Summary The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the Degenerin/Epithelial (DEG/ENaC) family [1]. PPK is specifically expressed in nociceptive, Class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses [2, 3]. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (a.k.a. CG8546, ppk26) [4]. Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK [5]. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons Balboa∷GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically co-expressing ppk transforms this punctate Balboa∷GFP expression pattern to the uniform distribution observed in its native cell-type. Furthermore, ppk-RNAi in Class IV neurons alters the broad Balboa∷GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus∷PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.

Summary Here, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons we identified 275 nociceptor-enriched genes. We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (ie. boilerman, fire dancer, oven mitt, trivet, thawb and bunker gear) or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.

SUMMARYHere, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, we identified 275 nociceptor-enriched genes.We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman, fire dancer, oven mitt, trivet, thawb, and bunker gear) or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.