by gene duplication are believed to have been inherited from the common ancestor of bony vertebrates. Here, we report five previously uncharacterized TAS1R members in vertebrates, named TAS1R4, 5, 6, 7, and TAS1Rcf, through a genome-wide survey of diverse taxa. For TAS1R2 and TAS1R3, mammalian and teleost fish genes were found to be paralogous. Phylogenetic analysis suggests that the bony vertebrate ancestor had nine TAS1Rs due to multiple gene duplications, and some TAS1Rs were lost independently in each lineage; ultimately, mammals and teleosts have retained only three TAS1Rs, whereas other lineages have retained more TAS1Rs. Functional assays and expression analysis in non-teleost fishes suggest that the novel T1Rs form heterodimers in taste receptor cells and contribute to the recognition of a broad range of ligands such as essential amino acids, including branched-chain amino acids, which were not previously considered as T1R ligands. These results highlight an unexpected diversity of taste sensations in both modern and the ancestors of vertebrates. The complex evolution of the taste receptor family might have enabled vertebrates to adapt to diverse habitats on Earth.

evolutionary biology↗

bioRxiv2022

Splashed E-box and AP-1 motifs cooperatively drive regeneration-response and shape regeneration abilities

Injury triggers genetic program to induce gene expression for regeneration. Several studies have recently reported the identification of regeneration-response enhancers (RREs) in zebrafish; however, it remains unclear whether a common mechanism operates in RREs. Here, we show that E-box and activator protein 1 (AP-1) motifs cooperatively function as RREs. We identified three RREs from the fn1b promoter by a search of conserved sequences and an in vivo transgenic assay for regeneration-response in zebrafish. Two of them derived from transposons displayed RRE activity only when combined with the -0.7 kb fn1b promoter, while another non-transposable element functioned as a standalone enhancer. A search for transcription factor-binding motifs and validation by transgenic assay revealed that both of E-box and AP-1 motifs are necessary and sufficient for RREs. Such RREs responded to variety of tissue injuries including zebrafish heart and Xenopus limb bud regenerations. Our findings highlight that regeneration is regulated by merging two activating signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome shaped regenerative capacities during evolution. SUMMARY STATEMENTThe study revealed that regeneration-response enhancer is composed of two transcription factor-binding motifs. The fidelity of regeneration-dependent gene expression is ensured by merging two activating signals evoked by injuries.

developmental biology↗

bioRxiv2022

The Modular μSiM: a Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support on-chip clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, we introduce an extension of our {micro}SiM (microdevice featuring a silicon-nitride membrane) platform. The modular {micro}SiM (m-{micro}SiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. We demonstrate the utility of the m-{micro}SiM by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and non-engineering, brain barriers focused laboratories. We develop and validate in situ and sampling-based assays of small molecule diffusion as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-{micro}SiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. We then demonstrate the ability to quickly reconfigure the m-{micro}SiM for co-culture and immune cell transmigration studies through addition of accessories and/or quick exchange of components. Because the development of modified components and accessories is easily achieved, custom designs of the m-{micro}SiM should be accessible to any laboratory desiring a barrier-style tissue chip platform.

bioengineering↗

bioRxiv2021

Placental mammals acquired the functional region and domain in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologues across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: (1) mouse NRK is localised at the plasma membrane via the CNH domain, where the CIR inhibits CK2. (2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN, and (3) leads to the inhibition of AKT signalling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

molecular biology↗

bioRxiv2021

Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood-brain barrier (BBB) properties in response to extrinsic signals, with Wnt/{beta}-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here we show that activation of Wnt signaling in hPSC-derived naive endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, and decreased PLVAP. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naive ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

neuroscience↗

bioRxiv2020

Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation

In animal gonads, transposable elements (TEs) are actively repressed to preserve genome integrity through the Piwi-interacting RNA (piRNA) pathway. In mice, piRNAs are most abundantly expressed in male germ cells, and form effector complexes with three distinct PIWI proteins. The depletion of individual Piwi genes causes male-specific sterility owing to severe defects in spermatogenesis with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the piRNAs loaded onto PIWIL1 changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'- ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses (ERVs). Although binding sites for the transcription factor A-Myb are identified in the transcription start site regions of the testis piRNA clusters, the piRNA clusters in the ovary show no well-defined binding motifs in their upstream regions. These results show that hamster piRNA clusters are transcribed by different transcriptional factors in the ovary and testis, resulting in the generation of sex-specific piRNAs. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes. Highlights- The size of PIWIL1-associated piRNAs changes during oocyte maturation - Phosphorylation of PIWIL3 in MII oocytes coincides with its association with small 19-nt piRNAs - Improved high-quality genome assembly and annotation identifies young endogenous retroviruses as major targets of piRNAs in hamster oocytes - PIWIL1- and PIWIL3-associated piRNAs share the 5'-ends of the common piRNA precursors in oocytes

molecular biology↗