Biology subjects

Hayashi, Y.

Publications and source records attributed to Hayashi, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Micropatterned neural induction with heat-inactivated extracellular matrix protein by on-demand high-speed laser

Microenvironmental heterogeneity in cultured cells can compromise cell quality, reduce experimental reproducibility, and weaken the confidence of cell therapeutic efficacy. Although micropatterned cell cultures are more homogeneous, conventional micropatterning methods lack flexibility. In this study, we develop a micropatterning technology by denaturing extracellular matrix (ECM) proteins in specific areas through heat inactivation using a high-speed laser via a light-responsive polymer layer. We have successfully seeded human induced pluripotent stem cells (hiPSCs) in flexible patterns and examine their neural induction in circular geometries of varying diameters. Size-dependent and cell-autonomous neural structures are formed on this substrate when hiPSCs differentiate into neural lineages in circles of different diameters. This self-organized pattern results from the mitotic orientation and localization of differentiating cells. Furthermore, teratogenic substances can modulate these patterns. Laser-induced heat inactivation of the ECM on culture substrates enables on-demand patterning, facilitating studies of cell-autonomous tissue formation, the effect of teratogenic substances, and precise tissue engineering in regenerative medicine. Table of ContentA lithography-, hydrogel-, and PDMS-free micropatterning technology is developed to enable flexible and rapid cell patterning directly onto culture substrates via high-speed laser irradiation. Using this technology, size-dependent, cell-autonomous tissue formation from human induced pluripotent stem cells were driven by mitotic orientation and spatial localization of differentiating cells. These self-organized structures can be applied for safety tests of teratogenic substances. ToC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/663334v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@22844aorg.highwire.dtl.DTLVardef@1b3938org.highwire.dtl.DTLVardef@149a748org.highwire.dtl.DTLVardef@85834c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Transcriptome comparison between the cultured and in vivo Chick Primordial Germ Cells by SMART-seq-based single cell RNA sequencing

Primordial germ cells (PGC), the precursors of the germline, have unique cellular characteristics to undergo long-distance migration to the embryonic gonads and have the potential to differentiate into somatic cells. Among the animal models studying PGC development, the chicken PGCs are an ideal model, since it is a rare model in which long-term PGC cultivation is applicable. Although the cultural applicability of chicken PGC makes it attractive for revealing the PGC character and its developmental processes, some differences from endogenous PGCs are known, such as the remarkable up-regulation of cell proliferation and a lesser ability to reach the gonads. Understanding these differences at the molecular level is crucial. To this end, we first performed SMART-seq-based single-cell RNA sequencing to compare transcriptomes between endogenous PGCs and cultivated PGCs. Our results revealed that PGC cultivation causes a shift from a MYC-dependent to a MYCN-dependent gene regulatory network in PGCs, suggesting that this reprogramming contributes to the acquisition of proliferation ability and stem cell characteristics in cultivated PGCs. Additionally, our results suggest that the MYCN-dependent gene regulatory network increases the risk of somatic differentiation, particularly in neural fate, in cultivated PGCs. In addition, our transcriptome analysis identified new cell populations that show molecular character as intermediate cell states between germline and pluripotent cells from the early embryonic stage. Thus, our study provides fundamental molecular information to understand both the effects of PGC cultivation and the developmental process of chicken PGCs.

developmental biology↗

bioRxiv2025

A selective top-down pathway from anterior cingulate cortex embeds a representation of saliency in hippocampus

