involvement of calcitonin and its receptor in the control of calcium

Involvement of Calcitonin and Its Receptor in theControl of Calcium-Regulating Genes and CalciumHomeostasis in Zebrafish (Danio rerio)

Anne-Gaelle Lafont ,1 Yi-Fang Wang,2 Gen-Der Chen ,3 Bo-Kai Liao ,1 Yung-Che Tseng ,1

Chang-Jen Huang ,3 and Pung-Pung Hwang1

1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan2Institute of Fishery Science, National Taiwan University, Taipei, Taiwan3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

ABSTRACTCalcitonin (CT) is one of the hormones involved in vertebrate calcium regulation. It has been proposed to act as a hypocalcemic factor,

but the regulatory pathways remain to be clarified. We investigated the CT/calcitonin gene–related peptide (CGRP) family in zebrafish

and its potential involvement in calcium homeostasis. We identified the presence of four receptors: CTR, CRLR1, CRLR2, and CRLR3. From

the phylogenetic analysis, together with the effect observed after CT and CGRP overexpression, we concluded that CTR appears to be a

CT receptor and CRLR1 a CGRP receptor. The distribution of these two receptors shows a major presence in the central nervous system

and in tissues involved in ionoregulation. Zebrafish embryos kept in high-Ca2þ-concentration medium showed upregulation of CT and

CTR expression and downregulation of the epithelial calcium channel (ECaC). Embryos injected with CT morpholino (CALC MO)

incubated in high-Ca2þ medium, showed downregulation of CTR together with upregulation on ECaC mRNA expression. In contrast,

overexpression of CT cRNA induced the downregulation of ECaCmRNA synthesis, concomitant with the downregulation in the calcium

content after 30 hours postfertilization. At 4 days postfertilization, CT cRNA injection induced upregulation of hypercalcemic factors, with

subsequent increase in the calcium content. These results suggest that CT acts as a hypocalcemic factor in calcium regulation, probably

through inhibition of ECaC synthesis. � 2011 American Society for Bone and Mineral Research.

KEY WORDS: CALCIUM; CALCITONIN; ZEBRAFISH; IONOREGULATION; RECEPTORS

Introduction

Ca2þ plays a crucial role in processes ranging from the

formation and maintenance of the skeleton to the temporal

and spatial regulation of neuronal function. Consequently, the

maintenance of extracellular Ca2þ concentration is of critical

importance for many vital functions of the animal body.(1) In the

model of Ca2þ reabsorption in mammalian kidneys, Ca2þ enters

the cell through an apical membrane via Ca2þ channels, then

binds to the calbindin binding protein, and eventually diffuses to

the basolateral membrane. From the cytosol, Ca2þ is extruded to

the blood through an ATP-dependent plasma membrane Ca2þ-ATPase (PMCA) and a Naþ/Ca2þ exchanger (NCX).(2) Recently,

several studies provided initial molecular physiological evidence

to support this Ca2þmodel in the gill cells of fish, which inhabit a

freshwater environment with fluctuating Ca2þ levels. In zebra-

fish, epithelial Ca2þ channels (ECaC), NCX1b, and PMCA2 have

been shown to be specifically expressed in a group of ionocytes

(mitochondria-rich [MR] cells) and be involved in transepithelial

Ca2þ uptake.(3–7)

Different hormones participate in the control and regulation

of calcium homeostasis. In mammals, the three main hormones

involved in the calcium regulation are parathyroid hormone

(PTH), vitamin D, and calcitonin (CT). Although vitamin D and

parathyroid hormone–related proteins seem to play a role in

teleost calcium regulation, the involvement of CT remains

debatable, with contradictory results having been published in

the literature. CT was discovered in the 1960 s as a new factor

involved in mammalian calcium regulation.(8) In mammals, this

32-amino-acid peptide is synthesized in thyroid C cells and acts

ORIGINAL ARTICLE JJBMR

Received in original form February 23, 2010; revised form September 4, 2010; accepted November 11, 2010. Published online November 23, 2010.

Address correspondence to: Dr. Pung-Pung Hwang, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

E-mail: [email protected]

Currrent address for A-G Lafont: Museum National d’Histoire Naturelle, UMR 7208 CNRS BOREA Biologie des Organismes et Ecosystemes Aquatiques, 7 rue Cuvier,

CP 32, 75231 Paris Cedex 05, France.

Additional Supporting Information may be found in the online version of this article.

Journal of Bone and Mineral Research, Vol. 26, No. 5, May 2011, pp 1072–1083

DOI: 10.1002/jbmr.301

� 2011 American Society for Bone and Mineral Research

1072

as a hypocalcemic and hypophosphatemic hormone. It has an

inhibiting action on osteoclast motility, proliferation,

and activity, and at the same time a stimulating effect on

osteoblast activity(9) CT also appears to inhibit intestinal Ca2þ

absorption and renal Ca2þ reabsorption in mammals, but this

function remains controversial.(10) In rabbit nephron,(11) CT-

stimulated Ca2þ reabsorption has been suggested to occur by

opening the low-affinity Ca2þ channel of the distal luminal

membrane, which subsequently stimulates the Ca2þ/Naþ

exchanger activity in the basolateral membranes; however,

the molecular physiological evidence for these pathways

remains unavailable.

