Dean Smith - US grants

The broad, long-term objectives of the research program are to define the basic molecular and cellular components required for chemosensory discrimination in Drosophila. This chemosensory model system has many parallels with vertebrate chemosensory systems, but is numerically simpler. The potent molecular genetic tools available in Drosophila combined with simple chemosensory behavior assays will allow us to gain insight into the principles of chemosensory information processing. The specific aims are to: 1. Identify, isolate and determine the biological function of a collection of gene products expressed exclusively in the chemosensory organs, 2. Identify the neuronal transmembrane odorant receptors in Drosophila, and characterize the expression patterns of individual members, and 3. Differentiate between several possible models by which odorant- binding proteins influence chemosensory behavior. Health relatedness: Understanding the molecular basis of chemosensation in Drosophila will provide insights into this process in related arthropods that transmit human diseases. This understanding will faciliate new approaches to control these diseases. The methods for achieving specific aim 1 are to utilize the enhancer trapping approach to identify, isolate, mutate and determine the biological function of a collection of genes we have identified that are expressed exclusively in the chemosensory organs. This method allows us to evaluate the biological function of these chemosensory-specific gene products in intact animals. The methods for achieving specific aim 2 are to use differential hybridization and single chemosensory neuron libraries to identify neuronal chemoreceptors. We will use antibodies to specific receptors and the promoters of receptor genes to drive LacZ reporter genes to map the spatial expression patterns of each gene. Identification of the neuronal receptors that mediate chemosensation is essential to our long-range goal of understanding the molecular basis of chemosensory discrimination. The methods for achieving aim 3 are to mis-express several members of the odorant-binding protein family with known ligand specificity in specific subsets of sensilla to sensitize subsets of chemosensory neurons to specific odorants and determine the resulting behavioral consequences.

This Small Business Innovation Research (SBIR) Phase II project will further the design, development, construction, and evaluation of a prototype fish freshness sensor based on the successful Phase I feasibility demonstration of using an array of semiconducting metal oxide chemiresistive sensors for quantitative fish freshness quality determination. The advantages of this approach to fish freshness monitoring is that the array data will provide information about the complex gases emitted by fish during degradation and will provide a basis for signal processing techniques to quantify the fish freshness.

The Phase II research is directed towards extending the Phase I demonstration of determining the freshness of Atlantic salmon to other species of fish and to a wider variety of fish handling procedures. The fish freshness sensor data will be compared with results from a gas chromatograph mass spectrometer and a sensory evaluation panel of trained individuals. A field-deployable prototype will be tested on location at fish processing plants to non-destructively determine the degree of degradation of fresh marine fin fish.

