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

Abstract

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca 2+ 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::EGFPmRNA 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 Ca 2+ . Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca 2+ signals from ~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 Ca 2+ signaling during the 24-48 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca 2+ imaging window by an additional 24 hours.

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

“…What we have called SP1 and SP2 in the -actin-aeq transgenic embryos, we previously described as "two distinct periods of Ca 2+ signaling" in zebrafish embryos that were injected with apoaequorin-mRNA (aeq-mRNA) to express aequorin transiently in the entire embryo (described by Cheung et al, 2006). We conducted a detailed comparison of SP1 and SP2 Ca 2+ signals during restricted time windows using both -actin-aeq transgenic embryos and aeq-mRNA injected embryos (n3 for each; see Fig.…”

“…In the zebrafish embryos by microinjecting an apoaequorin-mRNA (aeq-mRNA) into 1-cell stage embryos. Active aequorin was then reconstituted in vivo by incubating the embryos from the 64-cell stage with the coelenterazine co-factor (Cheung et al, 2006). Although this transient aequorin expression approach successfully extends the aequorin-based Ca 2+ imaging window by an additional ~24 hours (tõ 48 hpf), the aeq-mRNA is gradually degraded in the injected embryos resulting in a steady decline in the production of apoaequorin (Cheung et al, 2006).…”

“…Active aequorin was then reconstituted in vivo by incubating the embryos from the 64-cell stage with the coelenterazine co-factor (Cheung et al, 2006). Although this transient aequorin expression approach successfully extends the aequorin-based Ca 2+ imaging window by an additional ~24 hours (tõ 48 hpf), the aeq-mRNA is gradually degraded in the injected embryos resulting in a steady decline in the production of apoaequorin (Cheung et al, 2006). Furthermore, as the expression of aequorin is ubiquitous, and our current aequorin-based imaging platforms have no resolution in the z-axis, it is difficult to identify specific groups of cells, tissues or organ anlagen that are generating a particular signal in more complex, later stage embryos (Cheung et al, 2006).…”

“…Although this transient aequorin expression approach successfully extends the aequorin-based Ca 2+ imaging window by an additional ~24 hours (tõ 48 hpf), the aeq-mRNA is gradually degraded in the injected embryos resulting in a steady decline in the production of apoaequorin (Cheung et al, 2006). Furthermore, as the expression of aequorin is ubiquitous, and our current aequorin-based imaging platforms have no resolution in the z-axis, it is difficult to identify specific groups of cells, tissues or organ anlagen that are generating a particular signal in more complex, later stage embryos (Cheung et al, 2006). Thus, to follow-on from this transient, ubiquitous aequorin expression technique, we now report the successful generation of a transgenic zebrafish line that expresses apoaequorin targeted to the musculature using a muscle-specific -actin promoter.…”

“…With regard to the former, we developed a new line of transgenic zebrafish that express apoaequorin specifically in the trunk musculature. Active aequorin in these transgenic zebrafish embryos was then reconstituted by incubation in a solution containing the apoaequorin co-factor, coelenterazine (Cheung et al, 2006). While aequorin-based imaging allows for the continuous, non-disturbing visualization of intact zebrafish embryos for long-duration developmental studies (for example, during the entire development of the embryonic trunk musculature), a drawback of this technique is that it only allows for two-dimensional and low resolution Ca 2+ imaging.…”