Biology subjects

Yamanaka, Y.

Publications and source records attributed to Yamanaka, Y..

16 recordsLinked to original sources

bioRxiv2025

Increased tissue tension caused by depletion of CLDN3 in the non-neural ectoderm causes neural fold fusion defects in chick embryos

Neural tube morphogenesis provides a dynamic setting in which to study epithelial cell behaviours. Members of the claudin family of tight junction proteins regulate apical epithelial cell behaviors at all steps of neural tube development. We discovered that CLDN3, expressed in the non-neural ectoderm but not the neural ectoderm, is required to mediate neural fold fusion in chick embryos, particularly in the spinal region of the embryo. Depleting CLDN3 affects apical protein localization and apical domain morphology. Here, we used live imaging to re-examine the process of neural fold fusion in the cranial and spinal regions of the embryo and assessed biomechanical parameters of the non-neural ectoderm that are dependent on CLDN3. Our live imaging confirmed previous reports that unlike neural fold fusion in the cranial region and posterior neuropore, the spinal region does not depend on progressive fusion driven by "zippering" cell behaviors but instead fuses in a multi-step process where contact occurs simultaneously at multiple points along the anterior-posterior axis. We and others refer to the process of spinal neural fold fusion as "buttoning" to highlight the differences in cell behaviors from those observed during zippering (van Straaten et al., 1993). CLDN3-depletion decreased the rate of progression of neural fold buttoning within the spinal region. Using cell segmentation analyses we confirmed that CLDN3 depletion decreased the apical cell area of cells at the edges of the neural folds but not of lateral cells in the non-neural ectoderm. CLDN3 depletion increased pMLC staining within the apical domain of the cell, coinciding with a decrease in cell area, suggesting increased epithelial tension. Laser ablation studies revealed that the non-neural ectoderm of CLDN3-depleted embryos exhibits higher tension during neural fold fusion. We showed that treatment with the myosin II inhibitor blebbistatin is sufficient to partially rescue the neural fold fusion defects in CLDN3-depleted embryos. This work provides further evidence for the importance of non-neural ectodermal tissue tension in neural fold fusion and suggests that loss of CLDN3 may alter tissue tension through cytoskeletal regulation pathways within the apical domain. This work supports that CLDN3 contributes to neural fold fusion and epithelial tissue tension during neural fold fusion via modifications to the apical cytoskeleton. SummaryWe found that the tight junction protein Claudin-3 (CLDN3) plays a role in regulating tissue tension, by directing actomyosin contraction and apical cell shape/size changes essential for chick neural fold fusion.

developmental biology↗

bioRxiv2025

Mono-allelic p.R37H Dehydrodolichyl Diphosphate Synthase variants lead to protein glycosylation defects, aberrant lipid profiles and interneuron scarcity in a novel mouse model of progressive epileptic encephalopathy.

Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase (cis-PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, DhddsR37H+/-strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. DhddsR37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used "off-label" to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in DhddsR37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis-PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

neuroscience↗

bioRxiv2025

The microtubule-binding protein EML3 is required for mammalian embryonic growth and cerebral cortical development; Eml3 null mice are a model of cobblestone brain malformation

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

developmental biology↗

bioRxiv2025

Long-read RNA sequencing identifies isoform switching of a novel long non-coding RNA that promotes somatic cell reprogramming

Development of long-read RNA sequencing technologies has paved the way to the exploration of RNA isoform diversity and its relevance in regulating cell fate. However, identifying new functional isoforms is still very difficult. Here, we leverage long-read RNA sequencing to study changes in isoforms during somatic cell reprogramming and identify novel isoforms occurring throughout cell state transitions. We demonstrate tight regulation of non-coding isoforms and show that isoform switching plays previously overlooked functional roles and outcomes in gene regulation and cell fate changes. We uncover a novel long non-coding RNA, Snhg26, that undergoes isoform switching during reprogramming to enhance the conversion of differentiated cells towards the pluripotent state. Knock-down of Snhg26 in mouse and human pluripotency models reveals that it is important for pluripotency acquisition. Together, our study provides a resource to study full-length isoform usage during cell fate change. It also demonstrates the power of long-read sequencing to identify functionally relevant gene isoforms in the context of cell plasticity.

cell biology↗

bioRxiv2024

Aging-associated vacuolation of multi-ciliated cells in the distal mouse oviduct reflects unique cell identity and luminal microenvironment

