Biology subjects

Yoshida, J.

Publications and source records attributed to Yoshida, J..

13 recordsLinked to original sources

bioRxiv2026

Dysregulated flow responses drive pathogenesis in a cellular model of Sturge-Weber syndrome

Vascular diseases comprise a broad spectrum of conditions associated with organ dysfunction, yet how endothelial responses to mechanical forces drive their pathogenesis remains unclear. Sturge-Weber syndrome (SWS), a flow-associated vascular malformation caused by somatic GNAQ mutations, provides a model to investigate this question. Here, using isogenic induced pluripotent stem cell-derived endothelial cells (ECs), perfusable vascular models, and simplified systems under defined flow conditions, we show that SWS ECs exhibited maladaptive responses to laminar flow, including impaired flow alignment, reduced CLDN5 expression, and decreased transendothelial electrical resistance. Importantly, three-dimensional acoustic microscopy uncovered disturbed surface changes in SWS ECs, potentially contributing to changes in mechanosensing signaling. Transcriptomic and chromatin accessibility analyses revealed dysregulated flow-responsive gene programs, some of which were validated in human SWS samples. These findings provide insights into flow-dependent endothelial dysfunction and a framework for therapeutic modulation in SWS and other vascular disorders.

cell biology↗

bioRxiv2026

Bidirectional Extracellular Vesicle-Mediated Maternal-Embryonic Exchange in the Lecithotrophic Teleost Guppy

The placenta is defined as an organ that mediates the exchanges of nutrients, hormones, and other substances between the mother and embryo in viviparous animals. Its structure is diverse due to interspecific differences in fetal tissues and variation in the forms of maternal-fetal interfaces. Matrotrophic poeciliid teleosts, in which the embryo develops within the maternal ovarian follicle, possess functional placentas formed from maternal follicular and embryonic tissues. However, the physiological mechanisms underlying substrate exchange between mother and embryo remain unclear. Furthermore, although similar embryonic-maternal interfaces are observed in lecithotrophic poeciliids, it is unknown whether nutrient exchange occurs in these species. Therefore, this study investigated whether substance exchange occurs between the mother and embryo in the lecithotrophic teleost guppy (Poecilia reticulata) and identified the underlying physiological mechanisms. Histological analysis revealed that guppies have embryonic-maternal interfaces consisting of the maternal follicle and the embryonic yolk sac and pericardial sac. Additionally, tracking of 2,000 kDa fluorescein isothiocyanate-dextran injected into pregnant guppies confirmed its transport from mother to embryo. Immunofluorescence staining and electron microscopy revealed that substance transport from mother to embryo occurs via extracellular vesicles. Moreover, immunofluorescence staining and pharmacological experiments revealed exosome transport from embryo to mother. This study demonstrates that lecithotrophic guppies possess a functional placenta that mediates maternal-embryonic substrate transfer via extracellular vesicles. These findings provide fundamental insight into the evolution of placental strategies within the Poeciliidae family. Significance StatementViviparity, in which embryos develop within the maternal body, has evolved independently across diverse animal lineages. In lecithotrophic viviparity, embryos are thought to rely primarily on yolk-derived nutrients, with maternal-fetal exchange limited to small molecules such as gases. Here, using macromolecular tracer experiments and ultrastructural analyses in guppies, we show that large macromolecules (2000 kDa) are exchanged bidirectionally between mother and fetus via extracellular vesicles, despite the absence of direct tissue attachment. These findings challenge the conventional view of lecithotrophic viviparity and reveal a previously unrecognized mechanism of maternal-fetal communication. Our results suggest that extracellular vesicle-mediated exchange may represent a widespread and evolutionarily conserved strategy for maternal-fetal interaction across viviparous animals.

developmental biology↗

bioRxiv2025

High-throughput heterospheroid-based screening identifies drugs that reprogram tumor-associated macrophages

The tumor microenvironment (TME) provides a niche for immune evasion and immunotherapy resistance, in part, by recruiting pro-tumor M2-like macrophages. In the present study, using heterospheroids consisting of cancer cells and macrophages, we identified TAM activators, which are compounds that reprogram M2-like tumor-associated macrophages (TAMs) toward the antitumor M1-like phenotype. THP-1- or human peripheral monocyte-derived macrophages were co-cultured with liver cancer cells in an ultra-low attachment dish to generate heterospheroids. Cell surface marker expression and macrophage infiltration into the heterospheroids were assessed by flow cytometry and fluorescence microscopy, respectively. Lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma})-induced M1 marker expression was observed on the macrophages in the homospheroids; however, this induction was suppressed in heterospheroids. Microscopic imaging revealed that macrophage infiltration into the heterospheroids was decreased in the presence of LPS and IFN{gamma}, which prompted us to develop a high-content imaging screen. We identified two compounds [alprostadil (prostaglandin E1) and HX531] with TAM-activating activity. RNA-seq analysis revealed that HX531 modulated the immune and IFN response in cancer cells and cell division in macrophages. Moreover, alprostadil promoted the M1-like polarization of TAMs, increased tumor-infiltrating CD8+ T cells, and enhanced anti-PD-1 antibody therapeutic efficacy in a syngeneic mouse xenograft model. In conclusion, the heterospheroid culture recapitulates the immunosuppressive TME, which prevents the M1 polarization of TAMs. It provides a new platform for screening TAM activators and will enable the development of novel cancer immunotherapeutics when combined with high-content imaging analysis.

