Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1544-9
MOLECULAR AND GENOMIC PHYSIOLOGY
A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio Yusuke Kumai & Raymond W. M. Kwong & Steve F. Perry
Received: 1 February 2014 / Revised: 10 May 2014 / Accepted: 26 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The present study investigated the role of the transcription factor, glial cell missing 2 (gcm2), in Ca2+ regulation in zebrafish larvae. Translational gene knockdown of gcm2 decreased Ca2+ uptake and the density of ionocytes expressing the epithelial Ca2+ channel (ecac), and disrupted the overall Ca2+ balance. Ca2+ uptake and the expression of gcm2 messenger RNA (mRNA) were significantly elevated in larvae acclimated to low Ca2+ water (25 μM); the stimulation of Ca2+ uptake was not observed in fish experiencing gcm2 knockdown. Acclimation to acidic water (pH 4) significantly reduced whole-body Ca2+ content owing to reduced Ca2+ uptake and increased Ca2+ efflux. However, ecac mRNA levels and the density of ecac-expressing ionocytes were increased in fish acclimated to acidic water, and maximal Ca2+ uptake capacity (JMAX) was significantly increased when measured in control water (pH ~7.4). Acclimation of larvae to acidic water significantly increased gcm2 mRNA expression, and in gcm2 morphants, no such stimulation in Ca2+ uptake was observed after their return to control water. Overexpression of gcm2 mRNA resulted in a significant increase in the numbers of ecac-expressing ionocytes and Ca2+ uptake. These observations reveal a critical role for gcm2 in Ca2+ homeostasis in zebrafish larvae.
Keywords Glial cell missing 2,ECaC . Calcium sensing receptor . Acid water . Ionocytes Y. Kumai : R. W. M. Kwong : S. F. Perry (*) Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6 N5, Canada e-mail: [email protected] Present Address: Y. Kumai Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA
Introduction Calcium is central to numerous critical physiological functions in vertebrates including muscle contraction, neuronal excitation, bone mineralization, and intracellular signaling. Thus, Ca2+ levels in body fluids are maintained within a narrow physiological range [16]. For water-breathing aquatic animals, including zebrafish, Ca2+ is absorbed predominantly from the water (rather than from food) by specialized iontransporting cells (ionocytes) expressing apical membrane epithelial Ca2+ channel (ECaC), basolateral plasma membrane Ca2+-ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) [39]. Ca2+ conductance via ECaC is thought to constitute the ratelimiting step [39], and accordingly, endocrine regulation of Ca2+ uptake often involves altering the expression of ECaC. For example, in fish, cortisol acts as a hypercalcemic hormone by increasing the expression of ECaC [10, 32, 42], which, in turn, is related to the increase in ecac-expressing ionocytes [5, 6]. In addition to cortisol, several other calciotropic hormones have been identified in fish, including vitamin D [31, 32] stanniocalcin [45], calcitonin [28], and parathyroid hormone (PTH) [30]. Furthermore, recent studies indicate that the calcium-sensing receptor (CaSR) plays an important role in sensing ambient [Ca2+] and triggering physiological responses to maintain Ca2+ balance [25, 30, 33, 34]. The transcription factor, glial cell missing 2 (gcm2), may also contribute to Ca2+ balance. In mammals, gcm2 specifically regulates differentiation of the parathyroid gland [13]. While the loss of gcm2 did not affect serum concentration of PTH, the majority of gcm2 homozygous knockout mice suffered from mild hypocalcemia [13]. In addition to being a master regulator for parathyroid gland formation, a previous in silico analysis showed that there are gcm2-response elements within the promoter region of human CaSR [3], and another in vitro study demonstrated that gcm2 is able to transactivate CaSR expression in parathyroid-gland-derived cells [35].
Pflugers Arch - Eur J Physiol
Although zebrafish lack a parathyroid gland, gcm2 is expressed in the developing larvae as early as 10 h postfertilization (hpf) [17] where it is critically required for the proper development of pharyngeal cartilage [14] and formation of elaborated gill filaments [17, 37]. With respect to its function in osmoregulation, previous studies with zebrafish showed that gcm2 is required for the differentiation of H+ATPase-rich (HR) cells [4, 9] and that its knockdown induces compensatory increase in ionocytes expressing the Na+-Cl− co-transporter [43]; (see recent reviews [7, 20, 21]). Thus, despite a significant role for gcm2 in Ca2+ handling in mammals, there are no data supporting a similar role for gcm2 in any freshwater (FW) fish. In zebrafish larvae, the knockdown of gcm2 did not affect the density of Na+-K+-ATPase-rich cells (NaR cells) [4, 43], a subtype of ionocytes associated with Ca2+ uptake [7, 20]. However, Pan et al. [38] reported that only a subset of NaR cells express ecac while the results of other studies suggest that a subset of NaR cells express one or more isoforms of slc26 anion exchangers [1, 40]. Thus, further investigation is required to conclusively determine whether or not gcm2 is linked to the abundance of ecacexpressing cells. Given this background, the overarching hypothesis of the present study is that the transcription factor, gcm2, is critically involved in Ca2+ homeostasis in zebrafish larvae. The effects of gcm2 knockdown/overexpression on Ca2+ balance observed in the current study clearly support an evolutionarily conserved function of gcm2 as a regulator of Ca2+ homeostasis and introduce a new physiological function of gcm2 in FW fish.
