Biology subjects

Yoshihara, M.

Publications and source records attributed to Yoshihara, M..

13 recordsLinked to original sources

bioRxiv2026

Multi-layered regulatory networks driving human dopaminergic neuron differentiation

Neuronal differentiation requires coordinated regulation across chromatin organization, gene expression, protein abundance, and post-translational modifications. Using the LUHMES human dopaminergic neuronal differentiation model, we integrated proteome and phosphoproteome profiling with previously generated enhancer-promoter interaction maps from NET-CAGE and HiCap and transcriptomic analysis across three consecutive differentiation stages. Differentiation was accompanied by increased abundance and phosphorylation of proteins involved in axon guidance, cytoskeletal organization, and synaptic signaling, alongside repression of the cell cycle, DNA replication, and chromatin-associated programs. Phosphoproteome analysis further revealed extensive remodeling of signaling networks associated with neuronal maturation. Enhancer-promoter interaction analysis revealed substantially greater rewiring at enhancers than promoters and identified master and relay transcription factors regulated across multiple molecular layers. siRNA-mediated knockdown showed that transcription factors MYT1, ISL2, and NHLH2 are crucial for proper neuronal maturation, whereas LCOR acts as a negative regulator of differentiation. Integrative network reconstruction further nominated MEOX2 as a candidate enhancer-associated regulator of late dopaminergic maturation. Together, these findings provide a multi-layered view of regulatory networks governing human neuronal differentiation.

systems biology↗

bioRxiv2025

Transferring active inference to newly encountered prism-shifted environments

Active inference has been proposed as a unified explanation of perception and action. Previous work has shown that canonical neural networks that minimize shared Helmholtz energy are cast as performing active inference of the external environment. However, how animals flexibly adapt to newly encountered environments remains to be fully addressed. To investigate the brains generalizability and adaptability to new environments, this work develops canonical neural networks that employ multiple policy matrices in parallel. We demonstrate that the proposed model can recapitulate the prism adaptation--a form of visuomotor adaptation--under an arm-reaching task. Using policy matrices pretrained under various target positions, these networks could transfer previous experiences and exhibit faster adaptation to the prism-shifted environment than the naive networks. Furthermore, after-effects were observed following the removal of simulated prism glasses. These results suggest the biological plausibility and utility of the proposed model, providing insights into the adaptive capabilities of the brain.

neuroscience↗

bioRxiv2025

Hic2 fast-tracks iPS cell generation by suppressing KLF4-dependent epidermal detour

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is the most established cellular conversion by exogenous master transcription factors (TFs). A deeper understanding of this yet inefficient process is critical to extending our capability to control cellular identity for medical applications. Here we report 14 genes essential for efficient iPSC generation, but dispensable for self-renewal. Of those, overexpression of Hic2, a transcriptional suppressor highly expressed in PSCs, enhances iPSC generation [~]10-fold. This is achieved through a more direct transition towards pluripotency, bypassing an intermediate state with KLF4-dependent transient epidermal gene expression during iPSC generation. Mechanistically, HIC2 co-occupies these KLF4 targets and directly inhibits their expression. Our work demonstrates that master TFs necessary for cellular conversions can also activate obstructive genes during cellular reprogramming. We propose that identifying transcriptional suppressors against such side effects, like Hic2, can be a powerful strategy to achieve more efficient TF-mediated cell conversions.

developmental biology↗

bioRxiv2024

Comparative transcriptome and variant analyses of the pancreatic islets of a rat model of obese type 2 diabetes identifies a frequently distributed nonsense mutation in the lipocalin 2 gene

We have recently established the Zucker fatty diabetes mellitus (ZFDM) rat as a novel model of obese type 2 diabetes (T2D), originating from the obese Zucker fatty (ZF) rat harboring a missense mutation in the leptin receptor gene. Pathogenesis of dysfunction of the pancreatic islets and genetic factors of T2D in ZFDM rats remain unknown. Here, we perform comparative transcriptome and variant analyses of the pancreatic islets between the two strains. Among differentially expressed genes irrespective of obesity and glucose intolerance states, we identify a nonsense mutation, c.409C>T (p.Gln137X), in the lipocalin 2 (Lcn2) gene which encodes a secreted protein called neutrophil gelatinase-associated lipocalin, a well-known biomarker for inflammation. Interestingly, we find that the Lcn2 mutation is distributed widely in rat species, such as commonly used DA and F344 strains. We examine the Lcn2 mutation as a strong candidate gene for T2D in ZFDM rats by using genome editing of ZFDM rats in which the nonsense mutation is replaced with a wild-type nucleotide. We find that the genome editing well works but also observe that there is no significant difference in the development of T2D between genome-edited and original ZFDM rats. Finally, we perform a genetic linkage analysis by using backcross progeny between ZF and ZFDM rats and confirm that the Lcn2 mutation exhibits no significant association with the onset of T2D. Our data indicate that several rat strains would serve as Lcn2 deficient models, contributing to elucidate pathophysiological roles of Lcn2 in a wide variety of phenotypes.