pharmacology and toxicology↗

bioRxiv2025

Dynamic and context-dependent modulation of proprioceptive input in primate primary motor cortex

During voluntary movement, somatosensory input is attenuated--sensory gating--which may prevent the CNS from being overwhelmed by predictable afferent feedback not essential for motor control. In the cerebral cortex, sensory gating has been demonstrated in primary (S1) and higher-order sensory areas. The primary motor cortex (M1) is the major cortical output relay to the spinal cord and muscles, and proprioceptive feedback is crucial for shaping motor output, much as in S1. However, whether proprioceptive signals to M1 are attenuated during movement, and if so, why, remains unclear. We recorded somatosensory-evoked potentials (SEPs) in M1 of two monkeys performing a wrist task while electrically stimulating muscle and cutaneous afferents innervating forearm extensors and adjacent skin. Both local field potentials and single-neuron recordings revealed significant suppression of muscle-evoked SEPs during Active movement and Static hold phases, providing direct evidence that proprioceptive input to M1 is generally gated during motor execution, as previously reported for cutaneous input. Yet, amidst this suppression, SEPs in a subset of M1 neurons were preserved during Static hold, especially those evoked by antagonist muscle afferents. Because monkeys had to maintain precise joint angles for stable posture, these results suggest that proprioceptive signals from antagonist muscles--likely reflecting spindle activity in lengthening muscles--escape attenuation to provide information essential for joint angle control. Overall, our findings demonstrate that while proprioceptive input to M1 is broadly suppressed during motor output, specific afferent signals from antagonist muscles are selectively maintained in a context-dependent manner to support posture control.

neuroscience↗

bioRxiv2025

AgRP Neuronal Suppression by Spontaneous Movement Encodes Exercise Reinforcement

Hypothalamic agouti-related peptide (AgRP) neurons that drive hunger-related behaviors are activated during energy deficit, encode negative valence but can be inhibited even by perception of food without digestion. Here, we show that in head-fixed mice, discrete motor actions such as spontaneous walking and conditioned licking rapidly suppress AgRP neuron activity. This suppression persisted throughout movement and terminated upon cessation, which was independent of food perception or conditioned cues. The degree of suppression was positively correlated with movement vigor. While optogenetic enhancement of AgRP neuron suppression did not initiate movement, it promoted reinforcement of concurrent walking behavior. These findings unmask that spontaneous, low intensity physical activity can reduce AgRP neuron activity reinforcing concurrent movement/exercise with implications for systemic health benefits.

neuroscience↗

bioRxiv2023

Membrane molecule bouncer enables follicle fertilization in a viviparous teleost: Poecilia reticulata (guppy)

Fertilization is a fundamental mechanism of sexual reproduction. Generally, oocytes are ovulated from the ovarian follicles and contact the sperm outside the ovarian medulla. Unlike this, follicle fertilization which means the egg contact with sperm in the ovarian medulla without ovulation is known in the viviparous teleost species belonging to the Poeciliidae. In this study, we focused on a viviparous teleost species, Poecilia reticulata (guppy). Our sperm tracking assay indicated that the sperm reached the immature oocytes with a germinal vesicle, and the fertilized immature oocytes were presumed to contribute to littermates. The binding between immature oocytes and sperm is a specific trait in the guppy, which was not observed in Danio rerio (zebrafish) or Oryzias latipes (medaka). The loss- and gain-of-function assays indicated that bouncer plays a critical role in immature oocyte-to-sperm binding. This fertilization trait in immature oocytes may provide certain advantages for females with respect to nutrition or other gestation costs. Our findings shed light on the unique reproductive strategies of guppy and contribute to our understanding of the diverse reproductive mechanisms in vertebrates. Summary statementUnlike general vertebrates, guppys oocyte fertilizes with sperm in the ovarian medulla at the immature stages. The distinctive trait depends on the Ly6/uPAR protein bouncer.

