Biology subjects

Mizuno, S.

Publications and source records attributed to Mizuno, S..

At least 19 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

Oxygen-permeable microwell plates for high-throughput spheroid culture

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

bioengineering↗

bioRxiv2026

A patient-derived LMX1B variant causes tissue-specific manifestations of nail-patella syndrome in mice

Nail-patella syndrome (NPS) is a multisystem disorder caused by pathogenic variants in LMX1B and is characterized by dysplasia of the nails and patellae as well as extraskeletal complications such as progressive nephropathy and glaucoma. We generated a CRISPR/Cas9 knock-in mouse carrying the R252Q substitution, corresponding to a human LMX1B variant associated with renal-predominant disease. Phenotypic analysis revealed that homozygous mice were viable, but they displayed marked growth retardation and severe bilateral ocular opacity. Interestingly, while this model exhibited clear skeletal and ocular defects, the renal phenotype was relatively mild, although increased urinary albumin excretion, focal glomerular basement membrane abnormalities, and subtle changes in renal gene expression were detected. Beyond the classical NPS hallmarks, mutant mice also displayed midbrain morphological abnormalities, suggesting broader developmental consequences of this LMX1B variant. This patient-derived variant model not only recapitulates the pleiotropic features of NPS but also demonstrates organ-specific susceptibility to the R252Q substitution, providing a foundation for elucidating the complex molecular mechanisms underlying multisystem disease.

genetics↗

bioRxiv2026

Continuous Nuclear Export of p62/SQSTM1 Is Essential for Kidney Homeostasis

The selective autophagy receptor p62/SQSTM1 dynamically shuttles between the nucleus and cytoplasm through distinct nuclear localization and export signals, yet the physiological significance of this trafficking has remained unknown. Here, we generated mice carrying a deletion of the p62 nuclear export signal (dNES) to determine the in vivo role of p62 nuclear export. Homozygous dNES mice developed progressive podocyte injury, glomerulosclerosis, and fatal renal failure by 6-7 weeks of age, whereas heterozygous and dNES/- mice did not develop renal dysfunction. Loss of nuclear export caused constitutive nuclear accumulation of p62, accompanied by the formation of insoluble ubiquitin-positive aggregates and widespread alterations in the renal proteome, including activation of energy metabolism-related pathways and suppression of developmental programs. We previously demonstrated that the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) inhibits the nuclear export receptor XPO1, resulting in nuclear retention of p62 in cultured cells. The present findings provide in vivo evidence that continuous nuclear export of p62 is indispensable for maintaining kidney homeostasis and reveal that excessive nuclear accumulation, rather than cytoplasmic depletion, underlies p62-mediated toxicity. Collectively, these findings establish continuous nuclear export of p62 as an essential mechanism for maintaining kidney homeostasis. Significance StatementThe adaptor protein p62/SQSTM1 continuously shuttles between the nucleus and cytoplasm, but the physiological significance of this trafficking has remained unknown. Here, we show that disrupting the nuclear export signal of p62 causes progressive podocyte injury, glomerulosclerosis, and fatal kidney failure through excessive nuclear accumulation and aggregate formation. In contrast, dNES/+ and dNES/- mice remain healthy, demonstrating that excessive nuclear accumulation, rather than cytoplasmic depletion, drives disease. These findings identify continuous nuclear export as a fundamental mechanism that prevents toxic nuclear accumulation of p62 and preserves kidney homeostasis.

cell biology↗

bioRxiv2026

Visualization of neuronal morphology of the suprachiasmatic nucleus vasopressin neurons by Cre/FLPe-based genetic two-step sparse labelling - GT-SPARCL

Resolving the morphology of individual neurons in densely packed brain regions remains challenging. Sparse labeling is essential for delineating cellular architecture, yet achieving reproducible low-density labeling e.g. < 1% has been a persistent technical hurdle/problem. We present a genetically encoded two-step strategy, GT-SPARCL (Genetic Two-Step Sparse Cre/FLPe Combination Labeling), which leverages two independent stochastic recombination events to deliver stable, tunable low-density labeling in mice. As a proof of concept, we applied this method to visualizing arginine vasopressin (AVP) neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker composed of [~]10,000 neurons per side in mice. Using two-photon imaging of whole-mount SCN blocks, we reconstructed individual AVP neurons and uncovered previously under-resolved morphological heterogeneity. Based on axonal trajectories, we distinguished five structural types, including subclasses with commissural projections to the contralateral SCN and others extending projections beyond the nucleus. The majority ([~]70%) exhibited projections both within and outside the ipsilateral SCN, whereas the second most dominant subset ([~]20%) projected exclusively outside the SCN, representing an output-biased type. In contrast, neurons projecting exclusively within the ipsilateral SCN were exceptionally rare, suggesting that "dedicated" local-circuit AVP neurons do not form a major structural subtype. Collectively, our data indicate that AVP neurons are not structurally uniform but instead comprise diverse projection-defined subtypes, implying subtype-depen-dent contributions to intra-SCN communication, bilateral coupling, and circadian output. Beyond the SCN, our GT-SPARCL method may be applicable for achieving low-density labelling of neurons that can be defined by other specific Cre mouse lines.

