Biology subjects

Otsuka, K.

Publications and source records attributed to Otsuka, K..

9 recordsLinked to original sources

bioRxiv2026

A Col22a1-EGFP reporter reveals dynamic three-dimensional organization of the myotendinous junction

The myotendinous junction (MTJ) is a specialized extracellular matrix-rich interface that transmits contractile force generated by skeletal muscle to tendon, but its three-dimensional organization and dynamics remain poorly characterized. Here, we generate a Col22a1-EGFP knock-in mouse that enables direct visualization and quantitative analysis of MTJs from embryonic development to adulthood. Tissue-clearing and three-dimensional imaging reveal extensive variation in MTJ morphology and spatial distribution among skeletal muscles and between myofiber types. During embryonic MTJ formation, COL22A1-EGFP undergoes a striking transition from intracellular puncta within developing myofibers to extracellular accumulation at myofiber tips, associated with polarized Golgi organization. We further identify complex tendon protrusions that emerge during postnatal growth and connect tendon bodies with subsets of MTJs. These interfaces contain morphologically distinct Scx-lineage cells that express Col22a1, identifying a specialized tendon-associated population at the MTJ. Finally, muscle unloading and overload differentially alter COL22A1-EGFP signal intensity and area, respectively, demonstrating that the reporter provides a quantitative readout of MTJ responses to altered mechanical conditions. Thus, Col22a1-EGFP reveals previously unrecognized spatial, developmental, and adaptive features of the muscle-tendon interface and provides an experimental platform for investigating MTJ biology in vivo.

developmental biology↗

bioRxiv2026

GSDMD transcript levels are higher in endothelial than cardiomyocyte compartments across three human heart cohorts

Cardiac GSDMD research has largely centred on cardiomyocytes. We quantified donor-level endothelial-minus-cardiomyocyte differences in two single-nucleus cohorts and one GeoMx spatial cohort, retaining assay-specific scales. GSDMD was higher in endothelial compartments in 42/42, 38/38 and 27/32 donors and ranked, post hoc, at the 94.9th, 95.1st and 97.4th percentiles of the corresponding within-cohort signed-contrast distributions. Although positive direction and complete donor concordance were not unique transcriptome-wide, this hypothesis-led target retained high rank across markedly different reference backgrounds and assay modalities. A fixed eight-transcript panel included both endothelial- and cardiomyocyte-shifted members, arguing against a uniform panel-wide directional shift or simple detection failure. GeoMx provided the most discriminating transcriptome background but remained composition-sensitive. GSDMD transcripts are therefore recurrently higher in endothelial than cardiomyocyte compartments across human-heart cohorts, showing that a cardiomyocyte-only interpretation of human-heart GSDMD transcript signals is incomplete.

cell biology↗

bioRxiv2026

Spatial transcriptomics identifies a translayer architecture of pyroptosis-related transcription in systemic sclerosis skin

Anatomical-layer enrichment does not establish cellular attribution, so correspondence with major-cell-class expression must be tested gene by gene. Systemic sclerosis (SSc) skin is informative because inflammasome- and pyroptosis-related molecules have been reported across epidermal, fibroblast-associated and vascular contexts. Spatial transcriptomics of SSc and control skin, with same-donor single-nucleus profiles from 10 SSc donors, revealed gene-specific correspondence. Across 13 discovery sections, NLRP1, PYCARD and CASP4 were epidermally biased, whereas GSDMD was dermally biased. An independent 10-section SSc cohort supported epidermal NLRP1 (q = 0.0078) and PYCARD (q = 0.043), with directional, non-significant support for CASP4 and GSDMD. Despite epidermal NLRP1 bias, endothelial class-pseudobulk exceeded keratinocyte class-pseudobulk in all 10 donors. Restricted three-class reconstruction reproduced the NLRP1 direction in 1 and 0 of 10 donors under the two fixed dermal weightings and in 0-8 of 10 across a post hoc sweep of the dermal endothelial share; PYCARD and GSDMD were concordant in most donors at every weighting, and CASP4 in no more than 4 of 10. Thus, tissue layer and major cell class are gene-specifically non-equivalent axes of spatial interpretation.

immunology↗

bioRxiv2026

Folate-dependent one-carbon metabolism controls meiotic and post-meiotic epigenome remodeling in the male germline