developmental biology↗

bioRxiv2022

Origin, Prospective Identification, and Function of Circulating Endothelial Colony Forming Cells in Mouse and Human

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony forming cells (C-ECFCs) with proliferative and vasculogenic activity can be cultured; the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing reveals murine C-ECFCs emerge in the early postnatal period, display high vasculogenic potential, with enriched frequency of clonal proliferative cells compared to tissue-resident ECFCs, and are not committed to or derived from the bone marrow hematopoietic system but from tissue-resident ECFCs. In human subjects, C-ECFCs are present in the CD34bright cord blood mononuclear subset, possess proliferative potential and in vivo vasculogenic function in a naive or cultured state, and display a single cell transcriptome sharing some umbilical venous endothelial cell features like, higher Protein C Receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

cell biology↗

bioRxiv2022

Cerebellum Directly Modulates the Substantia Nigra Dopaminergic Activity

Evidence of direct reciprocal connections between the cerebellum and basal ganglia has challenged the long-held notion that these structures function independently. While anatomical studies have suggested the presence of cerebellar projections to the substantia nigra pars compacta (SNc), the nature and function of these connections (Cb-SNc) is unknown. Here we show that the Cb-SNc form monosynaptic glutamatergic synapses with both dopaminergic and non-dopaminergic neurons in the SNc. Optogenetic activation Cb-SNc axons in the SNc rapidly increases SNc activity, elevates striatal dopamine levels, and increases the probability of locomotion. During ongoing behavior, Cb-SNc axons are bilaterally activated prior to ambulation and unilateral lever manipulation. The Cb-SNc axons show prominent activation to water reward, and higher activation for sweet water, suggesting that the pathway also encodes reward value. Thus, the cerebellum directly, rapidly, and effectively modulates basal ganglia dopamine levels and conveys information related to movement initiation, vigor, and possibly reward processing.

neuroscience↗

bioRxiv2022

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

molecular biology↗

bioRxiv2022

Hippocampal CA1 represents action and reward events instantly compared to the superficial and deep layers of the lateral entorhinal cortex

The entorhinal cortex (EC) is the main interface between the hippocampus and the neocortex. The EC plays a critical role in learning and memory. We investigated the neuronal representation of behavioral events during operant learning in the hippocampal-entorhinal circuit of head-fixed rats. Both CA1 and lateral entorhinal cortex (LEC) neurons develop task-related activities after learning. Among diverse task-related activities, we compared the transient peak activities that represent action and reward and found a distinct difference in the timing of behavioral event representation between CA1 and LEC. CA1 represents action and reward events in close to real-time, whereas both the superficial and deep layers of the LEC showed delayed representation of those events. Our results suggest that subpopulations exist within which CA1 and LEC neurons process the information in a different order from the anatomically defined hippocampal-entorhinal circuit.

neuroscience↗

bioRxiv2022

Comparison of CRISPR/Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells

The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters, promoter-enhancer loops, and topologically associating domains. To analyze these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Kruppel-associated box zinc finger protein (KRAB-ZFP) gene cluster on chromosome 4 in mouse embryonic stem cells using CRISPR/Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN) with 70-bp homology arms; and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.

genomics↗

bioRxiv2022

Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived pseudoplacenta of the viviparous teleost Xenotoca eiseni

Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotenia, a hindgut-derived pseudoplacenta to receive the maternal supplement. However, the molecular mechanisms underlying viviparous non-mammalian animals remain elusive. We conducted this study to investigate the mechanism for nutrient absorption and degradation in trophoenia of X. eiseni. The tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of trophotaenia. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in trophotaenia. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in trophotaenia. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived pseudoplacenta.

zoology↗

bioRxiv2021

Inhibition of N-myristoyltransferase Promotes Naive Pluripotency in Mouse and Human Pluripotent Stem Cells

Naive and primed states are distinct states of pluripotency during early embryonic development that can be captured and converted to each other in vitro. To elucidate the regulatory mechanism of pluripotency, we performed a recessive genetic screen of homozygous mutant mouse embryonic stem cells (mESCs) and found that suppression of N-myristoyltransferase (Nmt) promotes naive pluripotency. Disruption of Nmt1 in mESCs conferred resistance to differentiation. Suppression of Nmt in mouse epiblast stem cells (mEpiSCs) promoted the conversion from the primed to the naive state. This effect was independent of Src, which is a major substrate of Nmt and is known to promote differentiation of mESCs. Suppression of Nmt in naive-state human induced pluripotent stem cells (hiPSCs) increased the expression of the naive-state marker. These results indicate that Nmt is a novel target for the regulation of naive pluripotency conserved between mice and humans.

developmental biology↗