Here I present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, I also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, I found that inhibition of TGFβ signaling blocked OS regeneration.

A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, then functional analysis of network clusters showed enrichment of stress response, signaling pathway and extracellular protease categories could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle.

Additional experiments were performed to identify precursor cells which contribute to OS regeneration. A population of cells expressing the pluripotency marker Piwi ( was observed to accumulate at the distal edge of regenerating OS blastemas, suggesting they may act as multi- or pluri-potent stem cells which contribute to siphon regeneration by differentiating into the functional tissues. Another population of Piwi expressing cells (PECs) has been identified in the transverse vessels of the branchial sac and these cells appear to increase in number following OS amputation. Expansion of the branchial sac (BS) PEC population following amputation could be inhibited by several pharmacological inhibitors of major conserved signaling pathways that I identified in the above-mentioned RNAseq study. This observation supports the hypothesis that signaling occurring within the regenerating OS is capable of inducing expansion of the PEC population in the BS, likely through the action of circulating PECs. However, the function of PECs and contribution to siphon regeneration remains unresolved.