In teleostean fish, CT has been suggested to be hypocalcemic,

but it has also been reported to show a hypercalcemic effect in

some cases.(12) CT was found to inhibit gill Ca2þ uptake(13,14) and

could be stimulated by treatment with high-calcium solution.(15,16) On the other hand, CT administration can produce

hypercalcemia concomitant with the increase in CT plasma

levels.(17) Divergent responses were also observed after transfer

from freshwater to seawater, with no change in eel plasma CT

concentration in spite of increased plasma Ca2þ level,(16)

whereas trout transfer resulted in a significant increase in

plasma CT concentration.(18)

Study of the mammalian CT gene has led to the discovery of a

new peptide called calcitonin gene–related peptide (CGRP).(19) In

teleostean fish, this neuropeptide could be involved in ionic

regulation.(20,21) In addition, a calcitonin gene (CALC) has been

identified in zebrafish, generating two different mRNA tran-

scripts coding for CT and CGRP by alternative splicing similar to

that in mammals.(22) CT and CGRP belong to a multigenic family,

together with amylin, adrenomedullin, intermedin, and calcito-

nin receptor–stimulating peptide (CRSP), and the members of

this family interact through the same family of G protein–

coupled receptors.(23) In mammals, CT receptor (CTR) and CT

receptor–like receptor (CRLR) share high sequence identity and

form specific receptors to this family when combined with

selective receptor activity–modifying proteins (RAMPs).(24) The

association of CTR and CRLR with three different RAMPs

determines the ligand specificity of the mammalian receptor.

In the pufferfish (Takifugu obscurus) four different receptors have

been cloned.(25) Their selective combination with five different

RAMPs determines their specificity. However, CT and CGRP

appear to interact with only two receptors, CTR and CRLR1.(25,26)

As described above, the regulatory pathways of CT in Ca2þ

homeostasis and the target transporters are still unclear. With the

advantage of a rich genetic database and well-developed

molecular physiological approaches, the expression and function

of Ca2þ transporters has been well studied in zebrafish.(3,4) In this

paper, we used zebrafish as a model for test our hypothesis that

CT controls the Ca2þ uptake mechanism by regulating the

expression of the related Ca2þ transporters (ECaC, NCX1b, and

PMCA2). Zebrafish CT, CGRP, and their possible receptors (CTR/

CRLRs) were identified and we focused on their actions. The use

of artificial freshwater (FW) with high or low Ca2þ concentrations,

together with the effects of CT gene overexpression or inhibition

on the regulation of several genes involved in the calcium

regulation, helped us investigate the involvement of CT in the

complex system that controls Ca2þ regulation.

Materials and Methods

Animals

Adult zebrafish (Danio rerio) obtained from stocks of the Institute

of Cellular and Organismic Biology at the Academia Sinica were

reared in circulating tap water or different artificial media

(described under Acclimation Experiments) at 288C under a

14/10 hour light-dark photoperiod. Eggs were collected at the

desired developmental stages of 1, 4, 30, 50, 80, 104, or 128 hours

postfertilization (hpf) and were anesthetized with buffered

MS222 (Sigma, St. Louis, MO, USA) following the guidelines of the

Academia Sinica Institutional Animal Care and Utilization

Committee (approval no.: RFiZOOHP2006086).

Molecular cloning and sequence analysis

All genes (CALC, CTR, CRLR1, CRLR2, and CRLR3) were predicted

from the Ensembl and NCBI databases. Full-length cDNAs were

then cloned and sequenced from adult zebrafish. Alignment of

the amino acid sequences was conducted by using ClustalW,

followed by manual optimization. Phylogenetic analyses were

carried out using the neighbor-joining method. One thousand

bootstrap replicate analyses were carried out with Mega3.1

software (Tempe, AZ, USA).

Preparation of total RNA

Appropriate amounts of zebrafish embryos and adult tissues

were collected and homogenized in Trizol reagent (Invitrogen,

Carlsbad, CA, USA) following the manufacturer’s protocol. The

total amount of RNA was determined by measuring the

absorbance at 230, 260, and 280 nm by spectrophotometry

(ND-1000, NanoDrop Technologies, Wilmington, DE, USA). The

RNA quality was controlled by running RNA denatured gel

electrophoresis.

RT-PCR

Total RNA extracted from zebrafish embryos (by pools of

25 embryos) and adult tissues (by pools of 6 individuals) was

subjected to removal of genomic DNA, cDNA synthesis, and then

to PCR amplification. Thirty cycles were performed for each

reaction. The amplicons were sequenced to confirm the desired

gene fragments. The primer sets for the PCR analysis are listed in

Supplemental Table 1.

Microinjection of cRNA and antisense morpholinonucleotide

The entire coding region of CT and of CGRP from zebrafish CALC

gene cDNAwere amplified by PCR and inserted into a pCS2þ or a

PCS2þ green fluorescent protein (GFP) XLT expression vector.

