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Miki Inoue, Takashi Baba, Fumiya Takahashi, Miho Terao, Shogo Yanai, Yuichi Shima, Daisuke Saito, Kei Sugihara, Takashi Miura, Shuji Takada, Mikita Suyama, Yasuyuki Ohkawa, Ken-Ichirou Morohashi, Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway., Communications biology, 10.1038/s42003-022-03941-5, 5, 1, 974-974, 2022.09, Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin β10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF.. |
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Yoshie Endo, Daisuke Asanuma, Shigeyuki Namiki, Kei Sugihara, Kenzo Hirose, Akiyoshi Uemura, Yoshiaki Kubota, Takashi Miura, Quantitative modeling of regular retinal microglia distribution., Scientific reports, 10.1038/s41598-021-01820-3, 11, 1, 22671-22671, 2021.11, Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia's morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.. |
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Mari Kawamura, Kei Sugihara, Hisako Takigawa-Imamura, Toshiyuki Ogawa, Takashi Miura, Mathematical Modeling of Dynamic Cellular Association Patterns in Seminiferous Tubules., Bulletin of mathematical biology, 10.1007/s11538-021-00863-x, 83, 4, 33-33, 2021.02, In vertebrates, sperm is generated in testicular tube-like structures called seminiferous tubules. The differentiation stages of spermatogenesis exhibit a dynamic spatiotemporal wavetrain pattern. There are two types of pattern-the vertical type, which is observed in mice, and the helical type, which is observed in humans. The mechanisms of this pattern difference remain little understood. In the present study, we used a three-species reaction-diffusion model to reproduce the wavetrain pattern observed in vivo. We hypothesized that the wavelength of the pattern in mice was larger than that in humans and undertook numerical simulations. We found complex patterns of helical and vertical pattern frequency, which can be understood by pattern selection using boundary conditions. From these theoretical results, we predicted that a small number of vertical patterns should be present in human seminiferous tubules. We then found vertical patterns in histological sections of human tubules, consistent with the theoretical prediction. Finally, we showed that the previously reported irregularity of the human pattern could be reproduced using two factors: a wider unstable wavenumber range and the irregular geometry of human compared with mouse seminiferous tubules. These results show that mathematical modeling is useful for understanding the pattern dynamics of seminiferous tubules in vivo.. |
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Kei Sugihara, Saori Sasaki, Akiyoshi Uemura, Satoru Kidoaki, Takashi Miura, Mechanisms of endothelial cell coverage by pericytes: computational modelling of cell wrapping and in vitro experiments., Journal of the Royal Society, Interface, 10.1098/rsif.2019.0739, 17, 162, 20190739-20190739, 2020.01, Pericytes (PCs) wrap around endothelial cells (ECs) and perform diverse functions in physiological and pathological processes. Although molecular interactions between ECs and PCs have been extensively studied, the morphological processes at the cellular level and their underlying mechanisms have remained elusive. In this study, using a simple cellular Potts model, we explored the mechanisms for EC wrapping by PCs. Based on the observed in vitro cell wrapping in three-dimensional PC-EC coculture, the model identified four putative contributing factors: preferential adhesion of PCs to the extracellular matrix (ECM), strong cell-cell adhesion, PC surface softness and larger PC size. While cell-cell adhesion can contribute to the prevention of cell segregation and the degree of cell wrapping, it cannot determine the orientation of cell wrapping alone. While atomic force microscopy revealed that PCs have a larger Young's modulus than ECs, the experimental analyses supported preferential ECM adhesion and size asymmetry. We also formulated the corresponding energy minimization problem and numerically solved this problem for specific cases. These results give biological insights into the role of PC-ECM adhesion in PC coverage. The modelling framework presented here should also be applicable to other cell wrapping phenomena observed in vivo.. |