Materials and methods Experimental animals Adult zebrafish (Danio rerio Hamilton-Buchanan 1822) were purchased from Big Al’s Aquarium Services (Ottawa, ON, Canada) and kept in the University of Ottawa Aquatic Care Facility where they were maintained in plastic tanks supplied with aerated, dechloraminated City of Ottawa tap water (see Table 1 for the ionic composition; pH ~7.5) at 28 °C. Fish were subjected to a constant 14 h L:10 h D photoperiod and fed daily until satiation with No. 1 crumble-Zeigler™ (Aquatic Habitats, Apopka, FL, USA). Embryos were collected and reared in 50-ml Petri dishes with dechloraminated City of Ottawa tap water supplemented with 0.05 % methylene blue. The Petri dishes were kept in incubators set at 28.5 °C. Dead embryos were removed, and water was changed daily. The experiments were conducted in compliance with guidelines from the Canadian Council of Animal Care (CCAC) and after the approval of the University of Ottawa Animal Care
Table 1 Ionic composition for normal and low-Ca2+ water (means±SE; in μM)
Normal Ca2+ Low Ca2+
Na+
K+
Ca2+
Mg2+
767±1.7 766±5.3
61.5±0.2 61.5±0.1
266±1.8 29.3±0.3
172±1.8 167±4.4
Committee (Protocol BL-226). All chemicals were purchased from Sigma unless stated otherwise. Preparation of acclimation water For acclimation experiments, larvae were raised in Ottawa tap water until 1 d post-fertilization (dpf) (for low Ca2+) or 2 dpf (for acidic water), and then raised in either low Ca2+ or acidic water until 4 dpf. Acidic water (pH ~4.0) was prepared by adding H2SO4 to Ottawa tap water, and low [Ca2+] water was prepared by adding NaCl, CaSO4 · 2H2O, MgSO4 · 7H2O, K2HPO4, and KH2PO4 to deionized water (see Table 1 for the ionic composition). Gene knockdown Expression of gcm2 and CaSR was knocked down by microinjecting previously validated morpholinos against gcm2 (5′-AAACTGATCTGAGGATTTGGACATG-3′) [4] and CaSR (5′-ACTTCAGATGAAACCTCATTGCTTC-3′) [25] using protocols as described in those studies. Briefly, suitable doses (1 ng for gcm2 and 4 ng for CaSR) of morpholinos were injected into one-cell stage embryos. A separate group of embryos collected on the same day were injected with a standard control morpholino (5′-CCTCTTAC CTCAGTTACAATTTATA-3′). All morpholinos were tagged with carboxyfluorescein on the 3′ end, and 24 h after the injection, embryos were screened for the ubiquitous presence of green fluorescence using fluorescence microscopy (SMZ1500 microscope; Nikon Instruments, Melville, NY). Only fluorescein-positive embryos were raised and used in the subsequent experiments. No significant developmental abnormalities were observed with any treatment. Unidirectional Ca2+ flux and determination of uptake kinetics To measure Ca2+ uptake, 12 larvae were transferred to a 2-ml tube, and 45Ca (in the form of CaCl2; Perkin Elmer) was added to reach 0.2 μCi/ml. Water samples (50 μl) were collected at the beginning and end of the 2-h flux period and excess water was stored to determine [Ca2+]. At the end of the flux period, larvae were killed by anaesthetic (tricaine methanesulfonate; MS-222) overdose and briefly rinsed in isotope-free water. Larvae (two larvae were pooled to make N=1) were then digested in Solvable® (Perkin Elmer, USA) overnight at
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65 °C. After complete digestion of larval fish, samples were supplemented with 500-μl glacial acetic acid and then mixed with 5-ml scintillation cocktail (Biosafe-II, RPI Corp., USA). Water samples were also supplemented with 5-ml scintillation cocktail. Radioactivity in all samples was determined using liquid scintillation counter (model LS-6500 Beckman Coulter, Co., Mississauga, ON, Canada). The concentration of Ca2+ in the water sample was measured using flame emission spectrophotometry (model AA260, Varian, Palo Alto, CA, USA). The rate of Ca2+ uptake (JCa in , pmol/fish/h) was calculated as follows: J Ca in ¼
F ; SA⋅n⋅t
where F = total incorporated radioactivity (disintegration per minute (DPM)), SA = specific activity of the medium (DPM/ pmol), n = number of larvae (typically two), and t = the duration of the incubation (h). DPM was calculated by the liquid scintillation counting program after taking quenching and counting efficiency into consideration. Ca2+ uptake kinetics were determined in control and acidic water at 4 dpf, using larvae raised for 48 h in either control or acidic water (48–96 h). For the kinetic analysis, ambient [Ca2+] was varied from 40 to 1,600 μM by dissolving appropriate amount of NaCl, CaSO4 · 2H2O, MgSO4 · 7H2O, K2HPO4, and KH2PO4 to deionized water; pH was adjusted as needed. A previous study [23] demonstrated that exposure