neuroscience↗

bioRxiv2026

TSUMUGI: a platform for phenotype-driven gene network identification from comprehensive knockout mouse phenotyping data

SummaryDeciphering complex organismal phenotypes requires elucidation of coordinated functions among multiple genes, yet this remains a fundamental challenge in functional genetics. The International Mouse Phenotyping Consortium (IMPC) has recently established a comprehensive phenotypic atlas based on systematic single-gene knockout mouse lines, providing an unprecedented resource for gene-phenotype associations in mammals. However, extracting phenotype-associated multiple gene relationships remains challenging. Here, we present TSUMUGI, a platform that identifies a phenotype-driven gene network by leveraging gene-phenotype associations from the IMPC. TSUMUGI enables users to initiate analyses from a user-specified phenotype or gene list and interactively explore a gene network. In addition, TSUMUGI highlights human disease-associated genes, supports flexible network filtering, and allows seamless export of a gene network for downstream analyses. By linking shared phenotypic signatures to putative functional gene modules, TSUMUGI provides a framework for systematic interpretation and hypothesis generation of complex organismal phenotypes. Availability and implementationThe web application is available online at https://larc-tsukuba.github.io/tsumugi/. The command-line tools are distributed via PyPI (https://pypi.org/project/TSUMUGI) and Bioconda (https://bioconda.github.io/recipes/tsumugi/README.html). Source code and documentation are hosted at GitHub (https://github.com/akikuno/TSUMUGI-dev) and archived on Zenodo (https://doi.org/10.5281/zenodo.18464478) under the MIT license.

bioinformatics↗

bioRxiv2026

Sparkling Water Co-Consumption Enhances Oxytocin-Linked HR Synchrony and Social Connection in Live Sports Spectatorship

Live sport spectatorship can promote social connection, yet scalable, low-burden strategies to strengthen bonding in real-world spectator settings remain limited. We here tested the hypothesis that co-consuming sparkling water during spectating attenuates hunger and fatigue and strengthens enjoyment and perceived unity, potentially via HR synchrony linked to oxytocin dynamics. In an individually randomized field experiment at a collegiate womens basketball home game (sparkling water: n = 21; plain water: n = 19), spectators consumed 200 mL of sparkling water or plain water in synchrony before tip-off and before the end of halftime, with additional ad libitum intake of the assigned beverage permitted thereafter. We assessed acute freshness and hunger, repeated fatigue and enjoyment, interpersonal HR synchrony from the first half to 15-min post-game, perceived unity toward players and fellow spectators, and salivary oxytocin levels. Relative to plain water, sparkling water increased freshness and reduced hunger after intake, attenuated post-game fatigue, and enhanced enjoyment and perceived unity. HR synchrony was preserved in association with attenuated post-game fatigue after the game, and sustained synchrony was associated with higher perceived unity. Oxytocin decreased more in the sparkling-water group, and this decrease was associated with preserved HR synchrony as well as with changes in unity toward fellow fans. These findings support our hypothesis and suggest that co-consuming sparkling water serves as an alcohol-free, low-burden ritual to enhance social connection during and after live sport events. Highlights- Sparkling water increased freshness and reduced hunger after intake in spectators. - Sparkling water attenuated the post-game increase in subjective fatigue. - Sparkling water increased enjoyment and perceived unity. - Post-game HR synchrony was preserved in association with lower fatigue and higher unity. - Oxytocin decreased more with sparkling water and tracked HR synchrony and fan unity. - Co-consuming sparkling water is a low-burden, alcohol-free ritual for social connection.

neuroscience↗

bioRxiv2025

Sparkling Water Limits Cognitive Fatigue with Stable Pupil Diameter during Real World Esports Training

