* Molecular and Environmental Toxicology Center, and School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705
Received May 27, 2004; accepted July 26, 2004
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ABSTRACT |
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Key Words: AHR2; ARNT2; TCDD toxicity; zebrafish; development.
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INTRODUCTION |
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PCDDs are lipophilic, persistent, bioaccumulative toxicants that have adversely affected feral fish populations of the most sensitive fish species to TCDD developmental toxicity, the lake trout (Cook et al., 2003). TCDD, the most potent PCDD, causes several toxic responses in developing teleost fish including yolk sac and pericardial edema, arrested growth, craniofacial malformations, ischemia, anemia, impaired swim bladder inflation, and mortality (Belair et al., 2001
; Dong et al., 2002
; Henry et al., 1997
; Peterson et al., 1993
; Tanguay et al., 2003
; Teraoka et al., 2002
; Walker and Peterson, 1994
). The zebrafish is an excellent model for studying the effects of TCDD on developing teleost fish due to its rapid growth, short generation time, high egg yield, and transparent embryos (Andreasen et al., 2002b
; Belair et al., 2001
; Bello et al., 2004
; Dong et al., 2002
; Henry et al., 1997
; Hill et al., 2003
, 2004
). Additionally, zebrafish developmental biology has been well characterized, the zebrafish genome is near completion, and numerous molecular and genetic techniques have been developed to study zebrafish gene function.
Components of the AHR signaling pathway have been characterized in zebrafish revealing important differences from what has been found in mammals. In contrast to the single mammalian gene for ahr, two distinct genes for the ahr, zfahr1 and zfahr2, have been identified in zebrafish (Andreasen et al., 2002a; Tanguay et al., 1999
). Conversely, multiple genes for arnt, arnt1, and arnt2, have been identified in mammals (Drutel et al., 1996
; Hirose et al., 1996
; Hoffman et al., 1991
; Li et al., 1994
) whereas only a single arnt gene with multiple splice variants, zfarnt2, has been identified in zebrafish (Hsu et al., 2001
; Tanguay et al., 2000
; Wang et al., 2000
). The presence of a single arnt gene in zebrafish is consistent with what has been found in most other fish species studied (Powell and Hahn, 2000
), although different fish species express different forms of ARNT. In killifish (Fundulus heteroclitus), like zebrafish, ARNT2 is the only form of ARNT yet detected (Powell et al., 1999
). In contrast, in rainbow trout (Oncorhynchus mykiss) and scup (Stenotomus chrysops) a different form of ARNT, likely an ortholog of ARNT1, predominates (Pollenz et al., 1996
; Powell and Hahn, 2000
). Interestingly, however, the recently sequenced Fugu rubripes genome appears to encode two forms of arnt (Rowatt et al., 2003
) suggesting that, at least in some fish species, two arnt genes do exist.
Experiments with an antisense morpholino targeted against zfAHR2 have shown that zfAHR2 is the receptor which mediates TCDD toxicity in the developing zebrafish embryo (Dong et al., 2004; Prasch et al., 2003
; Teraoka et al., 2003
). In vitro molecular evidence demonstrates that one form of zfARNT2, zfARNT2b, can function with zfAHR2 to induce XRE-driven transcription in COS-7 cells treated with TCDD, while other spice variants cannot. The zfAHR2/ARNT2b heterodimer also specifically recognizes XREs in gel shift assays (Tanguay et al., 2000
). In addition, zfAHR2 and zfARNT2a,b,c mRNAs have been shown to be co-localized in many of the same tissues as zfCYP1A after TCDD exposure (Andreasen et al., 2002b
). These results have suggested that zfARNT2b and zfAHR2 function to mediate TCDD developmental toxicity in the zebrafish. However, this has not been proven.
The purpose of the current study was to test the hypothesis that zfARNT2b functions as the dimerization partner for zfAHR2 in the zebrafish embryo to mediate the developmental toxicity caused by TCDD. Zfarnt2 morphant embryos which have transient knockdown of the zfARNT2 protein, and a line of insertional mutant zebrafish that lack expression of zfARNT2 were examined for responses to TCDD. Surprisingly, disrupting expression of zfARNT2 did not have a significant effect on endpoints of TCDD developmental toxicity suggesting that alternate dimerization partners for zfAHR2 exist in vivo.
