Hubert Amrein - US grants

RNA mediated transcriptional control is an emerging principle in gene expression. X-inactivation and genomic imprinting in mammals and dosage compensation in Drosophila represent prime examples where RNAs play a crucial role in gene expression. However, the molecular mechanisms involved in these processes are poorly understood. The long-term objectives of this project are to understand the regulatory roles of such nuclear RNAs in transcriptional regulation, and to elucidate the molecular mechanisms by which these RNAs associate with chromatin and change the DNA's propensity for gene expression. The general strategy of the proposed research is to investigate RNA mediated mechanisms in gene expression using the genetically established system of Drosophila. We have recently isolated two male-specific genes, roX1 and roX2, that function as regulatory RNAs in the male nucleus. roX1 and roX2 RNA co-localize with the male-specific lethal proteins (MSLs) to the single male X- chromosome and are likely to play a role in dosage compensation, a mechanism analogous to mammalian X-inactivation. The major interest in the proposed research will be directed towards understanding the function and regulation of the roX RNAs in male Drosophila. To accomplish these goals, we propose the following four specific aims: (1) Induce and characterize mutations in roX2 and investigate genetic interactions between roX1 and roX2; (2) Determine the parameters for X-chromosome localization and define structural components assoicated with the roX RNAs; (3) Study the consequences of ectopic roX RNA expression on the regulation of linked genes in an in vivo model system; (4) Determine the regulatory mechanism underlying male-specific roX expression and define essential cis-regulatory elements. The roX genes of Drosophila provide an ideal model system to investigate the roles of RNAs in transcriptional regulation and should advance our understanding of the principles that govern RNA mediated gene regulation.

One of the most fundamental processes in biology is the establishment of the male and female gender. Gender choice is generally determined by regulatory genes controlling sexual differentiation, physiology and behavior. Sexual behaviors are of particular interest: first, they can vary widely between the two sexes within a species or/and between same-sex individuals from closely related species. Consequently, these behaviors play a central role in the fitness and evolutionary success of a species. Second, sexual behaviors are robust, innate and in general complex, involving many sensory modalities. Therefore, they can serve as model behaviors for investigations into complex neural circuits.
Drosophila represents one of the best-characterized higher animals at the genetic and behavioral level. Moreover, the involvement of vision, chemoreception and audition offers ample opportunity to study this behavior from a variety of different angles. Recently, direct evidence for a pheromone receptor in male courtship was provided through the identification of a male-specific gustatory receptor gene, Gr68a. This gene is essential for the identification of a female partner during the male mating ritual. The goal of the presented study is to identify and characterize other pheromone receptors involved in male courtship; these receptors are likely to be found among the seven additional Gr genes closely related to GR68a and are proposed to control other aspects of male courtship. The proposed research will establish a framework of structural and molecular components that mediate an essential behavior common to most animals. Moreover, it will provide novel insights into how chemosensory inputs are transduced to the brain and integrated with other sensory inputs to generate a complex neuronal circuit.
Efforts to recruit graduate and undergraduate are very successful (two undergrads, Meister and McNealy, co-authored a paper in 2001). Currently, a postdoc, three graduate and five undergraduate students are working in the PI's lab. Findings are disseminated through publications in international journals and scientific conferences. Moreover, they have also been reported in mainstream media, including BBC (Leading Edge, radio science news program).