Capped CT mRNAs and CGRP mRNAs (cRNAs) were synthesized

using a mMessage mMachine kit (Ambion, Austin, TX, USA) from

a linearized vector containing the respective cDNAs and were

checked for their concentration and quality. The morpholino

oligonucleotide (MO) sequences were obtained from Gene Tools

(Philomath, OR, USA). The 25-nucleotide MOs designed against

the zebrafish CALC gene included the initiation codon ATG

(5’-CATGGTCCCCTTAAGATGCTCAGCT-3’ and were diluted in

CALCITONIN AND CA REGULATION IN ZEBRAFISH Journal of Bone and Mineral Research 1073

sterile water to obtain a 3mM stock solution. The 25-nucleotide

standard control oligo was used as the control, 5’-CCTCTTACCT-

CAGTTACAATTTATA-3’. This standard control oligo has no target

and no significant biological activity. The MO and cRNA solutions

containing 0.1% phenol red were injected into 1- to 2-cell stage

zebrafish embryos at 4 ng/embryo and 250 pg/embryo, respec-

tively, using an IM-300 microinjector system (Narishige, Tokyo,

Japan).

Dot blot analysis

To confirm the effectiveness of the injected cRNAs and MOs, the

protein levels of CT and CGRP in injected embryos were

examined by dot blot analysis. Using a narrow-mouth pipette tip,

25mg of total protein (in a volume of 2.5mL) from a crude extract

of zebrafish head (prepared from 100 embryos) was spotted onto

the nitrocellulose membrane (Millipore, Billerica, MA, USA). After

drying, the membrane was blocked with 5mL of PBST buffer

(140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mMK2HPO4, pH

7.4, and 0.05% Tween 20) plus 3% BSA. After PBST washing, the

membranes were probed with anti-CT or anti-CGRP antibody

(1.5 mg/mL in PBST) for 1 hour at room temperature. After a

second PBST washing, the membranes were incubated with

secondary antibody conjugated with horseradish peroxidase

(HRP) (Jackson Immunoresearch Laboratories, Avondale, PA,

USA) at a 1:5000 dilution for 1 hour at room temperature. After

thorough washing, the signals were detected using enhanced

chemiluminescence (ECL) (NEN Life Science Products, Boston,

MA, USA).

For dot blot analysis, a rabbit antisalmon CT (T-4026) and a

rabbit antimouse CGRP antibodies (T-4031) (Peninsula Labora-

tories, San Carlos, CA, USA) were used. The CT antiserum is raised

against salmon CT that differs in 4 of 32 amino acids. Antisalmon

CT antibody has been demonstrated to be efficient in different

teleost species.(27,28) The CGRP antiserum is raised against rat

CGRP peptide that differs from the zebrafish sequence in 9 of 37

amino acids. It has been successfully used in zebrafish.(29)

Acclimation experiments

High-Ca2þ (2.00mM) and low-Ca2þ (0.02mM) artificial fresh-

waters were prepared with double-deionized water (Milli-RO60;

Millipore) supplemented with adequate CaSO4 � 2H2O, MgSO4�7H2O, NaCl, K2HPO4 and KH2PO4.

(30) Except for Ca2þ, all the otherion concentrations of the media were the same as in the local

tap water ([Naþ]¼ 0.5mM, [Mg2þ]¼ 0.16mM, [Kþ]¼ 0.3mM).

Variations in the ion concentrations were maintained within

10% of the predicted values by examination with an atomic

absorption spectrophotometer (Hitachi Z-8000, Tokyo, Japan).

Zebrafish fertilized eggs were transferred to the different

media (high-Ca2þ [HiCa] and low-Ca2þ [LoCa]) and incubated

at 288C until sampling. Adult zebrafish were also transferred to

HiCa and LoCa media, and sampling was conducted 2 weeks

after the transfer. Fish were not fed during the 2 weeks of

acclimation.

Quantitative real-time RT-PCR

Quantitative real-time RT-PCR (qPCR) was performed using an

ABI Prism 7000 sequence detection system (Applied Biosystems,

Foster City, CA, USA) in a final volume of 20mL containing 10mL

of 2x SYBR Green master mix (Applied Biosystems), 100 nM

primer pair, and 5 ng cDNA. The standard curve of each gene was

checked in a linear range with b-actin as an internal control. The

primer sets for the qPCR were designed using Primer Express

software (v. 2.0.0, Applied Biosystems) and are listed in

Supplemental Table 1. The calculation of relative mRNA levels

was based on the comparative Ct method.(31)

Measurement of whole-body Ca2þ content

Twenty-five zebrafish embryos were briefly rinsed in deionized

water, pooled as one sample, and weighed. Two hundred mL of

HNO3 (13.1 N) was added to the samples for digestion at 608Covernight. Digested solutions were then diluted with double-

deionized water and subjected to atomic absorption spectro-

photometry.

Statistical analysis

Data are presented as means� SEM. Differences between

groups were assessed using Student’s t-test or ANOVA.

Results

Characterization of zebrafish CALC, CTR, CRLR1, CRLR2,CRLR3

From NCBI and Ensembl databases, four members of the

calcitonin-like receptor family, CTR (CAN88589), CRLR1

(NP_001004010), CRLR2 (XP_001920035), and CRLR3

(XP_001340713), were identified by cloning and sequencing.