The female reproductive organs present with the earliest aging characteristics, such as a decline in fertility and estrous cyclicity. While age-related changes in the ovary are well-documented, it is unclear if any age-associated changes occur in the other female reproductive organs, such as the oviduct/fallopian tube. The recent recognition of the distal end of the fallopian tube as the tissue of origin of high-grade serous tubal ovarian carcinomas (HGSCs), and that its patient demographic is strongly biased to postmenopausal women, motivated us to investigate age-associated changes in this organ. At the distal end of aged oviducts in mice, we found vacuolated multi-ciliated cells (MCCs) with a severely apically displaced and deformed nucleus. This phenotype was unique to the distal oviduct epithelium -infundibulum (INF) and ampulla (AMP). Ovariectomy did not affect the timeline of MCC vacuolation, suggesting little involvement of ovulation and hormonal regulation. MCC vacuolation was induced in hypoxia or hydroxyurea treatments in in vitro organotypic culture of all oviduct regions, not limited to the INF/AMP epithelium. This suggests high oxygen demand in MCCs, compared to other cell types, and a uniquely stressed INF/AMP epithelial microenvironment in vivo. We found that the blood circulation of INF/AMP depended on the ovarian artery, different from the rest of the oviduct epithelium and its circulation declined along with ovarian activities. We conclude that a decline in local blood circulation and distinct cellular identity of the INF/AMP epithelium caused age-associated MCC vacuolation, reflecting its mild, chronically stressed microenvironment.

physiology↗

bioRxiv2022

Reconstituting human somitogenesis in vitro

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches1-5, a system that robustly recapitulates human somitogenesis in both space and time remains missing. Here, we introduce a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis, which we termed Axioloids, that captures accurately the oscillatory dynamics of the segmentation clock as well as the morphological and molecular characteristics of segmentation and sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF/WNT signaling gradients and Retinoic Acid (RA) signaling components. We identify an unexpected critical role of RA signaling in the stabilization of forming segments, indicating distinct, but also synergistic effects of RA and extracellular matrix (ECM) on the formation and epithelialization of somites. Importantly, comparative analysis demonstrates striking similarities of Axioloids to the human embryo, further validated by the presence of the HOX code in Axioloids. Lastly, we demonstrate the utility of our Axioloid system to study the pathogenesis of human congenital spine diseases, by using patient-like iPSC cells with mutations in HES7 and MESP2, which revealed disease-associated phenotypes including loss of epithelial somite formation and abnormal rostrocaudal patterning. These results suggest that Axioloids represent a promising novel platform to study axial development and disease in humans.

developmental biology↗

bioRxiv2021

Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant

ObjectiveOver 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:-glucosaminide N- acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme). ResultsHgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat- Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine. InterpretationAltogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

animal behavior and cognition↗

bioRxiv2021

An improved molecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under pathological conditions.

Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes of their conformation and biological functions. Disturbed LLPS homeostasis in MLOs cause fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes of the degree of crowding in MLOs. However, it has not been investigated well due to lack of an appropriate method. To address this, we developed a genetically-encoded molecular crowding sensor CRONOS that senses the degree of macromolecular crowding in MLOs using fluorescence resonance energy transfer (FRET) system. CRONOS is a very bright biosensor with wider dynamic range and detect changes in the macromolecular volume fraction better than the previously reported mCer-mCit sensor in solution. By fusing to scaffold protein of each MLO, we successfully delivered CRONOS to MLO of interest and detected previously undescribed difference of the degree of crowding in each MLO. If not tagged, CRONOS localized to interstitial space of MLOs, giving us the crowding information of inspace. CRONOS also detected changes of degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for determination of molecular crowding and detection of pathological changes in MLOs in live cells.

biochemistry↗

bioRxiv2021

Modeling High-grade serous ovarian carcinoma using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing

Ovarian cancer remains the most lethal gynecological cancer today. High-grade serous ovarian carcinoma (HGSC) is the most common and lethal type of ovarian cancer and is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing. We mutated tumor suppressor genes associated with HGSC in two different combinations; Brca1, Tp53, Pten with/without Lkb1 and successfully generated HGSC, however, with different latencies and pathophysiology. By utilizing Cre lineage tracing in our system, we visualized peritoneal micrometastases in an immune-competent environment. Because our strategy is flexible in selecting mutation combinations and targeting areas, it would be useful for generating ovarian cancer mouse models. Significance StatementHigh grade serous ovarian cancer (HGSC) is the most common ovarian malignancy but our knowledge of early tumorigenesis is still quite limited due to late diagnosis of patients. We developed a new strategy of generating mouse ovarian cancer models using a combination of in vivo electroporation and CRISPR-mediated genome editing. We demonstrated that a combination of three tumor suppressor gene mutations (Brca1, Tp53 and Pten) is sufficient to develop HGSCs. Interesting, an additional mutation in Lkb1 drastically changed tumor latency, penetrance and pathophysiology through changing its cell-of-origin. Our strategy is highly flexible in selection of mutation combinations and targeting areas in immune competent mice and useful to study early tumorigenesis of ovarian cancer.

