Innexin2 Regulates Limb Regeneration in Procambarus clarkii: Insights from Transcriptome and DNA Methylation Analyses

Limb regeneration is one of the most fascinating phenomena in life sciences. Although Procambarus clarkii exhibits remarkable regenerative capabilities, the molecular mechanisms underlying its limb regeneration remain poorly understood. To address this, we investigated the transcriptome data and DNA methylation levels during the limb regeneration process in Procambarus clarkii. We employed fluorescence in situ hybridization (FISH), RNA interference (RNAi), and mTOR inhibitors to validate the impact of key genes and pathways. Our findings from the weighted gene co-expression network analysis (WGCNA) suggest that Innexin2 may be associated with limb regeneration. Whole-genome bisulfite sequencing (WGBS) analysis revealed changes in methylation levels between the 0 dpa and 15 dpa stages of limb regeneration, while KEGG enrichment analysis highlighted the mTOR signaling pathway. Furthermore, both Torin1 and Rapamycin, as mTOR inhibitors, demonstrated that this pathway is required for limb regeneration. Overall, our study provides insights into the molecular regulatory mechanisms underlying limb regeneration in the Procambarus clarkii, offering valuable data support for future research in regeneration biology.