to soft (low [Ca2+]) acidic water raises Na+ efflux which could be lethal to adult zebrafish. Thus, to minimize the impact of excess Na+ loss in the present study, the flux period for Ca2+ uptake was shortened from 2 to 1 h. For measurement of Ca2+ efflux, 4 dpf larvae were loaded with 45Ca by incubating them for 6 h in media containing 2 μCi 45Ca/ml. Subsequently, larvae were washed in isotopefree water and divided (in groups of 10) among 1.5-ml flux chambers. An initial water sample (250 μl) was collected immediately after fish were transferred, and samples were collected at 20-min intervals over 1 h. Subsequently, larvae were killed with MS-222 overdose and rinsed in isotope-free deionized water. Larvae were digested overnight at 65 °C with either Solvable® or 80 μl 1 N HNO3 (five larvae were pooled for each sample) to measure total radioactivity and wholebody Ca content. Water and tissue samples prepared using Solvable® were handled as they were in the uptake measurement as described above. The samples prepared with 1 N HNO3 were supplemented with deionized water (to a final volume of 1.5 ml), briefly centrifuged (12,000g), and the supernatant was collected to measure Ca2+ concentration (see above), and average Ca content was calculated. The rate of Ca2+ efflux (JCa out, pmol/fish/h) was calculated as follows:
J Ca out ¼
1 dW ; SA F ⋅n dt
where SAF = internal specific activity of average Ca2+ (DPM/ pmol), N = number of larvae in flux chamber, W = total radioactivity of the medium (DPM), and t = duration of measurement (h). Backflux correction was not used because the external specific activity was less than 5 % of internal specific activity. To determine whole-body Ca content, larvae were killed with M-222 overdose and briefly rinsed with deionized water (ten larvae were pooled to make one sample). After excess water was removed, larval tissue was digested overnight at 65 °C in 80 μl of 1 N HNO3 and handled as described above to calculate the whole-body Ca content. Real-time PCR The effects of gcm2 knockdown, exposure to acidic or low [Ca2+] water and exogenous treatment with cortisol (for conditions of cortisol treatment, see below) on messenger RNA (mRNA) expression of Ca2+ transporters (epithelial Ca2+ channel or ecac, plasma membrane Ca2+ ATPase or pmca2, and Na+/Ca2+ exchanger or ncx1b), several known regulators for Ca2+ homeostasis (calcium sensing receptor or casr, stanniocalcin or stc1, parathyroid hormone or pth1, and calcitonin or ct), and gcm2 were determined by real-time PCR. Total RNA was extracted from 4 dpf larvae (ten larvae were pooled to make one sample) by TRIzol® (Life Technologies) according to manufacturer’s instructions. Complimentary DNA was synthesized with 1 μg of RNA using RevertAid H-minus reverse transcriptase (Thermo, USA) after treatment with DNase I (Biolabs, USA) according to manufacturer’s instructions. Real-time PCR was performed using a Bio-Rad CFX96 qPCR system with Brilliant III SYBR Green Master Mix (Agilent Technologies, USA). All real-time (RT)-qPCR was performed using the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 20 s and 58 °C for 20 s, with final extension for 5 min at 72 °C. The expression levels of all genes of interest were normalized to 18S using methods described by Pfaffl [41] (for the list of primers, see Table 2). In situ hybridization Whole mount in situ hybridization was performed to quantify the changes in the numbers of ecac positive cells. In situ hybridization was performed as detailed in Kwong et al. [25]. In brief, 1-phenyl-2-thiourea (PTU)-treated fish at 4 dpf were fixed with 4 % paraformaldehyde overnight at 4 °C and washed several times with phosphate-buffered saline plus 0.1 % Tween-20 (PBST) before gradual dehydration
Pflugers Arch - Eur J Physiol Table 2 Primer sets used in the present study Gene
Sequence
Efficiency (%)
Reference
ecac (qPCR)
FWD 5′-TCCTTTCCCATCACCCTCT-3′ REV 5′-GCACTGTGGCAACTTTCGT-3′ FWD 5′-TGGCTCAGGATGCAGAACAG-3′ REV 5′-TAGGGTCCCAGCATCTCGAA-3′ FWD 5′-AAGCAGTTCAGGGGTTTAC-3′ REV 5′-CAGATCATTGCCTTGTATCA-3′ FWD 5′-TAAAGTGGCAGCGATACAGG-3′ REV 5′-CAGATCAAGGCGAAGATGG-3′ FWD 5′-AAATGCCCAAACAACTCCTG-3′ REV 5′-GGTTTGATGCCTTCACGATT-3′ FWD 5′-CCAGCTGCTTCAAAACAAACC-3′ REV 5′-ATGGAGCGTTTTCTGGCGA-3′ FWD 5′-TCATAAGCATGTGGAGCTGAGGCA-3′ REV 5′-ACGATGGGTTCATGAGCTTCTCCA-3′ FWD 5′-CTACGAGGCGAGAAGATTGCTT-3′ REV 5′-TGGATACGTCTGCAGCTTGTG-3′ FWD 5′-TCCCTGTGGTTATGACTTTGCA-3′ REV 5′-TGGACTTGAGCCATGAGACACT-3′ FWD 5′-ATGTCCAAATCCTCAGATCAGTTT-3′ REV 5′-TCAGTATTCCCCGCTGTCAT-3′ FWD 5′-GGCGGCGTTATTCCCATGACC-3′ REV 5′-GGTGGTGCCCTTCCGTCAATTC-3′
109.8
[32]
N/A
[25]
96
[32]
111.2
[32]
113
[25]
113.4
[45]
98
[25]
108.2
[28]
106.5
