A microRNA-mRNA Expression Network during Oral Siphon Regeneration in Ciona [microRNA]

Among the Chordates tunicates demonstrate the highest capacity for regeneration, ranging from full-body regeneration in colonial ascidians to appendage regeneration in solitary ascidians. Here we present a parallel study of mRNA and microRNA expression at three stages of oral siphon regeneration in the solitary ascidian Ciona robusta (a.k.a., Ciona intestinalis), and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed (DE) across the course of regeneration, we also identified 57 novel microRNAs, several of which are among the most highly DE. Analysis functional categories identified transcripts related to stress responses and apoptosis enriched at the wound healing stage, various signaling pathways including Wnt and TGF-ß enriched during early regrowth, and negative regulation of extracellular remodeling proteases as enriched in late stage regeneration. Additionally, comprehensive 3’-UTR binding site prediction and probability of conserved targeting for all C. robusta microRNAs (including those identified here) was calculated using TargetScanS. A microRNA-target network was subsequently constructed by calculating Pearson correlation coefficients for all predicted microRNA-mRNA target pairs expressed during regeneration. Network based clustering associated one or more microRNAs and their targets into 22 non-overlapping groups. Functional analysis of mRNA targets in each network cluster showed that enrichment of stress response, signaling pathway and extracellular remodeling categories associated with specific stages could also be associated with specific microRNAs. Finally, predicted targets of the miR-9 network cluster suggest a role in regulating differentiation and proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle. This work represents a significant advance in the prediction of microRNA effects on appendage regeneration and provides a foundation for investigating evolutionary conservation of microRNAs during regeneration in chordates. Overall design: microRNA profiles of Ciona robusta oral siphons during regeneration