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MATERIALS AND METHODS |
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Waterborne TCDD exposure of embryos. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) of >99% purity was obtained from Chemsyn, Lenexa, KS and dissolved in DMSO. Newly fertilized eggs, approximately 34 hours post-fertilization (hpf) were exposed to vehicle (0.1% DMSO) or TCDD for 1 h in glass scintillation vials with gentle rocking. Following the 1 h static exposure, embryos were rinsed with water and maintained in vehicle/TCDD-free water for the remainder of experiments. When appropriate, fish were anesthetized with tricaine (MS222; Sigma 1.67 mg/ml).
Morpholinos. The zfarnt2 morpholino (zfarnt2-MO), obtained from Gene Tools (Philomath, OR), was designed with sequence complementary to zebrafish ARNT2 cDNA (GenBank accession #AF219988). The zfarnt2-MO (5' AGCGGCTGGTGTTGCCATATTGCCT 3') overlapped the translation start site of zfARNT2 by extending from 7 bp upstream to 15 bp downstream of the AUG start codon and therefore blocked expression of all splice forms of zfARNT2. The Gene Tools standard control morpholino (5' CTCTTACCTCAGTTACAATTTATA 3') was used as a control (control-MO). Morpholinos were diluted to 0.15 mM in 1x Danieau's solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6) as described (Nasevicius and Ekker, 2000). Embryos were injected with approximately 10 ng of either the zfarnt2-MO or control-MO at the 12 cell stage and allowed to develop for approximately 2 h, after which damaged and unfertilized embryos were discarded. As the zfarnt2-MO was fluorescein tagged at the 3' end, injection success was assessed by fluorescence microscopy. Only zfarnt2-MO injected embryos exhibiting fluorescence at 3 hpf were used.
To assess the effect of the zfarnt2-MO on edema formation embryos were either uninjected or injected with the control-MO or zfarnt2-MO and exposed individually to either vehicle or 0.4 ng/ml TCDD according to the standard 34 hpf TCDD exposure protocol. At 72 hpf embryos were assessed for pericardial edema formation as previously described (Carney et al., in press; Prasch et al., 2003
). The extent of pericardial edema in individual zebrafish was estimated using digital photographs in which the cross sectional area of the pericardium was determined using the area measurement function of Scion Image. All pictures were taken at 10X magnification with the embryo in a lateral orientation at the same resolution. Eight embryos were assessed for each of the six treatment groups.
Western blotting. To evaluate zfARNT2 protein expression in embryos, Western blotting was performed using the polyclonal anti-ARNT2 M-20 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, cat # sc-8078). Protein levels were assessed in three treatment groups: uninjected, injected with control-MO, or injected with zfarnt2-MO. At 24 and 72 hpf 35 embryos from each treatment group were manually dechorionated, manually deyolked and pooled together. Tissue was disrupted by adding 50 µl of 1X SDS (63 mM Tris-HCl pH 6.8, 10% glycerol, 5% ß-mercaptoethanol, 3.5% sodium dodecyl sulfate) and homogenizing with a pestle. After boiling for 10 min the entire sample was resolved by SDS polyacrylamide gel electrophoresis on a 6% gel and transferred to a nitrocellulose membrane. The membrane was stained with Ponceau S to ensure even amounts of protein were loaded. Detection of the zfARNT2 protein was carried out by blocking the membrane for 1 h in TBS-T (25 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% dry milk. The membrane was washed one time in TBS-T before addition of ARNT2 M-20 antibody diluted 1:1000 in TBS-T containing 1% dry milk. After 2 h incubation the antibody was removed and the membrane washed three times in TBS-T. The membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech) diluted 1:10,000 in TBS-T containing 1% dry milk for 1 h. The membrane was washed three times in TBS-T before chemiluminescent detection using the ECL Western blotting analysis system (Amersham). In vitro translated zfARNT2b was produced from a pBKCMV expression construct (Tanguay et al., 2000) using the TNT Coupled Rabbit Reticulocyte Lysate System (Promega) as described by the manufacturer.
