The transcriptomics and differential gene expression of freezing and thawing in a freeze-tolerant vertebrate

Total body freezing is a highly specialized and rare winter survival strategy that is undertaken by few vertebrates. The freeze-tolerant wood frog ( Rana sylvatica ) can survive freezing of up to 70% of its body water and remain frozen for months. The full set of molecular mechanisms directing the complex, quick freeze-thaw process in wood frogs remains unknown. Here, we use RNA-Seq data to investigate the gene expression profile in livers of wood frogs from Connecticut, USA as they experience a single freeze-thaw episode. We take a fine time-scale approach and investigate gene expression as frogs enter, exit, and recover from freezing. We found 887 differentially expressed genes across experimental conditions. The highest number of differentially expressed genes corresponded to the time point at which animals reached a full-freezing state, a pattern indicative of the late onset and rapid nature of the freezing response. Upon full-freezing, processes related to metabolism as well as membrane and cell integrity were highly regulated. This pattern showcases the need to depress metabolic rate, while simultaneously mobilize and regulate transport of cryoprotectant molecules into cells. Upon thawing, animals actively recovered by increasing expression of genes related to immunity, heat shock responses, and antioxidant defense. Our findings provide an overview of gene expression patterns in a freeze-tolerant vertebrate, and evidence for the response being unique to each time point of the freeze-thaw process. The immediate and substantial regulation of genes at the onset of freezing is consistent with the freeze initiation hypothesis of freeze tolerance. We identify the genes and processes involved and suggest candidate genes for further study of their exact molecular mechanisms. Knowledge of recovery responses after thawing will be especially beneficial to advancing cryopreservation methods of organs and cells.