Prolonged esports play induces cognitive fatigue with reduced executive function and pupil constriction as a neurobiomarker of prefrontal activity across expertise levels. Since this pupil-linked cognitive fatigue is mitigated by sparkling water intake in casual players, we hypothesized that sparkling water prevents cognitive fatigue and sustains esports performance in esports athletes under real training conditions that reflect competitive practice. To test this hypothesis, nineteen hardcore esports players participated in a randomized crossover trial. Each completed two 3-hour sessions of head-to-head virtual football matches while consuming either sparkling water or plain water. Subjective fatigue, enjoyment, and executive function (flanker task) were assessed, and pupil diameter, heart rate, and salivary cortisol were continuously monitored. In-game performance was also recorded. Accuracy declined and pupil constriction progressed under the plain water condition, whereas executive accuracy was preserved and pupil diameter was maintained over the three-hour session in the sparkling water condition. Subjective ratings of fatigue and enjoyment increased similarly in both conditions. Heart rate, including both mean and peak values, and salivary cortisol concentrations showed no significant differences between conditions. In terms of in-game performance, goals, passes, and interceptions did not differ between conditions, whereas the number of fouls was lower and the number of shots was higher in the sparkling water condition. These findings demonstrate that sparkling water mitigates cognitive fatigue and sustains esports performance in esports athletes under real training conditions. As a caffeine- and sugar-free beverage, sparkling water may serve as a practical, low-risk intervention for managing cognitive fatigue and supporting daily practice in modern esports settings.

neuroscience↗

bioRxiv2025

Clonal Dynamics in the Male Germline from Development to Next Generation

Germ cells secure the continuity and evolutionary potential of species. Yet how reproductive success is allocated among the founder cells of the mammalian male germline, the primordial germ cells (PGCs), remains unknown. Here, we quantitatively traced individual PGCs, non-invasively barcoded in mouse embryos, across their establishment, lifelong maintenance within the testis, spermatogenesis, and transmission to the next generation. We found that highly skewed clonal contributions arise from a very early stochastic bottleneck during migration to the developing testes and remain stable throughout adult reproductive life. Reproductive success during adulthood was proportional to the embryonically established PGC clone size. Mathematical modeling shows that the patchy compartmentalization of PGC clones within the extended thin seminiferous tubules maintain clonal diversity and safeguard against the expansion of clones that gain a selective advantage. These findings uncover fundamental principles governing the development, stability, and evolutionary transmission of the mammalian germline.

developmental biology↗

bioRxiv2025

Sparkling Water Consumption Mitigates Cognitive Fatigue during Prolonged Esports Play

Prolonged esports play induces cognitive fatigue, characterized by diminished executive function with pupil constriction. Players often rely on caffeinated or sugary drinks to combat fatigue, but regular use poses health risks. Sparkling water, a sugar- and caffeine-free beverage, stimulates brainstem and prefrontal activity via sensory pathways mediated by transient receptor potential (TRP) channels in the throat. We here tested the hypothesis that sparkling water mitigates cognitive fatigue during prolonged esports play. Fifteen young adult players participated in a randomized crossover trial, each completing two 3-hour sessions of a virtual football game while consuming either sparkling water or plain water. Subjective fatigue, enjoyment, and executive function (via a flanker task) were measured at baseline and hourly, while pupil diameter and heart rate were monitored continuously. Blood glucose and salivary cortisol were also assessed periodically. Compared to plain water, sparkling water significantly attenuated increases in subjective fatigue, enhanced enjoyment, and preserved executive function, along with preventing pupil constriction. Heart rate, blood glucose, and salivary cortisol levels did not differ between conditions. Notably, players committed fewer in-game fouls with sparkling water, while offensive and defensive performance remained unchanged. These findings demonstrate that sparkling water alleviates both subjective and objective signs of cognitive fatigue during prolonged esports play, supporting our hypothesis. This non-caffeinated intervention may help sustain inhibitory control and promote fair behavior, offering a safe and sustainable strategy for managing mental fatigue in modern life. HighlightsO_LISparkling water alleviates cognitive fatigue and boosts enjoyment during esports. C_LIO_LISparkling water prevents pupil constriction linked to cognitive fatigue. C_LIO_LISparkling water keeps players alert without raising blood glucose or cortisol. C_LIO_LISparkling water reduces fouls in virtual football without impairing performance. C_LIO_LISparkling water offers a caffeine- and sugar-free option to manage mental fatigue. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/662064v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1e9058dorg.highwire.dtl.DTLVardef@16b7110org.highwire.dtl.DTLVardef@cbe294org.highwire.dtl.DTLVardef@1886d36_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Spatiotemporal dynamics of CD73 in mouse retina under physiological conditions