DESCRIPTION (provided by applicant): Chemosensory perception provides all organisms, from bacteria to humans, with essential information about the chemical composition of the external world. In insects and vertebrates, this 'chemical world' is generally perceived by two distinct sensory modalities, gustation and olfaction. Our long-tem objective is to understand how animals recognize chemical cues present in their environment and to investigate how these cues regulate feeding behaviors. Behavioral and electrophysiological studies have indicted that Drosophila possesses a well-developed sense of taste that can detect a large number of chemically diverse substrates (ligands). The broad impact of genetics in virtually all disciplines of biology has made Drosophila an extremely valuable model system in molecular and behavioral neurobiology. Its role has been of particular significance in uncovering the logic of chemosensory perception, because its chemosensory systems exhibit many parallels with those of vertebrates/mammals, and because it also serves as a model system for insects, many of which have a direct impact on human prosperity and health. Drosophila gustatory receptor neurons (GRNs) express putative seven transmembrane receptors (Gustatory Receptors or GRs) that detect soluble ligands. Activation of GRNs is propagated to taste centers in the CNS, which translate sensory input into various behavioral outputs. These behavioral taste responses can be broadly divided into acceptance behavior or avoidance behavior. Recent work in several laboratories has shown that acceptance and avoidance behaviors are mediated by two molecularly distinct subpopulations of GRNs (sweet and bitter neurons), each expressing different sets of GRs. Interestingly, individual neuron subpopulations express partially overlapping but not identical members of putative bitter-sensing GRs, suggesting that flies can discriminate distinct qualities of bitter taste. Molecular-genetics approaches, combined with behavioral and electrophysiological studies have also led to the identification of sugar taste receptors, which are also expressed in complex and overlapping sets of sweet neurons. Finally, these studies also established evidence that taste receptors are multimeric complexes composed of different GRs. Yet, despite all this progress, many basic questions about the taste receptors themselves, and about how detection of chemicals in taste organs is translated in the percept of a taste quality in the brain, remain unanswered. This application will investigate some of these questions. We propose to determine membrane topology and mode of signaling of GRs. Furthermore, we will investigate the heteromeric composition of sugar receptors using molecular genetic and behavioral analyses, and lastly, we shall test whether flies have the ability to discriminate between different flavors within the bitter taste modality. PUBLIC HEALTH RELEVANCE: Feeding is the most basic and essential of all behaviors. It has an immediate impact on human health and fitness. This grant will investigate the molecular function and structure of taste receptors, and the feeding behaviors that are mediated by these receptors. For these studies, we will use the genetically amenable model system of Drosophila, whose taste sensory system shares many basic principles with that of mammals, including humans.

[unreadable] DESCRIPTION (provided by applicant): Our long-term objective is to elucidate how social behaviors, universal features of animals with complex nervous systems, are genetically controlled through the various sensory systems in an organism. As a general strategy, we will utilize Drosophila melanogaster as a model system to elucidate the roles of pheromones and their receptors in social behaviors, such as courtship, mating, aggression etc. In most mammals and insects, the recognition of contact and/or volatile pheromone signals plays a central role in these behaviors. In Drosophila and other insects, members of two families of seven-transmembrane receptors, encoded by the olfactory (Or) and gustatory receptor (Gr) genes, are thought to recognize pheromones. Stimulation of these receptors leads to the activation of neural ensembles in the CNS, which are thought to receive also input from other sensory modalities (i.e. visual and auditory). Finally, a complex neural circuit must integrate the information from these diverse sensory channels and control the elaborate behavioral displays during courtship, aggression and other social interactions between individuals. To understand how such complex, integrated circuits operate is one of the main scientific challenges in modern, molecular neurobiology. We have previously identified a bona fide pheromone receptor, which plays a major role in male courtship. This receptor is encoded by Gr68a, a member of a Gr subfamily, which includes five other Gr genes - Gr32a, Gr39a.a, Gr39a.b, Gr39a.c and Gr39a.d. The central hypothesis of our proposal is that all these Gr genes encode pheromone receptors with specific roles in diverse social behaviors in Drosophila. We shall take a reverse genetic approach to elucidate the roles of these receptors. We will generate knock-out alleles for all six Gr pheromone receptor genes, generate Gr mutant fly strains and perform extensive behavioral analyses with these flies to elucidate Gr gene functions in courtship activation, courtship suppression, aggression, female attraction and other social behaviors. We shall map the axonal projections of Gr-expressing, pheromone-sensing neurons and identify neuronal targets that form synapses with their axons. Finally, we shall identify the main chemical compounds that are recognized by these GR pheromone receptors, and hence, represent the cues that activate these neural circuits. PUBLIC HEALTH RELEVANCE: This grant will investigate the specific role of pheromone receptors in complex social behaviors including sexual behavior, aggression and rejection, using the Drosophila as a model system. The proposed studies will employ extensive genetic analyses, behavioral experiments and neuroanatomical investigations to identify the function of these receptors, with the goal to reveal links between receptors and behavior. Our work will significantly expand our rather poor knowledge of pheromone-guided complex behavior and neural circuits that control them, a hot topic in the field of behavioral and molecular neuroscience. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Evaluation of nutrient food content is essential for the regulation of energy metabolism and feeding behavior in most animals. The model system Drosophila melanogaster and mammals share many of the nutrient sensing pathways that have been characterized thus far. For example, Drosophila and mammals release insulin or insulin-like peptides in response to the consumption of food, especially carbohydrates. Likewise, they can sense amino acids and proteins in food, and they share an ability to suppress feeding on amino acid mixtures or proteins that lack one or more essential amino acids. Nutrients are sensed mostly by the gastrointestinal system, but also by the brain. For example, numerous mammalian hypothalamic neurons can sense changes in external glucose concentration, but their function in the regulation of feeding and metabolism are poorly understood. The long-term objective of our research is to identify and characterize the molecular and neuroanatomical components that sense nutrients in the brain and propagate these events to regulate feeding and energy metabolism, using Drosophila as a model system. We recently identified the Gustatory receptor 43a (Gr43a) as a novel, electrogenic nutrient sensor in the Drosophila brain. Gr43a is narrowly tuned to the sugar fructose. Upon carbohydrate feeding, fructose concentration in the hemolymph increases several fold, which then leads to the activation of a small number of Gr43a expressing brain neurons. Behavioral experiments showed that Gr43a regulates feeding behavior in a satiation dependent manner: in hungry flies, it promotes feeding, while in satiated flies, it suppresses it. These observations lead us to propose that Gr43a stimulation by fructose activates a neural pathway, which is modulated by a satiation-dependent signal to generate distinct feeding behaviors. To elucidate the mechanism of the opposing behavioral outcomes of Gr43a activation, it will be essential to identify the signaling events that act downstream of Gr43a in the brain, and to identify the neuronal targets with which the Gr43a brain neurons communicate. Based on preliminary data, we propose that Gr43a brain neurons transmit their activity via the neuropeptide corazonin, the functional ortholog of mammalian gonadotropin releasing hormone. In addition, we have identified several other candidate effectors and modulators of Gr43a activity, and we shall use molecular genetic approaches to determine their specific roles in feeding promotion and suppression. Finally, we shall expand the neural circuitry activated by the Gr43a brain sensory neurons by characterizing crzR expressing neurons and identifying their targets. These studies will provide a framework for the molecular and cellular logic of a novel electrogenic nutrient sensor, which can be modulated by satiety signals to generate opposing behavioral outputs.

