Genetics of alcohol consumption in Drosophila melanogaster.

Individual variation in alcohol consumption in human populations is determined by genetic, environmental, social and cultural factors. In contrast to humans, genetic contributions to complex behavioral phenotypes can be readily dissected in Drosophila, where both the genetic background and environment can be controlled and behaviors quantified through simple high-throughput assays. Here, we measured voluntary consumption of ethanol in ∼3000 individuals of each sex from an advanced intercross population derived from 37 lines of the Drosophila melanogaster Genetic Reference Panel. Extreme quantitative trait loci mapping identified 385 differentially segregating allelic variants located in or near 291 genes at P < 10-8 . The effects of single nucleotide polymorphisms associated with voluntary ethanol consumption are sex-specific, as found for other alcohol-related phenotypes. To assess causality, we used RNA interference knockdown or P{MiET1} mutants and their corresponding controls and functionally validated 86% of candidate genes in at least one sex. We constructed a genetic network comprised of 23 genes along with a separate trio and a pair of connected genes. Gene ontology analyses showed enrichment of developmental genes, including development of the nervous system. Furthermore, a network of human orthologs showed enrichment for signal transduction processes, protein metabolism and developmental processes, including nervous system development. Our results show that the genetic architecture that underlies variation in voluntary ethanol consumption is sexually dimorphic and partially overlaps with genetic factors that control variation in feeding behavior and alcohol sensitivity. This integrative genetic architecture is rooted in evolutionarily conserved features that can be extrapolated to human genetic interaction networks.

Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior.

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild-derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]-element insertion mutants in Sema-5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild-type control. We observed significant variation in olfactory responses to benzaldehyde among F1 genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome-wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry-go-round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema-5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome-wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.

Functional dissection of Odorant binding protein genes in Drosophila melanogaster.

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.

Variation in genetic architecture of olfactory behaviour among wild-derived populations of Drosophila melanogaster.

Odour-guided behaviour is a quantitative trait determined by many genes that are sensitive to gene-environment interactions. Different natural populations are likely to experience different selection pressures on the genetic underpinnings of chemosensory behaviour. However, few studies have reported comparisons of the quantitative genetic basis of olfactory behaviour in geographically distinct populations. We generated isofemale lines of Drosophila melanogaster from six populations in Argentina and measured larval and adult responses to benzaldehyde. There was significant variation within populations for both larval and adult olfactory behaviour and a significant genotype x sex interaction (GSI) for adult olfactory behaviour. However, there is substantial variation in the contribution of GSI to the total phenotypic variance among populations. Estimates of evolvability are orders of magnitude higher for larvae than for adults. Our results suggest that the potential for evolutionary adaptation to the chemosensory environment is greater at the larval feeding stage than at the adult reproductive stage.

The genetic architecture of odor-guided behavior in Drosophila melanogaster.

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a coisogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.

Functional genomics of odor-guided behavior in Drosophila melanogaster.

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.

Characterization and differential expression of a human gene family of olfactomedin-related proteins.

Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. Olfactomedin was originally identified as the major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons. Homologues were subsequently found also in other tissues, including the brain and in species ranging from Caenorhabditis elegans to Homo sapiens. Most importantly, the TIGR/myocilin protein, expressed in the eye and associated with the pathogenesis of glaucoma, is an olfactomedin-related protein. The prevalence of olfactomedin-related proteins among species and their identification in different tissues prompted us to investigate whether a gene family exists within a species, specifically Homo sapiens. A GenBank search indeed revealed an entire human gene family of olfactomedin-related proteins with at least five members, designated hOlfA through hOlfD and the TIGR/myocilin protein. hOlfA corresponds to the rat neuronal AMZ protein. Phylogenetic analyses of 18 olfactomedin-related sequences resolved four distinct subfamilies. Among the human proteins, hOlfA and hOlfC, both expressed in brain, are most closely related. Northern blot analyses of 16 human tissues demonstrated highly specific expression patterns: hOlfA is expressed in brain, hOlfB in pancreas and prostate, hOlfC in cerebellum, hOlfD in colon, small intestine and prostate and TIGR/myocilin in heart and skeletal muscle. The link between TIGR/myocilin and ocular hypertension and the expression of several of these proteins in mucus-lined tissues suggest that they play an important role in regulating physical properties of the extracellular environment. Future studies can now assess whether other members of this gene family, like TIGR/myocilin, are also associated with human disease processes.

