Connecting the Transcriptome, microRNAs, and the Proteome in Tail Regeneration of the Green Anole Lizard, Anolis carolinensis

As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anole lizards regenerate their tails through a process that involves differential expression of hundreds of genes, including those related to wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. This complex process involves formation of new hyaline cartilage, spinal cord, muscle, vasculature, and skin. To investigate gene expression in regenerating tissue, we performed whole transcriptome, microRNA transcriptome, and proteome analysis of regenerating tail tip and base, skeletal muscle, and brain in Anolis carolinensis. We identified 350 novel and 196 known microRNA precursor families and over 2,000 proteins. Differentially expressed microRNAs in the regenerating tail include those previously reported to regulate regeneration and stem cell proliferation, as well as novel microRNAs. MicroRNA target prediction algorithms often have varying target lists, and their false positive and false negative rates are difficult to assess. Therefore, we used coordinated mRNA and microRNA expression patterns along with proteomic data to confirm the presence of predicted targets of down-regulated microRNAs in the regenerating tail. These findings identify microRNAs and their targets that play a coordinated role in regulating the regenerative process.