Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos

Through the injection of f-aequorin and the use of a photon imaging microscope, we have previously reported that a rhythmic series of intercellular Ca2+ waves circumnavigate zebrafish embryos over a 10 h period during gastrulation and axial segmentation. These waves first appear at about 65% epiboly and continue to arise every 5-10 min up to at least the 16-somite stage. In response to our publication, it was suggested that the waves may be an artefact caused by dechorionation of the embryos and would not be observed during the development of intact embryos (i.e. those with chorions). Here we demonstrate (again initially by aequorin imaging) that the rhythmic intercellular Ca2+ waves that traverse the blastoderm margin can also be observed in embryos that have an intact chorion. In addition, the appearance time, propagation pathway, velocity, duration and Ca2+ rise of the waves, as well as the interwave interval and the timing of wave onset, are approximately the same in both dechorionated embryos and those with an intact chorion. Furthermore, by loading intact embryos with Ca2+-green dextran at the single-cell stage and then using scanning confocal microscopy to obtain high-resolution images, we confirm the presence of circumferential Ca2+ waves and show that they pass through a population of deep cells located at the blastoderm margin. The confirmation of these pan-embryonic Ca2+ waves in zebrafish further corroborates our earlier suggestion that such waves might play a fundamental role in normal embryonic patterning during the gastrula period.