Genotyping zfarnt2 mutants. Part of the tail was removed from each euthanized larvae and two primer sets were used for genotyping. The 1715C-5 (5' CGGAAATGTCGCTGTTGTTAGTTGTG3') and 1715C-3 (5' GAACTGAGTTTGCGCGTTTGAGAC 3') set was used to distinguish zfarnt2/ mutants from wild type (WT) and from heterozygous (HET) larvae. The 1715C-5 and SFGR (5' TGCGATGCCGTCTACTTTGA 3') set was then used to distinguish WT from HET larvae. The sequences for 1715C-5 and 1715C-3 flank the knockout viral insertion and SFGR lies within the insert. DNA was extracted in buffer (0.01 M Tris, 2 mM EDTA, 0.2% Triton-X, 0.2 mg/ml Proteinase-K) at 55°C for 2.5 h, and heated at 100°C for 10 min before centrifugation. Sequences were amplified by PCR using GoTaq polymerase (Promega) and PCR products were examined by gel electrophoresis.
Assessment of TCDD developmental toxicity. Sexually mature adult fish, heterozygous for the zfarnt2 mutation, were pair mated and the clutch of embryos was divided into five groups with ten embryos per group. One group was exposed to vehicle and the other four groups were exposed to graded concentrations of TCDD (0.2, 0.4, 0.8, or 1.6 ng TCDD/ml) according to standard TCDD exposure protocol. All of the embryos in each group were examined at 96 hpf for three hallmark endpoints of TCDD developmental toxicity: pericardial edema, reduction in peripheral blood flow, and reduction in lower jaw length.
Pericardial edema was assessed as described above. As in index of peripheral blood flow, red blood cell (RBC) perfusion rates were measured by counting the number of RBCs passing through an intersegmental vein located in the posterior quarter of the trunk during a 7.5 s period using time lapse recording (Carney et al., 2004; Prasch et al., 2003
). To determine the extent of reduced lower jaw growth, the distance between the anterior edge of the lower jaw and the anterior edge of the eye was measured. This lower jaw to eye gap was measured from digital photographs of embryos mounted in lateral orientation. Using Scion Image a straight vertical line was first drawn along the anterior edge of the eye. A second straight horizontal line was then drawn from the anterior edge of the lower jaw to intersect the vertical line. Length of the horizontal line was the measure of lower jaw to eye gap. If the lower jaw had grown to extend in front of the eye the measure was given a negative value. If the lower jaw remained behind the eye the measure was given a positive value.
After all endpoints were measured the embryos were genotyped as described above except that the entire embryo was collected for DNA extraction. For these experiments n = 1 is defined as embryo(s) of the same genotype which came from a single breeding pair and were in the same vehicle or TCDD exposure group. When multiple embryos from the same exposure group had the same genotype, the values obtained for those embryos were averaged to obtain a single value. Embryos were obtained from six distinct adult breeding pairs to obtain a final n value of six for each dose of TCDD.
Whole mount immunolocalization of zfCYP1A. Eggs were collected from six heterozygous breeding pairs and exposed to either vehicle or 1.6 ng/ml TCDD. At 96 hpf embryos were anesthetized, fin clipped for genotyping, and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Tissue-specific expression of zfCYP1A was determined using the monoclonal antibody Mab1-12-3 (Park et al., 1986) as previously described (Andreasen et al., 2002b
; Carney et al., 2004
; Prasch et al., 2003
).
Statistics. For the dose response experiments significance was determined between the three genotypes at each dose of TCDD using a factorial ANOVA followed by the Fisher LSD test. Levene's test for homogeneity of variances was performed before the ANOVA and all data passed. For the zfarnt2-MO experiment data could not be transformed to pass Levene's test and therefore significance was determined for appropriate comparisons using a pair wise t-test assuming unequal variances. All statistical analysis was performed using the Statistica 6.0 software package. Results are presented as mean ± SE; level of significance was p (0.05).
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RESULTS |
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Representative photographs showing the lack of protection by the zfarnt2-MO are also shown (Fig. 1C). The uninjected embryo shows an increase in the size of the pericardial sac after TCDD exposure when compared to the vehicle-treated embryo because edema fluid is beginning to accumulate. This increase in pericardial area is also seen in the TCDD-treated zfarnt2 morphant. The zfarnt2 morphants were observed through 120 hpf (data not shown) and continued to develop all of the overt signs of TCDD toxicity observed in WT embryos (further increase in pericardial edema, yolk sac edema, reduction in peripheral blood flow), although at later times there was likely zfARNT2 protein being expressed because the effectiveness of the morpholino was decreasing.