According to the phylogenetic analysis of CTR and CRLR

sequences (Fig. 1), the four zebrafish receptor sequences can

be assigned individually to four monophyletic groups represent-

ing teleost CTR, teleost CRLR1, teleost CRLR2, and teleost CRLR3.

However, the existence of three subtypes of CRLR receptors

appears to be conserved among the teleost clade.

Tissue distribution and expression during embryonicdevelopment

RT-PCR analysis indicated that CT, CGRP, CTR, CRLR1, CRLR2, and

CRLR3 showed early expression before 1 day postfertilization

(dpf) and continued to be expressed throughout development

(Fig. 2A).

As for quantitative real-time RT-PCR analysis (Fig. 2B, C), CGRP

transcript was detected in several tissues (gill, heart, muscle, skin,

spleen) of adult zebrafish but was mostly present in the central

nervous system (brain, eye). In contrast, CT has been detected

only in one tissue, at the ultimobranchial body (UBB). Four of the

receptors presented a ubiquitous distribution in the different

tissues. The highest levels of CTR and CRLR1 cDNA were detected

in the brain, CRLR2 in the spleen, and CRLR3 in the heart. Both CTR

and CRLR1 were detected in osmoregulatory organs such as gill,

intestine, kidney, and skin.

1074 Journal of Bone and Mineral Research LAFONT ET AL.

Fig. 1. Phylogenetic tree of CTR and CRLR sequences of vertebrates. The amphioxus, drosophila, and oyster sequences of CRLR-like receptors were chosen

as outgroups to root the tree. The tree was constructed from alignment of the amino acid sequences, using the neighbor-joining method. The robustness

of the branching has been evaluated by bootstrap analysis, and values after 1000 trials are indicated on this consensus tree. The teleost CRLR and CTR

groups are indicated in light gray. Zebrafish CRLR1, CRLR2, CRLR3, and CTR are highlighted with black background. The accession numbers of the

sequences are as follows: Danio rerio CRLR3 (XP_001340713), Takifugu obscurus CRLR3 (BAE45314), Homo sapiens CRLR (NP_005786), Canis familiaris CRLR

(XP_545560), Bos taurus CRLR (NP_001095577), Sus scrofa CRLR (BAC54960),Mus musculus CRLR (NP_061252), Rattus norvegicus CRLR (NP_036849), Gallus

gallus CRLR (XP_421850), Taeniopygia guttata CRLR (XP_002192337), Xenopus laevis CRLR (NP_001080206), Xenopus tropicalis CRLR (AAI21844), Anolis

carolinensis CALCRL (ENSACAT00000001135), Danio rerio CRLR1 (NP_001004010), Gasterosteus aculeatus CRLR1 (ENSGACT00000003126), Paralichthys

olivaceus CRLR1 (BAA92816), Oryzias latipes CRLR1 (ENSORLT00000018106), Tetraodon nigroviridis CRLR1 (ENSTNIT00000011910), Takifugu obscurus CRLR1

(BAE45312), Takifugu rubripes CALCRL (ENSTRUT 00000016812), Oncorhynchus gorbuscha CRLR1 (CAD48406), Danio rerio CRLR2 (XP_001920035), Oryzias

latipes CRLR2 (ENSORLT00000000113), Gasterosteus aculeatus CRLR2 (ENSGACT00000019726), Tetraodon nigroviridis CRLR2 (ENSTNIT00000013717),

Takifugu obscurus CRLR2 (BAE45313), Takifugu rubripes CRLR2 (ENSTRUT00000030033), Danio rerio CTR (CAN88589), Gasterosteus aculeatus CTR

(FAA00374), Oryzias latipes CTR (FAA00375), Tetraodon nigroviridis CTR (FAA00373), Takifugu obscurus CTR (BAE76018), Takifugu rubipes CTR

(NP_001098689), Rana catesbeiana CTR (BAC77166), Taeniopygia guttata CTR (XP_002194777), Gallus gallus CTR (XP_425985), Anolis carolinensis CTR

(ENSACAT00000013279), Homo sapiens CTR (NP_001733), Canis lupus CTR (BAG68688), Mus musculus CTR (AAK56132), Rattus norvegicus CTR (AAA03030),

Bos taurus CTR (NP_001069737), Tursiops truncates CTR (ENSTTRT00000007261), Sus scrofa CTR (NP_999519), Branchiostoma floridae CRLR-like

(XP_002239593), Drosophila melanogaster CRLR-like (NP_725278), Crassostrea gigas CRLR-like (CAD82836). The unit of scale bar is the number of amino

acid substitutions per site.


Effect of CT and CGRP cRNA overexpression on receptormRNA expression

To identify the specificity of the four different cloned receptors,

the effects of CT or CGRP cRNA injection were analyzed by

quantitative RT-PCR. First, the effectiveness of the injected cRNAs

was tested by measuring the protein levels in embryos injected

with CT and CGRP cRNAs using dot blot analysis. The protein

expression levels of CT and CGRP in cRNA-injected embryos at 4

dpf were higher than those in the respective wild type controls

(Fig. 3A).

