Paul A. Garrity - US grants

DESCRIPTION (provided by applicant): I am interested in the molecular mechanisms directing axons to their correct targets. Insights into axon target selection mechanisms may help us protect or reestablish neuronal connections compromised by illness or injury. My lab uses the Drosophila melanogaster eye's photoreceptors (R-cells) to study how axons choose their correct targets. The axons of different R-cell subtypes project to targets in distinct layers of the brain. The signals that specify R-cell axon targeting are unknown, and how these signals stop R-cell axons at the correct target layer is not understood. Through genetic screens, we have found that mutations in two genes, slit and Ptp69, disrupt target selection in a related fashion. Slit encodes an extracellular protein that can act as a signal to guide axons, and Ptp69d encodes a receptor protein tyrosine phosphatase. We will use slit and Ptp69d to investigate the mechanisms determining R-cell axon target choice. We will: 1. Examine slit's role in R-cell axon target selection. Slit loss-of-function causes one subtype of R-cell axons to grow through their normal target layer and into other target layers. We will test the hypothesis that Slit is a signal controlling layer-specific termination of R-cell axons. We will identify the location of the Slit signal and the cells in which it functions using genetic and molecular experiments. We will test Slit's effect on R-cell axon behavior both in vivo, through ectopic expression, and in vitro, using cultured R-cells. 2. Identify substrates of Ptp69d. Ptp69d loss-of-function causes R-cell axons to terminate in inappropriate target layers. Ptp69d acts in a phosphatase-dependent fashion, but its substrates are unknown. We will identify candidate substrates from the fly nervous system biochemically using a Ptp69d substrate-trap mutant that can bind but not dephosphorylate its targets. We will further study those candidate substrates that disrupt axon targeting when inhibited with RNAi. These candidates will be tested for genetic and biochemical interactions with Ptp69d and roles in R-cell axon targeting. 3. Examine interactions between slit, Ptp69d, and robo-family members in R-cell axon target selection. Overexpression of the Slit receptors Robo and Robo2 causes R-cell axon target layer selection defects similar to slit loss-of-function. We will examine loss-of-function mutations in robo-family members for R-cell axon targeting defects and examine potential interactions between slit, Ptp69d, and robo-family members in R-cell axon target selection. We will also examine potential downstream signaling pathways by examining interactions between these genes and potential downstream effectors.

[unreadable] DESCRIPTION (provided by applicant): Neural circuits provide the physical infrastructure that supports behavior, and a major goal in neuroscience is to understand how circuits function to control behavior. At present, genetic strategies for disrupting circuit function within an intact animal focus on altering the function of single cells. However, most neurons synapse with multiple cells, and there is currently no general strategy for specifically altering just a subset of a neuron's contacts. The goal of this project is to create a genetic system for intentionally disrupting a subset of a neuron's connections, permitting the dissection of neural circuits at the level of the single contact or the single synapse. We propose to achieve our goal in Drosophila by creating artificially engineered regulators of growth cone movement that can be used to intentionally sculpt patterns of neuronal connectivity. The initial aim of the project involves characterizing a series of artificially engineered ligand/receptor pairs to determine which are able to influence growth cone navigation in the fly without unintended side effects. The second aim of the project involves characterizing the ability of these selected ligand/receptor pairs to alter the connectivity of an individually identified neuron to its identified targets, examining the consequences using histology and electrophysiology. The final aim of the project involves applying the ligand/receptor system to a well- characterized neural circuit for learning and memory, using the ligand/receptor system to dissect the functional importance of specific connections within the neural circuit. The tools developed in this work will be immediately usable by other investigators to manipulate various neural circuits in Drosophila. The tools created here could be used in other cell types for other purposes, such as the disruption of cell migration or cell contact. In addition, while this proposal will provide proof-of-principle development in the fly, variations on the strategy developed here should be feasible in mammals and could have applications as both experimental and engineering tools. The wiring pattern of the nervous system provides the physical framework for human behavior, including such fundamental processes of relevance to human health such as learning and memory. This proposal proposes to develop new ways to manipulate the wiring of the nervous system in order to study how it controls behavior. These developments will lead to new insights into learning and memory and potential applications of the basic strategy developed in this grant could be used to alter connections for therapeutic purposes. [unreadable] [unreadable]