? DESCRIPTION (provided by applicant) Appropriate food choices are critical for the acquisition of nutrients for growth, energy expenditure and reproduction. Taste perception is the primary sensory modality for the identification of nutritious compounds, such as carbohydrates, proteins, salts and fats, as well as harmful and toxic compounds, such as alkaloids, terpenoids and phenols, chemicals perceived as bitter by humans. Drosophila has served as an important model system to dissect the molecular basis of many sensory processes, including taste perception. The genomic/genetic resources are excellent and researchers have an impressive array of molecular-genetic, electrophysiological and imaging tools at their disposal, while the system is also amenable to elegant behavioral assays. Even though mammals and insects express evolutionarily distinct sets of taste receptors, the overall organization of taste sensory systems and especially the logic of taste coding are similar. Thus, the fly provides an opportunity to uncover basic mechanisms of taste perception, as well as organizing principle of their neural circuits. The Drosophila genome harbors 68 Gustatory receptor (Gr) genes, a subfamily of eight is thought to encode all putative sugar receptors. However, a model has emerged over the last few years that posits that only two heterodimeric sugar receptors (Gr5a/Gr64a and Gr5a/Gr64f) mediate most or all of sweet taste. We have tested this hypothesis using specific knock-in/expression alleles, as well as `sugar blind' Drosophila strains. These studies let us conclude that two major premises of this model need revision: First, Gr64a is not a major mediator of sweet taste and second, all sugar Gr proteins participate as units of functional sweet taste receptors. Moreover, while sugar receptors do function as multimeric complexes, Gr composition for most taste receptors is unknown. This gap in knowledge will be systematically filled with a methodical rescue strategy. We will also build on our recent discovery that sweet taste cells in the fly are not exclusively tuned to sugars, but also respond t fatty acids, another important nutritional component for almost all animals, including fruit flies.In preliminary studies, we have identified the first receptor gene necessary for fatty acid taste, a member of the Ionotropic receptor gene family. We will investigate the role of other members if this gene family by studying both their expression, as well as their function in fatty acid taste. Lastly, we discovered an entirely new taste modality, tuned to carboxylic acids but not acid in general. Carboxylic acids are generic components present in many fruits. We will determine the response profile of taste cells that specifically respond to these food components, and possibly to amino acids, and search for the receptors that recognize these compounds using a targeted candidate gene approach.

