William Daniel Tracey

Duke University Medical Center | DUMC · Department of Anesthesiology

Evolutionary arms races between predator and prey can lead to extremely specific and effective defense mechanisms. Such defenses include venoms that deter predators by targeting nociceptive (pain-sensing) pathways. Through co-evolution, venom toxins can become extremely efficient modulators of their molecular targets. The venom of velvet ants (Hyme...

Nociception in fruit fly ( Drosophila melanogaster ) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva...

The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious fo...

Nociception is the sensory modality by which animals sense stimuli associated with injury or potential tissue damage. When Drosophila larvae encounter a noxious thermal, chemical, or mechanical stimulus, they perform a stereotyped rolling behavior. These noxious stimuli are detected by polymodal nociceptor neurons that tile the larval epidermis. Al...

In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have...

When undisturbed, Drosophila larvae move forward through their environment with sweeping waves of caudal to rostral muscle contraction [1, 2]. In stark contrast, nociceptive sensory stimuli (such as attacks by parasitoid wasps) trigger the larvae to roll across the substrate by corkscrewing around the long body axis [3, 4]. While studies have descr...

The detection and processing of noxious sensory input depends on the proper growth and function of nociceptor sensory neurons in the peripheral nervous system. In Drosophila melanogaster , the class IV (cIV) multidendritic dendritic arborization (md-da) neurons detect noxious stimuli through their highly branched dendrites that innervate the epider...

Female mosquitos require a specific ion-channel protein to sense the presence of fresh water in which they can lay their eggs.

Inhibition of nociceptor activity is important for the prevention of spontaneous pain and hyperalgesia. To identify the critical K⁺ channels that regulate nociceptor excitability, we performed a forward genetic screen using a Drosophila larval nociception paradigm. Knockdown of three K⁺ channel loci, the small conductance calcium-activated potassiu...

A comprehensive understanding of the molecular machinery important for nociception is essential to improving the treatment of pain. Here, we show that the BMP signaling pathway regulates nociception downstream of the E3 ubiquitin ligase highwire (hiw). hiw loss of function in nociceptors caused antagonistic and pleiotropic phenotypes with simultane...

Multiple hiw alleles show insensitivity in thermal nociception. The hiwND8, hiwΔN and hiwΔC mutants all showed a longer latency to respond to a 46°C thermal probe than controls (w1118). n = 63, 46, 53 and 39 respectively. *** p < 0.001 (Steel’s test versus control). All error bars represent standard error. (PDF)

Overexpression of wnd leads to an up-regulation of nuclear pMad. (A-C) Representative pictures of pMad immunoreactivity in nociceptors of controls (ppk-GAL4 UAS-mCD8::GFP/+), wild-type wnd OE (ppk-GAL4 UAS-mCD8::GFP/UAS-wnd) and kinase-dead wnd (wndKD) OE (ppk-GAL4 UAS-mCD8::GFP/UAS-wndKD). (A’-C’) Split images for pMad signals. Scale bars represen...

All numerical data. This spreadsheet contains all numerical data that underlie figures, graphs and summary statistics presented in this article. (XLSX)

Phenotypes of UAS transgene were GAL4 driver-dependent. UAS-hiw△Ring and UAS-tkvQD were crossed to w1118 and tested in thermal nociception. The UAS strains that caused thermal hypersensitivity when crossed to ppk-GAL4 did not show significant hypersensitive phenotypes when crossed to w1118 in comparison to controls (ppk-GAL4 x w1118). n = 95, 70 an...

Expressing mad1 alone or hiw△Ring and mad1 did not cause thermal insensitivity. In comparison to controls (ppk-GAL4 x w1118, n = 115), animals expressing mad1 alone in nociceptors (ppk-GAL4 x UAS-mad1, n = 63, p > 0.99 versus control) and ones expressing hiwΔRing and mad in nociceptors (ppk-GAL4 UAS-hiwΔRing x UAS-mad1, n = 61, p > 0.14 versus cont...

Incubation at 30°C causes insensitivity to noxious heat. (A and B) Control animals (ppk-GAL4 UAS-mCD8::GFP; tub-GAL80ts x w1118) and tkvQD animals (ppk-GAL4 UAS-mCD8::GFP; tub-GAL80ts x UAS-tkvQD) with or without 22 hour heat induction were tested in thermal nociception. (A) After the 22 hour incubation at 30°C, both genotypes showed delayed respon...

Sexual differences in the responsiveness to optogenetic nociceptor stimulations. In control animals (ppk-GAL4 UAS-ChR2::YFP x w1118), males showed a lower responsiveness to optogenetic nociceptor stimulations compared to females. n = 191 and 211. All error bars represent standard error. (PDF)

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a populati...

Summary table of behavioral responses to dose-dependent optogenetic activation. Number of animals displaying bending, rolling, pausing, or crawling during optogenetic activation of ppk1.9-Gal4 or 412-Gal4 neurons across different levels of activation.