Fig. 2. CTR, CRLR1, CRLR2, CRLR3, CT, and CGRP mRNA expression during embryonic development and tissue distribution. mRNA expression patterns

during embryonic development were analyzed by RT-PCR (A) in zebrafish embryos from 3hours postfertilization (hpf) to 72 hpf. RT-PCR was performed on

3 different sets of samples. Zebrafish b-actin was used as the internal control. The CTR, CRLR1, CRLR2, CRLR3 (B), and CT, CGRP (C) expression patterns in

different tissues (B: brain; E: eye; G: gill; H: heart; I: intestine; K: kidney; M: muscle; O: ovary; Sk: skin; Sp: spleen; T: testis; UB: ultimobranchial body) were

determined by quantitative real-time RT-PCR in adult zebrafish. Values are the mean� SEM (n¼ 6–7). Zebrafish b-actin was used as the internal control.


CT cRNA injection induced a 2.5-fold increase in the expression

level of CTR at 30 hpf, with no significant variation observed

for CRLR1, CRLR2, and CRLR3 (Fig. 3B). At 4 dpf, a significant

increase for both CTR and CRLR1 mRNAs was observed. In the

same way, CGRP cRNA injection induced a 2-fold increase

only in the expression levels of CRLR1 at 30 hpf, whereas both

CTR and CRLR1 levels of expression were significantly increased

at 4 dpf (Fig. 3C). On the other hand, both CT and CGRP cRNA

injection had no effect on the expression of both CRLR2 and

CRLR3.

Effect of environmental calcium concentrations on CT,CGRP, CTR, CRLR1, and ECaC mRNA expression

To test the potential role of CT in teleost calcium regulation,

acclimation to artificial FW containing different concentrations of

Ca2þ was performed. The expression levels of CT and CTR were

significantly increased in the zebrafish embryos in HiCa

compared to those in LoCa; however, the level of ECaC was

significantly decreased in HiCa (Fig. 4A). Thus, HiCa medium

induced a downregulation of ECaC, which is involved in the Ca2þ

uptake concomitant with an upregulation of CT and CTR. This

suggests a potent role of CT and CTR in calcium regulation. In

contrast, acclimation to a different medium of Ca2þ showed no

modification in the levels of CGRP and CRLR1 mRNA expression

(Fig. 4A).

In adult zebrafish, acclimation to HiCa for 14 days also

caused an upregulation of mRNA expression for both CT and

CTR (UBB and gills, respectively) compared to LoCa (Fig. 4B).

However, 2-week acclimation of adult zebrafish to HiCa induced

Fig. 3. Effect of CT and CGRP cRNA overexpression on mRNA expression

of the 4 cloned receptors, as quantified by qPCR. One- to two-cell stage

zebrafish embryos were injected with 250 pg/embryo of CT or CGRP

cRNA. (A) CGRP and CT protein levels in cRNA-injected embryos and wild

type control at 4 days postfertilization (dpf) were analyzed by dot blot

with anti-CGRP and anti-CT antibodies, respectively. PBS represents a

blank control without protein. (B,C)mRNA expression of the 4 receptors in

embryos injected with CT (B) or CGRP (C) cRNA was examined by qPCR at

30 hpf and 4 dpf. Values are the mean� SEM (n¼ 6). The values were

normalized to b-actin. �Indicates a significant difference from the control

(Student’s t-test, p< .05).

Fig. 4. Effect of different Ca2þ concentration in artificial FW on CT, CGRP,

CTR, CRLR1, and ECaC mRNA expression. (A) Zebrafish embryos were

maintained in high-Ca2þ (HiCa: 2.00mM) and low-Ca2þ (LoCa: 0.02mM)

artificial freshwater. CT, CTR, CGRP, CRLR1, and ECaC mRNA expresions

were analyzed by qPCR at 30 hpf and 4 dpf. (B), Adult zebrafish were

acclimated to HiCa and LoCa artificial freshwater for 14 days, and mRNA

expression of CT in the ultimobranchial body (UBB) and of CTR and ECaC

in the gills was examined by qPCR. Values are the mean� SEM (n¼ 6).

The values were normalized by b-actin. �Indicates a significant differencefrom the control (Student’s t-test, p< .05).


a downregulation of ECaC mRNA expression in the gills

when compared to LoCa (Fig. 4B). Therefore, CT, CTR,

and ECaC regulation appear to depend on the Ca2þ concentra-

tion in the external environment, in the same way as in

embryos.

Injection of plasmid GFP-CT or -CGRP with or without MO

To further support our hypothesis, we blocked the synthesis of

CT in zebrafish using specific CALC MO and examined the

consequences on ECaC synthesis. Efficiency of CALC MO was

tested by the injection of CT cRNA coupled with GFP. Strong

expression of GFP at 4 dpf was observed, representing the

expression of CT (Fig. 5A, B). At the same time, we proceeded

with the co-injection of the same CT cRNA coupled to GFP with

CALC MO, resulting in the absence of GFP expression (Fig. 5C, D).

These results suggest that CALC MO can target endogenous CT

mRNA and eventually block the synthesis of its proteins.

Furthermore, CT protein expression levels in 4-dpf embryos

injected with CALCMO or control MOwere examined by dot blot

analysis (Fig. 5E). CT protein levels in CALC MO–injected embryos

were lower than those in control MO-injected embryos (Fig. 5E).