? DESCRIPTION (provided by applicant): The ability to identify different chemicals is a ubiquitous feature of most animals, from primitive roundworms to mammals. The most important feeding decisions animals have to make is to discriminate between palatable tasty food chemicals, such as sugars, and aversive, bitter tasting and possibly toxic chemicals. A second more elaborate process is the identification of food best suited for a specific condition, such as overall nutrition status (hunger vs. satiation), anticipated need for major energy expenditure (locomotion), a developmental stage or reproductive phase. To accommodate these needs, gustatory systems have evolved sensors for the identification of different types of nutrients. In this application, we will investigate the functions of a highly conserved Drosophila Gustatory receptor (Gr) gene subfamily in sensing biomolecules. Specifically, we present strong evidence that implicates the Gr28 gene family (Gr28a, Gr28b.a-b.e) in the perception of RNA through its ribose moiety. We show that Drosophila larvae are highly attracted to RNA/ribose, a preference entirely dependent on the presence of the Gr28 genes. Using a novel Ca2+ indicator (CaMPARI), we establish that RNA, ribose and uridine activate taste neurons expressing Gr28a. We also show that RNA, but not DNA is necessary for normal growth and survival during larval development. This is the first association of Gr genes with a direct chemosensory function in the detection of large biomolecules. We hypothesize that the Gr28 proteins recognize RNA precursor and other ribose containing compounds both externally and internally. Thus, this proposal will likely establish a molecular mechanism not only for the detection of exogenous RNA related nutrients, but also for the previously reported roles for these receptors in light and temperature sensing. Drosophila has been the major non-vertebrate model system in the study of taste sensory perception, as it provides a range of molecular genetic tools that can be employed in both cellular and whole animal assays, such as electrophysiological recordings and Ca2+ imaging on taste neurons and behavioral analyses. While the receptors (at least for sugar and bitter compounds) are evolutionarily not conserved between mammals and insects, the organization of the gustatory systems and the logic of taste coding in these diverse animal phyla are remarkably similar. Moreover, the use of taste receptors in postprandial nutrient sensing (either in the gut or the brain) has been reported in both systems, and likely plays important roles in feeding regulation. Thus, this work will have a significant impact on fundamental principles of conserved chemosensory processes.

Insects are the most abundant class of animals, next to vertebrates. For example, the biomass of termites alone equals that of humans, the most abundant mammal. There are about 1 million named insect species and approximately another 5 million yet to be classified, compared to about 66,000 species of vertebrates. While overall beneficial to our ecosystem, some insects have considerable negative impact on human health. Disease vectors, mostly flies and mosquitoes, are major transmitter of microbes that cause devasting human diseases, including yellow fever, dengue, malaria and zika. These insect vectors kill close to a million people each year, sicken hundreds of millions more and incur billions of dollars annually in costs for treatment and lost productivity. Other insect species are agricultural pests and consume crops and fruits of cultivated plants, leading to famine in many parts of the world. In light of these facts, a better understanding of insect biology and behavior, in particular chemosensory behavior, is paramount for developing specific and effective strategies for population control of harmful pests. Drosophila melanogaster, with its array of experimental tools, is uniquely suited to uncover the basic principles underlying these behaviors. Like mammals and other insects, Drosophila depend on chemosensory systems to navigate their external world appropriately. The sense of taste is particularly important to identify food sources and avoid harmful chemicals. To assure that all essential food chemicals are consumed, insects have evolved appetitive taste receptors for the three major macronutrients, proteins, carbohydrates and fats. Intriguingly, Drosophila larvae, in contrast to adult flies, can also sense ribonucleosides and RNA in their food. These chemicals represent an essential resource required to support rapid growth and survival during the fast-growing larval stages. Larvae employ a small number of closely related taste receptors, the Gustatory Receptors (Grs) 28 to detect these chemicals. The Gr28 genes are among the most conserved insect taste receptor genes, homologs of which are found in all insect genomes, from flour beetles to honeybees to mosquitoes. These observations suggest that the Gr28 genes have a conserved role, namely to detect RNA and ribonucleosides in insects. Remarkably, some of the Gr28 genes have been implicated in temperature and light sensing, expanding their role to sensory pathways beyond taste. Thus, an in-depth understanding of the function of receptors for RNA and ribonucleosides is of considerable interest, especially because they are broadly conserved in diverse insect species, from disease vectors (mosquitoes and flies), to agricultural pests (beetles, grasshoppers) and ecologically beneficial pollinators (honeybees). Exploiting the ability of insects to sense RNA and ribonucleosides via specific taste receptors may provide new opportunities to develop strategies for control of harmful insects.