Biology subjects

Tamura, M.

Publications and source records attributed to Tamura, M..

11 recordsLinked to original sources

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

Meso2EM: a cross-scale CLEM workflow linking mesoscale functional imaging to targeted electron microscopy

Meso2EM is a correlative light and electron microscopy workflow that transfers neurons selected from mesoscale functional images to targeted electron microscopy. We recorded Ca{superscript 2} signals from layer 2/3 neurons across a contiguous 3 x 3 mm cortical field in awake mice and reidentified a selected neuron after fixation and tangential sectioning. Lectin-labeled vascular architecture served as a shared landmark across in vivo two-photon imaging, confocal microscopy, laboratory micro-CT of resin-embedded tissue, and block-surface scanning electron microscopy, guiding focused-ion-beam scanning electron microscopy to the target cell body. The same progressive-targeting principle also supported serial ATUM-SEM reconstruction of an in vivo-tracked dendrite and serial transmission electron microscopy of optically selected dendrites from a patch-clamp-recorded Martinotti cell. Meso2EM therefore provides a practical route for preserving target identity across large changes in scale and specimen state while restricting electron-microscopy acquisition to a selected region.

neuroscience↗

bioRxiv2025

Three-dimensional and molecular brain atlas of the hagfish reveals the evolutionary origin and early diversification of the vertebrate brain

Hagfish and lampreys form the clade cyclostomes, the earliest branching lineage of the vertebrates. Although hagfish are generally assumed to have experienced intense modifications, its brain development shows certain ancestral patterns that are secondarily lost in the lamprey. Therefore, hagfish provides key insights into the evolutionary origin and early diversification of the vertebrate brain. However, neuroanatomical information of the hagfish brain is substantially limited today due to its sources based on old-fashioned methodology alone. Here, we provide a detailed brain atlas of the adult hagfish, based on three-dimensional reconstruction and expression analysis of genetic markers for major neuronal transmitters. Through our analyses, we found the following key characteristics. First, the dopaminergic system appears to be distributed more broadly than previously thought in the hagfish brain, implying that dopamine is involved in various neural functions. Second, the previously suggested "primordial cerebellum" area in hagfish shows notable affinity to the cerebellum-like octavolateral structures of the jawed vertebrate hindbrain. Last, the gene expression profile suggests a hippocampus-equivalent brain region in hagfish, that is, the ventrolateral subnucleus of the central prosencephalic complex (NCvl). This study highlights conserved and diversified neuroarchitecture of the hagfish brain, providing a pivotal reference for further studies.

evolutionary biology↗

bioRxiv2025

Whole-mount acetylcholinesterase (AChE) staining reveals unique motor innervation of the lamprey oral region: with special reference to the evolutionary origin of the vertebrate jaw

The evolutionary change of the trigeminal nerve-innervation pattern is essential to reveal the mechanism underlying jaw acquisition. However, the homology of the branches between gnathostomes (jawed vertebrates) and cyclostomes (living jawless vertebrates) remains unclear. In this study, we focused on a sub-branch called the ramus subpharyngeus, which belongs to the second branch of the lamprey trigeminal nerve and projects to the lower lip, investigating whether it contains motor components. To visualize motor fibers, we performed acetylcholinesterase (AChE) staining, a histochemical method that visualizes intrinsic activities of the catabolic enzymes produced by motor neurons and muscle fibers. As a result, we found AChE staining signals that correspond to the innervation course of the ramus subpharyngeus. To confirm that the signals are not produced by the motoneuronal somata nor muscle fibers, we conducted gene expression analysis by in situ hybridization. The results support that the signals mark the motor fibers. Based on these results, we propose that the lamprey oral apparatus is chiefly controlled by the second (i.e., pre-mandibular) branch of the trigeminal nerve and further suggest that a drastic reorganization of the anterior craniofacial region occurred during the acquisition of the vertebrate jaw.

developmental biology↗

bioRxiv2024

Synergistic reinforcement learning by cooperation of the cerebellum and basal ganglia

The cerebral cortex, cerebellum and basal ganglia are essential for flexible learning in mammals. Although traditionally thought to operate under different learning rules, recent evidence suggests that both the basal ganglia and the cerebellum may employ reinforcement learning mechanisms. This raises the question of how these structures coordinate when a common reward prediction error mechanism is active. To address this issue, we first examined output signals from the basal ganglia and cerebellum following the activity of the cerebral cortex. We recorded single-neuron activity from the output regions of the cerebellum and basal ganglia - the cerebellar nuclei (CN) and substantia nigra pars reticulata (SNr) - in both male and female ChR2 transgenic rats. Neurons in the CN and SNr exhibited distinct temporal response patterns; notably, the fast excitatory response in the CN, driven by mossy fiber input, was synchronized with the inhibitory response in the SNr, mediated via the direct pathway. Using these experimental findings together with connectome data, we developed both a semi-realistic spiking network model and a reservoir-based reinforcement learning model. In the latter model, successful learning depended on synaptic plasticity in both the cerebellum and basal ganglia with a temporal precision on the order of 10 ms. Furthermore, cortical {beta}-oscillations enhanced learning and optimal reinforcement learning occurred when the output of cerebellar and basal ganglia signal phase-locked at the frequency of cortical oscillation. Taken together, our results suggest that the coordinated output of the cerebellum and basal ganglia, driven by tightly tuned cortical input, underlies brain-wide synergistic reinforcement learning. Significance StatementThe cerebral cortex, cerebellum, and basal ganglia support learning. Recent research suggests that both the basal ganglia and cerebellum use a similar learning process called reinforcement learning, which involves predicting rewards. To understand how these brain regions work together, we recorded brain activity in rats while photo-stimulating the cerebral cortex. We found that two types of responses in the cerebellum and basal ganglia were synchronized, which might help activate the cerebral cortex. A computer model showed that precise timing of signals from both the cerebellum and basal ganglia is important for learning. This timing was important only when the cerebral cortex worked in a specific frequency range. Our findings suggest that coordinated brain activity enhances learning.