We then looked at the effect of the CALCMO injection on ECaC

synthesis and observed a significant 1.6-fold increase at 30 hpf

and 4 dpf (Fig. 5F). This result suggests an inhibitory effect of CT

on ECaC expression.

Effect of CALC MO injection on CTR, CRLR1, and ECaCmRNA expression in high-calcium medium

The observed downregulation of ECaC and upregulation of CTR

in HiCa (Fig. 4) were further tested with CALC MO injection. CALC

MO injection induced a significant decrease of CTR expression

and a significant increase of ECaC in HiCa medium compared to

the control injected with control MO (Fig. 6). These data suggest

that blocking CT synthesis induced a downregulation of its

receptor (CTR) and stopped the inhibition of ECaC synthesis. We

observed no significant changes in the expression of CRLR1.

Effect of CT or CGRP overexpression on the related geneexpression and calcium regulation

To investigate the mechanisms of CT action in calcium

regulation, CT cRNA overexpression was performed to observe

its effect on different genes implied in calcium homeostasis

(Fig. 7A). At 30 hpf and 4 dpf, CT cRNA injection induced a

significant 2-fold decrease in the expression of ECaC and a

concomitant 3-fold upregulation of stanniocalcin (STC). These

data indicate an inhibition of Ca2þ uptake. NCX1b and PMCA2

involved in the extrusion of Ca2þ in the blood showed no

significant modification at 30 hpf; however, a significant 2.5-fold

increase in the transcripts was found at 4 dpf. In the same way,

the hypercalcemic factors PTHR1, PTHR2, PTHR3, and VDR

presented no modification in their regulation at 30 hpf but

showed a significant increase (2- to 4-fold) after 4 dpf. On the

other hand, CGRP cRNA injection induced no significant variation

in the expression of ECaC, STC, NCX1b, PMCA2, PTH1R, PTH2R,

PTH3R, and VDR throughout the experiment (data not shown),

strengthening a different role for CT and CGRP in zebrafish.

Finally, to confirm this potential role of CT on the calcium

homeostasis, the effect of CT overexpression on whole-body

calcium content was observed (Fig. 7B). At 30 hpf and 50 hpf,

overexpression of CT induced a significant decrease in calcium

Fig. 5. Effect CALC MO injection on ECaC mRNA expression in FW.

(A–D) Zebrafish embryos were injected with CT GFP cRNA (250pg/

embryo) and GFP expression was observed in the head (A) and the tail

(B). When embryos were co-injected with CT GFP cRNA (250 pg/embryo)

and CALC MO (4 ng/embryo), GFP expression was abolished, as shown in

the head (C) and the tail (D). (E) CT protein levels in CALC MO–injected

embryos and control at 4 dpf were analyzed by dot blot with anti-CT

antibody. PBS, blank control without protein. (F) ECaC mRNA expression

in CALC MO-injected embryos and control was quantified by qPCR at

30 hpf and 4 dpf. Values are the mean� SEM (n¼ 5). The values were

normalized by b-actin. �Indicates a significant difference from the control

(Student’s t-test, p< .05).

Fig. 6. CTR, CRLR1, and ECaCmRNA expression in high-calcium medium,

with or without CALC MO injection. Zebrafish embryos, maintained in

high-Ca2þ (HiCa; 2.00mM) artificial medium, were injectedwith CALCMO

(4 ng/embryo). CTR, CRLR1, and ECaCmRNA expression was quantified by

qPCR at 30 hpf and 4 dpf. Values are the mean� SEM (n¼ 6). The values

were normalized by b-actin. �Indicates a significant difference from the

control (Student’s t-test, p< .05).


concentration when compared to the controls. In contrast, from

105 hpf CT overexpression induced a significant increase in the

calcium concentration. Thus, CT overexpression induced a short-

term downregulation in the calcium concentration followed by a

long-term upregulation. However, CGRP cRNA injection induced

no significant modification in whole-body calcium concentration

throughout the experiment (data not shown), strengthening the

hypothesis that, in contrast to CT, CGRP may not be involved in

the calcium homeostasis.

Discussion

The present phylogenetic tree analysis shows that CTR, CRLR1,

CRLR2, and CRLR3 sequences belong to four independent

monophyletic groups. Each zebrafish CTR gene belongs to the

same group of the respective orthologs from other vertebrates,

suggesting that this receptor could play similar function in

zebrafish. The situation of the CRLR genes is more complicated,

because in teleosts three subtypes have been described.

However, in the flounder and the pufferfish, CGRP has

been demonstrated to interact specifically with the CRLR1

receptor,(25,26,32,33) suggesting that this CRLR1 gene in zebrafish

could serve as the CGRP receptor.

This phylogenetic analysis was supplemented by a functional

study. Injection of specific CT cRNA or specific CGRP cRNA was

performed on zebrafish embryos and the effect on the regulation

of the four receptors was analyzed (Fig. 3). The functional study

supported the phylogenetic analysis and leads to consideration

that CTR is the CT receptor and CRLR1 is the CGRP receptor.

However, at 4 dpf, a significant increase could be noticed in both

CTR and CRLR1 expression after CT or CGRP cRNA overexpression.