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Tetsuhisa Otani, Thanh Phuong Nguyen, Shinsaku Tokuda, Kei Sugihara, Taichi Sugawara, Kyoko Furuse, Takashi Miura, Klaus Ebnet, Mikio Furuse, Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity, The Journal of cell biology, 10.1083/jcb.201812157, 218, 10, 3372-3396, 2019.10, Tight junctions (TJs) establish the epithelial barrier and are thought to form a membrane fence to regulate epithelial polarity, although the roles of TJs in epithelial polarity remain controversial. Claudins constitute TJ strands in conjunction with the cytoplasmic scaffolds ZO-1 and ZO-2 and play pivotal roles in epithelial barrier formation. However, how claudins and other TJ membrane proteins cooperate to organize TJs remains unclear. Here, we systematically knocked out TJ components by genome editing and show that while ZO-1/ZO-2-deficient cells lacked TJ structures and epithelial barriers, claudin-deficient cells lacked TJ strands and an electrolyte permeability barrier but formed membrane appositions and a macromolecule permeability barrier. Moreover, epithelial polarity was disorganized in ZO-1/ZO-2-deficient cells, but not in claudin-deficient cells. Simultaneous deletion of claudins and a TJ membrane protein JAM-A resulted in a loss of membrane appositions and a macromolecule permeability barrier and in sporadic epithelial polarity defects. These results demonstrate that claudins and JAM-A coordinately regulate TJ formation and epithelial polarity.. |
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Soon Ki Han, Xingyun Qi, Kei Sugihara, Jonathan H. Dang, Takaho A. Endo, Kristen L. Miller, Eun Deok Kim, Takashi Miura, Keiko U. Torii, MUTE Directly Orchestrates Cell-State Switch and the Single Symmetric Division to Create Stomata, Developmental Cell, 10.1016/j.devcel.2018.04.010, 45, 3, 303-315.e5, 2018.05, Precise cell division control is critical for developmental patterning. For the differentiation of a functional stoma, a cellular valve for efficient gas exchange, the single symmetric division of an immediate precursor is absolutely essential. Yet, the mechanism governing this event remains unclear. Here we report comprehensive inventories of gene expression by the Arabidopsis bHLH protein MUTE, a potent inducer of stomatal differentiation. MUTE switches the gene expression program initiated by SPEECHLESS. MUTE directly induces a suite of cell-cycle genes, including CYCD5;1, in which introduced expression triggers the symmetric divisions of arrested precursor cells in mute, and their transcriptional repressors, FAMA and FOUR LIPS. The regulatory network initiated by MUTE represents an incoherent type 1 feed-forward loop. Our mathematical modeling and experimental perturbations support a notion that MUTE orchestrates a transcriptional cascade leading to a tightly restricted pulse of cell-cycle gene expression, thereby ensuring the single cell division to create functional stomata. Stomata, small valves on the plant epidermis, are made of two guard cells surrounding a pore. Han et al. show that the transcription factor MUTE orchestrates gene regulatory circuits to switch cells to a differentiation state, then ensures that only a single symmetric division occurs to create a functional stoma.. |
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Kei Sugihara, Koichi Nishiyama, Shigetomo Fukuhara, Akiyoshi Uemura, Satoshi Arima, Ryo Kobayashi, Alvaro Köhn-Luque, Naoki Mochizuki, Toshio Suda, Hisao Ogawa, Hiroki Kurihara, Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling, Cell Reports, 10.1016/j.celrep.2015.10.051, 13, 9, 1814-1827, 2015.12, Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC) behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon.. |
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Satoshi Arima, Koichi Nishiyama, Toshiyuki Ko, Yuichiro Arima, Yuji Hakozaki, Kei Sugihara, Hiroaki Koseki, Yasunobu Uchijima, Yukiko Kurihara, Hiroki Kurihara, Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement, Development, 10.1242/dev.068023, 138, 21, 4763-4776, 2011.11, Angiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic 'cell-mixing'. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the 'cell-mixing' phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via γ-secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.. |
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Makoto Kurachi, Junko Kurachi, Fumiko Suenaga, Tatsuya Tsukui, Jun Abe, Satoshi Ueha, Michio Tomura, Kei Sugihara, Shiki Takamura, Kazuhiro Kakimi, Kouji Matsushima, Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration, Journal of Experimental Medicine, 10.1084/jem.20102101, 208, 18, 1605-1620, 2011.08, Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8+ T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8+ T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3-/- antigen-specific CD8+ T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8+ T cells. Early after infection, CXCR3-/- antigen-specific CD8+ T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-α are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3-/- CD8+ T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8+ T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8+ T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.. |
