Uncovering early thyroid hormone signalling events through temperature-mediated activation of molecular memory in the cultured bullfrog tadpole tail fin

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing a crucial role in postembryonic development. The best-studied mechanism of TH response is through modulating gene expression, however TH's involvement in coordinating the early steps in the TH signal transduction pathway is still poorly understood. The American bullfrog, Rana [Lithobates] catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development in the form of metamorphosis of the tadpole into a frog can be experimentally induced by TH exposure. The rate of TH-induced metamorphosis can be modulated by temperature where sufficiently cold temperatures (5 degrees C) completely halt precocious metamorphosis. Interestingly, when premetamorphic tadpoles exposed to exogenous THs at 5 degrees C are shifted to permissive temperatures (24 degrees C), their metamorphic rate exceeds that of TH-exposed tadpoles at the permissive temperature. This suggests that a molecular memory of TH exposure is retained at 5 degrees C even after THs are cleared at this low temperature. However, the molecular memory machinery is poorly understood. Herein we use RNA-seq analysis to identify potential components of the molecular memory in cultured tail fin that allows for the recapitulation of the molecular memory phenomenon. Eighty-one gene transcripts were TH-responsive at 5 degrees C compared to matched controls indicating that the molecular memory is more complex than previously thought. Many of these transcripts encode transcription factors including thyroid hormone-induced B/Zip, thibz, and a novel kruppel-like factor family member, klfX. Actinomycin D and cycloheximide treatment had no effect on their TH induction suggesting that a change in transcription or translation is not required. Rather a change in RNA stability may be a possible mechanism contributing to the molecular memory. The ability to manipulate temperature and TH response in cultured organs provide an exciting opportunity to further elucidate the early TH signalling mechanisms during postembryonic development.