Differential expression of G proteins in the mouse olfactory system.

Transmembrane signaling events at the dendrites and axons of olfactory receptor neurons mediate distinct functions. Whereas odorant recognition and chemosensory transduction occur at the dendritic membranes of olfactory neurons, signal propagation, axon sorting and target innervation are functions of their axons. The roles of G proteins in transmembrane signaling at the dendrites have been studied extensively, but axonal G proteins have not been investigated in detail. We used immunohistochemistry to visualize expression of alpha subunits of G(o) and G(i2) in the mouse olfactory system. G(o) is expressed ubiquitously on axons of olfactory receptor neurons throughout the olfactory neuroepithelium and in virtually all glomeruli in the main olfactory bulb. In contrast, expression of G(i2) is restricted to a sub-population of olfactory neurons, along the dorsal septum and the dorsal recess of the nasal cavity, which projects primarily to medial regions of the olfactory bulb, with the exception of glomeruli adjacent to the pathway of the vomeronasal nerve. In contrast to the overlapping expression patterns of G(o) and G(i2) in the main olfactory system, neurons expressing G(o) and those expressing G(i2) in the accessory olfactory bulb are more clearly separated, in agreement with previous studies. Vomeronasal axons terminating in glomeruli in the rostral region of the accessory olfactory bulb express G(i2), whereas those projecting to the caudal region express G(o). Characterization of the expression patterns of G(i2) and G(o) in the olfactory projection is essential for future studies aimed at relating transmembrane signaling events to signal propagation, axon sorting and target innervation.

Molecular evolution of olfactomedin.

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum-targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.

Epistatic interactions between smell-impaired loci in Drosophila melanogaster.

Odor-guided behavior is a polygenic trait determined by the concerted expression of multiple loci. Previously, P-element mutagenesis was used to identify single P[lArB] insertions, in a common isogenic background, with homozygous effects on olfactory behavior. Here, we have crossed 12 lines with these smell impaired (smi) mutations in a half-diallel design (excluding homozygous parental genotypes and reciprocal crosses) to produce all possible 66 doubly heterozygous hybrids with P[lArB] insertions at two distinct locations. The olfactory behavior of the transheterozygous progeny was measured using an assay that quantified the avoidance response to the repellent odorant benzaldehyde. There was significant variation in general combining abilities of avoidance scores among the smi mutants, indicating variation in heterozygous effects. Further, there was significant variation among specific combining abilities of each cross, indicating dependencies of heterozygous effects on the smi locus genotypes, i.e., epistasis. Significant epistatic interactions were identified for nine transheterozygote genotypes, involving 10 of the 12 smi loci. Eight of these loci form an interacting ensemble of genes that modulate expression of the behavioral phenotype. These observations illustrate the power of quantitative genetic analyses to detect subtle phenotypic effects and point to an extensive network of epistatic interactions among genes in the olfactory subgenome.

Evolution of olfactomedin. Structural constraints and conservation of primary sequence motifs.

