Figure 1.
Zebrafish mr and gr expression profiles.
Determined by RT-PCR, mr and gr mRNA in various tissues of adults (A), and during developmental stages of embryos (B). β-actin was used as the internal control.
Figure 2.
Ca2+ influx and gene expressions of Ca2+ regulation-related genes.
Ca2+ influx (A) and mRNA expression (B) of 3-dpf zebrafish embryos acclimated to low- (0.02 mM Ca2+) or high-Ca2+ (2.00 mM Ca2+) artificial fresh water. mRNA expression analyzed by qPCR and values were normalized to β-actin. Values are the mean ± SEM (n = 4∼6). *Significant difference (Student's t-test, p<0.05).
Figure 3.
Effects of exogenous cortisol in 3-dpf zebrafish embryos.
Ca2+ content (A), Ca2+ influx (B) and mRNA expressions (C). mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7).
Figure 4.
Effects of exogenous cortisol on ecac-expressing cells in 3-dpf zebrafish embryos.
In situ hybridization analysis indicated ecac signals (A) and density of ecac-expressing cells (B). abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7). Scale bar 100 µm.
Figure 5.
Specificity and effectiveness of MR MO and GR MO.
MR and GR cRNA (with GFP fusion) were injected into embryos respectively (A, B), and embryos coinjection of MR/GR MO with cRNA (C, D). Western blot were used to detect GR and MR protein expressions in wild type (WT) and the MO-injected embryos at 3 dpf (E).
Figure 6.
Effects of MR MO and GR MO in 3-dpf zebrafish embryos.
Ca2+content (A), Ca2+ influx (B), and mRNA expressions (C). mRNA expressions were analyzed by qPCR and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6 or 7).
Figure 7.
Effects of MR MO and GR MO on ecac-expressing cells in 3-dpf zebrafish embryos.
In situ hybridization analysis indicated ecac signals (A) and density of ecac-expressing cells (B). abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Value are the mean ± SEM (n = 6 or 7). Scale bar 100 µm.
Figure 8.
Effects of MR MO and GR MO on zebrafish embryos with cortisol treatment.
Ca2+ influx (A) and ecac mRNA expression (B) were analyzed in 3-dpf zebrafish embryos injected with GR MO or MR MO with cortisol treatment. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 9.
Effects of GR cRNA on GR-SB MO-injected zebrafish embryos.
Ca2+ influx (A) and ecac mRNA expression (B) were also analyzed in 3-dpf zebrafish embryos injected with GR-SB MO or GR-SB MO with GR cRNA. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 10.
Effect of MR MO and GR MO on ecac mRNA expression with low Ca2+ treatment.
ecac mRNA expression were analyzed in 3-dpf zebrafish embryos injected with GR MO or MR MO with low Ca2+ (0.02 mM; LCa) treatment. mRNA expressions were analyzed by qPCR, and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM. (n = 6∼8).
Figure 11.
Effects of exogenous cortisol on mRNA expressions of the vitamin D3-related genes.
qPCR was used to analyze mRNA expression and values were normalized to β-actin. (A) mRNA expressions in 1-dpf zebrafish embryos. (B) mRNA expressions in 3-dpf zebrafish embryos. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6).
Figure 12.
Effects of MR MO and GR MO on mRNA expression of the vitamin D3-related genes.
(A) mRNA expressions in 1-dpf zebrafish embryos. (B) mRNA expressions in 3-dpf zebrafish embryos. mRNA expression was analyze by qPCR and values were normalized to β-actin. abcIndicate a significant difference (p<0.05) using Tukey's multiple-comparison test following one-way ANOVA. Values are the mean ± SEM (n = 6).