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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.
FIGURE 1 (a) Schematic morphological change in Xenopus laevis embryo during the early embryogenesis from the fertilized egg to stage 11 (mid-gastrula) from Xenopus illustrations (Zahn et al. 2022), except for the stage-11 animal-pole panel, which was drawn by the authors. (b) Schematic of AFM with an upright optical microscope. Xenopus embryo was immobilized in a semi-cylindrical-groove agarose well and cultured in 1/10× Steinberg's solution. The underside morphology of the embryo was observed through a mirror placed beneath the gel plate, using the optical microscope. (c) Schematic of embryo placed in a semi-cylindrical well in a top view (upper figure) and side view (lower Figure). The embryo was slightly contacted on the edge of the agarose well (orange) and then gently held using a dulled glass capillary (green). (d) A microscope image of a developing embryo at around stage 12, viewed from the vegetal pole using the mirror. The yolk plug of the embryo is denoted by the arrowhead. After checking the stage with the image, the animal hemisphere was measured by AFM.
FIGURE 2 (a) Schematic morphology (upper) and an optical microscopic image (lower) of a Xenopus laevis embryo at stage 6. AFM-measured region is indicated by an open rectangle (red). The AFM mapping images of H (b) and E (c), and θ (d) estimated from (b). (e) Plots of averaged H and E values in the region denoted by open dashed-line rectangles (b–d, red). The black arrow indicates the position of the cell–cell boundary. The local regions where stiffness in the apical cytoplasmic region exceeded that at the cell–cell boundary are indicated by red arrowheads. The topography is plotted by flattening the tilt of the height image.
FIGURE 3 (a) Schematic morphology of Xenopus laevis embryo at stage 8 and an optical microscopic image, with AFM measured region indicated by an open square (red). Time-lapse mapping images of H (b), E (c), and θ (d) estimated from (b), where each mapping started at 0 min (left), 9 min (middle), and 24 min (right). Observed cell division is denoted by asterisks in (b). (e) Graph showing stiffness changes of individual cells over time. The colors in the graph correspond to those of the cells shown in AFM images in the upper figure.
FIGURE 4 (a) Schematic morphology of Xenopus laevis embryo, viewed from the animal pole (Animal) and the vegetal pole (Vegetal) at stage 10. The AFM-measured region is indicated by an open square (red). AFM mapping images of H (b), E (c), and θ (d) estimated from (b). (e) Histograms of E values in the apical cytoplasmic region (blue) and the cell–cell boundary (red), shown in (c). (f) Plots of E values versus cell area (apical cell size) in individual cells, observed in (b) and (c), respectively. The coefficient of determination was 0.036.
FIGURE 5 (a) An optical microscopic image and schematic image (inset) of Xenopus laevis embryo, viewed from the vegetal pole. AFM-measured regions are indicated by open squares: I and II are in the yolk plug, whereas III is in the animal region. AFM mapping images of H (b), E (c), and θ (d) estimated from (b). Measured regions of apical cytoplasmic regions (red) and cell–cell boundaries (blue) (e) segmented using Cellpose. Quantifications of stiffness in different measured regions in yolk plug (f) and animal hemisphere (g). Data are mean ± SD from 23 cells (g) and 34 cells (h) from a single embryo. For data analysis, Wilcoxon signed-rank test was used to determine p-values. ***p < 1.0 × 10−4.
