Introduction of aequorin into zebrafish embryos for recording Ca²⁺ signaling during the first 48 h of development

Ca²⁺ signals, whether transient pulses, propagating waves, or long-duration, steady gradients, are generally considered to play an important role in the pattern-forming events that occur during vertebrate development. One vertebrate that has long been a favorite of embryologists because of its ex utero development and the optical clarity of its embryos is the zebrafish, Danio rerio. Using the bioluminescent Ca²⁺ reporter aequorin, distinct Ca²⁺ signals have been reported for at least the first 48 h of zebrafish development, with signals becoming progressively more complex as the embryo develops. Here we provide a general introduction to aequorin and its use in monitoring Ca²⁺ signals and discuss methods for introducing aequorin into zebrafish embryos.