Elissa Anyon Hallem - US grants

DESCRIPTION (provided by applicant): The olfactory system in animals from Drosophila to humans is able to detect and distinguish among a remarkable number of odors. This ability depends on large and highly diverse families of odor receptors whose members are expressed in subsets of olfactory receptor neurons and which confer upon these neurons their odor specificities. Despite the fundamental role of odor receptors in normal olfactory function, very little is known about the functions of individual receptors in any species. This study will undertake an in vivo analysis of odor receptor function in the Drosophila antenna. The contribution of individual odor receptors to receptor neuron response properties will be examined, thus furthering our understanding of both olfactory coding and the molecular basis of odor-receptor interactions.

DESCRIPTION (provided by applicant): Parasitic nematodes are a major health concern worldwide, and current strategies for preventing or eliminating nematode infections are insufficient. One possible control strategy is to interfere with the ability of nematodes to locate their hosts. The long-term goal of my research is to better understand how parasitic nematodes locate their hosts, using the free-living nematode C. elegans and the insect-parasitic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae as models for human-parasitic nematodes. Carbon dioxide is an important host-seeking cue for many parasitic nematodes, yet little is known about the mechanism of CO2 response in nematodes. The overall goal of this proposal is to further our understanding of how parasitic nematodes respond to CO2. I have shown that C. elegans displays acute CO2 avoidance, and I have identified neurons and signaling pathways that are required for this response. I have also found that analogous neurons mediate CO2 attraction in H. bacteriophora. I will now further elucidate the signaling pathways and neural networks that mediate CO2 response in nematodes using molecular, genetic, and neurobiological approaches. I will test the hypothesis that the neural circuits that mediate CO2 response in parasitic nematodes are similar to the neural circuits that mediate CO2 response in free-living nematodes, but contain modifications that reflect the host-seeking requirements of parasites. Parasitic nematodes cause extensive morbidity and mortality worldwide. A better understanding of how parasitic nematodes find and infect their hosts will enable the development of new strategies for preventing nematode infections.

Parasitic nematodes are a major health concern worldwide, and current strategies for preventing or eliminating nematode infections are insufficient. One possible control strategy is to interfere with the ability of nematodes to locate their hosts. The long-term goal of my research is to better understand how parasitic nematodes locate their hosts, using the free-living nematode C. elegans and the insect-parasitic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae as models for human-parasitic nematodes. Carbon dioxide is an important host-seeking cue for many parasitic nematodes, yet little is known about the mechanism of CO2 response in nematodes. The overall goal of this proposal is to further our understanding of how parasitic nematodes respond to CO2. I have shown that C. elegans displays acute CO2 avoidance, and I have identified neurons and signaling pathways that are required for this response. I have also found that analogous neurons mediate CO2 attraction in H. bacteriophora. I will now further elucidate the signaling pathways and neural networks that mediate CO2 response in nematodes using molecular, genetic, and neurobiological approaches. I will test the hypothesis that the neural circuits that mediate CO2 response in parasitic nematodes are similar to the neural circuits that mediate CO2 response in free-living nematodes, but contain modifications that reflect the host-seeking requirements of parasites.

This NSF IDBR award is made to Prof. Katsushi Arisaka and collaborators at the University of California, Los Angeles, to develop a Bessel beam line confocal microscope. The goal of this project is to develop a system enabling the recording of three-dimensional cell structure in vivo, in real-time, with exceptional penetrating depth, and with minimal damage to the targeted sample. The proposed system will advance scientific understanding by facilitating cellular observation and systems level biologic analysis. Significantly, this system will enable the unprecedented high-speed recording of cell dynamics at super-resolution, in a temporal range permitting observation of developmental phenomena.

The broader impact is a cost-effective, easily configurable and user-friendly 3D imaging system for use by scientists towards the in vivo structural characterization of dynamic biological samples over a timespan of days. This collaboration will generate an available and reproducible microscope that significantly advances obtainable information concerning embryo development, dynamic neural network function, cellular differentiation and regulation, while enabling high-volume 3D cell imaging. This project directly integrates educational and research goals through incorporation of microscope development into a novel, interdisciplinary laboratory course developed by Dr. Arisaka at UCLA. Thus, development and construction of the system will serve as an educational platform directly fostering student learning. Moreover, the system will be housed in the Advanced Light Microscope Facility at the California NanoSystems Institute (CNSI), resulting in widespread availability to the extended scientific community.