Olfactomedin is a glycosylated extracellular matrix protein originally identified at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently localized throughout the mammalian central nervous system. Although olfactomedin homologues have been identified in fish, frog, rat, mouse and human, its function is still unknown. As a first step toward elucidating the function of olfactomedin, sequences of teleost, amphibian and human homologues were compared to identify invariant, and hence, potential functionally important motifs. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedin in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homo-polymer formation, such as the CXC motif near the amino terminus, are conserved as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, alignment of a region of 53 amino acids of fish, frog, rat and human olfactomedin reveals seven invariant residues including a negatively charged cluster of aspartic and glutamic acid residues. Molecular evolutionary genetic analysis reveals an accelerated rate of nucleotide substitutions in the mammalian lineage. The evolutionary rate at the protein level, however, is constant, indicating that evolution of olfactomedin is constrained by structural limitations. Whereas considerable evolutionary divergence is evident between fish, frog and mammalian olfactomedins, olfactomedins of rat and human show 98% amino acid sequence identity. It appears that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian and mammalian lineages. The apparent evolutionary pressure toward conservation of primary structure supports the notion that olfactomedin has an important function in the mammalian nervous system.

Pheromone regulated production of inositol-(1, 4, 5)-trisphosphate in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a G alpha(q/11)-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.

Quantitative genetic variation of odor-guided behavior in a natural population of Drosophila melanogaster.

Quantitative genetic variation in behavioral response to the odorant, benzaldehyde, was assessed among a sample of 43 X and 35 third chromosomes extracted from a natural population and substituted into a common inbred background. Significant genetic variation among chromosome lines was detected. Heritability estimates for olfactory response, however, were low, as is typical for traits under natural selection. Furthermore, the loci affecting naturally occurring variation in olfactory response to benzaldehyde were not the same in males and females, since the genetic correlation between the sexes was low and not significantly different from zero for the chromosome 3 lines. Competitive fitness, viability and fertility of the chromosome 3 lines were estimated using the balancer equilibrium technique. Genetic correlations between fitness and odor-guided behavior were not significantly different from zero, suggesting the number of loci causing variation in olfactory response is small relative to the number of loci causing variation in fitness. Since different genes affect variation in olfactory response in males and females, genetic variation for olfactory response could be maintained by genotype x sex environment interaction. This unusual genetic architecture implies that divergent evolutionary trajectories for olfactory behavior may occur in males and females.

Effects of single P-element insertions on olfactory behavior in Drosophila melanogaster.

Single P-element (P[lArB]) insertional mutagenesis of an isogenic strain was used to identify autosomal loci affecting odor-guided behavior of Drosophila melanogaster. The avoidance response to benzaldehyde of 379 homozygous P[lArB] element-containing insert lines was evaluated quantitatively. Fourteen smell impaired (smi) lines were identified in which P[lArB] element insertion caused different degrees of hyposmia in one or both sexes. The smi loci map to different cytological locations and probably are novel olfactory genes. Enhancer trap analysis of the smi lines indicates that expression of at least 10 smi genes is controlled by olfactory tissue-specific promoter/enhancer elements.

Signal integration in the nervous system: adenylate cyclases as molecular coincidence detectors.

Integrating multiple incoming messages simultaneously and discriminating 'meaningful' signals from spontaneous neural activity represent central problems to the nervous system. One mechanism by which signal integration and signal-to-noise resolution are achieved is the formation of temporal coincidence circuits by interacting transduction pathways. Signal integration via temporal coincidence detection is exemplified most readily by the way in which neural adenylate cyclases are regulated. This review will discuss the role of adenylate cyclases as coincidence detectors in the nervous system with special focus on adenylate cyclase type III, an isoenzyme that is found in large quantities in olfactory receptor neurons. The notion that olfactory transduction might also utilize an adenylate-cyclase-mediated temporal coincidence circuit strengthens the idea that signal integration via temporal-coincidence pathways is a universal feature of all neural adenylate cyclases.

Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium.

