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Fukuda, N.

Publications and source records attributed to Fukuda, N..

11 recordsLinked to original sources

Developmental emergence of spatiotemporal coordination in cerebellar Purkinje cell populations

Coordinated activity across neuronal populations is fundamental to brain function, yet how such network-level organization emerges during development remains incompletely understood. Here, we performed whole-cerebellar calcium imaging at cellular scale in zebrafish larvae to examine the developmental maturation of Purkinje cell population dynamics. Visual stimulation recruited large, spatially organized populations of Purkinje cells whose responses depended on inferior olive input and were associated with optokinetic behavior. Notably, in the absence of stimuli, Purkinje cells formed transient assemblies exhibiting distance-dependent coordination. During development, long-range coordination progressively emerged, extending initially local correlations into distributed population-wide coordination. Early enucleation, but not dark rearing, disrupted the developmental refinement of long-range coordination and induced aberrant population clustering, suggesting that early retina-dependent signals contribute to cerebellar network development. Together, these findings reveal key organizational features underlying the developmental emergence of coordinated cerebellar population dynamics and suggest that early retina-dependent signals shape population-level organization.

neuroscience↗

Transcriptomic analysis of the juvenile to adult transition in the mouse corpus callosum

The corpus callosum, a major white matter tract in the brain, undergoes age-related functional changes. To extend our investigation of age-related gene expression dynamics in the mouse corpus callosum, we compared RNA-seq data from 2-week-old and 12-week-old wild-type C57BL/6J mice and identified the differentially expressed genes (e.g., Serpinb1a, Ndrg1, Dnmt3a, etc.) between these ages. Interestingly, we found that genes highly expressed in myelinating oligodendrocytes were upregulated in 12-week-old mice compared to 2-week-old mice, while genes highly expressed in oligodendrocyte precursor cells (OPCs) and newly formed oligodendrocytes were downregulated. Furthermore, by comparing these genes with the datasets from 20-week-old and 96-week-old mice, we identified novel sets of genes with age-dependent variations in the corpus callosum. These gene expression changes potentially affect key biological pathways and may be closely linked to age-related neurological disorders, including dementia and stroke. Therefore, our results provide an additional dataset to explore age-dependent gene expression dynamics of oligodendrocyte lineage cells in the corpus callosum.

neuroscience↗

Investigating EMT-mediated resistance to EGFR tyrosine kinase inhibitors in NSCLC using innovative organoid models

BackgroundEpithelial-mesenchymal transition (EMT) has emerged as a key mechanism underlying resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, the intricacies of EMT-mediated resistance, driven by tumor microenvironment (TME) interactions, remain enigmatic. This study aimed to probe EMT-induced resistance in NSCLC using innovative in vitro organoid models. MethodsWe generated organoids by co-culturing EGFR-mutant NSCLC cells (HCC827, H1975), mesenchymal stem cells and endothelial cells. Drug susceptibility was compared between organoids and spheroids (cancer cells only) using EGFR TKIs - Gefitinib, Afatinib, Osimertinib. EMT marker (E-cadherin, ZEB1) expression was analyzed via immunofluorescence and western blotting. The effects of Bevacizumab and miR200c on overcoming resistance were also investigated. ResultsThe study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included the decrease of E-cadherin and an increase in ZEB1, both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the VEGF cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. ConclusionsThis study offered vital insights into EMT-driven EGFR TKI resistance, highlighting the utility of organoid models in evaluating resistance modulated by TME interactions. Our findings reveal promising directions for overcoming EMT-mediated resistance involving Bevacizumab and miR200c, warranting further in vivo validation.

cancer biology↗

In vivo long-term voltage imaging by genetically encoded voltage indicator reveals spatiotemporal dynamics of spinal cord neuronal populations during development

One of the central questions in neural development is how individual neurons assemble functional networks. To address this, it is essential to elucidate how coordinated activity emerges during development. However, tracking the functional maturation of neuronal populations over time remains challenging, as it requires long-term, non-invasive monitoring of membrane potential dynamics. Here, we developed a voltage imaging approach for zebrafish embryos using genetically encoded voltage indicators (GEVIs), enabling fast, direct and cell-type-specific measurements of membrane potentials from defined neuronal populations in a non-invasive manner. Using this approach, we detected coordinated voltage changes in spinal motor neurons with high spatiotemporal resolution. Depolarization and hyperpolarization events were observed at the population, single-cell, and subcellular levels. Notably, long-term voltage imaging revealed the early emergence and progressive maturation of membrane potential dynamics, characterized by increased firing rate, coupling strength and axonal outgrowth. This optical approach constitutes a significant advancement in the study of neural development, providing a powerful tool for investigating the spatiotemporal dynamics of neuronal populations in vivo.

neuroscience↗

Validity of freehand three-dimensional ultrasound system in measurement of three-dimensional surface shape of shoulder muscles

Freehand three-dimensional ultrasound (3DUS) system is a promising technique for accurately assessing muscle morphology. However, its accuracy has been validated mainly in terms of volume by examining lower limb muscles. This study aimed to validate 3DUS in the measurements of 3D surface shape and volume by comparing them with MRI measurements while ensuring the reproducibility of participant posture by focusing on the shoulder muscles. The supraspinatus, infraspinatus, and posterior deltoid muscles of 10 healthy males were scanned using 3DUS and MRI while secured by an immobilization support customized for each participant. A 3D surface model of each muscle was created from the 3DUS and MRI methods, and the agreement between them was assessed. For the muscle volume, the mean difference between the two models was within -0.51 cm3. For the 3D surface shape, the distances between the closest points of the two models and Dice similarity coefficient were calculated. The results showed that the median surface distance was less than 1.21 mm and Dice similarity coefficient was larger than 0.85. These results suggest that, given the above error is permitted, 3DUS can be used as an alternative to MRI in measuring volume and surface shape, even for the shoulder muscles.

