Mapping Single‐Cell Mechanics in the Early Embryogenesis of <i>Xenopus laevis</i> 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, as commonly observed in epithelial monolayers in vitro. In contrast, in the vegetal hemisphere during gastrulation, cell-cell boundaries exhibited relatively low stiffness compared with the cytoplasmic regions. Additionally, microscale 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.