Temperature is a ubiquitous environmental variable with major effects on physiology and behavior, and animals exhibit strong temperature-dependent orientation behaviors. However, the mechanisms that govern such orientation behaviors remain largely unknown. In this proposal, we propose to characterize the circuitry and the molecular mechanisms that guide this temperature-regulated behavior using molecular genetics in Drosophila. The aims of this proposal are to: Aim 1: Identify the neurons governing thermotactic behavior. We will identify and assess the function of neurons involved in thermotaxis by using genetic approaches to ablate and inhibit the function of specific neurons. Aim 2: Examine the molecular properties of regulators of thermotaxis and test the hypothesis that candidate thermosensory neurons act by triggering repulsion from extreme temperatures. Aim 3: Investigate the molecular basis of differences in themosensory behavior and candidate thermal sensors. Aim 4: Identify additional regulators of themotaxis using a combination of RNAi-based and conventional genetic screens. The goal of this research is to understand the molecular mechanisms and neural circuits that control animal behavior, by focusing on the molecules and neuronal pathways involved in sensing and responding to environmental stimuli. Our long-term experimental goal is to obtain a molecular and cellular explanation of how Drosophila undergoes directed migration guided by temperature differentials. Our results should provide insight into how animals process sensory information in order to discriminate between subtle gradations in sensory input, a topic of relevanceto human perceptual disorders like agnosia resulting from stroke or dementia and hallucination associated with mental illness. Our work will also enhance understanding of the basic mechanisms of temperature sensation. Temperature perception is highly relevant to the perception of pain, and our studies should provide insights into how such somatosensory information is sensed and processed.

Temperature sensing is critical for survival: animals must avoid conditions that would freeze or burn them and choose the best conditions for growth and reproduction. To achieve these goals, animals possess highly sensitive temperature sensors. However, the mechanisms by which these sensors operate at the molecular level are largely unknown. Furthermore, it is unknown whether differences in these sensors contribute to the adaptation of different animals to different environments. This research project,entitled "The molecular basis of thermal preference variation in Drosophila", addresses these basic questions by combining molecular, genetic and physiological approaches to study the thermal sensors of Drosophila fruit flies from different climates, ranging from temperate regions to the Mojave Desert. By comparing the molecular properties of thermal sensors from different fruit fly populations and examining how these properties affect behavioral responses to temperature, this study will provide fundamental insights into how thermal sensors respond to temperature at the molecular level and how these sensors contribute to variations in animal behavior. As closely related thermal sensors are found in vertebrates as well as in other invertebrate species, results from these studies will have direct implications for thermal sensing and behavior across a wide range of animals. In addition to the scientific impact, this project also incorporates an experiential learning opportunity for undergraduates, directly engaging students in scientific research examining natural variation in temperature sensing among Drosophila populations.

DESCRIPTION (provided by applicant): Manipulating the activity of identified neurons in brain circuits is essential for studying how they are organized and how they produce behavior. Manipulating circuit activity can also be used to treat neurological and psychiatric diseases including Parkinson's Disease, depression and epilepsy. Optogenetic tools have revolutionized our ability to manipulate neuronal activity. We propose to create a set of complementary thermogenetic tools. These have the potential to reliably provide stronger levels of activation and may be activated with less invasive stimulus delivery systems. At present, such tools are used exclusively in flies. We propose to modify these tools to optimize them for use in mammalian systems.