For maximizing survival, animals must accurately memorize the structure of their environment. This is achieved by stabilizing and enriching hippocampal place cells near salient features, while the representation of neutral locations incrementally drifts with time. However, the circuit mechanisms by which top-down and bottom-up inputs selectively embed saliency into the hippocampal spatial map remain poorly understood. Here we identified a specific top-down input from the anterior cingulate cortex (ACC) to the dysgranular zone of the retrosplenial cortex (RSCd) that becomes active exclusively at reward locations. Activity of these ACC neurons is both necessary and sufficient for shaping the hippocampal saliency map. Unlike dopaminergic neurons in the ventral tegmental area, which respond to reward irrespective of the context, this class of ACC neurons encodes reward-context associations. Their optogenetic activation induces a reduction in locomotor speed, a behavioral correlate of saliency detection, while their inhibition disrupts the formation of the hippocampal place map. This work not only provides the first mechanistic insight into how context-dependent saliency is integrated in a top-down fashion into the hippocampal map to guide adaptive behavior but also challenges the classical view of memory consolidation theory where memory unidirectionally moves in bottom-up fashion from the hippocampus to the neocortex.

neuroscience↗

bioRxiv2025

Identification of an early-heading mutant in Indonesian native rice cultivar: 'Gemdjah Beton'

Rice is one of the worlds most widely cultivated field crops and a short-day adapted plant. It possesses a complex genetic regulatory mechanism for heading date determination. Gemdjah Beton (GB) is an Indonesian rice cultivar that takes approximately 17 weeks to flower after being transplanted into a rice field in Japan. We isolated early-heading mutant line GB-10 (flowers around two to three weeks early in the field). Under long-day (LD) conditions, GB-10 flowered approximately two weeks earlier than GB. Under short-day conditions, the heading date between GB-10 and GB plants shows no apparent difference. In agreement with the heading date, Hd3a and RFT1 expressions show around a 10-time relative transcript quantity difference under LD conditions in GB-10 compared to GB. Interestingly, Ghd7 expression was significantly reduced in GB-10, which may trigger the Hd3a and RFT1 activation. A bulk segregation analysis indicated that a single nucleotide variant on chromosome 7 was genetically linked to the early heading phenotype of GB-10 lines. Taken together, we reported the successful isolation of GB-10 as an early-heading mutant.

plant biology↗

bioRxiv2025

Region- and stage-specific cortical plasticity shapes the multifaceted process of habit formation

Habits are essential for sustaining adaptive behaviors but can also lead to maladaptive behaviors such as compulsive and addictive disorders. They emerge through a shift in decision-making from motivation-driven strategy to an automatic one. During this, the level of habit execution, such as frequency and duration, is superficially maintained despite a decline in motivational drive, raising the question of how the amount of execution is maintained even when decision-making strategies undergo substantial changes. By developing a unique paradigm capable of inducing habit formation within a defined time window in male mice, we found that shift in decision-making and amount of habit execution are independently controlled by plasticity in distinct cortical pathways. Erasure of each plasticity selectively altered the decision-making strategy or the execution level without affecting the other. These findings reveal a dual regulatory model for habit, providing new insights into the neurocircuit mechanisms underlying both adaptive and maladaptive habits.

neuroscience↗

bioRxiv2025

Repressive S-adenosylmethionine biosynthesis status inhibits transcription of HeT-A retrotransposon in the germline of Drosophila.

S-adenosylmethionine (SAM) is the principal cellular donor of methyl moiety in the methylation reaction and regulates gene expression by regulating methylation-related cellular events, such as epigenetic status. Although SAM biosynthesis affects a variety of biological phenomena including disease and aging, whether cell-specific SAM biosynthesis status is present and how it contributes to cellular function are largely unknown. Here, we firstly showed that the Drosophila germline in gametogenesis has a repressive SAM biosynthesis status through the observation of SAM synthetase (Sam-S), a key enzyme for SAM biosynthesis. In addition, our study showed that germline-unique repressive SAM biosynthesis status contributes to inhibition of retrotransposon expression; enhancement of SAM biosynthesis in germline caused excessive expression of retrotransposons including HeT-A, a telomere-specific retroelement, as the most affected target. We found that the promoter activity of HeT-A is enhanced in SAM increased condition with increased accumulation of 6mA DNA methylation, the major DNA methylation modification in the Drosophila genome. Interestingly, the enhanced 6mA enrichment and gene expression in enriched loci were not correlated in other retrotransposons or structural genes. Taken together, our results suggest that SAM-deficient status in the germline uniquely regulates HeT-A transcription via 6mA methylation modification. Thus, our study provides a new understanding of how germline unique metabolic status contributes to regulation of the retrotransposon.

