Fig. 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.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: 4-cell to Adult
Fig. 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).
Fig. 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).
Fig. 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.

EXPRESSION / LABELING:
Gene:
Fish:
Condition:
Anatomical Term:
Stage: Protruding-mouth
Fig. 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).
Fig. 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).

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage: Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Protruding-mouth
Fig. 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.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage: Protruding-mouth
Fig. 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).

EXPRESSION / LABELING:
Gene:
Fish:
Condition:
Knockdown Reagents:
Anatomical Term:
Stage: Protruding-mouth
Fig. 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).
Fig. 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).

EXPRESSION / LABELING:
Genes:
Fish:
Condition:
Anatomical Term:
Stage Range: Prim-5 to Protruding-mouth
Fig. 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).

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage Range: Prim-5 to Protruding-mouth
Fig. S3

Co-localization of gr mRNA by in situ hybridization with anti-NKA using immunocytochemical analysis of zebrafish gill cryosections. (A) in situ hybridization of gr mRNA; (B) immunocytochemical staining of NKA. Arrow indicated gr mRNA and NKA protein signals at similar area. Scale bar 5 μm.
Acknowledgments
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