cancer biology↗

bioRxiv2020

Anatomical and cellular heterogeneity in the mouse oviduct-- its potential roles in reproduction and preimplantation development

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive tissue that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are basic coiling patterns and turning points in the coiled mouse oviduct can serve as reliable landmarks for luminal morphological regionalities. We identified previously unrecognized anatomical structures in the isthmus and uterotubal junction (UTJ) that likely play important roles in reproduction. Interestingly, during ovulation, the isthmus was transiently plugged by a thick mucus, keeping the oocytes within the ampulla. Preimplantation embryos travelled along the oviduct and formed a queue within small compartments of the UTJ before uterine entry. Taken together, the oviduct luminal epithelium had highly diverse luminal structures with distinct cell populations reflecting its complex functions in reproduction.

physiology↗

bioRxiv2020

Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis

Owing to technical advances in single cell biology, the appreciation of cellular heterogeneity has increased, which has aided our understanding of organ function, homeostasis and disease progression. The oviduct (also known as the fallopian tube in humans) is the distal-most portion of the female reproductive tract. It is essential for reproduction and the proposed origin of high grade serous ovarian carcinoma (HGSOC). In mammals, the oviduct is morphologically segmented along the ovary-uterus axis into four evolutionally conserved regions. It is unknown however if there is a diversification of epithelial cell characteristics between these regions. In this study, we identified transcriptionally distinct populations of secretory and multiciliated cells restricted to the distal and proximal regions of the oviduct. We demonstrated that these distal and proximal populations are distinct lineages specified early in Mullerian duct development and are maintained separately. These results aid our understanding of epithelial development, homeostasis and initiation of disease from the oviduct.

developmental biology↗

bioRxiv2020

Prom1 expression does not mark a stem/progenitor population in the mouse oviduct epithelium

The oviduct or fallopian tube is the site of fertilization and preimplantation embryonic development. The epithelium lining the oviduct consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis still remains poorly understood. CD133/Prom1 has been used to identify adult stem cell populations in various organs and often associated with cancer stem cell property. Using a Cre-recombinase based lineage tracing strategy, we found that CD133/Prom1 expression was not associated with a stem/progenitor population in the oviduct but marked a sub population of multiciliated and secretory cells which did not propagate. Interestingly, Prom1 expressing secretory cells rapidly transition to multiciliated cells and progressively migrate to the tips of epithelial folds in the ampulla. Our results show that CD133/Prom1 expression cannot be used as a progenitor/stem cell marker in the mouse oviduct.

developmental biology↗

bioRxiv2020

Human iPSC-derived hypertrophic chondrocytes reveal a mutation-specific unfolded protein response in chondrodysplasias

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, utilizing human chondrodysplasia-specific iPSCs, we examined the UPR caused by mutations in MATN3 or COL10A1. In growth plate-like structures formed from iPSC-derived sclerotome in vivo, the hypertrophic zone was disrupted, and induced hypertrophic chondrocytes in vitro showed varying levels of ER stress depending on the mutation. Autophagy inducers and chemical chaperones succeeded in reducing ER stress only in some mutants, while transcriptome analysis revealed many mutation-specific changes in genes involved in apoptosis, metabolism, and protein trafficking. In this way, our system has allowed the precise evaluation of the UPR caused by each mutation, opening up new avenues for treatment of individual chondrodysplasia patients.

cell biology↗

bioRxiv2019

Isolation of an archaeon at the prokaryote-eukaryote interface

The origin of eukaryotes remains enigmatic. Current data suggests that eukaryotes may have risen from an archaeal lineage known as \"Asgard archaea\". Despite the eukaryote-like genomic features found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear due to the lack of cultured representatives and corresponding physiological insight. Here we report the decade-long isolation of a Lokiarchaeota-related Asgard archaeon from deep marine sediment. The archaeon, \"Candidatus Prometheoarchaeum syntrophicum strain MK-D1\", is an anaerobic, extremely slow-growing, small cocci ([~]550 nm), that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexities have been proposed for Asgard archaea, the isolate has no visible organella-like structure. Ca. P. syntrophicum instead displays morphological complexity - unique long, and often, branching protrusions. Based on cultivation and genomics, we propose an \"Entangle-Engulf-Enslave (E3) model\" for eukaryogenesis through archaea-alphaproteobacteria symbiosis mediated by the physical complexities and metabolic dependency of the hosting archaeon.

microbiology↗

bioRxiv2019

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system1. Since murine and human embryos differ in their sizes, geometries, and nutrients, we use in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments2-4. In this way we recapitulate the murine and human segmentation clocks in vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock5,6, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.

developmental biology↗

bioRxiv2019

Modeling the Human Segmentation Clock with Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation1. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types2,3, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human iPSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton.

developmental biology↗