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It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.
Figure 1. Schematic to show the SIET procedure. (a) An initial 3-point calibration was performed using three ‘ion standard solutions’, with a known concentration of H+. In this case, calibration was performed at pH 6, 7 and 8. (b) An initial background recording was performed in the centre of a scanning chamber in the absence of an experimental embryo. (ci) Experiments were performed in a scanning chamber made of a 35-mm glass-bottomed microwell dish with pieces of silicon elastomer in the centre. (cii) Typically, the plane of scan was set at ˜200 µm above the equator of the embryo. (ciii, civ) Images to show a representative sample scan around the embryo. Visualization at the (ciii) top and (civ) bottom of the embryo allows for precise positioning of the ion-selective microelectrode (ISM) close to the embryo surface. The ↔ symbol indicates the eight measuring positions around the circumference of embryos. (d) Recalibration and (e) background scans were also performed at the end of each experiment. (f) Scanned embryos were photographed at 3–4 dpf to ensure that they had developed normally. AP, VP, D, V, Ant., and Pos. are animal pole, vegetal pole, dorsal, ventral, anterior, and posterior, respectively. Scale bars, 1 mm (ci, f) and 500 µm (cii).
Figure 2. SIET measurements showing the H+ fluxes recorded around the equatorial circumference of X. laevis embryos from stages 9 to 12 (i.e. ˜7–13.25 hpf). H+ fluxes were measured in eight positions around embryos, as shown in the schematic on the upper right corner of panel (a). Fluxes were measured at (a) stage 9; (b) stage 10; (c) stage 11; and (d) stage 12 at an elevation of ˜200 µm above the embryonic equator. The data represent the mean ± standard error of the mean (SEM) of n = 5 embryos for each stage measured. The reference point (Ref) was measured at a distance of ˜5 mm away from the embryo. In (c) and (d), the asterisks indicate that H+ efflux data acquired around the embryo were significantly lower (P < 0.05) than those acquired on the dorsal side. Statistical significance was tested by two-way ANOVA and Tukey’s honest significant difference test.
Figure 3. Comparison of H+ flux data in different locations around the equatorial circumference of X. laevis embryos between stages 9 to 12 (i.e. ˜7–14.25 hpf). The data shown in Fig. 2 were also plotted as a radial column chart to allow comparison between the stages for each measurement position. The results show that, at stage 12, the H+ effluxes at all the measuring positions except the right side are significantly higher (P < 0.05) than those at the same side at stage 9 and stage 10. Similarly, the H+ effluxes on the right dorsal and dorsal sides at stage 12 are significantly higher (P < 0.05) than that on the same side at stage 11. Statistical significance was tested by two-way analysis of variance (ANOVA) test and Tukey’s honestly significant difference test.
Figure 4. The effect of the V-ATPase blocker, bafilomycin A1 on the gross morphology and expression of the Zic3 gene in embryos at the neurula stage. (a, b) Gross morphology and (c, d) Zic3 gene expression of embryos treated with either (a, c) 0.4% DMSO or (b, d) 0.5 µM bafilomycin A1, from ˜7–13.25 hpf (i.e. equivalent to stages 9–12 in untreated, normally developing embryos). Embryos were fixed at ˜24 hpf (i.e. equivalent to stage 22 in untreated, normally developing embryos), and top-illuminated images were acquired either (a, b) immediately or (c, d) after in situ hybridization was conducted to detect Zic3 mRNA. Panels (ai), (aii), (bi) and (bii) show different embryos. In (ai, aii), the white, blue, black, and yellow arrowheads indicate the unfused and fused neural folds, eye anlage, and cement gland, respectively, whereas in (bi, bii), the purple arrowheads indicate the yolk plug. In (c), T, D, M and R indicate the telencephalon, diencephalon, mesencephalon and rhombencephalon, respectively. AP, VP, D, V, Ant., and Pos. are animal pole, vegetal pole, dorsal, ventral, anterior, and posterior, respectively. Scale bars, 500 µm.
