Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

Structured Odorant Response Patterns across a Complete Olfactory Receptor Neuron Population

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Faster-X Evolution of Gene Expression in Drosophila

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.     

Sensory Integration Regulating Male Courtship Behavior in Drosophila

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.     

Redundant Mechanisms for Regulation of Midline Crossing in Drosophila

During development, all neurons have to decide on whether to cross the longitudinal midline to project on the contralateral side of the body. In vertebrates and invertebrates regulation of crossing is achieved by interfering with Robo signalling either through sorting and degradation of the receptor, in flies, or through silencing of its repulsive activity, in vertebrates. Here I show that in Drosophila a second mechanism of regulation exists that is independent from sorting. Using in vitro and in vivo assays I mapped the region of Robo that is sufficient and required for its interaction with Comm, its sorting receptor. By modifying that region, I generated new forms of Robo that are insensitive to Comm sorting in vitro and in vivo, yet still able to normally translate repulsive activity in vivo. Using gene targeting by homologous recombination I created new conditional alleles of robo that are sorting defective (robo^SD). Surprisingly, expression of these modified proteins results in phenotypically normal flies, unveiling a sorting independent mechanism of regulation.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

The Zonal Organization of Odorant Receptor Gene Choice in the Main Olfactory Epithelium of the Mouse

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Molecular Mechanisms Underlying Motor Axon Guidance in Drosophila

Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.     

Odorant Receptor Polymorphisms and Natural Variation in Olfactory Behavior in Drosophila melanogaster

