Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by similar to 24 hours. Thus, it is currently not possible to image Ca2+ signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca2+. Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca2+ signals from similar to 2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca2+ signaling during the 24-48 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca2+ imaging window by an additional 24 hours.