developmental biology↗

bioRxiv2025

Intestinal fructose metabolism/GLP-1/β-cell axis counteracts hyperglycemia after short-term fructose ingestion

Fructose ingestion increases circulating GLP-1 and insulin, yet the specific contributions of these hormonal responses to glycaemic control remain incompletely defined. We hypothesised that fructose metabolism in intestinal L-cells triggers GLP-1 secretion, which then potentiates insulin secretion and counteracts fructose-induced hyperglycaemia. To test this hypothesis, we systematically characterised metabolic responses across multiple mouse strains after 24 h ad libitum fructose ingestion. In both lean (NSY.B6-a/a) and obese diabetic (NSY.B6-Ay/a) mice, fructose elevated plasma insulin, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP). The insulin response was preserved in GIP receptor-deficient mice (Gipr-/-) but was abolished in proglucagon-deficient mice (Gcg-/-) by pharmacological GLP-1 receptor antagonism, indicating a requirement for GLP-1, but not GIP. Across strains, fructose-induced insulin response correlated with attenuation of post-fructose glycaemia, consistent with insulin being essential for suppressing fructose-induced hyperglycaemia. To explore the mechanism underlying fructose-induced GLP-1 secretion, we combined ATP-sensitive potassium channel-deficient mice (Kcnj11-/-), GLUTag L-cell line, and metabolic tracing of 13C-labelled fructose in freshly isolated intestinal crypts. These complementary approaches support a model in which fructolysis increases the ATP/ADP ratio in L-cells, closes KATP channels, and stimulates GLP-1 secretion. In obese diabetic mice, increased fructolytic flux and a higher ATP/ADP ratio were associated with elevated GLP-1 levels, further corroborating this model. Collectively, our findings indicate that intestinal fructose metabolism drives GLP-1 secretion required to potentiate insulin secretion, thereby establishing a gut-pancreas axis that counter-regulates fructose-induced hyperglycaemia. KEY POINTS SUMMARYO_LIFructose ingestion acutely increases plasma insulin levels, but the underlying mechanisms and physiological significance remain elusive. C_LIO_LIOur study demonstrates that short-term (24h) fructose ingestion in mice elevates both insulin and glucagon-like peptide 1 (GLP-1) levels in the blood, with the plasma insulin response being GLP-1-dependent. C_LIO_LIWe found that fructose metabolism in intestinal L-cells triggered GLP-1 secretion by increasing the ATP/ADP ratio and closing ATP-sensitive K+ channels (KATP channels). C_LIO_LIThis intestinal fructose metabolism/GLP-1/{beta}-cell axis plays a crucial role in preventing fructose-induced hyperglycaemia, an effect that is compromised in obese diabetic mice. C_LIO_LIThese insights highlight the previously unclear metabolic responses following short-term fructose ingestion and their importance in glucose homeostasis. C_LI 1. ABSTRACT FIGURE LEGENDThis study investigated the hormonal effects of short-term fructose consumption in mice, allowing them ad-lib access to fructose solution for 24 h. Fructose metabolism in intestinal L-cells increases the intracellular ATP/ADP ratio, leading to GLP-1 secretion via KATP channel closure and Ca2+ influx. GLP-1 promotes insulin secretion from pancreatic {beta}-cells. The fructose metabolism/GLP-1/insulin pathway is essential for mitigation of fructose-induced hyperglycaemia. Figures were drawn using BioRender.com.

physiology↗

bioRxiv2025

Glucagon dysfunction in the liver induces hyperplasia of PP cells and the production of glucagon and pancreatic polypeptide double-positive cells