genetics↗

bioRxiv2023

Epigenetic heterogeneity of the Notch signaling components in the human developing retina

ObjectiveThe regulation of receptor expression is crucial for fine-tuned signal transduction. Notch signaling is a key signaling pathway involved in retinal development. Although the involvement of this signaling pathway in the differentiation of retinal ganglion cells has been documented, less is known about its involvement in earlier stages of retinal progenitor cell differentiation. To clarify the timing of Notch receptor expression in undifferentiated retinal progenitor cells and elucidate the possible involvement of chromatin remodeling in the regulation of Notch receptor expressions, we re-analyzed publicly available human fetal retina single-cell RNA-seq and ATAC-seq data. ResultsOn days 59, 74, and 78, we observed NOTCH1 mRNA expression in early retinal progenitor cells, which diminished at later stages of differentiation. Integration of single-cell RNA-seq and ATAC-seq revealed that chromatin remodeling in part of the NOTCH1 locus was accompanied by transitions in its mRNA expression. Importantly, chromatin accessibility in the region upstream of NOTCH1 depended on the differentiated cell types. These results suggest that chromatin remodeling may be involved in the differential expression of NOTCH1, although another type of Notch mRNA expression regulation may exist.

developmental biology↗

bioRxiv2023

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway

Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cells immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and a mutant with altered cilia (RFX2 -/-), we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

neuroscience↗

bioRxiv2022

B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening by pioneering factors

Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

cell biology↗

bioRxiv2021

Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

DUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent DUX4 expression in cellular models. We report here that a transient DUX4 expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

cell biology↗

bioRxiv2020

Ectopic histone clipping in the mouse model of progressive myoclonus epilepsy

We establish cystatin B (CSTB) as a regulator of histone H3 tail clipping in murine neural progenitor cells (NPCs) and provide evidence suggesting that epigenetic dysregulation contributes to the early pathogenesis in brain disorders associated with deficient CSTB function. We show that NPCs undergo regulated cleavage of the N-terminal tail of histone H3 at threonine 22 (H3T22) transiently upon induction of differentiation. CSTB-deficient NPCs present premature activation of H3T22 clipping during self-renewal mediated by increased activity of cathepsins L and B. During differentiation, the proportion of immature committed neurons undergoing H3T22 clipping is significantly higher in CSTB-deficient than in wild-type NPCs, with no observable decline within 12 days post-differentiation. CSTB-deficient NPCs exhibit significant transcriptional changes highlighting altered expression of nuclear-encoded mitochondrial genes. These changes are associated with significantly impaired respiratory capacity of differentiating NPCs devoid of CSTB. Our data expand the mechanistic understanding of diseases associated with CSTB deficiency.

pathology↗

bioRxiv2020

Differentiation of ciliated human midbrain-derived LUHMES neurons

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing. LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES, neuronal cilia biology can be investigated along a complete timeline: from proliferation through differentiation to mature neurons. Summary StatementWith LUHMES, a ciliated human neuronal cell model, the underlying "neurobiology" of cilia and ciliopathies can be investigated along a complete time line: from proliferation through differentiation to mature neurons.

neuroscience↗

bioRxiv2019

Balancing Selection at HLA-G Modulates Fetal Survival, Preeclampsia and Human Birth Sex Ratio

The population sex ratio is thought to be maintained through balancing selection on rare phenotypes. However, empirical evidence for genetic influence has thus far proven elusive. We combined 1000 Genomes data and large cohorts to study human sex ratios. We found underrepresentation of male offspring in preeclampsia, a serious pregnancy disorder with uncertain pathogenesis. Genetic variation of fetal human leukocyte antigen G (HLA-G), regulating maternal anti-fetal immune responses, was found to be under balancing selection. Sex-linked downregulation of HLA-G and upregulation of interferon alpha-1 (IFNA1) expression contribute to loss of fetal immunotolerance in preeclampsia and suggest hydroxychloroquine as a treatment option. Our findings indicate that an evolutionary trade-off between fetal immunotolerance and protection against infections promotes genetic diversity in HLA-G, thereby maintaining human sex ratios. One Sentence SummaryFetal HLA-G modulates human sex ratio.

evolutionary biology↗

bioRxiv2019

Single cell map of the human ovarian cortex.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. We analysed on a single cell level the transcriptome and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our single cell mapping reveals transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data does not support the existence of germline stem cells in adult human ovaries thereby reinforcing the dogma of a limited ovarian reserve.

cell biology↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