Neuroendocrine Coupling of Interoceptive Bacteria-Derived Cues to Foraging Behavior in C. elegans

Reviewer #1 (Public Review):

Summary:
Here, Boor et al focus on the regulation of daf-7 transcription in the ASJ chemosensory neurons, which has previously been shown to be sensitive to a variety of external and internal signals. Interestingly, they find that soluble (but not volatile) signals released by food activate daf-7 expression in ASJ, but that this is counteracted by signals from the ASIC channels del-3 and del-7, previously shown to detect the ingestion of food in the pharynx. Importantly, the authors find that ASJ-derived daf-7 can promote exploration, suggesting a feedback loop that influences locomotor states to promote feeding behavior. They also implicate signals known to regulate exploratory behavior (the neuropeptide receptor PDFR-1 and the neuromodulator serotonin) in the regulation of daf-7 expression in ASJ. Additionally, they identify a novel role for a pathway previously implicated in C. elegans sensory behavior, HEN-1/SCD-2, in the regulation of daf-7 in ASJ, suggesting that the SCD-2 homolog ALK may have a conserved role in feeding and metabolism.

Strengths:
The studies reported here, particularly the quantitation of gene expression and the careful behavioral analysis, are rigorously done and interpreted appropriately. The results suggest that, with respect to food, DAF-7 expression encodes a state of "unmet need" - the availability of nearby food to animals that are not currently eating. This is an interesting finding that reinforces and extends our understanding of the neurobiological significance of this important signaling pathway. The identification of a role for ASJ-derived daf-7 in motor behavior is a valuable advance, as is the finding that SCD-2 acts in the AIA interneurons to influence daf-7 expression in ASJ.

Weaknesses:
A limitation of the work is that some mechanistic relationships between the identified signaling pathways are not carefully examined, but this provides interesting opportunities for future work. A minor weakness concerns the experiment in which daf-7 is conditionally deleted from ASJ. This is an ideal approach for probing the function of daf-7, but these experiments seem to be carried out in the well-fed, on-food condition in which control animals should express little or no daf-7 in ASJ. Thus, the experimental design does not allow an assessment of the role of daf-7 under conditions in which its expression is activated (e.g., in animals exposed to un-ingestible food). An additional minor issue concerns the interpretation of the scd-2 experiments. The authors' findings do support a role for scd-2 signaling in the activation of daf-7 expression by un-ingestible food, but the data also suggest that scd-2 signaling is not essential for this effect, as there is still an effect in scd-2 mutants (Figure 4B).

Reviewer #2 (Public Review):

Summary:
In this work, Boor and colleagues explored the role of microbial food cues in the regulation of neuroendocrine-controlled foraging behavior. Consistent with previous reports, the authors find that C. elegans foraging behavior is regulated by the neuroendocrine TGFβ ligand encoded by daf-7. In addition to its known role in the neuroendocrine/sensory ASI neurons, Boot and colleagues show that daf-7 expression is dynamically regulated in the ASJ sensory neurons by microbial food cues - and that this regulation is important for exploration/exploitation balance during foraging. They identify at least two independent pathways by which microbial cues regulate daf-7 expression in ASJ: a likely gustatory pathway that promotes daf-7 expression and an opposing interoceptive pathway, also likely chemosensory in nature but which requires microbial ingestion to inhibit daf-7 expression. Two neuroendocrine pathways known to regulate foraging (serotonin and PDF-1) appear to act at least in part via daf-7 induction. They further identify a novel role for the C. elegans ALK orthologue encoded by scd-2, which acts in interneurons to regulate daf-7 expression and foraging behavior. These results together imply that distinct cues from microbial food are used to regulate the balance between exploration and exploitation via conserved signaling pathways.

Strengths:
The findings that gustatory and interoceptive inputs into foraging behavior are separable and opposing are novel and interesting, which they have shown clearly in Figure 1. It is also clear from their results that removal of the interoceptive cue (via transfer to non-digestible food) results in rapid induction of daf-7::gfp in ASJ, and that ASJ plays an important role in the regulation of foraging behavior.

The role of the hen-1/scd-2 pathway in mediating the effects of ingested food is also compelling and well-interpreted. The use of precise gain-of-function alleles further supports their conclusions. This implies that important elements of this food-sensing pathway may be conserved in mammals.

Weaknesses:
What is less clear to me from the work at this stage is how the gustatory input fits into this picture and to what extent can it be strongly concluded that the daf-7-regulating pathways that they have identified (del-3/7, 5-HT, PDFR-1, scd-2) act via the interoceptive pathway as opposed to the gustatory pathway. It follows from the work of the Flavell lab that del-3/7 likely acts via the interoceptive pathway in this context as well but this isn't shown directly - e.g. comparing the effects of aztreonam-treated bacteria and complete food removal to controls. The roles of 5-HT and PDFR-1 are even a bit less clear. Are the authors proposing that these are entirely parallel pathways? This could be explained in better detail.