neuroscience↗

bioRxiv2023

Granulomatous inflammatory responses are elicited in the liver of PD-1 knockout mice by de novo genome mutagenesis

Aimsprogrammed death-1 (PD-1) is a negative regulator of immune responses. Upon deletion of PD-1 in mice, symptoms of autoimmunity developed only after they got old. In a model experiment in cancer immunotherapy, PD-1 was shown to prevent cytotoxic T lymphocytes from attacking cancer cells that expressed neoantigens derived from genome mutations. Furthermore, the larger number of genome mutations in cancer cells led to the more robust anti-tumor immune responses after the PD-1 blockade. In order to understand the common molecular mechanisms underlying these findings, we hypothesize that we might have acquired PD-1 during evolution in order to avoid/suppress autoimmune reactions against neoantigens derived from mutations in the genome of aged individuals. Main methods: to test the hypothesis, we introduced random mutations into the genome of young PD-1-/- and PD-1+/+ mice. We employed two different procedures of random mutagenesis: administration of a potent chemical mutagen N-ethyl-N-nitrosourea (ENU) into the peritoneal cavity of mice and deletion of MSH2, which is essential for the mismatch-repair activity in the nucleus and, therefore, for the suppression of accumulation of random mutations in the genome. Key findingswe observed granulomatous inflammatory changes in the liver of the ENU-treated PD-1 knockout (KO) mice, but not in the wild-type (WT) counterparts. Such lesions also developed in the PD-1/MSH2 double KO mice, but not in the MSH2 single KO mice. Significance: the results we obtained support our hypothesis: PD-1 probably functions to avoid/suppress inflammatory responses against neoantigens derived from genome mutations in aged individuals.

immunology↗

bioRxiv2023

Rapamycin-sensitive mechanisms confine the growth of fission yeast below the temperatures detrimental to cell physiology

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to protein denaturing and heat damage to other cellular macromolecules. We here report that, in the presence of the macrolide compound rapamycin, the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that normally arrest its growth. Rapamycin is a potent inhibitor of the protein kinase complex TOR Complex 1 (TORC1), and consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance. These results suggest that TORC1, a well-established growth promoter, restricts the high-temperature growth of fission yeast and that compromised TORC1 signaling allows cell proliferation at higher temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens in S. pombe, which identified Sck1 and additional factors that appear to suppress cell proliferation at high temperatures. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology. Thus, growth arrest at high temperatures may not directly result from heat damage to cellular components essential for proliferation and viability. Significance StatementThe immunosuppressant rapamycin is a specific inhibitor of the protein kinase Target Of Rapamycin (TOR), and the drug is known to extend the lifespan of diverse eukaryotic organisms. In this study, we have found that rapamycin confers heat resistance on fission yeast, allowing its proliferation above the normal permissive temperatures. This unexpected observation suggests that TOR, which is known as a growth-promoting kinase, is inhibitory to cell proliferation at high temperatures. Our genome-wide screens have identified additional genes whose deletion leads to improved growth under heat stress. Thus, cells may have mechanisms that restrict proliferation even below the temperatures deleterious to their physiology.

cell biology↗

bioRxiv2021

Combinations of Peptides Synergistically Activate the Regenerative Capacity of Skin Cells In Vitro

OBJECTIVETo explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination in different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared to the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations. METHODSMicroarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment. RESULTSMicroarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared to individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS, and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, a second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism whereby peptide synergies may accelerate skin repair. CONCLUSIONThrough combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.

molecular biology↗

bioRxiv2020

Autotoxin-mediated voluntary triage in starved yeast community

When organisms face crises, such as starvation, every individual should adapt to environmental changes (1, 2), or the community alters their behaviour (3-5). Because a stressful environment reduces the carrying capacity (6), the population size of unicellular organisms shrinks in such conditions (7, 8). However, the uniform stress response of the cell community may lead to overall extinction or severely damage their entire fitness. How microbial communities accommodate this dilemma remains poorly understood. Here, we demonstrate an elaborate strategy of the yeast community against glucose starvation, named the voluntary triage. During starvation, yeast cells release some autotoxins, such as leucic acid and L-2keto-3methylvalerate, which can even kill the cells producing them. Although it may look like mass suicide at first glance, cells use epigenetic "tags" to adapt to the autotoxin inheritably. If non-tagged latecomers, regardless of whether they are closely related, try to invade the habitat, autotoxins kill them and inhibit their growth, but the tagged cells can selectively survive. Phylogenetically distant fission and budding yeast (9) share this strategy using the same autotoxins, which implies that the universal system of voluntary triage may be relevant to the major evolutional transition from unicellular to multicellular organisms (10).

microbiology↗

bioRxiv2019

Soft Windowing Application to Improve Analysis of High-throughput Phenotyping Data

MotivationHigh-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximises analytic power while minimising noise from unspecified environmental factors.\n\nResultsHere we introduce \"soft windowing\", a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant p-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft windowed and non-windowed approaches, respectively, from a set of 2,082 mutant mouse lines. Our method is generalisable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.\n\nAvailability and ImplementationThe method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin.

bioinformatics↗

bioRxiv2018

Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via elongation and expansion processes. In the first stage, synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.

developmental biology↗