[unreadable] DESCRIPTION (provided by applicant): Building upon the network foundation established by BRIN, INBRE proposes to expand and to develop Hawaii's competitive biomedical research capacity. The expansion will center on three thematic projects exploring the cellular basis of immunological and neurological diseases from the perspective of immunology, cell biology, and developmental biology. Each project will be led by a well-established senior investigator who will mentor junior investigators at both the lead and the affiliated baccalaureate institutions. This will extend into the state's community colleges where participating faculty will collaborate with established researchers at the lead institution. Each investigator - senior, junior, and Outreach - will recruit and mentor undergraduate and graduate students as well. The development will concentrate on not only individual research careers but also the network's overall approach to competitive research. This involves the establishment of rigorous standards and performance expectations coupled with attentive mentoring to assist network investigators and students in meeting these challenging criteria. Organizationally, INBRE will [unreadable] consist of four major cores: Administrative, Research, Bioinformatics, and Outreach. The Administrative [unreadable] Core will provide overall administrative support, including maintenance of the network's web site, evaluation efforts, and training and mentoring workshops and seminars. The Research Core will be comprised of the three thematic research projects with a Core leader to ensure project coordination. The Outreach Core extends the research thrust into the community colleges by promoting research and student participation through collaborations with the lead and the baccalaureate institutions. The Bioinformatics Core will also provide unique opportunities by emphasizing academic work force development. To facilitate this, the Core will be housed within the University of Hawaii at Manoa's Department of Information and Computer Sciences. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The broad, long-term objective is to genetically dissect RNA interference in Drosophila and identify the molecular components mediating this important process. Double-stranded RNA (dsRNA) induces potent cellular responses that compose an essential defense against deleterious RNAs, including viruses, viral replication intermediates and transposable elements. In many systems, dsRNA triggers RNA interference (RNAi); a dramatic and sequence specific destabilization of transcripts homologous to the dsRNA trigger. Normal cellular genes can be silenced using RNAi making this technique an important new tool to elucidate function of orphan gene products. Although the phenomenon is remarkably similar in diverse organisms, the precise mechanisms mediating RNAi are not fully understood. Genetic screens have identified several important components required for RNAi in Arabidopsis, Neurospora and C. elegans. However, these screens have not been saturating and recovered only viable mutations. Mutant screens for RNAi defective mutants in Drosophila have not been possible because of variable penetrance of RNAi suppression. Recently we solved this difficulty with a novel transgene design that effectively silences genes in adult tissues. These studies set the groundwork for the genetic screen proposed here designed to elucidate the molecular basis for RNAi in Drosophila. In .Specific Aim 1 we will isolate mutants defective and enhanced for RNAi suppression of the eye color pigment transporter WHITE using the FLP/FRT recombination system. The advantages of this screen are: 1. mutants affecting RNAi will be easily identified as eye color mutants, 2. homozygous mutant clones will be restricted to the compound eye, therefore we will recover mutations in viable and in potentially lethal genes required for RNAi not identified in other systems, 3. We will recover both suppressor and enhancer mutations affecting RNAi. In Specific Aim 2 we will use complementation, deficiency mapping, sequence analysis of mutated candidate genes and germline transformation rescue to identify the genes responsible for the mutant phenotypes observed in our screen. Completion of the studies will enhance our understanding of RNAi in Drosophila and broaden our understanding of RNAi in general. Recovery of enhancer of RNAi mutants may provide enhanced genetic backgrounds to facilitate research on orphan gene function in Drosophila in the post-genomic era. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The role of the T1 neuronal circuit in pheromone behaviors Research Summary Understanding the molecular and cellular basis of behavior is of great importance, both for the academic interest of understanding the substrates underlying behavior, and for the potential clinical relevance of understanding human behavior and mental illness. We propose to study a simple behavior paradigm in a genetically tractable animal model system; volatile pheromone-triggered behaviors in Drosophila melanogaster. 11-cis- vaccenyl acetate (cVA) is a male-produced pheromone that mediates aggregation and sexual behavior in Drosophila. cVA detection occurs through a small number of olfactory neurons located in trichoid sensilla on the antenna. Neurons within a subset of trichoid sensilla (the T1 sensilla) are exquisitely tuned to cVA and appear to be the primary cVA perception pathway. However, recent work also implicates a distinct set of trichoid neurons expressing Or65a in cVA-mediated behavior and learning. To begin to dissect the contribution of these two pathways in cVA-induced behavior, we will examine the effect of the loss or the constitutive activation of the T1 pathway on behavior. We propose two specific aims. First, we will create flies expressing a dominant, activating variant of the extracellular pheromone-binding protein LUSH that will allow us to manipulate the activity in this neuronal circuit. Second, we will use flies expressing activated LUSH or mutants with reduced T1 activity to dissect the role of increased or decreased activity in the T1 circuit on behavior. The results of these studies will provide new insights into the role of the T1 neuronal circuit in cVA-induced behavior. PUBLIC HEALTH RELEVANCE Pheromones trigger social behaviors in animals that are genetically hard wired, such as reproduction and aggression. In insects, pheromones guide mating behavior and aggregation to food sources. Therefore, understanding how pheromones are perceived and decoded by the insect nervous system could be of great practical benefit to control insects that transmit diseases or destroy agriculture. We study pheromone signaling in the Drosophila, where we can apply genetic analysis to behavior. The pheromone cVA is important for mating and aggregation in fruit flies, and appears to be sensed through two different neuronal circuits. Here, we will use genetic manipulations to activate or inactivate these pathways to elucidate the behavioral contributions of each to mating and aggregation behaviors. The expected results from these studies will help us understand how pheromone perception occurs in insects in general. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The broad long-term objective of this research is to elucidate the function of the volatile pheromone-detecting neurons expressing orphan odorant receptors. Volatile pheromones trigger or modulate social behaviors including mating. This work is significant because understanding pheromone biology in Drosophila will translate into new methods to control reproduction in insects that transmit human diseases and destroy crops and will shed new insights into the circuits guiding social behavior in an animal model system. Using a multifaceted approach that includes genetics, chemistry, electrophysiology, behavior and imaging we will identify ligands for these receptors, determine the behavioral effects of inactivating and activating these individual neural circuits, and map the downstream circuits activated by these neurons. The first aim is to determine the behaviors influenced through neurons expressing Or83c, which we have identified as a receptor for farnesol. The second aim is to identify ligands that trigger changes in activity in ten orphan trichoid receptors using the 'empty trichoid neuron' system, localize the endogenous neurons expressing these receptors, and characterize their downstream signal transduction requirements. The third aim is to identify behaviors modulated by activation or inactivation of these circuits triggered by odorant stimulation of these receptors. Aim 4 is to map the synaptic targets of the second order projection neurons innervating the trichoid glomeruli using the MARCM approach to begin to trace the neuroanatomy of these behavioral circuits. The completion of the studies will advance our understanding of volatile pheromone biology in an important model system, will identify new targets to manipulate insect behavior, and will guide future studies on pheromone signaling in more complex animal model systems. PUBLIC HEALTH RELEVANCE: Pheromones mediate social behaviors in insects. Insects carry human diseases that kill millions of people every year; malaria alone accounts for over one million deaths annually. Many insect behaviors, including mating, are triggered by pheromones, therefore understanding pheromone biology will provide new targets to manipulate pheromone signals, with the long-term goal of blocking mating in pathogenic insects. We propose to explore the function of orphan pheromone receptors to understand what they do and how they work and what neuronal circuits they activate. The results of these studies will guide future social behavioral experiments in more complex vertebrate animals.