Animals perceive and discriminate among a vast array of sensory cues in their environment. Both genetic and environmental factors contribute to individual variation in behavioral responses to these cues. Here, we asked to what extent sequence variants in six Drosophila melanogaster odorant receptor (Or) genes are associated with variation in behavioral responses to benzaldehyde by sequencing alleles from a natural population. Sequence analyses showed signatures of deviations from neutrality for Or42b and Or85f, and linkage disequilibrium analyses showed a history of extensive recombination between polymorphic markers for all six Or genes. We identified polymorphisms in Or10a, Or43a, and Or67b that were significantly associated with variation in response to benzaldehyde. To verify these associations, we repeated the analyses with an independent set of behavioral measurements of responses to a structurally similar odorant, acetophenone. Association profiles for both odorants were similar with many polymorphisms and haplotypes associated with variation in responsiveness to both odorants. Some polymorphisms, however, were associated with one, but not the other odorant. We also observed a correspondence between behavioral response to benzaldehyde and differences in Or10a and Or43a expression. These results illustrate that sequence variants that arise during the evolution of odorant receptor genes can contribute to individual variation in olfactory behavior and give rise to subtle shifts in olfactory perception.RESEARCHERS in many scientific fields have long appreciated that different animal species perceive the world differently. In fact, these differences are so striking that new disciplines have arisen to study the adaptations of sense organs to the environment (e.g., Ali 1978; Lythgoe 1979; Dusenbery 1992). Differences in sensory perception exist not only between species, but also between populations of a single species and between individuals within a population. What is the underlying genetic architecture for individual variation in sensory perception?Olfaction provides an excellent model for examining the underlying genetic mechanisms that result in variation in behavior. In both vertebrates and invertebrates, odorants are detected by families of odorant receptors expressed in populations of olfactory receptor neurons (ORNs), whose activation elicits a distinct spatial pattern of glomerular activity in the brain (Buck and Axel 1991; Vassar et al. 1994; Mombaerts et al. 1996; Laissue et al. 1999; Gao et al. 2000; Vosshall et al. 2000; Bhalerao et al. 2003; Wang et al. 2003). This combinatorial code allows for discrimination of a diverse repertoire of odorants.Drosophila melanogaster has a relatively simple olfactory system with only 60 odorant receptor (Or) genes (Vosshall and Stocker 2007) compared to ∼1000 in the mouse (Zhang and Firestein 2002; Zhang et al. 2004). The 60 genes are located throughout the genome, and 2 of these genes are alternatively spliced for a total of 62 identified proteins (Clyne et al. 1999; Gao and Chess 1999; Vosshall et al. 1999; Robertson et al. 2003). Furthermore, clusters of Ors throughout the genome suggest several recent gene duplication events (Robertson et al. 2003).The response spectra of individual ORNs have been extensively characterized using extracellular electrophysiological recordings from single sensilla on the antennae and maxillary palps. Recordings from basiconic sensilla on the antenna identified classes of neurons with distinct olfactory response profiles organized as two to four neurons in each sensillum with specific neuronal combinations occurring in distinct spatial regions of the antenna (de Bruyne et al. 1999, 2001).The majority of ORNs express a unique odorant receptor in addition to the highly conserved coreceptor, Or83b (Jones et al. 2005). Studies of a null mutant of Or83b implicated this receptor in positioning odorant receptor proteins in the sensory dendrites (Larsson et al. 2004; Benton et al. 2006). Odorant receptors in Drosophila have an atypical membrane topology with a cytoplasmic N terminus and an extracellular C terminus (Benton et al. 2006). Specific domains in the third cytoplasmic loops of two odorant receptors, Or22a and Or43a, have been implicated to interact with the third loop of Or83b (Benton et al. 2006). Drosophila odorant receptors act as ligand-gated nonselective cation channels formed by a dimeric complex between a unique Or and the Or83b coreceptor (Sato et al. 2008; Wicher et al. 2008).Several studies have examined ligand specificities of individual odorant receptor proteins and demonstrated that they respond to diverse and overlapping suites of ligands. Response profiles for many receptors have been characterized using the Gal4/UAS system to drive expression of individual odorant receptors in a mutant ORN lacking expression of its endogeneous receptor, followed by electrophysiological recording (Dobritsa et al. 2003; Hallem et al. 2004; Hallem and Carlson 2006). In addition, misexpression studies of Or43a resulted in a reduction of behavioral avoidance responses to benzaldehyde (Stortkuhl et al. 2005). This result combined with electrophysiological recordings from ORNs and heterologous expression in Xenopus oocytes further functionally characterized the odorant response profiles of this receptor (Wetzel et al. 2001) and identified several Or43a ligands, such as fruit- derived odorants benzaldehyde, cyclohexanone, cyclohexanol, and benzyl alcohol (Stortkuhl and Kettler 2001; Hallem et al. 2004).Despite advances in our understanding of odor coding, the molecular mechanisms responsible for variation in olfactory perception remain poorly understood. D. melanogaster is especially amenable to conducting such studies given its quantitatively simple olfactory system and since large numbers of genetically identical individuals can be reared in a common environment and these individuals can be subjected to simple, rapid, and highly reproducible quantitative behavioral assays Anholt and Mackay 2004). Here, we examine how molecular variation in odorant receptors contributes to variation in olfactory behavior in inbred lines derived from a natural population of D. melanogaster. We focused our analyses on six odorant receptors, Or7a, Or10a, Or42b, Or43a, Or67b, and Or85f, which have been shown by electrophysiology (Stortkuhl and Kettler 2001; Hallem et al. 2004; Stortkuhl et al. 2005; Hallem and Carlson 2006), through heterologous expression systems (Wetzel et al. 2001), or by calcium imaging studies (Wang et al. 2003) to respond to benzaldehyde. Significant variation in behavioral responses to benzaldehyde has been observed previously in this population and was normally distributed as is typical for a quantitative trait influenced by multiple genes (Wang et al. 2007). Here, we report associations between olfactory behavior and sequence variants in three Or genes. To validate the reliability of these associations we measured responses to a structurally similar odorant, acetophenone, in the same population, and showed that the associations with variation in responses to both odorants are largely similar with occasional molecular polymorphisms associated with variation in response to only one, but not the other odorant. These observations illustrate how sequence variants that arise during the evolution of Or genes can contribute to individual variation in olfactory behavior, how polymorphisms can give rise to subtle shifts in olfactory perception, and how naturally arising mutations within a population can combine to generate broad individual variation in sensory perception.     

The Gemin Associates of Survival Motor Neuron Are Required for Motor Function in Drosophila

Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2–8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3^ΔN mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3^ΔN overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3^ΔN, we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3^ΔN-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3^ΔN. Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.     

Extremely Sparse Olfactory Inputs Are Sufficient to Mediate Innate Aversion in Drosophila

Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction.     

Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged Drosophila

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.