Furthermore, CT cRNA injection had no significant effect on CGRP

mRNA expression, and vice versa: CGRP cRNA injection had no

effect on CT mRNA expression (data not shown). The interaction

between CT and specific CGRP receptor and between CGRP

and specific CT receptor, observed at 4 dpf, could be similar to

the situation describes in mammals. In mammals, CGRP has

a hypocalcemic activity similar to that of CT, but only at

supraphysiologic levels.(34) This effect is likely to be related to the

Fig. 7. Effect of CT cRNA overexpression on calcium transporter mRNA expression and whole-body calcium content. Zebrafish embryos maintained in FW

were injected with CT cRNA (250 pg/embryo). (A) ECaC, STC, NCX1b, PMCA2, PTH1R, PTH2R, PTH3R and VDRmRNA expression was quantified by qPCR (A) at

30 hpf and 4 dpf. Values are the mean� SEM (n¼ 6). The values were normalized by b-actin. (B) Calcium content in CT cRNA-injected embryos was

measured at 30, 50, 80, 105, and 128 hpf. Values are the mean� SEM (n¼ 5). �Indicates a significant difference from the control (Student’s t-test, p< .05).


cross-reactivity of CGRP with specific osteoclast CT receptors, for

which CGRP has a much lower affinity.(35) At 4dpf, the high

concentrations of each peptide could lead to a decrease in the

affinity between ligand and specific receptor, with a saturation of

the receptor that could lead in turn to cross-reactivity of CT on

CRLR1, and of CGRP on CTR. Concerning CRLR2 and CRLR3, we

suggest that they could have lower affinities to CT and CGRP or

represent receptors for other peptides of the same family,

such as adrenomedullin and amylin. This issue remains to be

investigated. In the pufferfish, a functional analysis of these

receptors has demonstrated their implication in specific

interactions with various adrenomedullin peptides characterized

in this species.(25)

We looked at the CT distribution and observed that this

peptide was synthesized only in the UBB (Fig. 2C). This situation is

common to all the vertebrates studied so far, with synthesis of CT

in the UBB for the nonmammalian vertebrates and in the thyroid

C cells for the mammals. CTR, however, presented a more

widespread distribution, with an important presence in the

central nervous system (brain and eye) and in osmoregulatory

tissues (gill and kidney) (Fig. 2B). In mammals, CT plays a major

function in calcium homeostasis. This peptide can interact with

CTR in bones, lungs, kidney, the gastrointestinal tract, and the

central nervous system (CNS).(9,36,37) In sauropsids, specific CT

receptors have been identified in organs related to calcium

regulation, such as the shell gland and the kidneys.(38,39) Very

little information exists on CTR in teleosts, especially its tissue

distribution. However, in the European eel, specific CT binding

sites have been detected in the brain, gill, heart, and kidneys,

similar to the present study.(40) In pufferfish, a CTR sequence has

been cloned from heart RNA,(26) but the situation in other organs

was not investigated. In vertebrates, CT appears to perform a

common endocrine function, with production in UBB or thyroid,

and acts on CNS, heart, and osmoregulatory organs.

CGRP and CRLR1 showed more ubiquitous distribution and

were highly expressed in the central nervous system (Fig. 2B, C).

In mammals, the major location of CGRP and its specific

receptor is in the central and peripheral nervous system.(41–43)

The wide distribution of CGRP in the brain suggests a function as

neuromediator or neuromodulator and involvement in various

brain functions.(44–46) CGRP appears to play a similar role as

neuromediator or neuromodulator in sauropsids and amphi-

bians.(37) In the European eel, a phylogenetically ancient species

among teleosts, CGRP was described to have an autocrine/

paracrine action in the central nervous system.(20,37) In zebrafish,

the function of CGRP in the CNS seems to be local paracrine

action, with the presence of both the peptide and its receptor. In

vertebrates, the autocrine/paracrine function of CGRP in the CNS

appears to be conserved among species. Furthermore, in a

mollusk phylum presenting real cerebralization, both cephalo-

pod CGRP and its specific binding sites were colocalized in the

CNS.(40,47) Therefore, the situation that we observed in zebrafish

is concordant with the current theory suggesting that this local

action of CGRP represents an ancient role in metazoa.

The role of CT in calcium homeostasis remains to be clarified in

teleosts, because contradictory conclusions have been reported

so far. In the eel and salmon, various studies have denied this

function, showing that exogenous CT administration had no

significant effect on the circulating calcium regulation.(48,49) In

contrast, exogenous CT administration in goldfish has resulted in

hypocalcemia, but only in young fish, suggesting that the role of

CT in calcium regulation could be related to the developmental

stage of the fish.(50) More recently, two different experiments

have emphasized the possible involvement of CT in the

inhibition of osteoclastic activity, similar to that in mammals,

suggesting a hypocalcemic role in calcium homeostasis in

teleosts.(51,52) However, there was no convincing molecular

physiological evidence to support this notion until the present

study.