[4]
ecac (in situ) pmca2 ncx1b casr stc1 pth1 ct gcm2 (qPCR) gcm2 (cRNA) 18S
N/A 98
[23]
FWD forward, REV reverse, N/A not applicable
using methanol. After rehydration with PBST, the fish were permeabilized in acetone for 20 min at −20 °C and then washed with PBST. The fish were pre-hybridized in a hybridization buffer supplemented with 500 μg/ml yeast tRNA and 50 μg/ml heparin (Sigma) for 2 h at 65 °C and then incubated with 100 ng of ecac RNA probe overnight at 65 °C. After serial washing with hybridization buffer and PBST, the fish were incubated in a blocking solution containing 10 % calf serum in PBST for 2 h before incubating with an alkaline phosphatase conjugated anti-dig antibody (1:2,000 dilutions at 4 °C overnight). Subsequently, the fish were washed with PBST and incubated in a NBT/BCIP staining buffer until the desired coloration intensity was obtained. Because relatively few cells stained positively, the total number of ecac-positive cells on yolk per larva was counted.
cells were counted. The average of those three counts was calculated. Exogenous treatment with cortisol Cortisol (hydrocortisone; H4001) was dissolved in dimethyl sulfoxide (DMSO) and larvae were exposed to 10 μM cortisol between 48- and 96-hpf. This dose of cortisol is known to affect osmoregulation in larval zebrafish [24, 27]. A separate group of larvae were exposed to DMSO as a vehicle control (final DMSO concentration in the medium did not exceed 0.1 %). Embryo medium containing cortisol/DMSO was changed daily. Ca2+ uptake was measured using 4 dpf larvae in the continued presence of cortisol/DMSO. Whole-body cortisol analysis
Overexpression of gcm2 Overexpression of gcm2 was performed as described by Chang et al. [4]. Overexpression of gcm2 was confirmed by vitally staining 4 dpf zebrafish larvae with Alexa-633 conjugated concanavalin A (conA; Life Technologies, USA), a specific marker for HR cells in larval zebrafish [24]. For each sham and gcm2 cRNA-injected larvae, three regions of interests (10,000 μm2 each) on the skin of yolk sac were randomly selected and conA-positive
Whole-body cortisol was measured using 4 dpf larvae acclimated to low pH (between 48- and 96-hpf) and low [Ca2+] water (between 24- and 96-hpf) with a cortisol EIA kit (Neogen, cat. #402710). Briefly, 25 larvae were pooled and homogenized in 200-μl 1× extraction buffer (a component of the EIA kit), and cortisol was extracted three times using 1 ml diethyl ether. The organic phase was collected and dried under continuous flow or N2, and the residue was re-suspended in 1× extraction buffer for EIA
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analysis. All samples were handled and analyzed according to the manufacturer’s instructions. Statistical analysis All statistical analyses were performed with SigmaPlot (v. 11, Systat Inc., Chicago, IL, USA). Data reported in Figs. 2b, 3, 6a, b, and 9c were analyzed with two-way ANOVA followed by Tukey’s post hoc test. Data reported in Figs. 8c and 9a were analyzed with one-way AOVA followed by Tukey’s post hoc test. All other data were analyzed using Student’s t test. Parameters (average±SEM) associated with kinetic analysis were determined by fitting the data to a one-site saturation curve using SigmaPlot. The number of solutions with different [Ca2+] (in which uptake was measured to determine uptake kinetics) was used as N to analyze difference in JMAX between control and acid-acclimated fish using Student’s t test [36]. When assumptions of normality or equal variance were violated, data were transformed using natural log- or square-root transformation. For all analyses, the level of statistical significance was set at p
MOLECULAR AND GENOMIC PHYSIOLOGY
A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio Yusuke Kumai & Raymond W. M. Kwong & Steve F. Perry
Received: 1 February 2014 / Revised: 10 May 2014 / Accepted: 26 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The present study investigated the role of the transcription factor, glial cell missing 2 (gcm2), in Ca2+ regulation in zebrafish larvae. Translational gene knockdown of gcm2 decreased Ca2+ uptake and the density of ionocytes expressing the epithelial Ca2+ channel (ecac), and disrupted the overall Ca2+ balance. Ca2+ uptake and the expression of gcm2 messenger RNA (mRNA) were significantly elevated in larvae acclimated to low Ca2+ water (25 μM); the stimulation of Ca2+ uptake was not observed in fish experiencing gcm2 knockdown. Acclimation to acidic water (pH 4) significantly reduced whole-body Ca2+ content owing to reduced Ca2+ uptake and increased Ca2+ efflux. However, ecac mRNA levels and the density of ecac-expressing ionocytes were increased in fish acclimated to acidic water, and maximal Ca2+ uptake capacity (JMAX) was significantly increased when measured in control water (pH ~7.4). Acclimation of larvae to acidic water significantly increased gcm2 mRNA expression, and in gcm2 morphants, no such stimulation in Ca2+ uptake was observed after their return to control water. Overexpression of gcm2 mRNA resulted in a significant increase in the numbers of ecac-expressing ionocytes and Ca2+ uptake. These observations reveal a critical role for gcm2 in Ca2+ homeostasis in zebrafish larvae.
