The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry

Abstract

Intracellular calcium ion (Ca2+) elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca2+/CaM-dependent protein kinase (CaMK-II) is identified as a necessary target of this Ca2+ elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six- and 12-somite stages, which is coincident with known left-sided Ca2+ elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (αKAP) that can hetero-oligomerize with and target active enzyme to membranes. αKAP, β2 CaMK-II and γ1 CaMK-II antisense morpholino oligonucleotides, as well as KV-targeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca2+ channel and γ1 CaMK-II; however, αKAP, β2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca2+-sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca2+ signals to the LPM.