The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca Signaling Events During the Early Development of Zebrafish (Danio rerio).

We have visualized many of the Ca signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca channel antagonists, Ca chelators, fluorescent dyes, RNA, and DNA.