Keywords Glial cell missing 2,ECaC . Calcium sensing receptor . Acid water . Ionocytes Y. Kumai : R. W. M. Kwong : S. F. Perry (*) Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6 N5, Canada e-mail: [email protected] Present Address: Y. Kumai Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA
Introduction Calcium is central to numerous critical physiological functions in vertebrates including muscle contraction, neuronal excitation, bone mineralization, and intracellular signaling. Thus, Ca2+ levels in body fluids are maintained within a narrow physiological range [16]. For water-breathing aquatic animals, including zebrafish, Ca2+ is absorbed predominantly from the water (rather than from food) by specialized iontransporting cells (ionocytes) expressing apical membrane epithelial Ca2+ channel (ECaC), basolateral plasma membrane Ca2+-ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) [39]. Ca2+ conductance via ECaC is thought to constitute the ratelimiting step [39], and accordingly, endocrine regulation of Ca2+ uptake often involves altering the expression of ECaC. For example, in fish, cortisol acts as a hypercalcemic hormone by increasing the expression of ECaC [10, 32, 42], which, in turn, is related to the increase in ecac-expressing ionocytes [5, 6]. In addition to cortisol, several other calciotropic hormones have been identified in fish, including vitamin D [31, 32] stanniocalcin [45], calcitonin [28], and parathyroid hormone (PTH) [30]. Furthermore, recent studies indicate that the calcium-sensing receptor (CaSR) plays an important role in sensing ambient [Ca2+] and triggering physiological responses to maintain Ca2+ balance [25, 30, 33, 34]. The transcription factor, glial cell missing 2 (gcm2), may also contribute to Ca2+ balance. In mammals, gcm2 specifically regulates differentiation of the parathyroid gland [13]. While the loss of gcm2 did not affect serum concentration of PTH, the majority of gcm2 homozygous knockout mice suffered from mild hypocalcemia [13]. In addition to being a master regulator for parathyroid gland formation, a previous in silico analysis showed that there are gcm2-response elements within the promoter region of human CaSR [3], and another in vitro study demonstrated that gcm2 is able to transactivate CaSR expression in parathyroid-gland-derived cells [35].
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Although zebrafish lack a parathyroid gland, gcm2 is expressed in the developing larvae as early as 10 h postfertilization (hpf) [17] where it is critically required for the proper development of pharyngeal cartilage [14] and formation of elaborated gill filaments [17, 37]. With respect to its function in osmoregulation, previous studies with zebrafish showed that gcm2 is required for the differentiation of H+ATPase-rich (HR) cells [4, 9] and that its knockdown induces compensatory increase in ionocytes expressing the Na+-Cl− co-transporter [43]; (see recent reviews [7, 20, 21]). Thus, despite a significant role for gcm2 in Ca2+ handling in mammals, there are no data supporting a similar role for gcm2 in any freshwater (FW) fish. In zebrafish larvae, the knockdown of gcm2 did not affect the density of Na+-K+-ATPase-rich cells (NaR cells) [4, 43], a subtype of ionocytes associated with Ca2+ uptake [7, 20]. However, Pan et al. [38] reported that only a subset of NaR cells express ecac while the results of other studies suggest that a subset of NaR cells express one or more isoforms of slc26 anion exchangers [1, 40]. Thus, further investigation is required to conclusively determine whether or not gcm2 is linked to the abundance of ecacexpressing cells. Given this background, the overarching hypothesis of the present study is that the transcription factor, gcm2, is critically involved in Ca2+ homeostasis in zebrafish larvae. The effects of gcm2 knockdown/overexpression on Ca2+ balance observed in the current study clearly support an evolutionarily conserved function of gcm2 as a regulator of Ca2+ homeostasis and introduce a new physiological function of gcm2 in FW fish.
