Biology subjects

Michiue, T.

Publications and source records attributed to Michiue, T..

4 recordsLinked to original sources

bioRxiv2025

Mapping single-cell mechanics in the early embryogenesis of Xenopus laevis using atomic force microscopy

During early embryonic development, cells undergo rapid cleavage divisions accompanied by morphological changes driven by mechanical cues. However, the spatiotemporal mechanics of regulative embryonic cells remains poorly understood. Here, we use atomic force microscopy (AFM) to map single-cell stiffness of Xenopus laevis embryos, a model of regulative development, from early cleavage (stage 6) to the onset of gastrulation (stage 11). To ensure stable AFM mapping, vitelline membrane-removed embryos were immobilized in custom grooved agarose wells and gently held using a dulled glass pipette. AFM observations revealed marked mechanical heterogeneity within the animal hemisphere: stiffness in apical cytoplasmic regions, which are defined as the central apical surface excluding cell-cell boundaries, varied among cells, regardless of size, indicating intrinsic variability. Cell-cell boundaries consistently showed high stiffness, suggesting strong adhesion, as commonly observed in epithelial monolayers in vitro. In contrast, in the vegetal hemisphere during gastrulation, cell-cell boundaries exhibited low stiffness, indicative of weaker adhesion. Additionally, micron-scale stiff inclusions were detected in the apical vegetal cytoplasm, with sizes comparable to those of yolk platelets. These findings demonstrate the capability of AFM to probe the microscale mechanical architecture of developing regulative embryos and to uncover regional mechanical asymmetries between the animal and vegetal hemispheres. Such asymmetries may contribute to key morphogenetic processes during early vertebrate development.

biophysics↗

bioRxiv2025

Identification of N-acetyl-rich heparan sulfate binding motifs and their role in expanding BMP distribution and signaling by Cerberus

Secreted signaling molecules called morphogens including BMP and Wnt provide positional information by their concentration gradient. Although binding to an extracellular matrix, heparan sulfate (HS), is essential for morphogen gradient formation, it is still unclear how different ranges of gradients can be achieved. We previously found that two types of HS, N-acetyl-rich HS (NAc-HS) and N-sulfo-rich HS (NS-HS), are differentially clustered on the cell membrane, and bind to Frzb and Wnt8, respectively. Furthermore, we demonstrated that Frzb expands Wnt8 distribution by transferring Wnt8 from NS-HS to NAc-HS. This indicates that NAc-HS or its unsulfated form, heparan, which has been considered as a precursor of HS including N-sulfo HS, has an essential role in morphogen distribution. Here, we identified a so-called BMP antagonist, Cerberus (Cer), as another NAc-HS-binding protein. Binding of Cer to NAc-HS was enhanced by knockdown and repressed by overexpression of ndst1, which catalyzes the conversion of NAc-HS to NS-HS, suggesting that Cer binds to NAc-HS and/or heparan as does Frzb. We then explored NAc-HS binding motifs using deletion or alanine-substitution mutants of Frzb and Cer, and revealed that clusters of basic amino acids, Lys and Arg, are critical for their binding to NAc-HS. Functionally, Cer was able to expand distribution and signaling ranges of BMP4 through relocation of BMP4 from NS-HS to NAc-HS as has been shown with Wnt8 and Frzb. For this Cer activity, its NAc-HS binding motifs were essential. These results suggest that NAc-HS clusters and NAc-HS binding motifs constitute an interacting unit to modulate extracellular distribution of BMP4 as well as Wnt8.

developmental biology↗

bioRxiv2021

Positive Feedback Regulation of fzd7 Expression Robustly Shapes Wnt Signaling Range in Early Heart Development

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when patterning differentiation of a thin region. Wnt is a morphogen that organizes cardiac development; especially Wnt6 patterns the cardiogenic mesoderm to induce differentiation of a thin pericardium in Xenopus. It is, however, unclear how Wnt6 can pattern such a thin tissue. In this study, we reveal that a Wnt receptor, frizzled7, is expressed in a Wnt-dependent manner in the prospective heart region, and that this receptor-feedback is essential for shaping a steep gradient of Wnt. In addition, the feedback imparts robustness against fluctuations of Wnt ligand production and allows the system to reach a steady state quickly. We also found a Wnt antagonist sFRP1, which is expressed at the opposite side of Wnt source, accumulates on a novel type of heparan sulfate (HS), N-acetyl-rich HS, which is highly presented in the outer of cardiogenic mesoderm, achieving local inhibition of Wnt signaling by restricting sFRP1 spreading. These two intricate regulatory systems restrict Wnt signaling and ensure reproducible patterning of a thin pericardium tissue.

developmental biology↗

bioRxiv2018

A novel cell segmentation method for developing embryos using machine learning

Cell segmentation is crucial in the study of morphogenesis in developing embryos, but it is limited in its accuracy. In this study we provide a novel method for cell segmentation using machine-learning, termed Cell Segmenter using Machine Learning (CSML). CSML performed better than state-of-the-art methods, such as RACE and watershed, in the segmentation of ectodermal cells in the Xenopus embryo. CSML required only one whole embryo image for training a Fully Convolutional Network classifier, and it took 20 seconds per each image to return a segmented image. To validate its accuracy, we compared it to other methods in assessing several indicators of cell shape. We also examined the generality by measuring its performance in segmenting independent images. Our data demonstrates the superiority of CSML, and we expect this application to significantly improve efficiency in cell shape studies.

developmental biology↗