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.

Imaging sequences and schematics showing the basic spatial wave types. The color scales indicate luminescent flux in [(photons per pixel per sec) x 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.