TCDD Toxicity in zfarnt2/ Mutants
One limitation of morpholino oligonucleotides is that they provide only a transient knockdown of protein levels. Therefore, it was possible that the zfarnt2 morphants developed endpoints of TCDD toxicity because there was still a small amount of zfARNT2 protein present that could act as a dimerization partner for zfAHR2. To determine this, a line of insertional mutants that are null for zfARNT2 was exposed to TCDD and examined for toxicity. RT-PCR performed on mutant embryos confirmed that they lack expression of three isoforms of zfARNT2 mRNA (Prasch et al., 2004). In order to thoroughly examine the response of zfarnt2/ embryos to TCDD, a dose response study was performed in which embryos were exposed to graded doses of TCDD and examined at 96 hpf, a time when three endpoints of TCDD toxicity are clearly manifested in WT embryos: pericardial edema, reduced peripheral blood flow, and reduced growth of the lower jaw.
First, zfarnt2+/+ (WT), zfarnt2+/ (HET), and zfarnt2/ embryos were examined for the formation of pericardial edema after exposure to graded doses of TCDD (Fig. 2A). At the lowest dose (0.2 ng TCDD/ml) there was only a small increase in pericardial edema in the WT and HET embryos. As the concentration of TCDD increased, both the frequency and magnitude of the edema increased so that at the highest concentration of TCDD (1.6 ng/ml) the pericardial area in WT and HET embryos was increased to approximately twice that observed in vehicle-treated embryos. As was seen with the zfarnt2-MO, the zfarnt2/ embryos had no protection against the formation of pericardial edema and showed the same response to TCDD as the WT and HET embryos. Representative photographs illustrating similar levels of pericardial edema formation in TCDD-treated WT and zfarnt2/ embryos are shown (Fig.2B).
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Although no obvious effects on TCDD toxicity were observed in the zfarnt2/ mutants, it remained a possibility that zfAHR2-mediated alterations in gene expression would be prevented in the zfarnt2/ mutants. To determine this, a marker of zfAHR2 pathway activation, zfCYP1A induction, was examined in whole mount embryos using the Mab1-12-3 antibody. In Figure 5 representative photographs of staining are shown for vehicle-treated WT (top panels), TCDD-treated WT (middle panels), and TCDD-treated zfarnt2/ embryos (bottom panels). Panels in column A show zfCYP1A staining in the trunk and panels in column B show staining in lateral views of the head. In WT vehicle-treated embryos little staining was observed in any of the tissues (Fig. 5, top panels). As previously reported (Andreasen et al., 2002b) WT TCDD-treated embryos show zfCYP1A immunofluorescence in the vasculature throughout the body, including the intersegmental arteries and veins (isa, isv) in the trunk, caudal vasculature (cv), and various brain vessels (bv). Staining was also observed in the anal and urinary pores (ap,up), heart (h), the branchial arches (ba), and structures of the lower jaw (lj). TCDD exposure induced a similar pattern of zfCYP1A protein induction in zfarnt2/ embryos (Fig. 5, bottom panels). The TCDD-treated zfarnt2/ embryos showed zfCYP1A induction in the vasculature throughout the body, the anal and urinary pores, the heart, the branchial arches, and the structures of the lower jaw indicating that the zfAHR2 signaling pathway was still functional in embryos lacking zfARNT2.
