Fig. 6

gpr161 knockdown disrupts Ca²⁺ handling which is essential for establishment of L–R asymmetry. (A) Defective Ca²⁺ handling resulting in elevated intracellular Ca²⁺ in gpr161 knockdown embryos surrounding the Kupffer′s vesicle. Pseudocolour scale for the intracellular Ca²⁺ signal as normal, intermediate and high intensity. The percentage of embryos exhibited such level of Ca²⁺ signal was listed in control, gpr161 knockdown, ncx1- and pmca-RNA rescue of the gpr161 knockdown embryos. (B) Removal of excessive Ca²⁺ by over-expression of 1 pg of Ca²⁺ pump pmca or Na⁺/Ca²⁺ exchanger ncx1 can rescue gpr161 knockdown phenotype. Cardiac looping defect in gpr161-MO (MO#24, 54.1 ± 4.0%; n = 368), rescued by pmca-RNA (32.0 ± 6.0%; n = 238), rescued by ncx1-RNA (34.0 ± 4.8%; n = 144) and results were from 4 injection experiments. n was number of total embryos. Error bars were ± SEM. t-test * and ^# indicated statistical significance, p < 0.01. All scale bars were 100 μm. (C) A model of gpr161 involved in Ca²⁺ handling and essential for L–R asymmetry for cardiac morphogenesis and visceral asymmetry in developing zebrafish embryos. Blocking gpr161 function led to defect in Ca²⁺ handling and resulted in elevated free cytosolic Ca²⁺ and loss of cardiac looping and abnormal chamber morphogenesis. The gpr161 knockdown can be rescued by removing excessive cytosolic free Ca²⁺ using over-expression of Ca²⁺ pump pmca and Na⁺/Ca²⁺ exchanger ncx1.