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

Abstract

SUMMARYIntracellular calcium ion (Ca 2+ ) 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, Ca 2+ /CaMdependent protein kinase (CaMK-II) is identified as a necessary target of this Ca 2+ 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 Ca 2+ 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 (aKAP) that can hetero-oligomerize with and target active enzyme to membranes. aKAP, b2 CaMK-II and g1 CaMK-II antisense morpholino oligonucleotides, as well as KVtargeted 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 Ca 2+ channel and g1 CaMK-II; however, aKAP, b2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca 2+ -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 Ca 2+ signals to the LPM.

CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia

“…Our findings suggest that CaMK-II promotes pronephric development by enabling both anterior migration and posterior morphogenesis, including ciliary stability. This is the first report of a direct downstream Ca 2+ -dependent target of PKD2 in the kidney and is consistent with the finding that CaMK-II is activated by PKD2 in ciliated cells of the zebrafish KV (Francescatto et al, 2010).…”

“…Our findings suggest that CaMK-II promotes pronephric development by enabling both anterior migration and posterior morphogenesis, including ciliary stability. This is the first report of a direct downstream Ca 2+ -dependent target of PKD2 in the kidney and is consistent with the finding that CaMK-II is activated by PKD2 in ciliated cells of the zebrafish KV (Francescatto et al, 2010).Activated CaMK-II has now been detected in multiple ciliated tissues during development including the KV, the inner ear and neuromasts. These sites of CaMK-II activation are consistent with known locations of prolonged Ca 2+ elevation in embryos of zebrafish and other species (Webb and Miller, 2003;Webb and Miller, 2007).…”

“…However, CaMK-II is expressed in every adult mammalian tissue (Tobimatsu and Fujisawa, 1989) and across all metazoan species (Tombes et al, 2003 ) CaMK-II has been detected in zebrafish embryos as early as the 10-somite stage (Francescatto et al, 2010), but it is also found in the forebrain, in a region corresponding to the olfactory placode, in the pronephric duct and cloaca, and at the base of inner ear hair cell kinocilia (Fig. 1).…”

“…A second method to evaluate whether activated kidney CaMK-II is dependent on PKD2 is through CaMK-II activity assays conducted in the presence and absence of Ca 2+ , as previously described (Francescatto et al, 2010). Prolonged Ca 2+ stimulation leads to autophosphorylation (P-CaMK-II), yielding an enzyme that is active in the absence of Ca -dependent activity that is Ca 2+ independent (autonomous) can be determined from cell lysates and kinase assays; this 'autonomy' rarely exceeds ~50%.…”

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish

“…lefty2 expression can be ectopically induced in the absence of nodal expression, or it can be abolished in the presence of nodal (Chocron et al, 2007;Shu et al, 2007). The expression of lefty1 and lefty2 is sometimes abolished in the presence of spaw in zebrafish morphants (Schneider et al, 2008;Lin and Xu, 2009;Francescatto et al, 2010;Zhang et al, 2012). The differential expression of lefty1 in notochord and lefty1 and lefty2 in brain and heart has been reported previously (Chocron et al, 2007).…”