An opioid-like system regulating feeding behavior in C. elegans

We think that this manuscript is of potentially significant interest and we would like to consider a revised version in which one key criticism needs to be addressed, which concerns the expression of nlp-24. The reporter analysis performed so far is insufficient and not interpretable due to the secreted nature of the reporter. We suggest to separate the nlp-24 sequence from the reporter by either an SL2 sequence or a self-cleaving 2A peptide (as demonstrated in the literature). This should reveal the site of expression of nlp-24 without the obscuring effect of secretion of the peptide-fused GFP. The SL2::GFP or 2A::GFP reporter cassette should be inserted into the rescuing nlp-24 genomic piece of DNA. The dependence of reporter gene expression on starvation should be tested.

It is also not clear why the authors do not discuss the previously reported expression of nlp-24 in the ASI neurons (Nathoo et al.). This finding suggests that nlp-24 may operate in an autocrine manner.

Thank you for your suggestion. We tested nlp-24::SL2::GFP as you commented. As you expected we could see ASI expression in nlp-24::SL2::GFP transgenic worms (Figure 7–figure supplement 2C). That suggest that nlp-24 may operate in an autocrine manner. We also added a starvation experiment (Figure 7–figure supplement 3). nlp-24 expression is increased by starvation. We didn’t see a big difference in nlp-24 expression in ASI neurons. Therefore we think that nlp-24 may also operate in an endocrine manner. These new data Figure 7–figure supplement 2C and Figure 7–figure supplement 3 are now discussed in the Results section (in the subsection headed “ASI neurons are required for opioid signaling and NPR-17 in ASI neurons is sufficient for opioid signaling”) of the revised manuscript.

Other minor editorial comments:

1) In Figure 1B the authors should indicate significance the way they did in Figure 1A: use lines to depict the comparisons made.

We did. We added lines in Figure 1B.

2) Some data that are in supplementary appears valuable enough to warrant inclusion of the data in the main figure: Figure 3–figure supplement 2, 3 and Figure 4–figure supplement 1, 2.

Figure 3–figure supplement 2, 3 became Figure 4. We made Figure 5 which included Figure 4C and Figure 4–figure supplement 1. And Figure 4–figure supplement 2 became Figure 9.

3) If existing, data should be added in Figure 6 on the following genotypes: npr-17(-), nlp-24 (overexpression) in wt background and in npr-17 mutant.

Unfortunately, we don’t have that data. We suspect npr-17 will show similar feeding behavior to wild type. And we thought that wild type behavior was already highly activated by starvation, so I assumed that nlp-24(O/E) would also show similar behavior with wild type worms.