Immunohistochemical double-staining for Ah receptor and ARNT in human embryonic palatal shelves

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix-PAS (HLH) proteins involved in transcriptional regulation. Polycyclic aromatic halogenated chemicals, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, bind to the AhR. In the cellular cytoplasm, the AhR exists as a complex with the heat shock protein HSP90 and other small peptides. This complex dissociates following ligand binding and then the ligand-bound AhR binds ARNT. The ligand-AhR-ARNT complex interacts with a specific, nuclear DNA sequence, the dioxin response element (DRE), altering transcription of a regulated gene. Studies in hepatoma cell lines indicate that both proteins are required for regulation of transcription. In this study, AhR and ARNT were localized immunohistochemically in human embryonic palatal cells and specific patterns of expression were seen for each protein. A double-staining protocol revealed that epithelial cells expressed both AhR and ARNT, but in mesenchyme and nasal spine cartilage individual cells were identified which expressed either AhR or ARNT. This heterogeneous pattern may be a means of suppressing transcriptional regulation and also suggests the existence of other, unidentified basic-helix-loop-helix partner(s). The heterogeneous expression pattern may also reflect a complex role for these HLH proteins as transcriptional regulators of embryonic development.