Oral Regeneration Is the Default Pathway Triggered by Injury in Hydra
Animals capable of whole-body regeneration can fully restore any tissue lost due to injury. Because different types of injury may require the regeneration of different structures, transcription during whole-body regeneration needs to dynamically adapt to meet the requirements specific to any given injury. However, the regulatory mechanisms responsible for modulating the transcriptional response to injury are poorly understood at the molecular level. We therefore used the highly regenerative cnidarian polyp, Hydra vulgaris, to better understand how transcription in wounded tissue responds to its surrounding tissue environment to enable the restoration of the original body plan. We comprehensively characterized changes in transcript abundance and chromatin accessibility during two types of regeneration, oral and aboral regeneration, and found that the initial transcriptional response to injury was the same regardless of the injury's tissue environment. This initial response included the activation of the canonical Wnt signaling pathway, which specifies oral tissue in cnidarians, likely through the direct upregulation of Wnt ligands by conserved injury responsive bZIP transcription factors. The duration of the injury-induced increase in Wnt signaling depended on the injury's tissue context, as Wnt signaling was activated only transiently during aboral regeneration but showed sustained activation during oral regeneration. Inhibiting TCF, the transcription factor downstream of canonical Wnt signaling, delayed the onset of oral- and aboral-specific transcription genome-wide, suggesting that proper regulation of canonical Wnt signaling is critical for both types of regeneration. Finally, we found that Wnt signaling was also activated by puncture wounds, and that removing pre-existing organizers induced these injuries to undergo ectopic head regeneration. These findings suggest that head regeneration is the default response to injury in Hydra, and that other wound repair outcomes are the result of inhibitory signals produced by the surrounding tissue environment. Furthermore, our work raises the possibility that Wnt signaling may be part of an ancient and conserved wound response program that predates the split of bilaterians and cnidarians over 500 million years ago. Overall design: Hydra vulgaris polyps were transversely bisected at the midpoint of their oral/aboral axis and allowed to regenerate for 0, 3, 8, or 12 hours. During regeneration, Hydra were either left untreated or were exposed to 5µM iCRT14, a TCF/ß-catenin inhibitor. The iCRT14 treatment also included a 2 hour pre-incubation in iCRT14 prior to amputation. Head and foot regenerating tips corresponding to ~1/3 of the total length of each regenerate were then isolated and used to prepare either RNA-seq or ATAC-seq libraries. ATAC-seq replicates were made up of ~15 pooled regenerating tips and RNA-seq replicates were made up of ~30 regenerating tips. Each ATAC-seq treatment group is made up of 3-5 biological replicates. Each RNA-seq treatment group is made up of 3 biological replicates.  There are a total of 71 ATAC-seq samples and 42 RNA-seq samples.