Environmental exposures can influence offspring health through epigenetic alterations in the male germline. Folate deficiency, a dietary perturbation that disrupts one-carbon metabolism and S-adenosylmethionine (SAM) production, has been linked to altered histone methylation and developmental abnormalities in offspring. However, when and how folate availability shapes the germline epigenome during spermatogenesis remains unclear. In this study, unbiased metabolomic profiling of spermatogenic cells uncovers stage-specific metabolic remodeling, including downregulation of serine- glycine-one-carbon (SGOC) metabolism in meiotic spermatocytes. Using a post-weaning folate-deficient mouse model, we investigate how folate availability influences germline epigenome establishment during spermatogenesis. Consistent with this metabolic transition, genome-wide chromatin accessibility profiling demonstrates extensive, stage-dependent remodeling under folate-deficient conditions, particularly in meiotic spermatocytes and post-meiotic spermatids. These accessibility changes display cell-type-specific genomic distributions and preferential localization to repressive chromatin compartments in post-meiotic cells. Histone modification analyses further reveal bidirectional redistribution of the active histone mark H3K4me3 in round spermatids. Although genome-wide distribution of the repressive mark H3K27me3 remains largely stable, folate deficiency alters its nuclear organization. Notably, a subset of H3K4me3 alterations established in post-meiotic cells is retained in mature sperm, providing a mechanistic link between paternal metabolic perturbation and the germline epigenome. Together, these findings demonstrate that folate availability shapes germline epigenome establishment through stage-specific metabolic and chromatin remodeling during spermatogenesis, revealing a metabolic basis for paternal environmental effects on the germline epigenome.

developmental biology↗

bioRxiv2024

Fatty acyl-CoA reductase FAR1 is essential for testicular seminolipid synthesis, spermatogenesis, and male fertility

Seminolipids are testis-specific ether glycolipids that are important for spermatogenesis. The fatty alcohol (ether-linked alkyl moiety) in ether lipids is generated from an acyl-CoA by fatty acyl-CoA reductase (FAR). To date, the diversity of the alkyl and acyl moieties in seminolipids, the specific stage of spermatogenesis during which seminolipids are produced, and the FAR isozyme (FAR1 or FAR2) involved in the synthesis of the alkyl moieties have remained largely unclear. Here, we demonstrated that Far1 is expressed in the mouse testis via quantitative RT-PCR analysis, whereas Far2 was barely detectable. In situ hybridization and quantitative RT-PCR analysis of spermatogenic cells separated via FACS revealed that Far1 is expressed in spermatogonia, spermatocytes, and spermatids. We generated Far1 knockout (KO) mice and found that male Far1 KO mice were infertile. In these mice, sperms were absent in the epididymides and the testes were small, with multinucleated cells and vacuoles in the seminiferous tubules. LC-MS/MS analysis showed that the vast majority of seminolipids (> 90%) in wild-type mouse testes contained C16:0 in both the alkyl and the acyl moieties. Seminolipids were present in all subclasses of spermatogenic cells in wild-type mice, but they were absent in Far1 KO mice. Instead, the production of non-ether, diacyl-type sulfogalactosyl lipids (sulfogalactosyl diacylglycerols) was induced in Far1 KO mice. In conclusion, the alkyl and acyl moieties of seminolipids in the testis are low in diversity, and Far1 is essential for seminolipid synthesis and spermatogenesis.

biochemistry↗

bioRxiv2023

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline

H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the proteins essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

developmental biology↗

bioRxiv2023

KRAB-zinc-finger proteins regulate endogenous retroviruses to sculpt germline transcriptomes and genome evolution

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.

genomics↗

bioRxiv2022

Classification of Extracellular Vesicles based on Surface Glycan Structures by Spongy-like Separation Media

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogenous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in-situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 m) of SPM. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling.

biochemistry↗

bioRxiv2020

Requirement for proper mitochondrial RNA processing in the restrictive control of cell proliferation during early lateral root morphogenesis

Although mechanisms that activate organogenesis in plants are well established, much less is known about the subsequent fine-tuning of cell proliferation, which is crucial for creating properly structured and sized organs. Here we show, through analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis. These mutants formed abnormally broadened (i.e., fasciated) LRs under high-temperature conditions due to excessive cell division. All TDF proteins localized to mitochondria, where they were found to participate in RNA processing: RRD1 in mRNA deadenylation, and RRD2 and RID4 in mRNA editing. Further analysis suggested that LR fasciation in the TDF mutants is triggered by reactive oxygen species generation caused by defective mitochondrial respiration. Our findings provide novel clues for the physiological significance of mitochondrial activities in plant organogenesis.

plant biology↗