Adenosine is essential to energy metabolism and neuromodulation in the central nervous system. The retina is a highly energy-demanding neural tissue, and dysregulation of adenosine signaling causes retinal diseases. However, the dynamics of ecto-5-nucleotidase (CD73), a key enzyme for extracellular adenosine generation, remain elusive. Here, we investigate its spatiotemporal profile from development to adulthood using two transgenic mouse lines. We found that CD73 is transiently expressed in the early astrocyte lineage (Embryonic day 16.5 to Postnatal day [P] 3), becomes prominent in the rod-photoreceptor lineage by P3, and appears in the inner nuclear layer from P7 onward. CD73 deletion delays rod-response recovery and shortens the implicit time in scotopic ERG under dim light. These findings indicate that light and other physiological cues influence CD73 expression, which has significant consequences for retinal function, thereby providing a foundation for exploring disease-related alterations in CD73 and designing therapies that restore adenosine homeostasis.

developmental biology↗

bioRxiv2025

Muc6-expressing gastric isthmus progenitors contribute to regeneration and metaplasia supported by myeloid-mesenchymal interactions

Gastric mucosal homeostasis is maintained by tissue-resident stem and progenitor cells residing in the isthmus region. Following mucosal injury, surviving cells contribute to regeneration, coinciding with characteristic pathological changes such as atrophic gastritis and metaplasia. To comprehensively understand the cellular dynamics involved in this process, we performed single-cell and spatial transcriptomics using newly generated transgenic mice. In human samples and mouse models, loss of gastric chief cells precedes, and even induces, loss of parietal cells during the progression of atrophy and metaplasia, validating the causal relationship underlying the decrease of these two lineages. Single-cell analysis confirmed robust stemness and metaplastic changes in the Muc6-expressing neck lineage following either chief or parietal cell ablation, and lineage-tracing experiments revealed that Muc6-expressing isthmus progenitors serve as a source of metaplasia and regeneration. Mechanistically, mucosal injury recruits IL-1-expressing myeloid cells, which stimulates NRG1 production in stromal fibroblasts, leading to mucosal proliferation and regeneration mediated by Myc activation in isthmus progenitors. These findings highlight the injury-responsible stem cell-like function of Muc6-expressing isthmal progenitors, which play a critical role in mucosal homeostasis and disease progression. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/648856v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@10fc2eaorg.highwire.dtl.DTLVardef@1c5b82corg.highwire.dtl.DTLVardef@1be747dorg.highwire.dtl.DTLVardef@d1d722_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Tff2 marks gastric corpus progenitors that give rise to pyloric metaplasia/SPEM following injury

In BriefTu et al. show that Tff2+ corpus isthmus cells are TA progenitors, and they, not chief cells, are the primary source of SPEM following injury. Upon Kras mutation, these progenitors directly progress to dysplasia, bypassing metaplasia, highlighting them as a potential origin of gastric cancer. HighlightsO_LITff2+ corpus cells are TA progenitors that give rise to secretory cells. C_LIO_LITff2+ progenitors, not chief cells, are the primary source of SPEM after injury. C_LIO_LIKras-mutant Tff2+ progenitors progress directly to dysplasia, bypassing metaplasia. C_LIO_LIMulti-omics analysis reveals distinct trajectories for SPEM and gastric cancer. C_LI O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/647847v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@48a2d1org.highwire.dtl.DTLVardef@b96642org.highwire.dtl.DTLVardef@1e89a4dorg.highwire.dtl.DTLVardef@1ea6a86_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG Pyloric metaplasia, also known as spasmolytic polypeptide-expressing metaplasia (SPEM), arises in the corpus in response to oxyntic atrophy, but its origin and role in gastric cancer remain poorly understood. Using Tff2-CreERT knockin mice, we identified highly proliferative Tff2+ progenitors in the corpus isthmus that give rise to multiple secretory lineages, including chief cells. While lacking long-term self-renewal ability, Tff2+ corpus progenitors rapidly expand to form short-term SPEM following acute injury or loss of chief cells. Genetic ablation of Tff2+ progenitors abrogated SPEM formation, while genetic ablation of GIF+ chief cells enhanced SPEM formation from Tff2+ progenitors. In response to H. pylori infection, Tff2+ progenitors progressed first to metaplasia and then later to dysplasia. Interestingly, induction of KrasG12D mutations in Tff2+ progenitors facilitated direct progression to dysplasia in part through the acquisition of stem cell-like properties. In contrast, Kras-mutated SPEM and chief cells were not able to progress to dysplasia. Tff2 mRNA was downregulated in isthmus cells during progression to dysplasia. Single-cell RNA sequencing and spatial transcriptomics of human tissues revealed distinct differentiation trajectories for SPEM and gastric cancer. These findings challenge the conventional interpretation of the stepwise progression through metaplasia and instead identify Tff2+ progenitor cells as potential cells of origin for SPEM and possibly for gastric cancer.

