ahr2hu3335 zebrafish, homozygous for a point mutation in ahr2, survive to adulthood and are functional AHR2 knockouts by all measures tested. The premature stop codon in residue 534 is predicted to result in a non-functional protein due to its truncated transactivation domain. Though we cannot exclude the possibility that some biological activity of a potential cryptic protein remains, we saw no evidence to support its presence. Analysis of ahr2hu3335 mRNA levels suggests that the mutant AHR2 transcript is at least partially degraded and the truncated protein may be present only at very low levels, if at all.
adult zebrafish exhibit notable fin and skeletal differences compared to wild type. We also observed a higher background of developmental abnormalities in ahr2hu3335
larvae. These phenotypes may not necessarily be due to the mutation; reduced spawning and small clutch sizes of ahr2hu3335
zebrafish limited the choice of embryos for experiments, whereas large wild type clutches allow for precise selection of high-quality embryos. Studies in both AHR-deficient and AHR ligand-treated mice provide strong evidence of an endogenous role of the receptor in female reproductive physiology. Deficiencies in maintaining pregnancy and surviving lactation have been reported in AHR knockout mice 
, and disruption of AHR function alters ovarian development, folliculogenesis, steroid hormone synthesis, ovulation and possibly reproductive senescence 
. In keeping with AHR knockout mouse models, ahr2hu3335
zebrafish are capable of producing viable embryos, but exhibit decreased reproductive success. It is important to note, however, that other ENU-induced mutations throughout the genome of this fish line could be responsible for observed phenotypic abnormalities. Zebrafish TILLING mutants require multiple outcrosses to reduce undesired mutations to background levels. Because outcrosses of the ahr2hu3335
line were in progress at the time of this study, it is premature to attribute all phenotypic abnormalities observed in ahr2hu3335
homozygotes to the mutation in ahr2
. Decreased reproductive capacity of homozygous mutants, as well as fin and jaw abnormalities may represent interesting models of endogenous AHR function and certainly warrant further investigation if they persist in the mutant line following further outcrosses.
In the present study, we used TCDD as a tool to investigate AHR2 function in the ahr2hu3335
line. We found that ahr2hu3335
embryos were resistant to TCDD-induced developmental toxicity at concentrations that cause severe malformations in ahr2+
embryos treated with 10 nM TCDD showed few signs of morbidity at 120 hpf. Transient AHR2 knockdown delays, but does not prevent, TCDD-induced mortality 
. Therefore it would be interesting to examine longer-term effects of TCDD exposure in future experiments with the ahr2hu3335
The most well-known biomarker of AHR activation is the induction of CYP1A expression. Among the suite of cytochrome P450 metabolizing enzymes in zebrafish, CYP1A, CYP1B1, CYP1C1 and CYP1C2 are elevated in response to AHR agonist exposure 
. In agreement with our developmental toxicity data, no elevation in CYP1A, CYP1C1 or CYP1C2 expression was observed in TCDD-exposed ahr2hu3335
embryos. Taken together, these data support the concept that AHR2 is not functional in this line. The notable, but statistically insignificant, increase in CYP1A expression following TCDD treatment in ahr2hu3335
embryos is likely due to TCDD activation of AHR1B, as further discussed below.
While the dependence of CYP1A activation by TCDD on AHR2 is well-established, studies with PAHs in zebrafish embryos have revealed diverse CYP1A expression patterns dependent on other AHR isoforms 
. This study represents the first time that an in silico-
based modeling approach was utilized to investigate ligand binding by all three receptors. Molecular docking with TCDD predicted that both AHR1B and AHR2, but not AHR1A, would bind TCDD due to substitutions in the binding pocket. In contrast to TCDD, in silico
modeling with leflunomide predicts favorable binding energies for all three zebrafish AHR isoforms. Interestingly, leflunomide docked into AHR1A with a different predicted conformation than in the other two receptors, but with equivalent affinity. This finding is particularly intriguing, as AHR1A is incapable of binding classical AHR ligands 
, is deficient in transactivation activity 
, and therefore was once considered non-functional.
We confirmed the AHR modeling results in vivo using CYP1A protein expression as a biomarker of AHR activation. In keeping with our mRNA expression and in silico modeling studies, TCDD-exposed ahr2hu3335 larvae were largely devoid of CYP1A protein expression observed in TCDD-exposed ahr2+ larvae. Leflunomide also induces strong vascular CYP1A protein expression in ahr2+ larvae, but unlike with TCDD, the ahr2hu3335 embryos exhibited striking leflunomide-induced CYP1A expression in the liver. This finding is in agreement with the modeling results. To tease apart AHR isoform-dependence of the residual CYP1A expression, we transiently knocked down the receptors individually and in combination in ahr2hu3335 larvae. We found AHR1B-dependent vascular induction and AHR1A-dependent liver induction of CYP1A expression. Knockdown of AHR1A and AHR1B in combination prevented all CYP1A induction. Taken together, these data suggest that, contrary to previous observations with TCDD, all three AHR isoforms are involved in leflunomide-induced CYP1A expression in zebrafish larvae.
