Biology subjects

Okawa, S.

Publications and source records attributed to Okawa, S..

12 recordsLinked to original sources

bioRxiv2025

Activated fatty acid synthesis pathway in macrophages propagates pathogenic fibroblast expansion after myocardial infarction

Metabolic pathways, such as fatty acid oxidation and oxidative phosphorylation, can modulate inflammatory cells. However, little is known about the effects of the fatty acid synthesis pathway in macrophages on inflammation and cardiac remodeling after myocardial infarction (MI). Using spatial metabolomics, here we show that cardiac macrophages residing in the infarct synthesize de novo fatty acids and increase the production of fatty acid enzymes including ACLY and FASN. Mice deficient in myeloid Acly and Fasn have improved cardiac function after MI and reduced fibrosis. Combining Cleavage Under Targets and Release Using Nuclease (CUT&RUN), RNA sequencing analysis of Acly-/-macrophages, and macrophage-specific in vivo gene silencing, we demonstrate that ACLY acetylates the promoter region of the upstream regulator Krt17, which drives the production of pro-fibrotic cytokines, including IL-33. Single-cell RNA sequencing of cardiac fibroblasts shows that the expansion of a population of fibroblasts (Fibroblast 5) expressing high levels of extracellular matrix genes after MI is confined in the absence of macrophage Acly. Finally, the analysis of spatial multi-omics data of human hearts with MI uncovers myofibroblasts with the Fibroblast 5 gene signature. These myofibroblasts are located near cardiac macrophages expressing high levels of ACLY. In summary, we show that macrophage ACLY and FASN are deleterious in MI pathogenesis.

immunology↗

bioRxiv2024

Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease driven by endothelial cell inflammation and dysfunction, resulting in the pathological remodeling of the pulmonary vasculature. Innate immune activation has been linked to PAH development; however, the regulation, propagation, and reversibility of the induction of inflammation in PAH is poorly understood. Here, we demonstrate a role for interferon inducible protein 16 (IFI16), an innate immune sensor, as a modulator of endothelial inflammation in pulmonary hypertension, utilizing human pulmonary artery endothelial cells (PAECs). Inflammatory stimulus of PAECs with IL-1{beta} up-regulates IFI16 expression, inducing proinflammatory cytokine up-regulation and cellular apoptosis. IFI16 mRNA stability is regulated by post-transcriptional m6A modification, mediated by Wilms tumor 1-associated protein (WTAP), a structural stabilizer of the methyltransferase complex, via regulation of m6A methylation of IFI16. Additionally, m6A levels are increased in the peripheral blood mononuclear cells of PAH patients compared to control, indicating that quantifying this epigenetic change in patients may hold potential as a biomarker for disease identification. In summary, our study demonstrates IFI16 mediates inflammatory endothelial pathophenotypes seen in pulmonary arterial hypertension.

cell biology↗

bioRxiv2024

Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In RPC-specific Phosphatase and tensin homolog conditional knock-out (Pten-cKO) and RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3) mice, glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants suppresses RPC proliferation and photoreceptor differentiation, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling by knocking-out Ctnnb1, encoding {beta}-catenin, phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies. IMPACT STATEMENTTransgenic and pharmacological approaches reveal developmental elevations in glycolytic flux have an instructive role in promoting rod photoreceptor differentiation and maturation via activation of Wnt signaling.

developmental biology↗

bioRxiv2024

Deep learning-driven neuromorphogenesis screenings identify repurposable drugs for mitochondrial disease

Mitochondrial disease encompasses untreatable conditions affecting tissues with high energy demands. A severe manifestation of mitochondrial disease is Leigh syndrome (Leigh), which causes defects in basal ganglia and midbrain regions, psychomotor regression, lactic acidosis, and early death. We previously generated isogenic pairs of Leigh cerebral organoids and uncovered defects in neuromorphogenesis. Here, we leveraged on this disease feature to devise drug discovery pipelines. We developed a deep learning algorithm tailored for cell type-specific drug repurposing to identify drugs capable of promoting neuronal commitment. In parallel, we performed a survival drug screen in yeast and validated the repurposable hits on branching capacity in Leigh neurons. The two approaches independently highlighted azole compounds, Talarozole and Sertaconazole, both of which lowered lactate release and improved neurogenesis and neurite organization in Leigh midbrain organoids. Hence, targeting neuromorphogenesis has led to identify potential new drugs for mitochondrial disease and could prove an effective strategy for further drug discovery.

