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In intact Xenopus embryos, an increase in intracellular Ca(2+) in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca(2+) increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca(2+) signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca(2+) transients, and neural induction to be explored. We have imaged Ca(2+) dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca(2+) transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type Ca(2+) channels. The accumulated Ca(2+) pattern correlated with the expression of the early neural precursor gene, Zic3. When the transients were blocked with pharmacological agents, the level of Zic3 expression was dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca(2+) -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca(2+) transients over time may play a role in controlling the expression pattern of Zic3.
Fig. 1. The changes in intracellular Ca2 that occur in explants during
gastrulation. (A) A representative example of a PMT trace obtained from
an aequorin-loaded explant. Data collection was started at 9 hpf (stage 10)
when the explant was prepared, and was ended at 13 hpf (stage 12). Two
components are visible on the trace: a slow-rising component and rapid
spikes. Emitted light is expressed in arbitrary (arb.) units. (B) The average
number of Ca2 transients observed from explants. The maximum number
of events occurs at stage 11. Mean SEM of five explants is shown.
Fig. 2. Isolated culture of separated ectoderm and mesoderm portions of the explant. (A, B) Representative temporal traces obtained for the separated
ectoderm and mesoderm portions. No signal was recorded from the isolated ectoderm (A), whereas a few signals were recorded from the isolated mesoderm
(B). (A , B ) At the end of the experiment, the ectoderm differentiated into a constricted ball of tissue (A ), while the mesoderm underwent convergence and
extension to form an elongated structure (B ).
Fig. 3. Representative example of the Ca2 signaling events observed in an explant for approximately 4.5 h starting at 11 hpf (A). Each panel represents 120 s of accumulated light. The area circled in yellow in (C, E, F, H, K, N, and R) highlights a repetitive signal at the dorsal ectoderm–mesoderm border. Color scale indicates luminescent flux in photons/pixel. E and M are ectoderm and mesoderm, respectively.
Fig. 4. The effect of nifedipine on Ca2 transients. Three representative
temporal traces from aequorin-loaded explants that were (A) maintained
under normal conditions (control) or treated with either (B) 150 M
nifedipine or (C) 300 M nifedipine. Nifedipine suppresses the Ca2
transients in a dose-dependent manner at 150 M, both the amplitude and
the number of transients are reduced when compared with the untreated
controls, and at 300 M all of the transients are completely inhibited.
Fig. 5. Detection of Zic3 by whole-mount in situ hybridization in whole control embryo and in explants. (A) Zic3 expression in stage 12 control embryo;
dorsal view, anterior up. (B) Isolated dorsal ectoderm (sibling control embryos at stage 12). (C–F) Four different open-faced explants showing the crescent
shape staining in the posterior-most region of the ectoderm; dorsal view, animal pole up. Scales bars in (A) 500 m, in (B–F) 200 u m.
Fig. 6. Comparison of the expression of Zic3 with the accumulated location of Ca2 transients. Representative aequorin-loaded (n 5) (A) untreated control
and (B) BAPTA-AM treated explants to show (ii) pseudo-3D profile of accumulated increase in [Ca2 ]i during the 8 h of data acquisition that corresponds
to gastrulation and (i) the subsequent expression pattern of Zic3, determined by in situ hybridization in the same explant. The Ca2 increase above the
background (5 photons) is outlined in black in (Ai) to show that this region correlates with the pattern of Zic3 expression. (C) Representative examples of
untreated control and BAPTA-AM-treated explants to show Zic3 expression (n 38, 4 independent experiments). E and M are ectoderm and mesoderm,
respectively.