These data demonstrate that there are concrete differences in ligand binding activity of the zebrafish AHRs, and that AHR1A is not a pseudogene as previously proposed, but rather has affinity for different ligand structures. While residual CYP1A expression has been observed in TCDD-treated AHR2-morphants, it was faint and vascular in nature, attributable to incomplete knockdown 
. Our immunohistochemical results with the ahr2hu3335
line suggest that mild vascular expression of CYP1A is induced via AHR1B, and can be effectively knocked down to background with morpholino injection. AHR1A-dependent CYP1A expression is seemingly incongruous with previous investigation of AHR1A function in vitro
, but the lack of a known AHR1A ligand limited previous efforts. The AHR1A-dependent CYP1A expression pattern we observed here is consistent with the reported AHR1A mRNA expression in the liver 
Putative AHR1A ligands could be identified with further in silico
modeling; work by Incardona and colleagues also offers clues with several PAHs that induce CYP1A expression independently of AHR2 
. Pyrene induced liver expression of CYP1A in an AHR1A-dependent manner 
, and more recently retene-induced CYP1A expression was shown to be incompletely dependent on AHR2 
. Here, we offer further evidence that AHR1A is a functional receptor in vivo
, though the transactivation requirements for this receptor remain to be elucidated. In vitro
data with AHR chimera proteins suggest that transactivation requirements of AHR1A differ from those of AHR2 
The presence of three apparently functional aryl hydrocarbon receptors in zebrafish raises several interesting questions: How do these receptors differ? What functions have led to their evolutionary conservation? And to what extent do the AHR1 receptors need to be considered in toxicological studies in zebrafish? While the presence of multiple AHRs certainly complicates study of receptor function in fish, subfunction partitioning among isoforms presents a unique opportunity to unravel the many physiological functions of the AHR that are conserved among vertebrates 
. As summarized in , the studies presented here add to a body of research demonstrating significant differences in receptor expression, ligand binding, and mRNA induction activity. With respect to transcript localization, AHR2 is widely distributed through most organs investigated in adult zebrafish, while AHR1A is mainly expressed in the liver, and to a lesser extent in the heart, kidney and swim bladder 
. AHR1B expression has yet to be fully characterized, but our CYP1A IHC results suggest that the isoform is widely distributed, but is expressed at much lower levels than AHR2. The subfunction partitioning of these receptors is not strictly locational. Overlapping expression of AHR2 and AHR1A has been previously described, and we also noted overlap in AHR2- and AHR1-dependent CYP1A expression patterns 
. A cell or tissue-level analysis may reveal more subtle localization differences, as has been implied in differential PAH-induced CYP1A patterns in endocardial and myocardial tissue 
. Little is yet known about the endogenous function of these receptors and their downstream transcriptional targets. If expression of AHR1A and AHR1B is limited, it may be difficult to detect significant changes in their transcriptional targets in whole embryo homogenate. As we have shown here, however, the ahr2hu3335
line will ease the study of the other two receptors by removing the overpowering transcriptional changes induced through AHR2. The three receptors together present an intriguing opportunity to unravel multiple regulatory functions that may be conserved in the mammalian AHR.
Summary of zebrafish AHR ligand binding, activity and expression.
This is the first report of CYP1A induction dependent on all three of the zebrafish AHRs. Toxicity mediated through the AHR1 receptors, however, has not, as of yet, been documented. Pyrene-induced liver toxicity and pericardial edema were reduced with AHR1A knockdown, but AHR2 knockdown prevented the majority of the chemical's developmental effects 
. In the case of TCDD and other similarly-structured HAHs, the small binding pocket of AHR1A prevents it from having a role in ligand-induced toxicological effects. AHR1A and AHR1B receptors may hold little importance in toxicological studies with these compounds. Indeed the studies presented here support the large body of previous research indicating that TCDD-induced early life stage toxicity is mediated through AHR2. Though some CYP1A and other downstream target induction may occur via AHR1B, any developmental abnormalities caused by this pathway are more subtle than those investigated to date. The possibility remains, however, that AHR1B may play a role in later life stage impacts of TCDD. These data warrant further investigation of the AHR isoforms with structurally diverse, less-well studied compounds. Ultimately, further bioinformatic and modeling efforts with zebrafish and mammalian AHRs could help determine the best model for human AHR activity, taking into account both ligand binding and receptor expression characteristics.
This was the first time that all three AHR isoforms were knocked down in developing zebrafish. Our findings suggest that, consistent with mammalian literature, AHR function is not required to complete development 
. Without full histological evaluations of the AHR1Amo/AHR1Bmo/ahr2hu3335
larvae at 120hpf, we cannot exclude non-lethal malformations, particularly hepatic abnormalities, which have been reported in AHR knockout mice 
. It may not be possible to fully answer the question of whether the AHR paralogues are required for hepatic development in zebrafish with the tools employed here, as the liver undergoes significant development after 72hpf, when morpholino efficacy is in decline. We therefore present the ahr2hu3335
line as a valuable resource available to the zebrafish research community, and suggest that development of both AHR1A and AHR1B (already requested by the research community) mutant lines would further extend the power of this model for investigating both the endogenous and ligand-mediated roles of the AHR in developing vertebrates.