PUBLICATION
Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development
- Authors
- Cheung, C.Y., Webb, S.E., Meng, A., and Miller, A.L.
- ID
- ZDB-PUB-060616-21
- Date
- 2006
- Source
- The International journal of developmental biology 50(6): 561-569 (Journal)
- Registered Authors
- Meng, Anming, Miller, Andrew L., Webb, Sarah E.
- Keywords
- aequorin, apoaequorin, coelenterazine, Ca2+, zebrafish
- MeSH Terms
-
- Aequorin/biosynthesis
- Aequorin/genetics*
- Animals
- Apoproteins/biosynthesis
- Apoproteins/genetics*
- Calcium Signaling/physiology*
- Imidazoles
- Microscopy, Fluorescence
- Pyrazines
- Recombinant Proteins/biosynthesis
- Recombinant Proteins/genetics
- Transfection
- Zebrafish/embryology*
- Zebrafish/metabolism
- PubMed
- 16741871 Full text @ Int. J. Dev. Biol.
Citation
Cheung, C.Y., Webb, S.E., Meng, A., and Miller, A.L. (2006) Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development. The International journal of developmental biology. 50(6):561-569.
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::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 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 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping