Watanabe K et al. (2022), Partial exogastrulation due to apical-basal pol...

ECB-ART-50410

Genes Cells 2022 Jun 01;276:392-408. doi: 10.1111/gtc.12934.

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Partial exogastrulation due to apical-basal polarity of F-actin distribution disruption in sea urchin embryo by omeprazole.

Watanabe K , Yasui Y , Kurose Y , Fujii M , Yamamoto T , Sakamoto N , Awazu A .

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Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half was higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.

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gRNAs referenced: rhoa gRNA1

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	FIGURE 1. Developmental stages of control and omeprazole‐treated Hemicentrotus pulcherrimus (HP) embryos. Typical bright‐field images of the gastrulation process (steps 1, 2, 4, and 5) of sea urchin embryos (scale bars: 30 μm). Red arrows indicate vegetal pole positions. Gastrulation did not progress after step 2 in the treated embryos, and the outward protrusion of the vegetal pole side was more pronounced at step 4. In step 5, gastrulation stopped incompletely without penetrating the archenteron, and “partial” exogastrulation was observed in more than half of treated embryos (bellow image). # and #’ refer to the total number of sampled embryos and the number of embryos with similar shape to the image, respectively
	FIGURE 2. Visualization of endodermal tissue and secondary mesenchyme cells (SMCs). (a) Endodermal tissue was stained for endogenous alkaline phosphatase (red arrows) in control (left) and treated embryos (right) in step 5, where stained part located inner region in control embryos while stained located outer region in treated embryos (scale bars: 30 μm). (b) Typical locations SMCs in treated embryo in step 4 where gcm, the marker of SMC, was stained (red arrow) (scale bars: 30 μm). # and #’ refer to the total number of sampled embryos and the number of embryos clearly stained similar to the image, respectively
	FIGURE 3. Whole embryonic distributions of actinin‐GFP, fibropellin‐1‐GFP, and pH. (a) Definitions of angle θ (0°–180°) from the vegetal pole (0°) to the animal pole (180°) along the circumference of the embryo cross section and apical and basal sides of cells (see also Figure S7) of confocal fluorescence microscopic images determined via actinin (actinin‐GFP: Green) intensity of embryos. (b–e) Average and 95% confidence intervals (error bars) of apical–basal ratios at angle θ obtained by n samples (left), and typical confocal fluorescence microscopic images (scale bars: 30 μm) (right) of actinin‐GFP intensities at step 1 (b) and step 2 (c), fibropellin‐1‐GFP intensities at step 2 (d), and pH indicator intensities at step 2 (e). In the left‐hand side images, blue and orange curves and bars represent the control and treated embryo values, respectively, where gray bars indicate significantly different average values between the control and treated embryos according to Welch's t‐test (p < .05) (see also Figure S1). Magenta curves in the right panel of (c) were included along the region with significant differences between the values of the control and treated embryos. The correlation coefficients of apical–basal ratios between actinin‐GFP intensities (c) and pH indicator intensities (e) in control and treated embryos were 0.56 and 0.64, respectively
	FIGURE 4. Effect of F‐actin regulator‐knockout on gastrulation. (a) Bright‐field images of gastrulation in control, RhoA‐knockout, and YAP1‐knockout embryos at selected time‐points. Primary mesenchyme cells and pigment cells were observed in all embryos suggesting that development did not stop. The knockout embryos did not form the structure like prism larva observed at 45 hpf in the control embryo (scale bars: 30 μm). # and #’ refer to the total number of sampled embryos and the number of embryos with similar shape to the image, respectively. (b and c) Average fluorescence intensities (arbitrary fluorescence units) and 95% confidence intervals (error bars) of intracellular pH indicator (b) and apical–basal ratio of pH indicator (c) of RhoA‐knockout embryos and control embryos as a function of angle θ. The indications of colors and θ are stated in Figures 3 and S1
	FIGURE 5. Coarse‐grained model and simulation of control, treated, and over‐polarized embryos. (a) Modeled cell lengths of apical and basal sides of control, treated, and over‐polarized embryos; lia,fin and lib,fin refer to the final length of the apical and basal sides of the ith cell, respectively (see Experimental procedures). Distributions of the final length of the former two models were determined based on the distributions of apical–basal ratios of actinin‐GFP intensities from control and treated embryos in step 2. The top center panel was a modification of Figure 3d. Red, green, and blue circles in the top panel and panel (b) represent pigment cells, wedge cells, and other cells, respectively. (b) Snapshots of the initial embryo shape at 18 and 26 hpf of the three models. (c) Definition of the roundness of the vegetal side of embryos from simulation results and imaging. (d and e) Roundness indices of vegetal sides of modeled control and treated embryos (d) and experimentally determined values (e). Blue and orange # refer to the sampling number of control and treated embryos at each time point, error bars indicated 95% confidence intervals, and * indicated that the roundness of control embryos was significantly larger than that of treated embryos according to Welch's t‐test (p < .05, see Figure S5b). (f) The ratio of apical/basal lengths obtained by experimental and simulation data, and p‐values of Welch's t‐test between the two types of data, where * and NS between two box plots indicate p < .05 and p > .05, respectively. Embryos at step 4 (late gastrula) and simulation final step were compared

References [+] :

Aihara, Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side. 2001, Pubmed, Echinobase