Temperature strongly affects physiology and behavior, and thermosensation is relevant to body temperature homeostasis and pain in humans, host seeking by insect disease vectors, and movement in all animals. Using D. melanogaster as a model system, we propose to define a novel molecular pathway for thermal sensing involving a previously unappreciated class of potential thermal sensing protein. This work will thus lead to the definition of a new molecular pathway for thermal sensing of potentially broad relevance for both basic biology and human health. In preliminary experiments, we have identified a critical regulator of themnosensory behavior. Furthermore, we have found that this protein is capable of conferring thermal sensitivity upon a neuron when ectopically expressed. Together our preliminary data suggest this protein may function as a previously unappreciated type of thermal sensor. We propose to extend these studies in three aims. 1) We will investigate the site of action and molecular nature ofthe putative thermal sensor in controlling thermotactic behavior. 2) We will examine how this putative thermal sensor affects temperature sensing by thermosensory neurons. 3) We will examine the molecular mechanisms by which the thermal sensor detects temperature by examining its potential signaling mechanism and the signaling pathways with which it may interact to transduce temperature infonnation. Together, these studies will provide fundamental new insights into the molecular mechanisms of thermal sensation. RELEVANCE (See instmctions): This proposal investigates the molecular mechanisms of thermal sensation. Thermosensation is critical for animal sun/ival and physiology. In humans, thermosensation is critical for pain, inflammation and tx)dy temperature regulation. Thus, thermosensory mechanisms are of biomedical relevance. In addition, themosensation is important for host-seeking by insect vectors of human diseases like malaria and West Nile. Thus the study of thermosensation is relevant to the control of insect-borne human disease.

Temperature has a major impact on animal physiology, and the ability to withstand daily and seasonal fluctuations in environmental temperature is critical for survival. This challenge is particularly acute for small insects, which include major agricultural pests and animal disease vectors, from aphids and flies to mosquitoes. The body temperatures of these animals closely follow the ambient temperature, and their sensitivity to temperature, especially cold, is a key factor in determining their geographic distributions. In this project, the researchers will probe the molecular and cellular pathways through which animals adjust their physiology to withstand cooling, using the fruit fly Drosophila as a model. The key systems this animal uses to sense that the environmental temperature is dropping will be identified. Next, the researchers will examine how the initial sensation of cooling is communicated to and processed by the brain. Finally, the investigators will examine how signals from the brain act to adjust the animal's physiology so that key organ systems are able to cope with environmental change. From an intellectual perspective, these studies will provide insight into how an animal's senses can modulate its physiology, and into how an animal can respond to environmental challenges. This work is designed with several broader impact objectives. These include training undergraduate, graduate and post-doctoral fellows in biological research, providing experiential learning opportunities to engage undergraduate students in scientific research, providing mentored teaching opportunities for graduate students and post-doctoral fellows, and providing publicly available resources for the research and educational communities.

Temperature affects all biological processes, and the ability to sustain physiological function despite fluctuations in body temperature is important for animal survival. In this proposal, the investigators will examine how animals withstand temperature fluctuations, particularly cooling, using the fruit fly Drosophila melanogaster as a model. The proposal uses a combination of molecular genetics, physiology and behavior to examine three main research goals. First, the cellular and molecular sensors through which cooling is detected to initiate physiological changes that confer cool tolerance will be identified and studied. Second, the cellular and molecular mechanisms involved in eliciting these physiological changes will be identified and examined. Third, the extent of inter-species variation and environmental plasticity in cool tolerance among multiple Drosophila species will be explored, identifying promising natural variants and regulatory strategies for further study. Together, these studies will provide insights into the cellular and molecular mechanisms by which animals sense temperature and modulate their thermotolerance to cope with fluctuations in environmental temperature.

? DESCRIPTION (provided by applicant): Animals detect multiple kinds of stimuli, including chemicals, force, light and temperature. Distinct sensory modalities often rely on related molecular receptors2. In some cases the same receptor participates in multiple sensory modalities. How does such polymodality emerge? How can it be regulated to enable sensory discrimination? We will probe these issues by studying two classes of polymodal receptors we have identified in the fruit fly Drosophila melanogaster whose polymodality is regulated by complementary molecular mechanisms. We propose to investigate these issues in three aims. 1) We will probe the molecular mechanisms by which the activity of a receptor for both moderate warming and aversive chemicals can be regulated in a cell-specific fashion to achieve appropriate sensitivity and specificity, and use these studies to investigate the molecular basis of thermal and chemical detection in both fruit flies and Anopheles mosquitoes. 2) We will investigate how a pair of receptors act together to mediate both thermosensation and hygrosensation, and how these distinct activities are regulated to confer appropriate behavioral responses to each stimulus in both fruit flies and Anopheles mosquitoes. 3) We will test the hypothesis that multiple related receptors can function together in a combinatorial fashion to create distinct classes of sensory receptors that mediate responses to distinct sensory modalities in different sensory neurons in both fruit flies and Anopheles mosquitoes. Together, these studies will provide fundamental insights into the molecular basis of sensory receptor polymodality, how sensory receptors detect thermal and chemical stimuli, and how sensory receptor activity can be regulated to achieve appropriate sensory sensitivity and specificity.

