Tissue absence initiates regeneration through Follistatin-mediated inhibition of Activin signaling

1) The major findings revolve around two TGFβ family members, follistatin and activin-1, and their role in regeneration, effects on cell proliferation, etc. The story is interesting but fairly simple and straightforward. Therefore, the paper would be improved by significant shortening to concentrate on the major findings. The authors are encouraged to rewrite the paper in shorter format and reduce the number of figures accordingly.

We have reworked the paper as suggested. We reduced the number of figures from seven to five, removing data from multiple experiments from the main figures. Focus is increased on the follistatin RNAi phenotype and we removed roughly 1/3 of the text from the manuscript.

2) The interpretation that activin-1(RNAi) significantly speeds up the normal regenerative response is not convincing. An alternative interpretation is that the mechanisms regulating homeostasis and regeneration may have become uncoupled in activin-1(RNAi). The increased ovo expression in animals 20 days after amputation is intriguing, suggesting that there is an elevated level of tissue homeostasis after a single round of RNAi and regeneration. Are there survival disadvantages for animals that have undergone long term activin-1(RNAi) coupled with amputation? The claim of speeding regeneration is semantic in nature and should be toned down.

We have simplified the manuscript and it now focuses more on the follistatin phenotype. With activin RNAi, we present suppression of the follistatin RNAi phenotype and increased ovo+ progenitor presence at amputation sites. The issue of speeding regeneration will be a good target for future work.

We also investigated the long-term survival of activin-1(RNAi) animals following amputation as suggested. We found that there was not an increased level of animal death over the period observed (∼1 month) in activin-1(RNAi) animals as compared to controls. This is now referenced in the main text.

3) The authors argue that activin is upstream of Smad4 by showing that Smad4(RNAi) causes an increase in apoptosis like activin(RNAi). Since Smad4(RNAi) causes a profound regeneration defect, the apoptotic increase could be secondary to this defect. Furthermore, it has been shown that activin can signal independent of Smad4 (Suzuki et al., Biochemical and biophysical research communications, 2010.) Are there any other upstream R-Smads capable of recapitulating the Activin defect or also rescue the Follistatin phenotype?

We removed the smad4 phenotype from the manuscript, as we now are leaving the apoptosis responses to wounding in activin deficient animals for more detailed future work.

4) The authors show adult homeostatic expression patterns for follistatin and activin, yet they have no apparent phenotype under homeostasis. One concern is that the RNAi conditions may not be effective enough to knock down these homeostatic levels of these factors. This would prevent them from making the conclusion that these factors have no effect normally. Activin especially seems like a good candidate to regulate the neoblast homeostatic niche. In order for the authors to conclude that knockdown of these factors has no effect in homeostasis, they should provide in situ data to demonstrate that the adult expression of these factors is indeed repressed by RNAi to a reasonable degree.

We performed the RNAi controls in homeostatic conditions as suggested. We used quantitative PCR to measure fst expression levels in unamputated fst(RNAi) animals and also in fst(RNAi) fragments 24h after amputation. In these two cases, fst expression was inhibited by RNAi to a similar extent. We performed the same experiment in unamputated activin-1(RNAi) animals and 24h activin-1(RNAi) regenerating fragments. Here as well, expression was inhibited by RNAi to a similar degree in both contexts. These data are now included in Figure 1–figure supplement 3 and Figure 4–figure supplement 3, respectively.

To further demonstrate that the effect of fst RNAi is specific to regeneration, we labeled amputated fst(RNAi) animals that had failed to regenerate with a combination of markers that identify differentiating neurons 20d after amputation. Strikingly, in these animals that produced no blastemas, new neurons were still being made in old tissues. Furthermore, irradiated animals (irradiation kills neoblasts and blocks tissue turnover) die within roughly 2 weeks of irradiation, whereas these amputated animals survive long-term despite failing to regenerate. These data demonstrate that fst(RNAi) animals that fail to produce new tissues at wound sites continue to replace aging cells for tissue turnover, indicating the specificity of requirement for fst under these experimental conditions for regeneration. These data are now included in Figure 1.

5) The generic controls for RNAi seem inadequate. The authors should perform rescue experiments and lack of off-target effects.

We performed and now present quantitative PCR experiments to measure the extent of both fst and activin-1 inhibition by RNAi in unamputated and regenerating animals (see point 4 above).

Furthermore, (in addition to previously presented in situ controls) we measured the extent of fst and activin-1 inhibition in fst; activin-1 double RNAi animals and observed that fst is inhibited at least as strongly in these double RNAi animals as in fst; control double RNAi animals. These data are presented in Figure 4–figure supplement 2.

To address the specificity of fst RNAi, we produced two additional RNAi constructs for inhibiting fst expression, with each construct spanning a non-overlapping portion of the fst gene. Both of these constructs caused a regeneration phenotype. These data are now presented in Figure 1–figure supplement 1.