A ryanodine receptor-dependent Cai2+ asymmetry at Hensen's node mediates avian lateral identity

Abstract

In mouse, the establishment of left-right (LR) asymmetry requires intracellular calcium (Cai2+) enrichment on the left of the node. The use of Cai2+ asymmetry by other vertebrates, and its origins and relationship to other laterality effectors are largely unknown. Additionally, the architecture of Hensen's node raises doubts as to whether Cai2+ asymmetry is a broadly conserved mechanism to achieve laterality. We report here that the avian embryo uses a left-side enriched Cai2+ asymmetry across Hensen's node to govern its lateral identity. Elevated Cai2+ was first detected along the anterior node at early HH4, and its emergence and left-side enrichment by HH5 required both ryanodine receptor (RyR) activity and extracellular calcium, implicating calcium-induced calcium release (CICR) as the novel source of the Cai2+. Targeted manipulation of node Cai2+ randomized heart laterality and affected nodal expression. Bifurcation of the Cai2+field by the emerging prechordal plate may permit the independent regulation of LR Cai2+ levels. To the left of the node, RyR/CICR and H+V-ATPase activity sustained elevated Cai2+. On the right, Cai2+ levels were actively repressed through the activities of H+K+ATPase and serotonin-dependent signaling, thus identifying a novel mechanism for the known effects of serotonin on laterality. Vitamin A-deficient quail have a high incidence of situs inversus hearts and had a reversed calcium asymmetry. Thus, Cai2+ asymmetry across the node represents a more broadly conserved mechanism for laterality among amniotes than had been previously believed.

The ATP-sensitive K+-channel (KATP) controls early left–right patterning in Xenopus and chick embryos

“…LR-relevant ion transporters include Ca 2+ channels (Garic-Stankovic et al, 2008; Hibino et al, 2006; McGrath et al, 2003; Pennekamp et al, 2002), KCNJ9 (Aw et al, 2008b) and KCNQ1 (Morokuma et al, 2008) potassium channels, as well as pumps, including the ER Ca 2+ pump (Kreiling et al, 2008), and H + pumps (Adams et al, 2006b), H + /K + exchangers (Levin et al, 2002), and Na + /K + exchangers (Ellertsdottir et al, 2006). …”

Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish

“…At later developmental stages, there is evidence that Ca 2+ signaling is essential in axial patterning and cell migration (Blaser et al, 2006; Kume et al, 1997; Slusarski et al, 1997a; Wallingford et al, 2001; Westfall et al, 2003a). Recent studies also implicate Ca 2+ signaling in the specification of left-right asymmetry during vertebrate embryogenesis (Garic-Stankovic et al, 2008; McGrath et al, 2003; Sarmah et al, 2005; Schneider et al, 2008; Takao et al, 2013; Yuan et al, 2015). …”

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

“…In fact, the involvement of PKD2 and of both motile and sensory cilia (two-cilia model) prompted the proposal that left-sided Ca 2+ elevation was an early asymmetric event (Tabin and Vogan, 2003). Sustained Ca 2+ elevations were subsequently detected on the left side of the embryonic node in mice (McGrath et al, 2003;Tanaka et al, 2005), chick (Garic-Stankovic et al, 2008;Raya et al, 2004) and zebrafish at the five-to eight-somite stage (Jurynec et al, 2008;Sarmah et al, 2005). In addition to PKD2, left-sided Ca 2+ release has been linked to ryanodine receptors (Garic-Stankovic et al, 2008;Jurynec et al, 2008) and inositol phosphate-dependent signals (Sarmah et al, 2005), implicating intracellular Ca 2+ amplification.…”

“…Sustained Ca 2+ elevations were subsequently detected on the left side of the embryonic node in mice (McGrath et al, 2003;Tanaka et al, 2005), chick (Garic-Stankovic et al, 2008;Raya et al, 2004) and zebrafish at the five-to eight-somite stage (Jurynec et al, 2008;Sarmah et al, 2005). In addition to PKD2, left-sided Ca 2+ release has been linked to ryanodine receptors (Garic-Stankovic et al, 2008;Jurynec et al, 2008) and inositol phosphate-dependent signals (Sarmah et al, 2005), implicating intracellular Ca 2+ amplification. Gap junctions may enable Ca 2+ to spread through target cells on the left side of the node (Hatler et al, 2009;Levin and Mercola, 1999) and H + K + ATPase may help maintain the driving force for Ca 2+ elevations (Garic-Stankovic et al, 2008).…”

“…In addition to PKD2, left-sided Ca 2+ release has been linked to ryanodine receptors (Garic-Stankovic et al, 2008;Jurynec et al, 2008) and inositol phosphate-dependent signals (Sarmah et al, 2005), implicating intracellular Ca 2+ amplification. Gap junctions may enable Ca 2+ to spread through target cells on the left side of the node (Hatler et al, 2009;Levin and Mercola, 1999) and H + K + ATPase may help maintain the driving force for Ca 2+ elevations (Garic-Stankovic et al, 2008). PKD2 targeted to endomembranes in KV cells may be more important than plasma membrane PKD2 for left-right asymmetry in zebrafish (Fu et al, 2008).…”