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DISCUSSION |
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Several mechanisms can be proposed to explain how activation of zfAHR2 by TCDD leads to developmental toxicity in zebrafish. One hypothesis is that toxicity is dependent on XRE binding and alterations in gene expression caused by the zfAHR2-ARNT heterodimer. The results of the current study demonstrate that, although zfARNT2b can function as a dimerization partner for zfAHR2 in vitro, there must be other protein(s) present in the developing embryo which can also function as a dimerization partner for zfAHR2 to cause XRE-dependent transcriptional activation and presumably toxicity upon TCDD exposure. In mammals two distinct arnt genes, arnt1 and arnt2, have been identified and characterized (Drutel et al., 1996; Hirose et al., 1996
; Hoffman et al., 1991
; Li et al., 1994
). Because of sequence similarity in the PAS domains, the ARNT1 and ARNT2 proteins have common dimerization partners and have been shown to have some functional redundancy. ARNT1 and ARNT2 have both been shown to dimerize with Hif1-
and function to regulate hypoxia responsive genes (Keith et al., 2001
; Maltepe et al., 2000
). Similarly, both ARNT1 and ARNT2 have been shown to dimerize with AHR to promote transactivation on XRE containing reporter constructs (Hirose et al., 1996
). Because neither the arnt1 nor arnt2 knockout mouse is viable (Hosoya et al., 2001
; Maltepe et al., 1997
) it has not been possible to thoroughly examine the role that each form of ARNT plays in mediating endpoints of TCDD exposure in mice. However, conditional disruption of ARNT1 in mice causes loss of TCDD dependent gene induction in the liver (Tomita et al., 2000
). It has also been demonstrated that TCDD's effect on lymphocyte development in the mouse is independent of ARNT2 (Laiosa et al., 2002
). This data, along with the expression patterns of ARNT1 and ARNT2 cDNA (Hirose et al., 1996
; Jain et al., 1998
) suggests that ARNT1 may be the physiological dimerization partner for AHR mediating many endpoints of TCDD toxicity in mice.
In addition to what has been found in mammals, evidence from other species also supports the idea that an additional ARNT-like protein is expressed in zebrafish. ARNT1 and ARNT2 homologs have been characterized in xenopus (Bollerot et al., 2001; Rowatt et al., 2003
), partial ARNT1 and ARNT2 sequences have been identified in the recently sequenced Fugu rubripes genome (Rowatt et al., 2003
), and genes encoding ARNT1 have been identified in rainbow trout (Pollenz et al., 1996
) and scup (Stenotomus chrysops) (Powell and Hahn, 2000
). Therefore, it seems likely that similar to what is observed in mammals, amphibians, and other fish species, zebrafish likely express an additional ARNT or ARNT-like protein which can form a functional heterodimer with zfAHR2 to mediate alterations in gene expression upon TCDD exposure. However, if and how this altered gene expression causes toxicity is still unclear. Recently it has been shown that blocking induction of the CYP1A protein in zebrafish provides no protection against TCDD developmental toxicity suggesting that altered regulation of other, currently unknown, genes may be important (Carney et al., 2004
).
An alternate hypothesis for the mechanism of TCDD toxicity is that the effects of zfAHR2 activation are independent of XRE-binding and transcriptional activation. The mammalian AHR has been shown to interact with a variety of cellular factors such as NF-B (Tian et al., 1999
), steroid receptors (Porterfield, 2000
; Safe et al., 1998
), and the retinoblastoma (Rb) protein (Ge and Elferink, 1998
; Puga et al., 2000
). If toxic responses to activated zfAHR2 were due to interactions with other proteins, embryos lacking zfARNT2 would be sensitive to TCDD even if alterations in gene expression through zfAHR2 were prevented. However, the pattern of zfCYP1A induction demonstrates that zfAHR2 pathway activation is intact in all tissues in the zfarnt2/ mutant embryos. Therefore, although interactions of the activated zfAHR2 with other proteins may play a role in causing TCDD developmental toxicity in zebrafish, this cannot be determined from the current study. In mice it has been demonstrated that nuclear localization of the AHR is required to generate TCDD-induced toxic responses suggesting that interactions with cytosolic proteins cannot be involved in causing at least some endpoints of toxicity in mice (Bunger et al., 2003
).
Clearly there is a gap in our present understanding of the AHR signaling pathway in fish. Although only a single form of ARNT has been identified in most fish species (Powell and Hahn, 2000), in zebrafish there must be other ARNT proteins expressed during early development that are functional with zfAHR2. Because differences in the proteins that make up the AHR signaling pathway may be an important factor contributing to differences in species sensitivity to TCDD, future efforts will be aimed at identifying additional zebrafish ARNT proteins to obtain a more complete understanding of how AHR signaling occurs in fish. In addition, the phenotype of the zfarnt2/ mutant fish will be more closely studied to determine what other roles this protein may be playing in the developing zebrafish embryo.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 53705-2222. Fax: (608) 265-3316. E-mail: repeterson{at}pharmacy.wisc.edu.
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