Understanding the mechanisms that regulate cellular identity and proliferation is crucial for elucidating cellular functions. Under normal conditions, pancreatic endocrine cells express only a single hormone, and their numbers are tightly regulated. Contrary to this general principle, our study revealed a significant increase in glucagon (GCG) and pancreatic polypeptide (PP) double-positive cells (GCG+ PP+ double-positive cells), along with hyperplasia of both PP and cells in proglucagon-deficient mice. Similarly, systemic glucagon receptor-deficient mice exhibited PP-cell hyperplasia and an increase in GCG+ PP+ double-positive cells, with enhanced PP-cell self-replication observed at 4 weeks and the appearance of GCG+ PP+ double-positive cells at 10 weeks. Liver-specific glucagon receptor-deficient mice induced similar effects, which were linked to hyperaminoacidemia. Elevated glutamine levels were found to promote GCG+ PP+ double-positive cell formation via mTOR signaling, suggesting a molecular mechanism driving pancreatic endocrine cell plasticity. Collectively, these findings indicate that increased plasma amino acid levels caused by impaired glucagon action in the liver promotes the proliferation of cells and PP cells, and disturbs their cellular identity maintenance.

cell biology↗

bioRxiv2024

Heterogeneous Effects of Src Inhibition on Determinants of Metastasis in Preclinical Models of Human Bladder Cancer

BackgroundPast work in preclinical models of solid tumors have implicated SRC in invasion and metastasis, but also demonstrated it inhibited bladder cancer metastasis. ObjectiveDetermine if the role of SRC in metastasis is dependent on bladder cancer molecular subtype membership. MethodsWe analyzed large public datasets, performed in vitro invasion and migration assays using small-molecule and doxycycline inducible SRC knock-down constructs, and in vivo experimental metastasis assays. ResultsLooking at large public datasets, we found SRC is upregulated in luminal papillary muscle invasive bladder cancer. Using the consensus classifier on RNA expression from 30 cell lines, we demonstrated that chemical SRC antagonists inhibited migration in luminal papillary cells but had little effect in basal/squamous lines. Conditional SRC knockdown inhibited migration in luminal papillary RT112 cells, whereas it increased migration and reduced proliferation in luminal papillary UM-UC6 cells. Regardless, these effects did not affect levels or sites of experimental metastasis in vivo. ConclusionsThe results support the conclusion that SRCs biological effects in bladder cancer are not primarily involved in promoting invasion and metastasis. Further work is required to define SRCs roles in luminal papillary bladder cancers.

cancer biology↗

bioRxiv2024

Plasma membrane H+-ATPase activation increases global transcript levels and promotes the shoot growth of light-grown Arabidopsis seedlings.

(1) Plant cell growth require the elongation of cells mediated by cell wall remodelling and turgor pressure changes. The plasma membrane (PM) H+-ATPase facilitates both cell wall remodelling and turgor pressure changes, by acidifying the apoplast of cells, referred to as acid growth. The acid growth theory is mostly established on the auxin-induced activation of PM H+-ATPase in non-photosynthetic tissues. However, how PM H+-ATPase affect the growth in photosynthetic tissues of Arabidopsis remains unclear. (2) Here, a combination of transcriptomics and cis-regulatory element analysis was conducted to identify the impact of PM H+-ATPase on global transcript levels and the molecular mechanism downstream of the PM H+-ATPase. (3) The PM H+-ATPase activation increased transcript levels globally, especially cell wall modification-related genes. The transcript level changes were in PM H+-ATPase-dependent manner. Involvement of Ca2+ was suggested as CAMTA motif was enriched in the promoter of PM H+-ATPase-induced genes and cytosolic Ca2+ elevated upon PM H+-ATPase activation. (4) PM H+-ATPase activation in photosynthetic tissues promote the expression of cell wall modification enzymes and shoot growth, adding a novel perspective of photosynthesis-dependent PM H+-ATPase activation in photosynthetic tissues to the acid growth theory that has primarily based on findings from non-photosynthetic tissues.