DESCRIPTION (provided by applicant): All animals live in constantly changing environments and are, therefore, subjected to fluctuations in critical environmental cues such as temperature. Given that all biochemical reactions are temperature sensitive to a certain extent, it is particularly critical for animals to compensate for these temperature changes in order to maintain steady internal conditions. In order to do so, animals must be able to detect small temperature changes and then trigger the appropriate homeostatic compensatory mechanisms. To date, research into the molecular and neuronal basis of temperature detection and thermotransduction, and the physiological mechanisms of temperature adaptation and compensation, have largely been pursued as independent lines of investigation. To obtain a full understanding of how animals respond appropriately to temperature changes, these intellectual issues must be brought together and studied as a whole. In this Program Project grant we bring together researchers experienced in issues of temperature detection and temperature compensation to ask how animals detect temperature changes and translate this information to effect compensatory changes in neuron function to maintain behavioral robustness. A particular strength of this proposal is the synergy among investigators exploring these issues in multiple systems; this diversity will elucidate common underlying principles that can be generalized ID other species. The overall questions being asked here are: 1) What are the molecular mechanisms by which animals detect temperature changes? 2) What are the neuronal mechanisms that encode information about temperature changes? 3) How do motor programs compensate for temperature fluctuations? 4) What defines the limits of the range in which these homeostatic mechanisms operate? 5) What are the common principles of temperature detection and compensation among species?

Project Summary / Abstract CRISPR-mediated genome editing is accelerating basic research and its application to improve human health. In traditional genetic model organisms, like the fruit fly Drosophila melanogaster, CRISPR has accelerated and simplified genetic manipulations. In organisms where genome modifications have been more challenging, like the malaria mosquito Anopheles gambiae, CRISPR-based genome editing presents new opportunities to study their biology and to disrupt disease transmission. Here we propose to simplify and expedite CRISPR-based genome editing for fly and mosquito researchers. We propose to achieve these goals in two aims: 1) We will develop and optimize a universal CRISPR toolkit for genome editing in Drosophila. In aims 1.a and 1.b, we will optimize a set of simple-to-use targeting vectors that will speed and simplify the implementation of CRISPR-Cas9 gene editing in Drosophila. In aim 1.c., we will modify these optimized vectors to generate a set of reagents that will allow investigators to readily create fluorescently labeled targeted knockouts and reporter knock-ins (including Gal4, GFP and epitope-tags) by simply combining gene-specific gRNAs with these universal reagents. Aim #2: Develop and optimize a uHITI toolkit for genome editing in Anopheles. In aim 2.a., we will optimize our targeting approach for use in A. gambiae. In aim 2.b., we will modify these optimized A. gambiae vectors to generate a set of reagents to allow investigators to create targeted knock-outs and reporter knock-ins (including the QF transcription factor, GFP, epitope-tags and a genetically encoded calcium indicator) by combining gene-specific gRNAs with these universal reagents. These tools will simplify and expedite the generation of targeted knock-outs and knock- ins by investigators studying Drosophila and Anopheles. In Drosophila, these tools will also increase the feasibility of creating genome-wide knock-out and knock-in collections. In Anopheles, these tools can expedite the introduction of complex gene cassettes into different genomic locations in order to disrupt vector populations.

