Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium

Abstract

The involvement of the calcium-sensing receptor (CaSR) in Ca(2+) homeostasis was investigated in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h posfertilization (hpf) and increased with development until plateauing at ∼48 hpf. At 4 dpf, CaSR mRNA was increased in fish acclimated to low Ca(2+) water (25 μM vs. 250 μM in normal water). Using immunohistochemistry and confocal microscopy, we demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich ionocytes enriched with Na(+)/K(+)-ATPase and epithelial Ca(2+) channel (ecac). Translational knockdown of the CaSR prevented 4 dpf larvae from regulating whole body Ca(2+) levels when exposed to a low Ca(2+) environment. Further, the increases in ecac mRNA expression and Ca(2+) influx, normally associated with exposure to low-Ca(2+) water, were prevented by CaSR knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability to regulate Ca(2+) when confronted with a low-Ca(2+) environment. Results from real-time PCR suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin (stc-1) remained elevated in the CaSR morphants following acclimation to low-Ca(2+) water. Overall, the results suggest that the CaSR is critical for Ca(2+) homeostasis in larval zebrafish exposed to low environmental Ca(2+) levels, possibly owing to its modulation of stanniocalcin mRNA expression.

Keywords: calcium homeostasis; calcium-sensing receptor; ecac; stanniocalcin; zebrafish.

Fig. 1.

A: mRNA expression of calcium-sensing receptor (CaSR) in various tissues of adult zebrafish and at different developmental stages of embryos and larvae (B); 18S was used as an internal control. C: effects of acclimation to normal (250 μM) or low (25 μM) Ca²⁺ water on the mRNA expression levels of CaSR in larval zebrafish at 4 days postfertilization (dpf). Data were normalized with 18S and were expressed relative to the fish maintained in normal Ca²⁺ water. *Statistical difference using Student's t-test, P < 0.05. Data are expressed as means ± SE; n = 6.

Fig. 2.

A: representative Western blot showing that the CaSR antibody yielded a single immunoreactive band at ∼100 kDa in the lysates of zebrafish larvae at 4 days postfertilization (dpf). B: a picture of a 4-dpf larval zebrafish with arrows, indicating the regions where CaSR immunostaining was observed. Fluorescent immunohistochemistry and confocal microscopy of the CaSR, showing the CaSR protein expression (green) in neuromasts on the head region (C) as well as on the tail (D). The inset image in C is a neuromast under higher magnification. CaSR is also expressed in the olfactory epithelium (D), ionocytes on the skin of the yolk sac (E), and corpuscles of Stannius (F, G). F and G: double immunostaining of CaSR (green) and Mitotracker (red) was performed. Scale bars = 50 μm.

Fig. 3.

Double fluorescence immunohistochemical confocal images of zebrafish larvae at 4 dpf, illustrating the presence of the calcium-sensing receptor (CaSR; green) in ionocytes on the skin of the yolk sac. Some CaSR-positive cells were enriched with Na⁺/K⁺-ATPase (NKA; red) (A–C) or mitochondria (Mitotracker staining, red) (D–F). The inset image in F shows membranous CaSR expression in a mitochondrion-rich cell under higher magnification. Cells labeled with arrows represent areas of colocalization. Scale bar = 20 μm.

Fig. 4.

Double immunohistochemistry and in situ hybridization images of zebrafish larvae at 4 dpf, illustrating the presence of the calcium-sensing receptor (CaSR) in ecac-expressing ionocytes on the skin of the yolk sac. Cells labeled with arrows represent areas of colocalization. A portion of ecac mRNA was colocalized with NKA (A–C)and CaSR-positive cells (D–F). Scale bar = 20 μm.

Fig. 5.

A: representative Western blot showing the protein expression of the calcium-sensing receptor (CaSR) in the lysates of sham fish and CaSR morphants (CaSR MO) at 4 dpf. β-actin was used as an internal control. B: subsequent quantitative analysis revealed that the protein expression level of the CaSR in the morphants was significantly reduced following morpholino knockdown (Student's t-test; P < 0.05). Data are expressed as means ± SE; n = 3. C: immunohistochemistry and confocal microscopy revealed that CaSR protein was expressed in ionocytes of sham fish (cells labeled with arrows), whereas its expression was virtually absent in CaSR MO.

Fig. 6.

Influx of Ca²⁺ (A) and whole body Ca²⁺ levels (B) in sham fish and calcium-sensing receptor morphants (CaSR MO) after acclimation to normal (250 μM) or low (25 μM) Ca²⁺ water. ^a,b,c,dBars labeled with different letters represent a statistical difference (two-way ANOVA, followed by a post hoc Holm-Sidak test; P < 0.05). Data are expressed means ± SE; n = 6.

Fig. 7.

A: mRNA expression of epithelial Ca²⁺ channel (ecac), Na⁺/Ca²⁺ exchanger (ncx1b) and plasma membrane Ca²⁺-ATPase (pmca2) in 4 dpf larval zebrafish after acclimation to normal (250 μM) or low (25 μM) Ca²⁺ water. *Significant difference between normal and low-Ca²⁺-treated fish (Student's t-test; P < 0.05). Values are expressed as means ± SE; n = 6. B: mRNA expression of ecac in sham fish and calcium-sensing receptor morphants (CaSR MO) after acclimation to normal or low Ca²⁺ water. ^a,b,c,dBars labeled with different letters represent a statistical difference (two-way ANOVA, followed by a post hoc Holm-Sidak test; P < 0.05). Data are expressed as means ± SE; n = 6.

Fig. 8.

The mRNA expression of parathyroid hormone-1 (pth-1) (A) and stanniocalcin-1 (stc-1) (B) in sham fish and calcium-sensing receptor morphants (CaSR MO) after acclimation to normal (250 μM) or low (25 μM) Ca²⁺ water. ^a,b,cBars labeled with different letters represent a statistical difference (two-way ANOVA, followed by a post hoc Holm-Sidak test; P < 0.05). Data are expressed as means ± SE; n = 6.