plant biology↗

bioRxiv2024

The role of protein shape in multiphasic separation within condensates

Liquid-liquid phase separation (LLPS) of biological macromolecules leads to the formation of various membraneless organelles. LLPS can not only form homogenous condensates but also multilayered and multiphase condensates, which can mediate complex cellular functions. However, the factors that determine the topological features of multiphase condensates are not fully understood. Herein, we focused on Ca2+/calmodulin-dependent protein kinase II (CaMKII), a major postsynaptic protein that undergoes various forms of LLPS with other postsynaptic proteins, and present a minimalistic computational model that reproduces these forms of LLPS, including a form of two-phase condensates, phase-in-phase (PIP) organization. Analyses of this model revealed that the competitive binding of two types of client proteins is required for the PIP formation. The PIP only formed when CaMKII had high valency and a short linker length. Such CaMKII proteins exhibited a low surface tension, a modular structure, and slow diffusion. These properties are consistent with the functions required by CaMKII to store information at the synaptic level. Thus, the computational modeling reveals new structure-function relationships for CaMKII as a synaptic memory unit.

neuroscience↗

bioRxiv2024

AgRP1 modulates breeding season-dependent feeding behavior in female medaka

Feeding and reproduction are known to be closely correlated with each other, and the seasonal breeders show breeding season-dependent feeding behavior. However, most model animals do not have definite breeding seasonality, and the mechanisms for such feeding behavior remain unclear. Here, we focused on female medaka (Oryzias latipes); they show breeding season-dependent feeding behavior, and their condition of breeding season can be experimentally controlled by day-length. We first demonstrated that, among previously reported feeding-related peptides (neuropeptides involved in feeding), agouti-related peptide 1 (agrp1) and neuropeptide y b (npyb) show higher brain expression under the breeding condition than under the non-breeding one. Combined with analysis of agrp1 knockout medaka, we obtained results to suggest that long day-induced sexually mature condition, especially ovarian estrogenic signals, increase the expressions of agrp1 in the brain, which results in increased food intake to promote reproduction. Our findings advance the understanding of neural mechanisms of feeding behavior for reproductive success.

neuroscience↗

bioRxiv2024

Intervening to preserve function in ischemic cardiomyopathy with a porous hydrogel and extracellular matrix composite in a rat myocardial infarction model

A variety of hydrogels have been developed for intramyocardial injection therapy after myocardial infarction. Some of these biomaterials have incorporated bioactive substances that promote local tissue regeneration and integration, while others have emphasized the mechanical role of the injectate in providing functional benefit. In this study, we incorporated two bioactive features, porosity, and extracellular matrix derived hydrogel (ECM), into a mechanically optimized, thermoresponsive, degradable hydrogel (poly(N-isopropylacrylamide-co-N-vinylpyrrolidone-co-MAPLA) copolymer) and evaluated whether injection of this biomaterial could abrogate the remodeling process in a rat ischemic cardiomyopathy model. After myocardial infarction by coronary artery ligation, rats were randomly divided into four groups: group NP (non-porous hydrogel) without either ECM or porosity, group PM (porous hydrogel) from the same synthetic copolymer with mannitol beads as porogens, and group PME with porosity and ECM digest added to the synthetic copolymer. A group with PBS injection alone served as a control. Intramyocardial injections were made 3 days after myocardial infarction. Serial echocardiography was assessed over time, and histological assessments were performed eight weeks after infarction. Results demonstrated improved echocardiographic function and neovascularization in the PME group compared to the other hydrogels and PBS injection. The PME group also demonstrated significant improvement in LV geometry and macrophage polarization (towards M2) compared to the PBS group, whereas these differences were not observed in the NP or PM groups versus the control. These results demonstrate that further functional improvement may be achieved in hydrogel injection therapy for ischemic cardiomyopathy by incorporating porosity and ECM digest, representing a combination of mechanical and biological effects.

bioengineering↗

bioRxiv2024

Arabidopsis ABCC4 encodes a cytokinin efflux transporter and is involved in root system development

