Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish

19 The involvement of the calcium-sensing receptor (CaSR) in Ca homeostasis was investigated 20 in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h post21 fertilization (hpf) and increased with development until plateauing at about 48 hpf. At 4 days 22 post-fertilization (dpf), CaSR mRNA was increased in fish acclimated to low Ca water (25 μM 23 versus 250 μM in normal water). Using immunohistochemistry and confocal microscopy, it was 24 demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on 25 the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry 26 and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich 27 ionocytes enriched with Na/K-ATPase and epithelial Ca channel (ecac). Translational 28 knockdown of the CaSR prevented 4 dpf larvae from regulating whole-body Ca levels when 29 exposed to a low Ca environment. Further, the increases in ecac mRNA expression and Ca 30 influx, normally associated with exposure to low Ca water, were prevented by CaSR 31 knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability 32 to regulate Ca when confronted with a low Ca environment. Results from real-time PCR 33 suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin (stc-1) 34 remained elevated in the CaSR morphants following acclimation to low Ca water. Overall, the 35 results suggest that the CaSR is critical for Ca homeostasis in larval zebrafish exposed to low 36 environmental Ca levels, possibly owing to its modulation of stanniocalcin mRNA expression. 37 38