neuroscience↗

bioRxiv2024

NEUROG2 regulates a human-specific neurodevelopmental gene regulatory program

Unique hallmarks of human neocortical development include slower rates of neurogenesis and the establishment of an extracellular matrix-rich, outer-subventricular zone that supports basal neural progenitor cell expansion. How gene regulatory networks have evolved to support these human-specific neurodevelopmental features is poorly understood. Mining single cell data from cerebral organoids and human fetal cortices, we found that NEUROG2 expression is enriched in basal neural progenitor cells. To identify and purify NEUROG2-expressing cells and trace their short-term lineage, we engineered two NEUROG2-mCherry knock-in human embryonic stem cell lines to produce cerebral organoids. Transcriptomic profiling of mCherry-high organoid cells revealed elevated expression of PPP1R17, associated with a fast-evolving human-accelerated regulatory region, oligodendrocyte precursor cell and extracellular matrix-associated gene transcripts. Conversely, only neurogenic gene transcripts were enriched in mCherry-high cortical cells from Neurog2:mCherry knock-in mice. Finally, we show that Neurog2 is sufficient to induce Ppp1r17, which slows human neural progenitor cell division, and Col13a1, an extracellular matrix gene, in P19 cells. NEUROG2 thus regulates a human neurodevelopmental gene regulatory program implicated in supporting a pro-proliferative basal progenitor cell niche and tempering the neurogenic pace. SUMMARY STATEMENTTranscriptomic analyses of NEUROG2-mCherry knock-in human embryonic stem cell-derived cerebral organoids reveal a link between NEUROG2 and extracellular matrix remodeling during human cortical development.

developmental biology↗

bioRxiv2023

Proneural genes form a combinatorial code to diversify neocortical neural progenitor cells

Neocortical neural progenitor cells (NPCs) are molecularly heterogeneous, yet the genes that confer distinct neuronal morphologies and connectivities during development are poorly understood. Here, we determined that a proneural gene combinatorial code diversifies cortical NPCs. By mining scRNA-seq data from murine embryonic and early postnatal cortices and generating trajectory inference models, we found that Neurog2 is predominant, and is transiently co-expressed with Ascl1 and/or Neurog1 during an apical-to-basal NPC transition state in NPCs with early pseudotime identities. To assess whether proneural gene pairs confer distinct properties, we first used Neurog2/Ascl1 reporter mice expressing unique reporters, revealing that NPCs have distinct cell division modes and cell cycle dynamics dependent on their proneural gene profile. To assess Neurog2/Neurog1 interactions, we used double knock-out mice and novel split-Cre transgenics crossed to a Rosa-diptheria-toxin-A line to delete double+ cells, showing Neurog1/Neurog2 are specifically required to generate early-born neurons and to maintain NPCs. Finally, in silico mutation of a cortical Neurog2-gene regulatory network and validation using Neurog1/Neurog2 mutant and deleter mice, identified Bclllb and Nhlh2, expressed in early-born neurons, as dependent on Neurog1/Neurog2. Our data explains how proneural genes act combinatorically to diversify gene regulatory networks, thereby lineage restricting NPCs and creating cortical neuronal diversity.

developmental biology↗

bioRxiv2022

Pten regulates endocytic trafficking of cell adhesion and signaling molecules to pattern the retina

The retina is an exquisitely patterned tissue, with neuronal somata positioned at regular intervals to completely sample the visual field. Cholinergic amacrine cells are spectacular exemplars of precision, distributing in two radial layers and tangentially, forming regular mosaics. Here, we investigated how the intracellular phosphatase Pten and the cell adhesion molecule Dscam cooperate to regulate amacrine cell patterning. Using double mutants to test epistasis, we found that Pten and Dscam function in parallel pathways to regulate amacrine cell positioning. Mechanistically, Pten regulates endocytic remodeling of cell adhesion molecules (Dscam, Megf10, Fat3), which are aberrantly redistributed in Pten conditional-knock-out (cKO) amacrine cells. Furthermore, extracellular vesicles derived from multivesicular endosomes have altered proteomes in PtencKO retinas. Consequently, Wnt signaling is elevated in PtencKO retinal amacrine cells, the pharmacological disruption of which phenocopies PtencKO patterning defects. Pten thus controls endocytic trafficking of critical cell adhesion/signaling molecules to control amacrine cell spacing. HIGHLIGHTSO_LIPten and Dscam act in parallel pathways to regulate amacrine cell spacing C_LIO_LIEndocytic remodeling of cell adhesion molecules is perturbed in PtencKO retinas C_LIO_LIExtracellular vesicle content is altered in PtencKO retinas C_LIO_LIPerturbation of Wnt signaling phenocopies defects in amacrine cell positioning C_LI eTOC BLURBPatterns in nature range from stereotyped distributions of colored patches on butterfly wings to precise neuronal spacing in the nervous system. Waddington proposed that built-in constraints canalize developmental patterns. Touahri et al. identified Pten-mediated endocytic trafficking of cell adhesion/signaling molecules as a novel constraint measure controlling retinal amacrine cell patterning.