Project Summary Molecular and cellular determinants of Drosophila larva thermotaxis How nervous systems detect and integrate multiple sensory cues to generate robust behaviors is a major question in neuroscience. Such integration is particularly salient in thermosensing, as animals are frequently required to integrate input from multiple thermoreceptor classes. Temperature's ubiquity also means input from other modalities (e.g., olfaction) is commonly received in the context of ongoing thermosensory stimulation. Achieving a comprehensive understanding of the molecular and circuit mechanisms underlying the integration of information from multiple sensors remains a challenge. We will address this challenge in the Drosophila larva. Its ease of genetic manipulation, synaptic-resolution connectome of thermosensory and olfactory processing areas, amenability to neuronal imaging, and stereotyped behaviors, all make it a favorable system for a comprehensive molecular and circuit level investigation of the mechanisms of sensory integration. We propose to achieve these goals in three aims: Aim 1) Establish the molecular and cellular receptors that provide thermosensory input In aims 1.a. and 1.b., we will identify the molecular basis of thermosensing by thermosensory neurons in the larval Dorsal Organ and examine their roles in guiding behavior through cell-specific inhibition and activation combined with high-resolution behavioral analysis. Aim 2) Probe the activities of the interneurons that process thermosensory input In aim 2.a., we will examine how thermosensory inputs act to modulate the neuronal activity of individually identifiable downstream projection neurons revealed from the larval antennal lobe connectome. This will establish the manner in which peripheral sensory input influences these second-order interneurons. In aim 2.b., we will investigate how thermosensory and olfactory systems interact in multi-sensory integration of chemical and thermal cues. Aim 3) Probe the functions of the interneurons that process thermosensory input In aim 3, we will determine the contribution of each projection neuron to thermotactic navigation through cell- specific inhibition and activation of individual PNs combined with high-resolution behavioral analysis. Taken together, these studies combine molecular genetics, physiology, and high resolution behavioral analyses to perform a comprehensive analysis of how this relatively small neural circuit processes multiple, distinct sensory inputs to control robust and flexible behaviors.

PROJECT SUMMARY Invertebrate Gustatory Receptors (GRs) are a large and evolutionarily diverse family of sensory receptors known to play important roles in invertebrate taste, smell and thermotransduction. Given the importance of these sensory modalities in host-seeking behavior in important humand disease vectors like mosquitoes, GR family members serve as potentially powerful targets for vector control agents. However, little is known about GR structure and function. We propose a physiological and biochemical analysis of members of two GR subfamilies: Gr43a and Gr28bD. These initial studies will serve as a precursor for a subsequent R01 to carry out structural and functional analyses of these GRs. We propose to achieve these goals in two aims: Aim #1: Identify and physiologically characterize multiple orthologs of Gr43a and Gr28bD. Unlike most GRs, Gr43a and Gr28bD orthologs can be functionally characterized in heterologous cells. In aim 1.a., we will express orthologs of these GRs from additional insect species, including disease vectors and extremophiles, in heterologous cells and characterize their physiological properties. This will enable a comparative analysis of sequence and function among each receptor class. Aim #2: Biochemically characterize multiple Gr43a and Gr28bD orthologs. We find Gr43a and Gr28bD orthologs can be partially purified from heterologous cells. In aim 2, we will expand this approach to incorporate additional orthologs characterized in aim 1 and optimize our purification protocol and explore key properties including oligomeric state and thermal stability in various membrane mimics. This will provide important biochemical information about GR complexes and identify orthologs best suited for subsequent structural analysis. The physiological characterization of multiple Gr43a and Gr28bD orthologs will enable direct examination of evolutionary variation and conservation in GR family function. The expression and purification of multiple family members will provide multiple candidates for biochemistry and structural determination, maximizing the likelihood of success of subsequent GR structural determinations.