This award is being made jointly by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Biomedical Engineering, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate).

Non Technical Abstract
The primary objective of this project is to acquire a BioSorter (Union Biometrica) for the University of California, Los Angeles (UCLA). The BioSorter is a unique large-particle flow cytometer that rapidly sorts objects based on patterns of fluorescence or fluorescence intensity, including objects that are too large or fragile for standard flow cytometry. The BioSorter enables automated high-throughput sorting of organisms such as free-living and parasitic worms; fly embryos and larvae; and stem cells, other mammalian cells, and cell clusters. The rapid and precise sorting capabilities of the BioSorter enable screening strategies that will greatly expand the scope and impact of diverse research and education programs at UCLA. The BioSorter will be operated as a core facility on the UCLA campus. Research and training programs that will utilize the BioSorter span the fields of parasitology, neurobiology, developmental biology, biochemistry, genetics, toxicology, and stem cell biology.

Technical Abstract
The BioSorter will be used by students of all levels (high school, undergraduate, and graduate) for cutting-edge research and training. Research programs enabled by the BioSorter include: studies of how human-parasitic worms locate hosts to infect; studies of how environmental toxins affect the reproductive system; studies of stem cell development and differentiation; studies of the molecular and cellular basis of molting; studies of natural variation in populations; studies of the role of stem cells in neural circuit function; studies of mammalian germline function; and studies of brain neurochemistry. Education programs enabled by the BioSorter include: introductory and advanced research training courses for undergraduate students using fruit fly genetics and development as a model system, and a summer research program for students from local high schools. We expect at least 135 undergraduate students and 12 high school students to use the BioSorter each year. Use of the BioSorter in research training courses will expose students to cutting-edge technology and greatly enhance their training experience.

DESCRIPTION (provided by applicant): This proposal aims to understand the functional organization of sensory neural circuits, and lies at the interface of neurobiology and parasitology. A fundamental question in neuroscience is how sensory input is transformed into behavioral output. We are addressing this question using nematode olfaction as a model system. Over a hundred species of nematodes are parasites of humans, and over a quarter of the world's population is infected with parasitic nematodes, making parasitic nematodes a major health problem worldwide. Many parasitic nematodes actively search for hosts by responding to host-emitted sensory cues, and one possible control strategy is to interfere with their ability to locate hosts. However, the sensory behaviors of parasitic nematodes remain largely unexplored. We propose to undertake an in-depth analysis of olfactory behaviors and olfactory neural circuit function in free-living and parasitic nematodes, with particular emphasis on the odor-driven host-seeking behaviors of the skin-penetrating human threadworm Strongyloides stercoralis. We will use calcium imaging and quantitative behavioral analysis in combination with single-cell ablation and targeted gene disruption to identify genes, circuits, and behaviors required for successful human parasitism. We will also compare the functional properties of olfactory neural circuits in S. stercoralis, the closely related rat-parasitic nematode Strongyloids ratti, and the free-living nematode Caenorhabditis elegans. We will leverage the conserved neuroanatomy but diverse behavioral repertoires of nematodes to gain fundamental insights into how the specific features of a neural circuit shape its behavioral output. These experiments will lead to important discoveries about the functional organization of sensory neural circuits, and will provide a foundation for understanding how sensory circuits can be modified as a result of learning and memory, aging, and disease. At the same time, these experiments will provide insight into how human parasites use sensory cues to target humans, thereby paving the way for the development of novel strategies for preventing harmful parasitic infections.

The overarching goal of this project is to address a fundamental question in neuroscience: how do sensory neural circuits support flexible behaviors? In nearly all animals, behavioral responses to sensory stimuli vary as a function of age, prior experience, nutritional state, and environment. However, the molecular and cellular mechanisms that enable animals to generate context-appropriate behaviors are poorly understood. This project will investigate the neural basis of behavioral flexibility using the response of the free-living nematode Caenorhabditis elegans to carbon dioxide (CO2) as a model system. It will elucidate the context-dependent changes in neural circuit function that determine whether CO2 is attractive, repulsive, or neutral to C. elegans. Because the survival of nearly all animals depends on the ability to adapt to changing internal and external conditions, this study will have broad implications for animal behavior. In addition, CO2 is a host cue for many harmful parasitic nematodes of humans, livestock, and plants. A better understanding of how nematodes respond to CO2 may enable the development of new strategies for preventing harmful nematode infections. In addition, CO2 response by C. elegans will be used as an educational platform for mentoring, teaching, and outreach activities designed to engage students of all levels in scientific research.