cell biology↗

bioRxiv2024

Afadin-deficient retinas exhibit severe neuronal lamination defects but preserve visual functions

Neural lamination is a common feature of the central nervous system (CNS), with several subcellular structures, such as adherens junctions (AJs), playing a role in this process. The retina is also heavily laminated, but it remains unclear how laminar formation impacts retinal cell morphology, synapse integrity, and overall retinal function. In this study, we demonstrate that the loss of afadin, a key component of AJs, in mice leads to significant pathological changes. These include the disruption of outer retinal lamination and a notable decrease as well as mislocalization of photoreceptors, their outer segments, and photoreceptor synapses. Interestingly, despite these severe impairments, we recorded small local field potentials, including the a- and b-waves. We also classified retinal ganglion cells (RGCs) into ON, ON-OFF, and OFF types based on their firing patterns in response to light stimuli. Additionally, we successfully characterized the receptive fields of certain RGCs. Overall, these findings provide evidence that retinal circuit function can be partially preserved even when there are significant disruptions in both retinal lamination and photoreceptor synapses. Our results indicate that retinas with severely altered morphology still retain some capacity to process light stimuli.

neuroscience↗

bioRxiv2024

KOnezumi-AID: A Software to Automate the Design of Multiplex Knockouts Using Target-AID

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, which can increase the risk of chromosomal rear-rangements, presenting a major obstacle when attempting to knockout multiple genes simultaneously. Base editing systems like Target-AID provide a safer alternative by enabling precise base modifications without requiring DNA double-strand breaks, making them a promising solution for addressing this challenge. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockouts highlights the need for a comprehensive system to design guide RNA (gRNA) for the simultaneous knockout of multiple genes. Here, we present KOnezumi-AID, a command-line tool designed to facilitate gRNA design for Target-AID-mediated genome editing. KOnezumi-AID supports gene knockout strategies by inducing premature termination codons or promoting exon skipping. It generates experiment-ready gRNA designs for both mouse and human genomes. Additionally, KOnezumi-AID offers batch processing capabilities, enabling rapid and precise gRNA design for large-scale genome editing projects such as CRISPR screening. In summary, KOnezumi-AID provides an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.

bioinformatics↗

bioRxiv2024

A novel genetic mouse model of fatal aortic dissection reveals massive inflammatory cell infiltration in the thoracic aorta

BackgroundAortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanism of AD, we generated a spontaneous AD mouse model. MethodsWe incorporated a novel missense variant (p.G234D) in FBN1, the gene for fibrillin-1, identified in a non-syndromic familial AD patient into mice using CRISPR/Cas9 system. We performed histopathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging and single-cell (sc)RNA-sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D protein to latent Tgf{beta} binding proteins (LTBPs), and signaling pathways in the mutant aortic wall were examined by western blot analysis. Results50% of the Fbn1G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. scRNA-sequencing, validated by immunostaining, revealed a significant increase in MHC class II-positive pro-inflammatory macrophages and monocytes at the site of intima tears with upregulation of MMP2/9 and marked disruption of elastic lamina. Subendothelial matrices, such as type IV collagen and laminin, expanded into the medial layer, where fibronectin expression was highly upregulated. Fbn1G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 and ICAM-1, all of which likely contributed to the infiltration of immune cells. Biochemically, FBN1G234D lost the ability to bind to latent TGF{beta} binding protein (LTBP)-1, -2, and -4, resulting in the downregulation of TGF{beta} signaling in the aortic wall. ConclusionsWe show that dynamic interactions involving endothelial cells (ECs) and macrophages/monocytes in the intima, where the ECM microenvironment is altered with the reduced TGF{beta} signaling, contributes to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for development of therapeutic strategies.

pathology↗

bioRxiv2023

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out (DKO) mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis. Significance StatementMYOD protein is not expressed in quiescent muscle stem cells but accumulates rapidly following muscle injury, leading to the proliferation of myogenic progenitors for differentiation. However, the direct targets of MYOD, aside from myogenin, which play roles in myogenic differentiation remain incompletely understood. Using previously established MyoD knock-in mice and single-cell RNA sequencing, we discovered that Dusp13 and Dusp27 are potential target genes of MYOD that promote myogenesis during muscle regeneration in adult mice.

cell biology↗

bioRxiv2023

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently.

bioengineering↗