Project Summary/Abstract The goals of this training program entitled ?Predoctoral Training in Cross-disciplinary Molecular and Cellular Biology? (CMCB) at Brandeis University are to produce rigorous, quantitative scientists with expertise in multiple disciplines, to provide trainees with the skills needed to succeed in diverse science-related careers, and to help trainees explore and pursue their career interests in an informed manner. This new program combines the complementary strengths of two prior Brandeis T32 programs: ?Genetic and Biochemical Mechanisms of Regulation, T32GM007122?, expiring after >40 years of NIGMS support, and ?Quantitative Biology, T32EB009419?, expiring after 10 years of NIBIB support. While both training programs had strong track records of student research productivity and career outcomes, we significantly rethought and revised core elements of training (based on student and faculty feedback) to better prepare students for a future in which interdisciplinary research is increasingly crucial. Innovations include: (1) increased quantitative training, through courses and an annual workshop, (2) revising the timing and goal of the qualifying exam, both to improve the training value and to better serve students from diverse scientific and personal backgrounds, (3) earlier implementation of Individual Development Plans and Thesis Committee Meetings to accelerate trainee career development and research progress, (4) introducing a secondary research advisor in a complementary discipline to facilitate interdisciplinary training, (5) a two week professional Externship in Year 4+, (6) new program self-assessment mechanisms, including semi-annual trainee feedback and the creation of an External Advisory Committee, and (7) formal training in and oversight of mentoring for all training faculty. Trainee appointments will be made at the end of Year 1, after students have completed one year of coursework, four 9- week laboratory rotations, and chosen a lab. In Year 2, trainees serve as teaching assistants for one course per semester, take a Proseminar course to help them craft their thesis research plan and defend it at their qualifying exam (end of Year 2), and to develop a career Individual Development Plan (IDP). In Year 3+, trainees take a final elective and focus on their research. They present their work at annual Departmental talks, have annual Thesis Committee Meetings focused on career planning and research progress. In Year 4+, they engage in a two-week career Externship and serve as mentors at the annual Quantitative Analysis workshop. Program outcomes and success will be measured by: (1) sustained research impact (reflected in trainee publications), (2) development of independent scientific thinking and communication (assessed through thesis proposals, annual Departmental talks and Thesis Committee Meetings), (3) active trainee engagement in their own career development (reflected in annual IDPs, discussions at Thesis Committee Meetings, and the new Externship program), and (4) trainee placement and long-term success in science-related careers. There are 29 CMCB training faculty, and 12 slots are requested (~6 trainees/year, with trainees supported in Years 2 and 3).

PROJECT SUMMARY/ABSTRACT Mosquito disease transmission relies on the insect?s ability to feed on human hosts, a behavior driven by host- associated sensory cues. Temperature and humidity are key short-range cues that promote the final stages of host approach and biting, but little is known about the molecular and cellular basis of mosquito responses to these cues, or whether these mechanisms are conserved among evolutionarily distant mosquitoes. We propose to address these knowledge gaps by: 1) Probing the molecular basis and evolutionary conservation of heat-seeking between the malaria vector Anopheles gambiae (An. gambiae) and the dengue vector Aedes aegypti (Ae. aegypti). 2) Investigating the (as yet unknown) molecular and cellular basis of mosquito humidity sensation in An. gambiae. We propose to achieve these goals in three aims: Aim 1) Probe the evolutionary conservation of heat seeking mechanisms. Mosquito blood-feeding is thought to have a common evolutionary origin, but whether the mechanisms that control heat-seeking are conserved across mosquitoes is an open question. We will test this conservation by comparing the roles of key receptors implicated in heat-seeking in multiple mosquito species in order to reveal whether and in what ways heat-seeking mechanisms are shared across vector mosquitoes. Aim 2) Determine the sensory specificities of candidate humidity receptor-expressing neurons in An. gambiae. Despite its importance for host seeking, mosquito humidity sensing is largely unexplored. We will test the hypothesis that mosquito humidity sensors rely on relatives of receptors important for sensing humidity in Drosophila. We will examine the stimulus sensitivities of the sensory neurons expressing these receptors and test the role(s) of these receptors in detecting sensory stimuli. Aim 3) Establish the behavioral roles of candidate hygroin An. gambiae. We will test how our candidate receptors for humidity and temperature contribute to the mosquito?s behavioral responses to humidity and temperature, as well as host seeking and blood feeding. We will also compare how host-seeking roles relate to homeostatic roles in helping modulate body temperature and hydration state. This work will identify molecular receptors and sensory neurons that detect temperature and humidity in vector mosquitoes and establish their roles in mosquito host seeking and blood feeding. As these behaviors supports disease transmission, these basic science findings have potential relevance for vector control efforts.