The broad long-term objective of this research is to determine the molecular mechanisms underlying detection of volatile odorants and pheromones in Drosophila melanogaster. This work is significant because insects transmit devastating diseases to humans and they use olfaction to find hosts and mates. Understanding these mechanisms will facilitate new approaches for its disruption. Drosophila is an excellent model system to reveal mechanisms underlying olfaction due to its wealth of genetic tools, including unbiased genetics screens. We recently found lipid translocation, mediated by the phosphatidylserine (PS) flippase dATP8B, is essential for normal odorant sensitivity. We are now leveraging this discovery to identify the components and pathways underlying lipid translocation and its relation to odorant sensitivity. This work represents a new path of investigation. Using a genetic screen, we identified a protein kinase C that genetically interacts with dATP8B and appears to transduce most of the effects of PS localization. We propose experiments to decipher how this kinase impacts olfaction. Aim 1 is to explore the role of PKC98E in olfaction by characterizing its localization in olfactory neurons, and evaluating the phenotype of null and dominant alleles to analyze the olfactory consequences. Aim 2 explores the role of conserved PKC phosphorylation sites in ORCO on olfactory sensitivity and receptor trafficking, and whether phosphorylation is odorant-dependent in vivo. Aim 3 is to identify additional components involved in this process from a pool of 35 pre-selected candidates to gain a more complete understanding of this mechanism. Successful completion of these aims will significantly advance our understanding of insect olfaction and may provide exciting insights into the role of lipid translocation in modulating olfactory signal transduction.