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BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood.
RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements.
CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.
Figure 1.
Imaging of calcium dynamics. (a) Emission filters and dichroic mirrors used here were designed to reflect laser light efficiently at 488 nm but transmit 90% of light at 505 nm, collecting a large fraction of the emission spectrum of calcium green. (b) The experimental design for the observation of calcium dynamics in the Xenopus DMZ. Embryos were injected with calcium green dextran at the 4-cell stage (1) and cultured to early gastrula stages. Open-face DMZ explants were then prepared (2) with deep cells apposed to the coverglass (3). (c) The confocal system used allows effective, long-term confocal time-lapse of living cells. The left panel shows a field of cells from a calcium green dextran-labeled DMZ explant after one 8 s scan; the right panel shows the same field of cells after 254 additional 8 s scans over 85 min. The mean fluorescent intensity was reduced by only 6%
Figure 2.
and Movies 1 and 2
Long-range intercellular calcium waves in the dorsal marginal zone. (a) Individual frames from confocal time-lapse of the large propagating calcium wave in Movie 1 (see supplementary material). Time points are indicated in white. Cell mixing produces a mosaic pattern of labeled (bright) and unlabeled (black) cells. A large calcium wave initiates (t = :00), travels ∼20 cell diameters (t = 1:00), and subsides over approximately eight minutes (t = 8:00). The scale bar indicates 50 μm in this and all subsequent figures. (b) The plot of δF/F0 for each of the areas shown in the colored boxes in the last panel of (a). The plot shows the propagatory nature of the rise in calcium levels and the more even recovery. (Movie 1) This movie shows the patterns of calcium release in a DMZ explant labeled on the left side with calcium green dextran; the right side is unlabeled and is black, though cells are present in the field of view. In this and all DMZ movies, the dorsal lip of the blastopore is at the bottom of the screen, and the dorsal midline runs vertically through the middle of the screen. The mediolateral axis is horizontal. As the movie begins, several small flashes of calcium release can be observed throughout the explant (described in Figure 4b). About halfway through the movie, a small intercellular calcium wave arises near the midline of the explant, followed closely by another, much larger calcium wave. Still frames depicting the large wave in this movie are presented in Figure 1a. (Movie 2) This movie shows a DMZ explant in which all the cells are labeled with calcium green dextran. This explant undergoes a small wave, then a larger wave, then another small wave. These waves are less dramatic than those in Movie 1, possibly due to less effective calcium green dextran loading
Figure 3.
and Movies 3 and 4
Short-range intercellular calcium waves in the DMZ. (a) A small wave arises (t = :00), propagates ∼10 cell diameters (t = 1:00), then dissipates over the following 2 minutes (t = 3:20). The scale bar indicates 50 μm. (a′) The plot of δF/F0 for each of the areas shown in the colored boxes in the last panel of (a). (Movie 3) This movie shows the wave from Figure 3a. (b) A small wave arises (t = :00), propagates ∼10 cell diameters (t = 1:00), then dissipates over the following 4 minutes (t = 5:00). (Movie 4) This movie shows the wave from Figure 3b. (b′) The plot of δF/F0 for each of the areas shown in the colored boxes in the last panel of (b). As with the larger waves, the plot shows a propagatory increase in calcium levels and a more even recovery
Figure 4.