To further investigate the situation in teleosts, we compared

the regulation of the CT/CGRP gene family and ECaC in fish

acclimated to different calcium concentrations (Fig. 4) or injected

with the calcitonin gene morpholinos, which blocked CT protein

synthesis (Figs. 5 and 6). Recently in zebrafish ECaC has been

cloned and characterized to be responsible for Ca2þ uptake in

the gill MR cells.(6) Our results lead us to conclude that there is

a hypocalcemic role for CT, probably through the inhibition

of ECaC synthesis. Furthermore, this study suggests that

this hypocalcemic role of CT could be conserved throughout

zebrafish development and still be present in adults.

On the other hand, the effects of overexpression of CT on

various genes involved in calcium homeostasis were observed

(Fig. 7A). Injection of CT cRNA induced a significant decrease in

ECaC mRNA expression, strengthening the notion that CT could

act as an inhibitor of ECaC synthesis. In zebrafish, ECaC is one of

the main proteins involved in calcium absorption, permitting

Ca2þ to enter the cell. Embryos kept in low-calcium medium

induced an upregulation of the whole-body calcium influx,

together with increased ECaC expression in the gill MR cells.(7)

Recently stanniocalcin has been characterized as a hypocalcemic

hormone in zebrafish embryos. It can negatively regulate ECaC

gene expression to reduce Ca2þ uptake.(53) In the present study,

CT cRNA injection caused an upregulation of STC mRNA

expression at both 30 hpf and 4 dpf. Thus, the inhibitory effect

of CT on ECaC synthesis could manifest through an upregulation

of STC.

The downregulation of ECaC expression should follow an

induced downregulation in Ca2þ uptake, resulting in decreased

calcium concentration as observed at 30 hpf and 50 hpf after CT

cRNA injection (Fig. 7B). However, from 105 hpf and until the end

of the experiment, the calcium content of the injected embryos

was upregulated compared to the controls. In mammals, calcium

uptake depends on the entry of Ca2þ through an epithelial

channel such as ECaC, and the diffusion through the basolateral

membrane and extrusion to the blood is via PMCA and NCX.

Among the several NCX and PMCA isoforms identified in

zebrafish, two (PMCA2 and NCX1b) have been specifically

characterized as key players, with ECaC, in the MR cells’ Ca2þ

uptake.(6) In the present study, CT cRNA injection induced the

upregulation of both NCX1b and PMCA2 at 4 dpf (Fig. 7A). The

effect of CT cRNA injection on the expression of mRNA coding

hypercalcemic factors such as vitamin D and PTH/PTHrP

receptors was also elucidated. In zebrafish, one vitamin D

receptor (VDR) and three parathyroid hormone receptors have

been identified.(54,55) PTH1R, PTH2R, and PTH3R constitute

common receptors to the PTH/PTHrP family in teleosts.(56) After


CT cRNA injection, we observed an upregulation of these four

receptors at 4 dpf (Fig. 7A). Altogether, the upregulation of

NCX1b, PMCA2, VDR, and PTHRs can explain the increase in the

calcium concentration observed at 105 and 128 hpf.

In mammals, the effect of vitamin D on PMCA and NCX

remains controversial.(1) In vitro, positive regulation of vitamin D

on PMCA has been shown, with upregulation of PMCA protein

expression.(57) In the rat, this protein has been demonstrated to

enhance PMCA stability and activity.(58) In the same way, PTH

seems to have an action on NCX regulation in the parathyr-

oidectomized rat kidney.(59) In teleosts, vitamin D and PTH/PTHrP

proteins have been described as hypercalcemic factors, as in

mammals. In the sturgeon, a chondrostean fish, PTHrP shows a

stimulation of calcium uptake concomitant with inhibition of

calcium efflux.(60) Therefore, the hypocalcaemia induced by CT

mRNA overexpression seems to have been counterbalanced by

PMCA2 and NCX1b synthesis, probably through the upregulation

of hypercalcemic hormones (vitamin D and PTH/PTHrP).

One of the factors that can trigger the response to a

modification in the calcium concentration is the calcium-sensing

receptor (CaSR). CaSR is a G protein–coupled receptor that binds

calcium ions and allows cells to react to changes of extracellular

concentrations.(61) In mammals, this factor plays a key role in the

regulation and secretion of the calciotropic hormones.(62–65)

CaSR has been shown to stimulate PTH secretion.(65)

Thus, in the present study we have shown a biphasic effect

of CT overexpression: a short-term hypocalcemic effect followed

by long-term hypercalcemia. This long-term consequence is

actually a compensatory form of hypercalcemia resulting from

the induction of hypercalcemic factors. For the first time we

demonstrate physiological evidence that CT is involved in

calcium regulation through a direct or indirect (via STC)

inhibition of ECaC synthesis in zebrafish. This action of CT

results in inhibition of Ca2þ uptake, in accordance with its

hypocalcemic role played in mammals. Using zebrafish as a

model, the present findings provide new insight into the

regulatory pathways mediated by CT, and thus contribute to our

understanding of the role of CT in vertebrate Ca2þ regulatory

mechanisms.

Disclosures

All the authors state that they have no conflicts of interest.

Acknowledgments

This study was financially supported by the grant to PPH from the

National Science Council and the postdoctoral fellowship to AGL

from Academia Sinica, Taiwan, ROC. We extend our thanks to Ms.

YC Tung and Mr. JY Wang for their assistance during the

experiments.

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