A novel culture method for Rana [Lithobates] catesbeiana olfactory epithelium reveals differential effects of transcriptional and translational inhibitors on the temperature-dependent induction of thyroid hormone-responsive transcripts.

Amphibian organ culture is invaluable for gaining insight into thyroid hormone (TH) action. The olfactory epithelium (OE) of Rana (aka Aquarana or Lithobates) catesbeiana tadpoles is a highly TH-sensitive tissue that regulates essential survival behaviors and undergoes drastic remodeling during TH-dependent metamorphosis. These features make it a valuable bioindicator for normal and disrupted TH action. To our knowledge, we developed and validated the first successful ex vivo culture method for anuran tadpole olfactory epithelium. Pairs of OE-containing principal cavities from premetamorphic tadpoles were excised, each cavity cut in half, and exposed to 10 nM 3,5,3'-triiodothyronine (T3) or solvent control at 24 °C for 24 h. Each half cavity showed consistent enrichment of the olfactory marker omp compared to back skin. Robust and reproducible TH responses were observed in individual, unpooled cultured olfactory epithelium (C-OE) as measured by reverse transcription-quantitative polymerase chain reaction of TH-responsive transcripts thra, thrb, and thibz. No spatial variation in hormone sensitivity within the principal cavity was observed supporting the use of repeated-measures statistics while capturing biological variation between animals. To probe early TH signaling events, C-OE and cultured back skin (C-skin) were treated with T3 in the presence of transcriptional and translational inhibitors at 24 °C or 4 °C. The cold condition represents a metamorphic stasis period during which the TH signal is initiated, but metamorphosis does not proceed. Notably, TH-induction of thibz at 4 °C was unaffected by transcriptional inhibition in both tissues, suggesting that mechanisms other than RNA synthesis may be driving the increase in transcript abundance.