Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation

Abstract

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.

Figure 1

Three patterns of calcium transients that appear between 50% and 75% epiboly are ventral marginal signal (A–F), yolk flash (G), and marginal hot spots (H). (A–F) The persistent ventral signal is shown in f-aequorin-loaded zebrafish embryos at approximately 50% epiboly. (A–C) Sixty seconds of accumulated luminescence. (D–F) Same data superimposed on corresponding bright-field images. In A and D, the embryo is viewed from the vegetal pole, in B and C it is viewed from the left side, and in E and F it is viewed from the ventral side. (G) A rapid increase in calcium in the exposed portion of the yolk cell appears as a brief “yolk flash,” shown in a 30-sec integration at the late shield stage. This image corresponds to the large spike at 100 min in Fig. 2A. A time-lapse imaging sequence of the yolk flash is shown in the supplementary material on the PNAS web site. (H) Multiple loci of persistent elevated calcium levels, or marginal hot spots, are shown in a 30-sec integration of photons from minute 135 in Fig. 2B. (I) Schematic showing orientation of G and H. D, dorsal; V, ventral; L, left; R, right; HS, hot spots. The color code indicates luminescent flux in the same units as in Figs. 2 and 3. The scale bar applies to all frames.

Figure 2

Periodic increases of intracellular calcium recur throughout gastrulation as waves that propagate around the blastoderm margin. (A) Regularity of the wave pulses is shown over 10 h of development from 30% epiboly to 12 somites. Stages are indicated in the gray box beneath the graph. The data stream was binned in 60-sec intervals for quantification. The abscissa indicates photon flux as the mean value of photons (10⁻²) per pixel per sec for an imaging field covering the entire embryo (approximately 14,500 pixels). Time is in min with t = 0 set at about 5 h after fertilization. The average detector background level (noise) of the imaging system is shown by the red line near the origin, representing approximately 10 “noise” photons per sec for the area covered by the embryo. Each calcium wave appears as a spike in the graph. Waves are arbitrarily divided into gastrulation waves before blastopore closure and tail bud pulses afterwards. The rise in luminescence between 60 and 100 min indicates the appearance of a prolonged locus of increased calcium on the ventral side of the blastoderm margin (see Fig. 1 A–F). Asterisks above the graph indicate the yolk flash shown in Fig. 1G and the waves in Figs. 2D and 3 A–C. (B and C) Three waves from the series in A are plotted at higher temporal resolution to demonstrate wave durations, interwave intervals, and temporal components. (B) The black trace represents whole-embryo luminescence, as in A, but quantified in 5-sec intervals. Red, blue, and green traces indicate spatial subsamples from locations indicated by the schematic embryo in D. (C) The whole-embryo luminescence data for the three waves shown in B (black trace) are aligned at the time of maximal pacemaker luminescence (red asterisk). Prewave pulse (PWP) and pacemaker pulse (PMP) time intervals are indicated. (D) Whole-embryo luminescence (black trace) and spatial subsamples (red, blue, and green) for the third wave shown in B and C are graphed at a higher temporal resolution. The imaging sequence for this wave is shown in Fig. 3A. The schematic inset shows the locations of the 400-pixel sampling regions and the pathway of wave propagation from the dorsal pacemaker hot spot (PM; arrows). Time-lapse imaging sequences for the data in a, b, and d are shown in the supplementary material on the PNAS web site.

Figure 3

Imaging sequences and schematics showing the basic spatial wave types. The color scales indicate luminescent flux in [(photons per pixel per sec) × 10⁻²]. (Bar = 200 μm.) The red asterisks and PM in the schematics indicates the dorsal midline pacemaker or wave initiating site. (A) Sequence of 30-sec data integrations showing a bidirectional propagating calcium wave occurring at about 90% epiboly (Fig. 2A, asterisk). Data from this wave are plotted in matching time intervals in Fig. 2D. The end of the interwave interval is shown in the first frame, the prewave pulse is in the second frame, and the dorsal pacemaker pulse and propagating wave are in the next four frames. The schematic shows a generalized bidirectional wave oriented as in the imaging frames. Blue dashed arrows indicate epibolic movements. (B) A unidirectional wave that required about 5 min to circumnavigate the blastoderm margin is shown in sequential 60-sec integrations starting with the pacemaker pulse. (C) During somitogenesis, periodic radial calcium wave fronts spread from the tail bud region. The images in C represent a 120-sec integration window moving along the data stream in 20-sec steps at the 10-somite stage. Time-lapse movies of these imaging sequences are shown in the supplemental material on the PNAS web site.