It would also be helpful to elaborate more on why the identified transcriptional positive feedback loop is predicted to extend roaming state duration - as opposed to some other mechanism of increasing roaming such as increased probability of roaming state initiation. This doesn't seem self-evident to me. Related to this point is the somewhat confusing conclusion that the effects of tph-1 and pdfr-1 mutations on daf-7 expression are due to changes in ingestion during roaming/dwelling. From my understanding (e.g. Cermak et al., 2020), pharyngeal pumping rate does not reliably decrease during roaming - so is it clear that there are in fact lower rates of ingestion during roaming in their experiments? If so, why does increased roaming (via tph-1 mutation) result in further increases in daf-7 expression in animals fed aztreonam-treated food (Fig 3B)? Alternatively, there could be a direct signaling connection between the 5-HT/PDFR-1 pathways and daf-7 expression which could be acknowledged or explained.

Reviewer #3 (Public Review):

Summary:
In this interesting study, the authors examine the function of a C. elegans neuroendocrine TGF-beta ligand DAF-7 in regulating foraging movement in response to signals of food and ingestion. Building on their previous findings that demonstrate the critical role of daf-7 in a sensory neuron ASJ in behavioral response to pathogenic P. aeruginosa PA14 bacteria and different foraging behavior between hermaphrodite and male worms, the authors show, here, that ingestion of E. coli OP50, a common food for the worms, suppresses ASJ expression of daf-7 and secreted water-soluble cues of OP50 increases it. They further showed that the level of daf-7 expression in ASJ is positively associated with a higher level of roaming/exploration movement. Furthermore, the authors identify that a C. elegans ortholog of Anaplastic Lymphoma Kinase, scd-2, functions in an interneuron AIA to regulate ASJ expression of daf-7 in response to food ingestion and related cues. These findings place the DAF-7 TGF-beta ligand in the intersection of environmental food conditions, food intake, and food-searching behavior to provide insights into how orchestrated neural functions and behaviors are generated under various internal and external conditions.

Strengths:
The study addresses an important question that appeals to a wide readership. The findings are demonstrated by generally strong results from carefully designed experiments.

Weaknesses:
However, a few questions remain to provide a complete picture of the regulatory pathways and some analyses need to be strengthened. Specifically,

1. The authors show that diffusible cues of bacteria OP50 increase daf-7 expression in ASJ which is suppressed by ingestible food. Their results on del-3 and del-7 suggest that NSM neuron suppresses daf-7 ASJ expression. What sensory neurons respond to bacterial diffusible cues to increase daf-7 expression of ASJ? Since ASJ is able to respond to some bacterial metabolites, does it directly regulate daf-7 expression in response to diffusible cues of OP50 or does it depend on neurotransmission for the regulation? Some level of exploration in this question would provide more insights into the regulatory network of daf-7.

2. The results including those in Figure 2 strongly support that daf-7 in ASJ is required for roaming. Meanwhile, authors also observe increased daf-7 expression in ASJ under several conditions, such as non-ingestible food. Does non-ingestible food induce more roaming? It would complete the regulatory loop by testing whether a higher (than wild type) level of daf-7 in ASJ could further increase roaming. The results in pdf-1 and scd-2 gain-of-function alleles support more ASJ leads to more roaming, but the effect of these gain-of-function alleles may not be ASJ-specific and it would be interesting to know whether ASJ-specific increase of daf-7 leads to a higher level of roaming. In my opinion, either outcome would be informative and strengthen our understanding of the critical function of daf-7 in ASJ demonstrated here.

3. The analyses in Figure 4 cannot fully support "We further observed that the magnitude of upregulation of daf-7 expression in the ASJ neurons when animals were moved from ingestible food to non-ingestible food was reduced in scd-2(syb2455) to levels only about one-fourth of those seen in wild-type animals (Figure 4D)...", because the authors tested and found the difference in daf-7 expression between ingestible and non-ingestible food conditions in both wild type and the mutant worms. The authors did not analyze whether the induction was different between wild type and mutant. Under the ingestible food condition, ASJ expression of daf-7 already looks different in scd-2(syb2455).

4. The authors used unpaired two-tailed t-tests for all the statistical analyses, including when there are multiple groups of data and more than one treatment. In their previous study Meisel et al 2014, the authors used one-way ANOVA, followed by Dunnett's or Tukey's multiple comparison test when they analyzed daf-7 expression or lawn leaving in different mutants or under different bacterial conditions. It is not clear why a two-tailed t-test was used in similar analyses in this study.