Materials and methods Experimental animals Adult zebrafish (Danio rerio Hamilton-Buchanan 1822) were purchased from Big Al’s Aquarium Services (Ottawa, ON, Canada) and kept in the University of Ottawa Aquatic Care Facility where they were maintained in plastic tanks supplied with aerated, dechloraminated City of Ottawa tap water (see Table 1 for the ionic composition; pH ~7.5) at 28 °C. Fish were subjected to a constant 14 h L:10 h D photoperiod and fed daily until satiation with No. 1 crumble-Zeigler™ (Aquatic Habitats, Apopka, FL, USA). Embryos were collected and reared in 50-ml Petri dishes with dechloraminated City of Ottawa tap water supplemented with 0.05 % methylene blue. The Petri dishes were kept in incubators set at 28.5 °C. Dead embryos were removed, and water was changed daily. The experiments were conducted in compliance with guidelines from the Canadian Council of Animal Care (CCAC) and after the approval of the University of Ottawa Animal Care
Table 1 Ionic composition for normal and low-Ca2+ water (means±SE; in μM)
Normal Ca2+ Low Ca2+
Na+
K+
Ca2+
Mg2+
767±1.7 766±5.3
61.5±0.2 61.5±0.1
266±1.8 29.3±0.3
172±1.8 167±4.4
Committee (Protocol BL-226). All chemicals were purchased from Sigma unless stated otherwise. Preparation of acclimation water For acclimation experiments, larvae were raised in Ottawa tap water until 1 d post-fertilization (dpf) (for low Ca2+) or 2 dpf (for acidic water), and then raised in either low Ca2+ or acidic water until 4 dpf. Acidic water (pH ~4.0) was prepared by adding H2SO4 to Ottawa tap water, and low [Ca2+] water was prepared by adding NaCl, CaSO4 · 2H2O, MgSO4 · 7H2O, K2HPO4, and KH2PO4 to deionized water (see Table 1 for the ionic composition). Gene knockdown Expression of gcm2 and CaSR was knocked down by microinjecting previously validated morpholinos against gcm2 (5′-AAACTGATCTGAGGATTTGGACATG-3′) [4] and CaSR (5′-ACTTCAGATGAAACCTCATTGCTTC-3′) [25] using protocols as described in those studies. Briefly, suitable doses (1 ng for gcm2 and 4 ng for CaSR) of morpholinos were injected into one-cell stage embryos. A separate group of embryos collected on the same day were injected with a standard control morpholino (5′-CCTCTTAC CTCAGTTACAATTTATA-3′). All morpholinos were tagged with carboxyfluorescein on the 3′ end, and 24 h after the injection, embryos were screened for the ubiquitous presence of green fluorescence using fluorescence microscopy (SMZ1500 microscope; Nikon Instruments, Melville, NY). Only fluorescein-positive embryos were raised and used in the subsequent experiments. No significant developmental abnormalities were observed with any treatment. Unidirectional Ca2+ flux and determination of uptake kinetics To measure Ca2+ uptake, 12 larvae were transferred to a 2-ml tube, and 45Ca (in the form of CaCl2; Perkin Elmer) was added to reach 0.2 μCi/ml. Water samples (50 μl) were collected at the beginning and end of the 2-h flux period and excess water was stored to determine [Ca2+]. At the end of the flux period, larvae were killed by anaesthetic (tricaine methanesulfonate; MS-222) overdose and briefly rinsed in isotope-free water. Larvae (two larvae were pooled to make N=1) were then digested in Solvable® (Perkin Elmer, USA) overnight at
Pflugers Arch - Eur J Physiol
65 °C. After complete digestion of larval fish, samples were supplemented with 500-μl glacial acetic acid and then mixed with 5-ml scintillation cocktail (Biosafe-II, RPI Corp., USA). Water samples were also supplemented with 5-ml scintillation cocktail. Radioactivity in all samples was determined using liquid scintillation counter (model LS-6500 Beckman Coulter, Co., Mississauga, ON, Canada). The concentration of Ca2+ in the water sample was measured using flame emission spectrophotometry (model AA260, Varian, Palo Alto, CA, USA). The rate of Ca2+ uptake (JCa in , pmol/fish/h) was calculated as follows: J Ca in ¼
F ; SA⋅n⋅t