neuroscience↗

bioRxiv2022

ChemPert: mapping between chemical perturbation and transcriptional response for non-cancer cells

Prior knowledge of perturbation data can significantly assist in inferring the relationship between chemical perturbations and their specific transcriptional response. However, current databases mostly contain cancer cell lines, which are unsuitable for the aforementioned inference in non-cancer cells. Here we present ChemPert (https://chempert.uni.lu/), a database consisting of 82270 transcriptional signatures across 167 non-cancer cell types, enabling more accurate predictions of perturbation responses and drugs compared to cancer databases in non-cancer cells. In particular, ChemPert correctly predicted drug effects for treating non-alcoholic steatohepatitis and novel drugs for osteoarthritis. Overall, ChemPert provides a valuable resource for drug discovery in non-cancer diseases.

bioinformatics↗

bioRxiv2021

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterisation and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

plant biology↗

bioRxiv2020

Proneural genes define ground state rules to regulate neurogenic patterning and cortical folding

Transition from smooth, lissencephalic brains to highly-folded, gyrencephalic structures is associated with neuronal expansion and breaks in neurogenic symmetry. Here we show that Neurog2 and Ascl1 proneural genes regulate cortical progenitor cell differentiation through cross-repressive interactions to sustain neurogenic continuity in a lissencephalic rodent brain. Using in vivo lineage tracing, we found that Neurog2 and Ascl1 expression defines a lineage continuum of four progenitor pools, with double+ progenitors displaying several unique features (least lineage-restricted, complex gene regulatory network, G2 pausing). Strikingly, selective killing of double+ progenitors using split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Finally, consistent with NEUROG2 and ASCL1 driving discontinuous neurogenesis and folding in gyrencephalic species, their transcripts are modular in folded macaque cortices and pseudo-folded human cerebral organoids. Neurog2/Ascl1 double+ progenitors are thus Notch-ligand expressing niche cells that control neurogenic periodicity to determine cortical gyrification. HIGHLIGHTSO_LINeurog2 and Ascl1 expression defines four distinct transitional progenitor states C_LIO_LIDouble+ NPCs are transcriptionally complex and mark a lineage branch point C_LIO_LIDouble+ NPCs control neurogenic patterning and cortical folding via Notch signaling C_LIO_LINeurog2 and Ascl1 expression is modular in folded and not lissencephalic cortices C_LI eTOC BLURBEmergence of a gyrencephalic cortex is associated with a break in neurogenic continuity across the cortical germinal zone. Han et al. identify a pool of unbiased neural progenitors at a lineage bifurcation point that co-express Neurog2 and Ascl1 and produce Notch ligands to control neurogenic periodicity and cortical folding.

developmental biology↗

bioRxiv2018

Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies

Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors1-3, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors4,5 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice6. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning7, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.

developmental biology↗

bioRxiv2017

Signaling-dependent control of apical membrane size and self-renewal in rosette-stage human neuroepithelial stem cells

In the early developing nervous system, self-renewing neural stem cells are polarized and maintain an apical domain facing a central lumen. The presence of apical membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis cells lose their polarization and the concomitant loss of the apical domain coincides with a loss of the stem cell identity. Very little is known about the molecular signals controlling apical membrane size. Here we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human ESCs to identify a conserved molecular signalling pathway initiated by lysophosphatidic acid (LPA) that controls apical membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This apical domain size increase occurs independently of effects on proliferation and involves a SRF-dependent transcriptional induction of junctional and apical membrane components.

neuroscience↗