Summary table of graph data and statistical testing for functional imaging and nociceptive experiments.Genotypes, number of animals tested, graph data and statistical testing presented for DnB GCaMP imaging and nociceptive behavior experiments.

Summary table of graph data and statistical testing for PMSI silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for PMSI silencing experiments.

Summary table of graph data and statistical testing for activation experiments. Genotypes, number of animals tested, graph data and statistical testing presented for 412-Gal4, 4051-Gal4 and off-target activation experiments.

Summary table of graph data and statistical testing for Goro functional imaging and behavior experiments. Genotypes and maximum ∆F/F0 per animal tested during Goro imaging experiments. Genotypes, number of animals tested, graph data and statistical testing presented for 412-Gal4+Goro-experiments.

Generate kymograph from curvature analysis.

Summary table of graph data and statistical testing for silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for R70F01∩412 silencing experiments and curvature analysis.

Summary table of graph data and statistical testing for silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for silencing experiments on nociceptive, gentle-touch and crawling assays.

Summary table of graph data and statistical testing for thermogenetic activation experiments. Genotypes, number of animals tested, graph data and statistical testing presented for thermogenetic activation experiments.

Summary table for output connectivity graph.Percentage of top hit neurons (>3 synapses with DnB) that fall into the category: premotor, motor, or nociceptive integrator neurons.

Larval body curvature analysis.

A comprehensive understanding of the molecular machinery important for nociception is essential to improving the treatment of pain. Here, we show that the BMP signaling pathway regulates nociception downstream of the E3 ubiquitin ligase highwire ( hiw ). Hiw loss of function in nociceptors caused antagonistic and pleiotropic phenotypes with simulta...

Inhibition of nociceptor activity is important for the prevention of spontaneous pain and hyperalgesia. To identify the critical K ⁺ channels that regulate nociceptor excitability we performed a forward genetic screen using a Drosophila larval nociception paradigm. Knockdown of three K ⁺ channel loci, the small conductance calcium-activated potassi...

Nociception, the sensory mechanism that allows animals to sense and avoid potentially tissue-damaging stimuli, is critical for survival. This process relies on nociceptors, which are specialized neurons that detect and respond to potentially damaging forms of energy — heat, mechanical and chemical — in the environment. Nociceptors accomplish this t...

The molecular mechanisms of sensing noxious mechanical force by nociceptive sensory neurons remain poorly understood. Traditional methods for probing mechanical nociception behavioral responses are labor intensive and involve the testing of one animal at a time. This time consuming process of behavioral testing has largely precluded large scale ana...

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 nocic...

Here, we describe a targeted reverse genetic screen for thermal nociception genes of 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 nocice...

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...

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 signif...

Dendrite shape is considered a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their function depends on these morphologies, remain unclear. Here, we demonstrate a requirement for the microtubule-severing protein katanin p60-like 1 (Kat-60L1) in regulating the elaborate...

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. W...

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. Paradoxically, fema...

Specialized somatosensory neurons detect temperatures ranging from pleasantly cool or warm to burning hot and painful (nociceptive). The precise temperature ranges sensed by thermally sensitive neurons is determined by tissue-specific expression of ion channels of the transient receptor potential(TRP) family.We show here that in Drosophila, TRPA1 i...

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...

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 involve...

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...

Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in fruit fly larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae "roll" with a motor pattern that is completely distinct from the style of locomoti...

Pain is a major health problem. Despite the importance of adequate pain modulation in relieving patients’ suffering, the available pharmacological agents for treating pain are limited. Existing pain-treating drugs that predominantly target the cyclooxygenase and opioid pathways are often inadequate in providing relief from pain and can have undesir...

A number of repellent compounds produced by plants elicit a spicy or pungent sensation in mammals . In several cases, this has been found to occur through activation of ion channels in the transient receptor potential (TRP) family . We report that isothiocyanate (ITC), the pungent ingredient of wasabi, is a repellent to the insect Drosophila melano...

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

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...

DNA-binding proteins are important for regulating gene expression during development. It is widely assumed that this regulation involves sequence-specific DNA binding by these transcription factors to cognate cis-regulatory sequences of their downstream target genes. However, studies in both the Drosophila and the mouse model systems have provided...

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,...

Runx1 (also known as AML1, Cbfa2 and Pebpa2b) and Cbfb encode a DNA-binding alpha subunit and the non-DNA-binding beta subunit of a mammalian core-binding factor (CBF). The discovery of RUNX1 and CBFB as genes rearranged in human leukemias prompted predictions that both genes would play important roles in normal hematopoiesis. These predictions wer...

The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically contro...

The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates th...

Thesis (Ph. D.)--State University of New York at Stony Brook, 1999. Includes bibliographical references (leaves 112-121).