where F = total incorporated radioactivity (disintegration per minute (DPM)), SA = specific activity of the medium (DPM/ pmol), n = number of larvae (typically two), and t = the duration of the incubation (h). DPM was calculated by the liquid scintillation counting program after taking quenching and counting efficiency into consideration. Ca2+ uptake kinetics were determined in control and acidic water at 4 dpf, using larvae raised for 48 h in either control or acidic water (48–96 h). For the kinetic analysis, ambient [Ca2+] was varied from 40 to 1,600 μM by dissolving appropriate amount of NaCl, CaSO4 · 2H2O, MgSO4 · 7H2O, K2HPO4, and KH2PO4 to deionized water; pH was adjusted as needed. A previous study [23] demonstrated that exposure to soft (low [Ca2+]) acidic water raises Na+ efflux which could be lethal to adult zebrafish. Thus, to minimize the impact of excess Na+ loss in the present study, the flux period for Ca2+ uptake was shortened from 2 to 1 h. For measurement of Ca2+ efflux, 4 dpf larvae were loaded with 45Ca by incubating them for 6 h in media containing 2 μCi 45Ca/ml. Subsequently, larvae were washed in isotopefree water and divided (in groups of 10) among 1.5-ml flux chambers. An initial water sample (250 μl) was collected immediately after fish were transferred, and samples were collected at 20-min intervals over 1 h. Subsequently, larvae were killed with MS-222 overdose and rinsed in isotope-free deionized water. Larvae were digested overnight at 65 °C with either Solvable® or 80 μl 1 N HNO3 (five larvae were pooled for each sample) to measure total radioactivity and wholebody Ca content. Water and tissue samples prepared using Solvable® were handled as they were in the uptake measurement as described above. The samples prepared with 1 N HNO3 were supplemented with deionized water (to a final volume of 1.5 ml), briefly centrifuged (12,000g), and the supernatant was collected to measure Ca2+ concentration (see above), and average Ca content was calculated. The rate of Ca2+ efflux (JCa out, pmol/fish/h) was calculated as follows:
J Ca out ¼
1 dW ; SA F ⋅n dt
where SAF = internal specific activity of average Ca2+ (DPM/ pmol), N = number of larvae in flux chamber, W = total radioactivity of the medium (DPM), and t = duration of measurement (h). Backflux correction was not used because the external specific activity was less than 5 % of internal specific activity. To determine whole-body Ca content, larvae were killed with M-222 overdose and briefly rinsed with deionized water (ten larvae were pooled to make one sample). After excess water was removed, larval tissue was digested overnight at 65 °C in 80 μl of 1 N HNO3 and handled as described above to calculate the whole-body Ca content. Real-time PCR The effects of gcm2 knockdown, exposure to acidic or low [Ca2+] water and exogenous treatment with cortisol (for conditions of cortisol treatment, see below) on messenger RNA (mRNA) expression of Ca2+ transporters (epithelial Ca2+ channel or ecac, plasma membrane Ca2+ ATPase or pmca2, and Na+/Ca2+ exchanger or ncx1b), several known regulators for Ca2+ homeostasis (calcium sensing receptor or casr, stanniocalcin or stc1, parathyroid hormone or pth1, and calcitonin or ct), and gcm2 were determined by real-time PCR. Total RNA was extracted from 4 dpf larvae (ten larvae were pooled to make one sample) by TRIzol® (Life Technologies) according to manufacturer’s instructions. Complimentary DNA was synthesized with 1 μg of RNA using RevertAid H-minus reverse transcriptase (Thermo, USA) after treatment with DNase I (Biolabs, USA) according to manufacturer’s instructions. Real-time PCR was performed using a Bio-Rad CFX96 qPCR system with Brilliant III SYBR Green Master Mix (Agilent Technologies, USA). All real-time (RT)-qPCR was performed using the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 20 s and 58 °C for 20 s, with final extension for 5 min at 72 °C. The expression levels of all genes of interest were normalized to 18S using methods described by Pfaffl [41] (for the list of primers, see Table 2). In situ hybridization Whole mount in situ hybridization was performed to quantify the changes in the numbers of ecac positive cells. In situ hybridization was performed as detailed in Kwong et al. [25]. In brief, 1-phenyl-2-thiourea (PTU)-treated fish at 4 dpf were fixed with 4 % paraformaldehyde overnight at 4 °C and washed several times with phosphate-buffered saline plus 0.1 % Tween-20 (PBST) before gradual dehydration
Pflugers Arch - Eur J Physiol Table 2 Primer sets used in the present study Gene
Sequence
Efficiency (%)
Reference
ecac (qPCR)
