Opposing chemosensory functions of closely related gustatory receptors

Reviewer #1 (Public Review):

Summary:
Ahn and Amrein characterize the expression of members of the Gr28 family of gustatory receptors in taste neurons in the Drosophila melanogaster larva, define the behaviorally-relevant ligands for these receptors, and use chemogenetic experiments to show, strikingly, that different neurons have opposite behavioral responses to the chemogenetic ligand. They go on to show what neurons need to be silenced to lose responses to bitters, and very nicely show what subunits of the Gr28 bitter receptors are necessary and sufficient for responses to bitters. This is a nice piece of work, rigorously carried out, that tackles the neurons and receptors that drive innate responses to tastants in Drosophila larvae.

Strengths:
1. The chemogenetic experiments in Figure 2 are cleanly done with very clear results, and the subsetting in Figure 2B further clarifies the cellular requirements for the behavior.
2. The rescue experiments with different Gr28 subunits in the Gr28 mutant are creative and clear.

Weaknesses:
1. The authors should define early and clearly that expression of Gr28 genes studied in this paper relies not on looking at the endogenous gene but at the expression of Gal4 under the control of enhancers from these loci. The Gal4 drivers are useful and important tools, but the possibility exists that their expression is not 100% congruent with the endogenous expression of these receptors. I would not require a comprehensive validation of the lines by RNA in situ hybridization compared to the Gal4 driver lines but would recommend the disclaimer be made and that the authors are more precise in talking about the expression of the marker rather than the expression of the specific receptor gene.

2. The important chemogenetic behavioral data would benefit from a clearer presentation including a cartoon to explain what the behavior is and how it is scored. Figure 2 is the key figure in this paper and it would be helpful if the figure were re-organized to guide the non-expert reader to the key result. I recommend labeling the positive controls Gr43a as "sweet" and Gr66a as "bitter" and perhaps organize the presentation to have the negative control at the left, then Gr28ba that had no effect, then group Gr28a with Gr43a for positive valence and Gr28bc with Gr66a for negative valence. I'm not sure what the value is of showing both 0.1 mM and 0.5 mM capsaicin, the text does not explain. The experiment in Figure 2B is important but non-experts will not understand what is being done here - can the authors please provide a cartoon like those in Figure 1 showing what cells are being subjected to chemogenetics and how this differs from Figure 2A?

3. The AlphaFold ligand docking in Figure 8 is conducted with Gr28bc monomers, which are unlikely to be the in vivo relevant structure, given that the related OR/ORCO ancestor structures are tetramers. I recommend that this component of the paper either be removed entirely or that the authors redo the in silico work using the AlphaFold-Multimer package reported by Hassabis and Jumper in 2022 https://www.biorxiv.org/content/10.1101/2021.10.04.463034v2. It will be interesting to see what a tetramer structure looks like with the ligand.

Reviewer #2 (Public Review):

This study investigates how genes in the Gr28 family of gustatory receptors function in the taste system of Drosophila larvae. Gr28 genes are intriguing because they have been implicated in taste as well as other functions, such as sensing temperature and ultraviolet light. This study makes several new findings. First, the authors show that four Gr28 genes are expressed in putative taste neurons, and these neurons can be largely divided into subsets that express Gr28a versus Gr28bc. The authors then demonstrate that these two neuronal subsets drive opposing behaviors (attraction versus avoidance) when activated. The avoidance-promoting neurons respond to bitter compounds and are required for bitter avoidance, and Gr28bc and Gr28ba were specifically implicated in bitter detection in these cells. Together, these findings provide insight into the complexity of taste receptor expression and function in Drosophila, even within a single receptor subfamily.

The conclusions are well-supported by the experimental data. Strengths of the paper include the use of precise genetic tools, thorough analyses of expression patterns, carefully validated behavioral assays, and well-controlled functional imaging experiments. The role of Gr28bc neurons is more thoroughly explored than that of Gr28a neurons. However, a previous study from the same lab (Mishra et al., 2018) showed that Gr28a neurons detect RNA and ribose, which are attractive to larvae. Presumably, this is the attractive response that is being recapitulated upon artificial activation of Gr28a neurons.

I only have one technical concern: In Figure 2B, the authors do not show confirmation that using Gr66a-lexA driving lexAop-Gal80 eliminates Gal4-driven gene expression in the desired cells (cells co-expressing Gr66a and Gr28a). This is important for interpreting the behavioral experiment in order to demonstrate that the Gr28a cells mediating attraction are distinct from Gr66a/Gr28bc cells.

Reviewer #3 (Public Review):

a) Important findings
- This study confirms that Gr28 subfamily members are expressed in distinct sets of taste neurons in Drosophila larvae, supporting previous findings (e.g., Kwon et al., 2011).
- Neurons expressing different members of the Gr28 family exhibit distinct behavioral responses when chemically activated with capsaicin.
- Silencing experiments reveal that neurons expressing Gr28bc are necessary for larval avoidance of four bitter compounds, whereas neurons expressing Gr28be are necessary for avoiding lobeline and caffeine.
- Inserting either Gr28ba or Gr28bc into the GR28 mutant line restored larval avoidance of denatonium.
- Calcium imaging experiments show that Gr28ba and Gr28bc are involved in sensing denatonium, while none of the GR28 family members are involved in detecting quinine.

b) Caveats
- The authors did not acknowledge that neurons expressing members of the GR28 family also express other Gr family members, which could potentially contribute to the detection and behavioral responses to the tested bitter compounds.
- Gal4 lines from various studies exhibit varying expression patterns, highlighting the necessity for improved reagents. These findings also suggest the importance of employing different Gal4 lines for each receptor to validate the results of the current study.
- Activating or silencing neurons pertains to the function of the neurons rather than the receptors.
- Inconsistency is observed in the use of different reagents across the experiments. Specifically, all six Gal4 lines were utilized in the Chemical Activation experiments, while only two lines were employed in the silencing experiments.
- The Alphafold structure prediction is exciting but lacks conclusive evidence.