Leclerc C et al. (2000), Imaging patterns of calcium transients during n...

XB-ART-10337

J Cell Sci 2000 Oct 01;113 Pt 19:3519-29. doi: 10.1242/jcs.113.19.3519.

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Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.

Leclerc C , Webb SE , Daguzan C , Moreau M , Miller AL .

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Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

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Species referenced: Xenopus laevis
Genes referenced: adm gmnn ncam1 nog odc1 zic3

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	Fig. 1. Changes in intracellular calcium that occur in the dorsal ectoderm during gastrulation. Measurements start at stage 8 (5 hours postfertilisation) and end at the neurula stage (15 hours postfertilisation). A representative embryo was injected with aequorin in an apical position of the dorsal micromeres at the 8-cell stage to localise it to the dorsal ectoderm during gastrulation. (A) Profile of aequorin-generated light over time. Emitted light was recorded with a luminometer and expressed in arbitrary (arb.) units proportional to the anode current of the photomultiplier. Two components can be observed, a single slow component and multiple rapid spikes (see text). (B) Diagram to show the injection site (left) and the subsequent location of the aequorin (right; shaded zone) during gastrulation. AP, animal pole; VP, vegetal pole; V, ventral; D, dorsal. (C) Schematic illustration of the developmental time course of this aequorin-injected embryo.
	Fig. 2. Luminescence profiles of [Ca2+]i in embryos injected with aequorin into different locations at the 8-cell stage. (A) Aequorin injected into ventral micromeres is located in the ventral ectoderm during gastrulation. When injected into equatorial locations of either (B) ventral or (C) dorsal micromeres, it becomes located in the ventral and dorsal mesoderm, respectively. (D) Aequorin injected into the macromeres is located in the endoderm during gastrulation. In each panel the inset illustrates the location of the injection site (left) and the subsequent location of the aequorin during gastrulation (on the right; shaded zone). Under all these injection protocols, no significant [Ca2+]i increase was detected. AP, animal pole; VP, vegetal pole; V, ventral; D, dorsal.
	Fig. 3. An example of Ca2+ transients that occur in the dorsal ectoderm from a representative embryo at stage 10.5. The aequorin-generated photon image represents 120 seconds of accumulated light and is superimposed on its corresponding Lucifer Yellow generated fluorescent image. These images show the different intensities of two transients occurring simultaneously but at two independent positions of the dorsal ectoderm. A diagram of the whole embryo shows its orientation and the red box indicates where the images were obtained on the embryo. AP, animal pole; VP, vegetal pole. Colour scale indicates luminescence flux in photons/pixel. Scale bar, 0.3 mm.
	Fig. 4. The changing pattern of localized [Ca2+]i transients that occur in the dorsal ectoderm during gastrulation. (A) Images of a representative embryo at stages 8-9, 10, 11, 12 and 13, on to which are superimposed yellow spots to mark the position of each localized Ca2+ transient observed throughout each stage. (B) Drawings reconstructed from the corresponding video images of A. (C) Drawings of animal-vegetal (upper) and sagittal (lower) sections of embryos at stages 9 and 10, respectively. Superimposing the images in A on the drawings in B illustrates the distribution of the positions of the localized calcium transients relative to the anteroposterior axis of the dorsal ectoderm and the blastopore lip observed at each developmental stage. The approximate extent of this distribution is illustrated by the red shaded region in B. AC, animal cap; NIMZ, noninvoluting marginal zone; IMZ, involuting marginal zone. Scale bar, 0.5 mm. (D) Histogram showing the amplitude of the transients versus developmental stages.
	Fig. 5. Distribution of the amplitude of the transient observed during gastrulation from stage 8 to stage 13, in the dorsal ectoderm. Integration time, 120 seconds. Two populations of transients can clearly be distinguished, centred at 25 and 275 photons/second, respectively.
	Fig. 6. A detailed view of a propagating Ca2+ transient that occurred in the dorsal ectoderm of a representative embryo at stage 11.5. (A) Sequence taken every 20 seconds to show the appearance and subsequent disappearance of a propagating Ca2+ wave. Each panel represents 120 seconds of accumulated light. Wave velocity, calculated from the spread of the wave over successive images, was estimated to be approximately 10 mm/second. (B) Diagram to illustrate the propagation pathway of the wave, both laterally (red arrows) and from the animal pole toward the vegetal pole (blue arrows) in a radial manner. (C) Temporal profile of the luminescence output generated by the wave shown in A. The inset shows the region of the embryo from which the light was collected. Note that after the transient the [Ca2+]i in participating cells did not return to its original resting level. Levels of calcium (arrows on C) are estimated from photon counting assuming (1) that the resting level of Ca2+ is 320 nM and (2) that [Ca2+]i varies as the square of the luminescence (Shimomura, 1995). The colour scale indicates luminescence flux in photons/pixel. Scale bar, 0.3 mm.
	Fig. 7. The effect of the L-type Ca2+ channel antagonist, R(+) BayK, on calcium signalling. (A-B) Luminometry profiles of calcium in embryos, (A) treated with R(+)BayK from stage 8 and (B) untreated (control). Aequorin was injected into the 2 dorsal micromeres at the 8-cell stage so that it was localised in the dorsal ectoderm during gastrulation. In the presence of R(+)BayK no Ca2+ signal was detected, compared to the significant Ca2+ activity (both a single slow rising component and multiple rapid transients) observed in the controls. (C) Comparative morphology of the R(+)BayK-treated embryo and the sibling untreated (control) embryo at stage 25.
	Fig. 8. LTC antagonists repress the expression of the neuralising genes Zic3 and geminin. (A-C) Embryos were treated with R(+)BayK (10 mM) or nicardipine (500 mM) from stage 8 to stage 12-12.5, and then fixed and in situ hybridisation performed at stages 12 or 24. Zic3 expression is dramatically reduced (A,B) and geminin expression is lost entirely (C) in these representative embryos, when compared to the untreated controls. (D) Representative embryos demonstrating the effect of long-term and short-term nicardipine treatment on Zic3 expression. Embryos were treated for either 4 hours (from stage 8 to stage 10) or 8 hours (from stage 8 to stage 12- 12.5) with 500 mM nicardipine, and the expression pattern of Zic3 visualised at stage 24. Maximal reduction of Zic3 expression occured when the LTCs were inhibited for the entire 8 hours of gastrulation. Bars, 0.5 mm.
	Fig. 9. Intracellular calcium increase is required for neural induction mediated by attenuation of BMP signals. Expression of the pan-neural marker NCAM in the animal caps was measured by RT-PCR. Stage 8-9 animal caps treated with noggin (A) or dissociated caps (B) differentiated into neural cells expressing NCAM, whereas R(+)BayK pretreated animal caps before noggin induction (R(+)BayK/noggin) and BAPTA-AM loaded caps before dissociation (Dissociated+BAPTA) show a dramatic reduction in NCAM expression. Intact cap, animal caps not treated with noggin or nor dissociated; sibling control embryos served as positive control (Embryo) and PCR on the same RNA without reverse transcription was done to check the absence of genomic DNA (-RT). ODC is a control with ornithine decarboxylase.