W. Daniel Tracey

painless, a Drosophila Gene Essential for Nociception

We describe a paradigm for nociception in Drosophila. In response to the touch of a probe heated above 38 degrees C, Drosophila larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. In a genetic screen for mutants defective in this noxious heat response, we identified the painless gene. Recordings from wild-type larval nerves identified neurons that initiated strong spiking above 38 degrees C, and this activity was absent in the painless mutant. The painless mRNA encodes a protein of the transient receptor potential ion channel family. Painless is required for both thermal and mechanical nociception, but not for sensing light touch. painless is expressed in peripheral neurons that extend multiple branched dendrites beneath the larval epidermis, similar to vertebrate pain receptors. An antibody to Painless binds to localized dendritic structures that we hypothesize are involved in nociceptive signaling.

Pickpocket Is a DEG/ENaC Protein Required for Mechanical Nociception in Drosophila Larvae

Highly branched class IV multidendritic sensory neurons of the Drosophila larva function as polymodal nociceptors that are necessary for behavioral responses to noxious heat (>39 degrees C) or noxious mechanical (>30 mN) stimuli. However, the molecular mechanisms that allow these cells to detect both heat and force are unknown. Here, we report that the pickpocket (ppk) gene, which encodes a Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunit, is required for mechanical nociception but not thermal nociception in these sensory cells. Larvae mutant for pickpocket show greatly reduced nociception behaviors in response to harsh mechanical stimuli. However, pickpocket mutants display normal behavioral responses to gentle touch. Tissue-specific knockdown of pickpocket in nociceptors phenocopies the mechanical nociception impairment without causing defects in thermal nociception behavior. Finally, optogenetically triggered nociception behavior is unaffected by pickpocket RNAi, which indicates that ppk is not generally required for the excitability of the nociceptors. Interestingly, DEG/ENaCs are known to play a critical role in detecting gentle touch stimuli in Caenorhabditis elegans and have also been implicated in some aspects of harsh touch sensation in mammals. Our results suggest that neurons that detect harsh touch in Drosophila utilize similar mechanosensory molecules.

Dendritic Filopodia, Ripped Pocket, NOMPC, and NMDARs Contribute to the Sense of Touch in Drosophila Larvae

BACKGROUND Among the Aristotelian senses, the subcellular and molecular mechanisms involved in the sense of touch are the most poorly understood. RESULTS We demonstrate that specialized sensory neurons, the class II and class III multidendritic (md) neurons, are gentle touch sensors of Drosophila larvae. Genetic silencing of these cells significantly impairs gentle touch responses, optogenetic activation of these cells triggers behavioral touch-like responses, and optical recordings from these neurons show that they respond to force. The class III neurons possess highly dynamic dendritic protrusions rich in F-actin. Genetic manipulations that alter actin dynamics indicate that the actin-rich protrusions (termed sensory filopodia) on the class III neurons are required for behavioral sensitivity to gentle touch. Through a genome-wide RNAi screen of ion channels, we identified Ripped Pocket (rpk), No Mechanoreceptor Potential C (nompC), and NMDA Receptors 1 and 2 (Nmdars) as playing critical roles in both behavioral responses to touch and in the formation of the actin-rich sensory filopodia. Consistent with this requirement, reporters for rpk and nompC show expression in the class III neurons. A genetic null allele of rpk confirms its critical role in touch responses. CONCLUSIONS Output from class II and class III md neurons of the Drosophila larvae is necessary and sufficient for eliciting behavioral touch responses. These cells show physiological responses to force. Ion channels in several force-sensing gene families are required for behavioral sensitivity to touch and for the formation of the actin-rich sensory filopodia.

Distinct in vivo requirements for establishment versus maintenance of transcriptional repression

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. Here we show, by using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos, which indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression.

Egg Laying Decisions in Drosophila Are Consistent with Foraging Costs of Larval Progeny

Decision-making is defined as selection amongst options based on their utility, in a flexible and context-dependent manner. Oviposition site selection by the female fly, Drosophila melanogaster, has been suggested to be a simple and genetically tractable model for understanding the biological mechanisms that implement decisions [1]. Paradoxically, female Drosophila have been found to avoid oviposition on sugar which contrasts with known Drosophila feeding preferences [1]. Here we demonstrate that female Drosophila prefer egg laying on sugar, but this preference is sensitive to the size of the egg laying substrate. With larger experimental substrates, females preferred to lay eggs directly on sugar containing media over other (plain, bitter or salty) media. This was in contrast to smaller substrates with closely spaced choices where females preferred non-sweetened media. We show that in small egg laying chambers newly hatched first instar larvae are able to migrate along a diffusion gradient to the sugar side. In contrast, in contexts where females preferred egg laying directly on sugar, larvae were unable to migrate to find the sucrose if released on the sugar free side of the chamber. Thus, where larval foraging costs are high, female Drosophila choose to lay their eggs directly upon the nutritious sugar substrate. Our results offer a powerful model for female decision-making.

The Ankyrin Repeat Domain of the TRPA Protein Painless Is Important for Thermal Nociception but Not Mechanical Nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.

Larval Defense against Attack from Parasitoid Wasps Requires Nociceptive Neurons

Parasitoid wasps are a fierce predator of Drosophila larvae. Female Leptopilina boulardi (LB) wasps use a sharp ovipositor to inject eggs into the bodies of Drosophila melanogaster larvae. The wasp then eats the Drosophila larva alive from the inside, and an adult wasp ecloses from the Drosophila pupal case instead of a fly. However, the Drosophila larvae are not defenseless as they may resist the attack of the wasps through somatosensory-triggered behavioral responses. Here we describe the full range of behaviors performed by the larval prey in immediate response to attacks by the wasps. Our results suggest that Drosophila larvae primarily sense the wasps using their mechanosensory systems. The range of behavioral responses included both “gentle touch” like responses as well as nociceptive responses. We found that the precise larval response depended on both the somatotopic location of the attack, and whether or not the larval cuticle was successfully penetrated during the course of the attack. Interestingly, nociceptive responses are more likely to be triggered by attacks in which the cuticle had been successfully penetrated by the wasp. Finally, we found that the class IV neurons, which are necessary for mechanical nociception, were also necessary for a nociceptive response to wasp attacks. Thus, the class IV neurons allow for a nociceptive behavioral response to a naturally occurring predator of Drosophila.

Molecules and Mechanisms of Mechanotransduction

To many animals, including humans, some of the best things in life are mechanical. Not only courtship and sex but also simple movements such as walking depend on the ability to transform mechanical energy in the form of touch, sound, and muscle tension into ionic currents. This ability is also

Balboa Binds to Pickpocket In Vivo and Is Required for Mechanical Nociception in Drosophila Larvae

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. 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 (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. 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 coexpressing 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.

Alternatives to Mammalian Pain Models 2: Using Drosophila to Identify Novel Genes Involved in Nociception

Identification of the molecules involved in nociception is fundamental to our understanding of pain. Drosophila, with its short generation time, powerful genetics and capacity for rapid, genome-wide mutagenesis, represents an ideal invertebrate model organism to dissect nociception. The fly has already been used to identify factors that are involved in other sensory systems such as vision, chemosensation, and audition. Thus, the tiny fruit fly is a viable alternative to mammalian model organisms. Here we present a brief primer on techniques used in screening for thermal and/or mechanical nociception mutants using Drosophila.