The directional and sequential flow of cytokinin in plants is organized by a complex network of transporters. Genes involved in several aspects of cytokinin transport have been characterized, but a large part of the elaborate system remains elusive. In this study, we have identified ABCC4 as a cytokinin efflux transporter gene. Using a transient expression system in tobacco leaves, we screened Arabidopsis transporter genes and isolated ATP-BINDING CASSETTE TRANSPORTER C4 (ABCC4). Further validation through drug-induced expression in Arabidopsis and heterologous expression in budding yeast revealed that ABCC4 effluxes the active form of cytokinins. During the seedling stage, ABCC4 was highly expressed in roots, and its expression was up-regulated in response to cytokinin application. Loss-of-function mutants of ABCC4 displayed enhanced primary root elongation, similar to mutants impaired in cytokinin biosynthesis or signaling, which was suppressed by exogenous trans-zeatin treatment. In contrast, overexpression of the gene led to suppression of root elongation. These results suggest that ABCC4 plays a role in the efflux of active cytokinin, thereby contributing to root growth regulation. Our findings contribute to unraveling the many complexities of cytokinin flow and enhance our understanding of the regulatory mechanisms underlying root system development in plants.

plant biology↗

bioRxiv2024

The yolk sac vasculature in early avian embryo provides a novel model for the analysis of cancer extravasation

Hematogenous metastasis, a key trait of cancer cells, involves a complex sequence of cell migration steps, including intravasation, circulation, arrest in capillary vessels, and extravasation. Among these steps, extravasation is challenging to image in amniotes like humans and mice due to its unpredictable timing and location, limiting our understanding of cellular and molecular mechanisms through imaging. Establishing a new cancer carrier model with high-resolution imaging capabilities in amniotes is crucial. In this study, we investigated the yolk sac vasculature (YSV) of early avian embryos (chickens and quail) as a new model for studying extravasation, offering excellent imaging capabilities. We examined the YSV structure and attempted fluorescent labeling to enhance visibility. We then injected mCherry-labeled HT-1080 cells into YSV and observed their behavior, revealing distinct morphologies and extravasation dynamics. Our findings suggest that the YSV model holds promise as a novel cancer carrier model for elucidating cellular and molecular mechanisms through imaging-based approaches.

cancer biology↗

bioRxiv2024

The universal labeling and sorting of avian primordial germ cell utilizing Lycopersicon Esculentum lectin

Avian species serve as vital resources in human society, with the preservation and utilization of these species heavily reliant on primordial germ cells (PGCs). However, methods for effectively isolating these rare cells alive from embryos remain elusive in avian species beyond chickens. Even within chicken species, existing techniques have shown limited efficiency. In our study, we present a rapid, simple, and cost-effective method for labeling and sorting circulating-stage PGCs across various avian species, including Carinatae and Ratitae, utilizing Lycopersicon Esculentum lectin. Significantly, this method demonstrates high sorting efficiency by identifying a wide range of PGC subtypes while preserving the proliferative potential of chicken PGCs. This approach is anticipated to make a significant contribution to the conservation, research, and agricultural industries related to birds worldwide.

cell biology↗

bioRxiv2024

Intraplacental injection of human iPSC-derived PDX1+ pancreatic progenitors prolongs Pdx1-deficient mice survival

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

cell biology↗

bioRxiv2024

Label-free imaging of cellular organization in living mammalian cells via external apodization phase-contrast microscopy

Developing techniques to visualize intracellular structures, which influence the spatiotemporal functionality of biomolecules, is essential for elucidating mechanisms governing cellular behavior. In this study, we demonstrate that label-free external apodization phase-contrast (ExAPC) microscopy serves as a valuable tool for the simultaneous observation of various intracellular structures with high spatiotemporal resolution, while successfully mitigating halo artifacts. Additionally, through quantitative analysis of images obtained by combining ExAPC microscopy with fluorescence microscopy, we identified distinct heterogeneities in biomolecular condensates, lipid droplets, and mitochondria. Our findings highlight the potential of ExAPC microscopy to provide detailed insights into alterations in intracellular structures associated with diverse cellular processes, corroborating the existing knowledge and potentially contributing to the discovery of novel cellular mechanisms.

cell biology↗