The techniques described here are the outcome of studies that have sought to define downstream targets of Ca²⁺ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.^1-3 The original discoveries of embryonic Ca²⁺ signaling were dependent on the use of natural and engineered Ca²⁺ indicators, such as aequorin⁴ and fura-2.⁵ Even with current technology, the detection of transient elevations of Ca²⁺ requires cumbersome analytical tools and does not reveal the targets of such Ca²⁺ signals.

This laboratory investigates Ca²⁺ signals that act through the Ca²⁺/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.⁶ CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.^7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca²⁺ levels have diminished due to its ability to autophosphorylate at Thr²⁸⁷. In this autophosphorylated state, CaMK-II remains active in a Ca²⁺/CaM-independent manner, until dephosphorylated.⁶ Thus, the localization of phosphorylated CaMK-II (Thr²⁸⁷) can identify cells in which natural, relevant Ca²⁺ elevations have occurred.

An antibody against autophosphorylated (P-Thr²⁸⁷) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.⁹ Zebrafish (Danio rerio) have seven CaMK-II genes^10,11 whose protein products contain a sequence of MHRQE[pT²⁸⁷]VECLK in this region.^10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca²⁺/CaM-independent activity.¹² Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.¹³

This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer’s Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.¹² This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.¹² In addition to the Kupffer’s Vesicle (KV), autophosphorylated (P-T²⁸⁷) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.^12,13 In the zebrafish kidney, P-T²⁸⁷-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.¹³ Finally, in the developing inner ear, P-T²⁸⁷-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.¹⁴ In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca²⁺ release and illuminated potential new signaling pathways.

These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T²⁸⁷-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.