Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development

Abstract

The aryl hydrocarbon receptor (AHR) is commonly known for its role in the adaptive metabolism of xenobiotics and in the toxic events that follow exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). Previously, we have demonstrated that the AHR and its heterodimeric partner, the AHR nuclear translocator (ARNT), play a role in the developmental closure of a hepatic vascular shunt known as the ductus venosus (DV). To investigate the mechanism of DV closure, we generated hypomorphic alleles of the Ahr and Arnt loci. Using these models, we then asked whether this vascular defect could be rescued by receptor activation during late development. By manipulating gestational exposure, the patent DV in AHR or ARNT hypomorphs could be efficiently closed by dioxin exposure as early as embryonic day 12.5 and as late as embryonic day 18.5. These findings define the temporal regulation of receptor activation during normal ontogeny and provide evidence to support the idea that receptor activation and AHR-ARNT heterodimerization are essential for normal vascular development. Taken in the broader context, these data demonstrate that similar AHR signaling steps govern all major aspects of AHR biology.

Fig. 1.

Generation of Ahr^fxneo mice. (A) Schematic diagram of the Ahr cDNA identifying important functional domains including the basic helix–loop–helix (bHLH) domain, the Per-Arnt-Sim (PAS) domain with A and B repeats, and the transactivation domain (TAD). (B) Schematic diagram illustrating the region surrounding the bHLH domain of the murine Ahr locus, the targeting construct, and the resulting hypomorphic Ahr^fxneo allele. Exon numbers reflect known coding exons. To generate the hypomorphic Ahr allele, the Neo gene was inserted upstream of exon 2, and both Neo and exon 2 were flanked with loxp sites. Dotted lines indicate regions of homology used for homologous recombination. Dashed lines indicate fragment sizes detected by the probe after digestion of genomic DNA with BamHI. Solid lines represent the fragment sizes generated by PCR genotyping of the wild-type and hypomorphic alleles using OL4064 as the forward primer and OL4088 as the reverse primer. (C) Schematic diagram of the Arnt^fxneo hypomorphic allele. (D) Southern blot of mouse tail biopsies showing bands of 7.4 kb and 6.0 kb, indicating the presence of the wild-type and mutant alleles, respectively. (E) PCR genotyping of tail biopsies showing bands of 106 bp and 140 bp, indicating the presence of the wild-type and mutant alleles, respectively.

Fig. 2.

The Ahr^fxneo allele is hypomorphic. (A) Western blot analyses showing AHR protein levels in cytosolic extracts from wild-type (wt) and Ahr^fxneo/fxneo (fxneo) mice prepared from liver, kidney, spleen, heart, and lung. Molecular mass reflects the Ahr^d allele. Standards prepared from dilutions of liver extracts from wild-type animals were included to estimate the amount of protein expressed in various tissues. ns, nonspecific protein band identified by reaction with preimmune sera (data not shown). (B) The Ahr^fxneo allele is hypomorphic in function. Liver microsomes from 12-week-old wild-type (+/+), Ahr^fxneo/fxneo, and Ahr^–/– mice were prepared 7 days after treatment with 100 μg/kg dioxin in DMSO or with DMSO alone. Microsomal isolations were incubated with ethoxyresorufin in the presence of NADPH. EROD activity was measured from vehicle- and dioxin-treated animals at an excitation of 510 nm and an emission of 590 nm. Fluorescent values were normalized to total protein levels. The wild-type and Ahr-null groups each contain five animals, whereas the Ahr^fxneo/fxneo groups each contain four animals. White bars, vehicle-treated animals; gray bars, dioxin-treated animals. Error bars indicate standard error. Those groups not sharing a superscript letter differ significantly at P ≤ 0.05.

Fig. 3.

Porto-caval shunting and patent DV in Ahr^fxneo hypomorphs mimic the Ahr-null liver vascular phenotype. Time-lapsed radiographs of the liver vascular system after injection of contrast agent into the portal vein of wild-type (A–C), Ahr^–/– (D–F), Ahr^fxneo/fxneo (G–I), and Arnt^fxneo/fxneo (J–L) mice. Arrows indicate key anatomical features of the liver: PV, portal vein; shIVC, suprahepatic inferior vena cava; BV, branching vessels.

Fig. 4.

Embryonic exposure to dioxin leads to DV closure in Ahr^fxneo and Arnt^fxneo hypomorphs. Timed matings of heterozygous (Ahr^fxneo/+) AHR hypomorph females and homozygous (Ahr^fxneo/fxneo) AHR hypomorph males were performed. At various stages of gestation, pregnant females were given an i.p. injection of 25 μg/kg dioxin or vehicle (DMSO) alone. The DV status of the resulting progeny was assessed by radiography or Trypan blue liver perfusion when the pups were 4–6 weeks old. Similar studies were performed with the ARNT hypomorphs and Ahr-null mice, and injections of pregnant females were performed at E16.5 and E18.5, respectively. Approximately half of the Ahr-null mice in the vehicle group received DMSO at E18.5, while the other half received no treatment during development. (A) Liver vascular architecture of an untreated Ahr^fxneo/fxneo hypomorph (Left) and an Ahr^fxneo/fxneo hypomorph after in utero exposure to dioxin at E14.5 (Right). (B) Frequency of DV closure in adult AHR Ahr^fxneo hypomorphs, Arnt^fxneo hypomorphs, and Ahr-null mice after in utero exposure to either dioxin or vehicle (DMSO) at various stages of gestation.