and Movies 5 and 6
Calcium waves and calcium flashes. (a) A high-magnification view of cells involved in the wave described in Figure 2. During the 4 minutes preceding the wave, very little movement is observed (t = :00–4:00). The yellow outline at t = 4:40 indicates the position of two cells just prior to the calcium wave. The calcium wave propagates from left to right across the field of cells between t = 5:00 and t = 5:40. As the wave moves across and begins to dissipate, cells move dramatically (compare initial position to cells at t = 7:20). As the calcium levels recover, the cells move dramatically in the opposite direction, beyond their original position (t = 8:00–12:00). The scale bar indicates 50 μm. (a′) The plot of δF/F0 for each of the areas shown in the colored boxes in the last panel of (a) shows that local changes in calcium levels reflects the pattern of calcium release and recovery in the overall wave (compare with Figure 2a′). (Movie 5) This movie shows the cells in Figure 4a. (b) A high-power view of cells involved in a calcium flash. Three cells dramatically increase calcium levels between t = :00 and t =:20. By t = 1:00, calcium levels are decreasing and return to baseline by t = 1:20. (b′) The plot of δF/F0 for each of the areas shown in the colored boxes in the last panel show that the rise in calcium levels does not propagate to other cells in the frame. (Movie 6) This movie shows the calcium flash in Figure 4b
Figure 5.
and Movies 7 and 8
The frequency of calcium waves. The graph plots waves/hour for different groups of explants; each point represents a single filmed explant. Calcium waves arise in DMZs at an average rate of 0.71/hour (see Movies 1–4 in supplementary material). No calcium waves were observed in explanted animal caps (see Movie 7 in supplementary material) or in ventral marginal zones (VMZ). Calcium waves were not inhibited by expression of Nxfz-8, though the frequency was reduced. Treatment with thapsigargin abolished calcium wave activity (see Movie 8 in supplementary material). A double asterisk indicates that the difference from wild-type is statistically significant to p < 0.05 by the Mann-Whitney U test. (Movie 7) This movie shows a representative time-lapse of an animal cap explant labeled with calcium green dextran. Small calcium flashes can be seen, but no calcium waves arise. (Movie 8) This movie shows a representative DMZ treated with thapsigargin; no calcium waves are observed
Figure 6.
Thapsigargin treatment inhibits convergent extension but does not affect mesodermal cell fate. (a) Control embryos at stage 12 have almost finished blastopore closure (dorsal view is shown; anterior at top). Xnot staining reveals long, narrow notochords. (b) Embryos treated with thapsigargin fail to close their blastopores by stage 12. Xnot staining indicates that notochords have failed to converge and extend. In a few cases (far right embryo), some convergent extension occurs and the anterior notochord elongates, though the posterior notochord remains broad. (c) Control embryos stained for Xnot. (d) Embryos treated with BHQ fail to close their blastopores, and notochords do not converge and extend, though Xnot is expressed strongly. (e) Control embryos hybridized to MyoD; somites are elongated along each side of the notochord. (f) Thapsigargin treated embryos express MyoD at normal levels, though somites fail to converge and elongate; MyoD expression domains remain short and broad. (g) Quantitation of convergent extension by measurement of the length-to-width ratio (mean ± standard error) of Xnot expression domains in control and thapsigargin-treated embryos. The total area of the Xnot expression domains differed by less than 3% between control and experimental embryos (h) Length-to-width ratios of Xnot expression domains in control and BHQ-treated embryos. Slight differences in stages account for differences in control LWRs in THAP and BHQ experiments (see [a] and [c])
Figure 7.
Thapsigargin inhibits convergent extension of DMZ explants without affecting gene expression. (a) Untreated control DMZ explants (n = 31) elongate significantly and change shape as a result of convergent extension, forming a rounded head and an elongated tail. (b) DMZ explants treated with thapsigargin (2 μM) fail to elongate, though subtle narrowing of the mesoderm is sometimes observed (n = 33). This variability is consistent with that of treated whole embryos. (c) BHQ treatment (10 μM) suppresses convergent extension (n = 18). (d) Quantitation of convergent extension of DMZ explants (mean LWR ± standard error). (e) RT-PCR demonstrates that thapsigargin treatment does not affect dorsovental patterning of the mesoderm in DMZ explants cultured to stage 22. (f) Thapsigargin does not affect dorsal cell fate specification in DMZ explants cultured to stage 12. (THAP indicates 10 thapsigargin-treated DMZs; CTL indicates 10 untreated DMZ explants; and −RT indicates no reverse transcriptase control). EF1α and actin serve as loading controls