FWD 5′-TCCTTTCCCATCACCCTCT-3′ REV 5′-GCACTGTGGCAACTTTCGT-3′ FWD 5′-TGGCTCAGGATGCAGAACAG-3′ REV 5′-TAGGGTCCCAGCATCTCGAA-3′ FWD 5′-AAGCAGTTCAGGGGTTTAC-3′ REV 5′-CAGATCATTGCCTTGTATCA-3′ FWD 5′-TAAAGTGGCAGCGATACAGG-3′ REV 5′-CAGATCAAGGCGAAGATGG-3′ FWD 5′-AAATGCCCAAACAACTCCTG-3′ REV 5′-GGTTTGATGCCTTCACGATT-3′ FWD 5′-CCAGCTGCTTCAAAACAAACC-3′ REV 5′-ATGGAGCGTTTTCTGGCGA-3′ FWD 5′-TCATAAGCATGTGGAGCTGAGGCA-3′ REV 5′-ACGATGGGTTCATGAGCTTCTCCA-3′ FWD 5′-CTACGAGGCGAGAAGATTGCTT-3′ REV 5′-TGGATACGTCTGCAGCTTGTG-3′ FWD 5′-TCCCTGTGGTTATGACTTTGCA-3′ REV 5′-TGGACTTGAGCCATGAGACACT-3′ FWD 5′-ATGTCCAAATCCTCAGATCAGTTT-3′ REV 5′-TCAGTATTCCCCGCTGTCAT-3′ FWD 5′-GGCGGCGTTATTCCCATGACC-3′ REV 5′-GGTGGTGCCCTTCCGTCAATTC-3′
109.8
[32]
N/A
[25]
96
[32]
111.2
[32]
113
[25]
113.4
[45]
98
[25]
108.2
[28]
106.5
[4]
ecac (in situ) pmca2 ncx1b casr stc1 pth1 ct gcm2 (qPCR) gcm2 (cRNA) 18S
N/A 98
[23]
FWD forward, REV reverse, N/A not applicable
using methanol. After rehydration with PBST, the fish were permeabilized in acetone for 20 min at −20 °C and then washed with PBST. The fish were pre-hybridized in a hybridization buffer supplemented with 500 μg/ml yeast tRNA and 50 μg/ml heparin (Sigma) for 2 h at 65 °C and then incubated with 100 ng of ecac RNA probe overnight at 65 °C. After serial washing with hybridization buffer and PBST, the fish were incubated in a blocking solution containing 10 % calf serum in PBST for 2 h before incubating with an alkaline phosphatase conjugated anti-dig antibody (1:2,000 dilutions at 4 °C overnight). Subsequently, the fish were washed with PBST and incubated in a NBT/BCIP staining buffer until the desired coloration intensity was obtained. Because relatively few cells stained positively, the total number of ecac-positive cells on yolk per larva was counted.
cells were counted. The average of those three counts was calculated. Exogenous treatment with cortisol Cortisol (hydrocortisone; H4001) was dissolved in dimethyl sulfoxide (DMSO) and larvae were exposed to 10 μM cortisol between 48- and 96-hpf. This dose of cortisol is known to affect osmoregulation in larval zebrafish [24, 27]. A separate group of larvae were exposed to DMSO as a vehicle control (final DMSO concentration in the medium did not exceed 0.1 %). Embryo medium containing cortisol/DMSO was changed daily. Ca2+ uptake was measured using 4 dpf larvae in the continued presence of cortisol/DMSO. Whole-body cortisol analysis
Overexpression of gcm2 Overexpression of gcm2 was performed as described by Chang et al. [4]. Overexpression of gcm2 was confirmed by vitally staining 4 dpf zebrafish larvae with Alexa-633 conjugated concanavalin A (conA; Life Technologies, USA), a specific marker for HR cells in larval zebrafish [24]. For each sham and gcm2 cRNA-injected larvae, three regions of interests (10,000 μm2 each) on the skin of yolk sac were randomly selected and conA-positive
Whole-body cortisol was measured using 4 dpf larvae acclimated to low pH (between 48- and 96-hpf) and low [Ca2+] water (between 24- and 96-hpf) with a cortisol EIA kit (Neogen, cat. #402710). Briefly, 25 larvae were pooled and homogenized in 200-μl 1× extraction buffer (a component of the EIA kit), and cortisol was extracted three times using 1 ml diethyl ether. The organic phase was collected and dried under continuous flow or N2, and the residue was re-suspended in 1× extraction buffer for EIA
Pflugers Arch - Eur J Physiol
analysis. All samples were handled and analyzed according to the manufacturer’s instructions. Statistical analysis All statistical analyses were performed with SigmaPlot (v. 11, Systat Inc., Chicago, IL, USA). Data reported in Figs. 2b, 3, 6a, b, and 9c were analyzed with two-way ANOVA followed by Tukey’s post hoc test. Data reported in Figs. 8c and 9a were analyzed with one-way AOVA followed by Tukey’s post hoc test. All other data were analyzed using Student’s t test. Parameters (average±SEM) associated with kinetic analysis were determined by fitting the data to a one-site saturation curve using SigmaPlot. The number of solutions with different [Ca2+] (in which uptake was measured to determine uptake kinetics) was used as N to analyze difference in JMAX between control and acid-acclimated fish using Student’s t test [36]. When assumptions of normality or equal variance were violated, data were transformed using natural log- or square-root transformation. For all analyses, the level of statistical significance was set at p