Preliminary studies conducted in the lab of the Principal Investigator demonstrated that CO2 response by C. elegans can be rapidly modulated by ambient oxygen (O2) levels and nutritional state such that CO2 can be either attractive, repulsive, or neutral. Modulation of the CO2 circuit may be attributable to changes in the extracellular signaling molecules used, changes in the interpretation of these signaling molecules by downstream neurons, or a combination of both. This proposal aims to distinguish among these possibilities using an integrated approach that includes molecular biology, genetics, calcium imaging, and behavioral analysis. By comparing the functional properties of the CO2 circuit under high vs. low O2 conditions and in well-fed vs. starved animals, and by elucidating the molecular pathways that regulate CO2 response under these different conditions, this work will pinpoint the specific molecular and cellular features of the CO2 circuit that determine the behavioral response to CO2. The results of this study will elucidate basic principles of circuit design that support flexible behavioral outputs. Many of the molecular pathways and circuit motifs that operate in nematodes also operate in other animals, including mammals. Thus, results from this study will be relevant to many other animal species.

PROJECT SUMMARY Skin-penetrating nematodes, including the human-parasitic threadworm Strongyloides stercoralis, infect nearly one billion people worldwide and are a major source of morbidity in low-resource settings. Infections can cause chronic gastrointestinal distress, stunted growth and cognitive impairment in children, and even death in the case of S. stercoralis infection. S. stercoralis has a complex life cycle that includes a parasitic generation inside the host and a free-living generation outside the host. In previous work, we showed that the detection of carbon dioxide (CO2) and host-emitted odorants is important for multiple stages of the S. stercoralis life cycle. Moreover, we showed that CO2 and many host-emitted odorants elicit life-stage-specific behavioral responses, such that the chemosensory preferences of the infective larvae are distinct from those of the non-infective life stages. However, the neural mechanisms that mediate these chemosensory responses have not yet been investigated. Here, we propose to elucidate the molecular, cellular, and circuit mechanisms of chemosensation in S. stercoralis. In Aim 1, we will elucidate the neural mechanisms that mediate CO2 response in S. stercoralis. We will also investigate how CO2 microcircuit function is modulated across life stages to generate life-stage- specific responses to CO2. In Aim 2, we will elucidate the neural mechanisms that mediate responses to host- emitted odorants in S. stercoralis. We will also investigate how olfactory microcircuit function is modulated across life stages. In Aim 3, we will address the molecular mechanisms of chemosensation. We will identify genes and signaling pathways that mediate responses to CO2 and host-emitted odorants in S. stercoralis. We will also identify molecular mechanisms that contribute to parasite-specific and life-stage-specific chemosensory responses. Taken together, our results will provide key insights into the chemosensory mechanisms that underlie the complex interactions of parasitic nematodes with their human hosts.

PROJECT SUMMARY Skin-penetrating nematodes, including the human threadworm Strongyloides stercoralis, are parasitic roundworms that infect nearly one billion people worldwide. They are intestinal parasites that cause chronic gastrointestinal distress as well as stunted growth and long-term cognitive impairment in children. Infections with S. stercoralis can be fatal for immunosuppressed individuals. Despite the health burden caused by parasitic nematodes, many aspects of their basic biology remain poorly understood. In particular, remarkably little is known about how parasitic nematodes respond to host-emitted sensory cues. In this proposal, we will investigate how parasitic nematodes respond to heat. Heat is emitted by all mammals and is a robust sensory cue for many parasites, including nematodes. We will investigate how heat triggers two different steps of the parasite-host interaction: host seeking, the process whereby the soil-dwelling infective larvae actively search for hosts to infect; and activation, the process whereby the infective larvae exit their developmentally arrested state and resume growth after entering the host. First, we will conduct a quantitative analysis of the temperature-driven host- seeking behaviors of S. stercoralis (Aim 1). Second, we will elucidate the molecular and cellular mechanisms that underlie these behaviors (Aim 2). Third, we will investigate the molecular and cellular mechanisms by which heat triggers activation (Aim 3). These experiments will leverage our recent development of a CRISPR-Cas9 system for targeted mutagenesis in S. stercoralis, allowing us to identify genes required for host seeking and activation. Together, our results will provide fundamental insights into how sensory responses shape the interactions of parasitic nematodes with their hosts.