bioengineering↗

The developing epicardium regulates cardiac chamber morphogenesis by promoting cardiomyocyte growth

The epicardium, the outermost layer of the heart, is an important regulator of cardiac regeneration. However, a detailed understanding of the crosstalk between the epicardium and myocardium during development requires further investigation. Here, we generated three models of epicardial impairment in zebrafish by mutating the transcription factor genes tcf21 and wt1a, and by ablating tcf21+ epicardial cells. Notably, all three epicardial-impairment models exhibit smaller ventricles. We identified the initial cause of this phenotype as defective cardiomyocyte growth, resulting in reduced cell surface and volume. This failure of cardiomyocytes to grow is followed by decreased proliferation and increased abluminal extrusion. By temporally manipulating its ablation, we show that the epicardium is required to support ventricular growth during early cardiac morphogenesis. By transcriptomic profiling of sorted epicardial cells, we identified reduced expression of FGF and VEGF ligand genes in tcf21-/- hearts, and pharmacological inhibition of these signaling pathways partially recapitulated the ventricular growth defects. Thus, the analysis of these epicardial-impairment models further elucidates the distinct roles of the epicardium during cardiac morphogenesis and the signaling pathways underlying epicardial-myocardial crosstalk.

developmental biology↗

Heat hypersensitivity of ryanodine receptor type 1 mutants implicated in malignant hyperthermia

Cellular heat-sensing is a universal strategy for avoiding thermal damage and adapting to environments by regulating thermogenic activities. If heat-sensing results in the acceleration of processes governing cellular thermogenesis, hyperthermia can occur. However, how this positive feedback loop contributes to hyperthermia development, especially the gap between heat-sensing and thermogenesis, remains largely unknown. Here, we show that an optically controlled local heat pulse induces an intracellular Ca2+ burst in cultured HEK 293 cells overexpressing ryanodine-receptor-type-1 (RyR1) mutants related to the life-threatening illness malignant hyperthermia (MH), and that the Ca2+ burst originates from heat-induced Ca2+-release (HICR) because of the mutant channels heat hypersensitivity. Furthermore, the heat hypersensitivity of the four RyR1 mutants was ranked, highlighting the complexity of MH. Our findings reveal the novel cellular heat-sensing mechanism, HICR, is essential for the functional positive feedback loop causing MH, suggesting a well-tuned HICR is fundamental for heat-mediated intracellular signaling.

biophysics↗

Automated high-throughput proteome and phosphoproteome analysis using paramagnetic bead technology

Recent developments in proteomics have enabled signaling studies where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is robust, versatile, high-throughput, and achieves higher sensitivity than classical protocols. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.

systems biology↗

TOMVi: A Tool for Overviewing Metadata Visualization

DNA sequencing technology has been improved drastically in the recent decades, which allows comprehensively analyze microbial community and gene expression. Simultaneously with the improvements, many tools and software for much amount of sequence data have been released. These analysis tools/software can produce statistic information. However, to interpret and to find the relationships and/or associations between the information and the experimental condition may be a complex task, especially for non-bioinformaticians. In this article, an open source software TOMVi (Tool for Overviewing Metadata Visualization) which visualized similarity and relationships between the samples corresponding to the experimental conditions was introduced. TOMVi allows researcher to interactively manipulate the composition data in each sample toward to each experimental condition such as location, time-series and others with graphical user interface (GUI), and to visualize the existence and behavior of each OTU. The figure illustrated by this tool can provide an intuitive suggestion but not always correct as statistically, and will be helpful to decide further analyzing tactics.\n\nThe TOMVi is available at http://github.com/Daisuke-Kyoui/tomvi.

bioinformatics↗

Genome-wide strategies reveal target genes of Npas4l associated with cardiovascular development in zebrafish

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we combined complementary genome-wide approaches including transcriptome analyses and chromatin immunoprecipitation (ChIP). The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes crucial for hematoendothelial specification such as etv2, tal1 and lmo2. We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

developmental biology↗

Genetic compensation is triggered by mutant mRNA degradation

Genetic compensation by transcriptional modulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems 1-3; however, whether and how such a response can be activated in the absence of protein feedback loops is unknown. Here, we develop and analyze several models of transcriptional adaptation in zebrafish and mouse that we show are not caused by loss of protein function. We find that the increase in transcript levels is due to enhanced transcription, and observe a correlation between the levels of mutant mRNA decay and transcriptional upregulation of related genes. To assess the role of mutant mRNA degradation in triggering transcriptional adaptation, we use genetic and pharmacological approaches and find that mRNA degradation is indeed required for this process. Notably, uncapped RNAs, themselves subjected to rapid degradation, can also induce transcriptional adaptation. Next, we generate alleles that fail to transcribe the mutated gene and find that they do not show transcriptional adaptation, and exhibit more severe phenotypes than those observed in alleles displaying mutant mRNA decay. Transcriptome analysis of these different alleles reveals the upregulation of hundreds of genes with enrichment for those showing sequence similarity with the mutated genes mRNA, suggesting a model whereby mRNA degradation products induce the response via sequence similarity. These results expand the role of the mRNA surveillance machinery in buffering against mutations by triggering the transcriptional upregulation of related genes. Besides implications for our understanding of disease-causing mutations, our findings will help design mutant alleles with minimal transcriptional adaptation-derived compensation.

genetics↗