The extracellular mucous matrix of olfactory neuroepithelium is a highly organized structure in intimate contact with chemosensory cilia that house the olfactory transduction machinery. Here we describe the molecular cloning and primary structure of olfactomedin, which is the major component of this extracellular matrix. Olfactomedin is expressed exclusively in olfactory neuroepithelium and its amino acid sequence shows no homologies to any known protein. This olfactory tissue-specific glycoprotein contains cysteines which form disulfide-linked polymers that constitute the primary architecture of the olfactory extracellular matrix. By analogy to other extracellular matrix proteins of the nervous system, olfactomedin may influence the maintenance, growth, or differentiation of chemosensory cilia on the apical dendrites of olfactory neurons.

Formation of the extracellular mucous matrix of olfactory neuroepithelium: identification of partially glycosylated and nonglycosylated precursors of olfactomedin.

Olfactomedin is the major glycoprotein of the extracellular mucous matrix of frog olfactory neuroepithelium. It is responsible for the primary architecture of this extracellular matrix by forming via intermolecular disulfide bonds polymers, which are covered with evenly spaced carbohydrate groups. To study glycosylation of olfactomedin, we raised antibodies against the mature protein and antibodies against a region adjacent to an N-linked glycosylation site near its amino terminus. The latter antibodies cannot bind when this site is glycosylated and reveal precursors of olfactomedin in the perinuclear regions of Bowman's glands. In contrast, antiserum against the mature protein stains acinar regions of glands and the ciliary surface. Enzymatic deglycosylation of olfactomedin shows stepwise removal of carbohydrate and reveals a 51-kDa deglycosylated form. Our results indicate that, prior to secretion, most, if not all, of the six potential N-linked glycosylation sites of olfactomedin are glycosylated with carbohydrate moieties of about 8-10 sugar residues.

Molecular neurobiology of olfaction.

Odor discrimination is mediated via dendritic cilia of olfactory receptor neurons. Odorants traverse the aqueous mucous interphase that lines the surface of the olfactory neuroepithelium and interact with odorant receptors, which are members of the superfamily of G-protein-linked receptors. These interactions trigger synthesis of second messengers, including cyclic AMP and inositol triphosphate. Cyclic AMP opens a cation channel to elicit the generator current, which depolarizes the cell and, ultimately, leads to action potentials. Inositol triphosphate opens a calcium channel in the ciliary plasma membrane. Calcium entering through both this channel and the cyclic nucleotide-gated channel modulates the response to odorants by amplifying the generation of cyclic AMP after binding to calmodulin. Calcium also is essential for desensitization of olfactory receptor neurons. Differential expression of odorant receptors of diverse ligand specificities by different olfactory neurons ensures that the structures and concentrations of odorants that reach the chemosensory surface are encoded as distinct patterns of neuronal activity, which are relayed to the brain where they take shape as characteristic odor sensations.

Olfactomedin: purification, characterization, and localization of a novel olfactory glycoprotein.

We have identified a novel glycoprotein expressed exclusively in frog olfactory neuroepithelium, which we have named "olfactomedin". Olfactomedin is a 57-kDa glycoprotein recognized by seven monoclonal antibodies, previously shown to react solely with proteins of olfactory cilia preparations. It undergoes posttranslational modifications, including dimerization via intermolecular disulfides and attachment of complex carbohydrate moieties that contain N-acetylglucosamine and beta-D-galactoside sugars. Olfactomedin strongly binds to Ricinus communis agglutinin I and has been purified to homogeneity by lectin affinity chromatography. Polyclonal rabbit antiserum raised against purified olfactomedin confirmed that it is expressed only in olfactory tissue. Immunohistochemical studies at the light microscopic and electron microscopic level show that olfactomedin is localized in secretory granules of sustentacular cells, in acinar cells of olfactory glands, and at the mucociliary surface. The massive production of olfactomedin and its striking deposition at the chemosensory surface of the olfactory neuroepithelium suggest a role for this protein in chemoreception.

Benzodiazepine receptors in the eye.

Central and peripheral benzodiazepine receptors were localized in the rat, monkey, and human eye by in vitro autoradiography. Central benzodiazepine binding sites, visualized with 3H-R015-1788, were enriched in the inner plexiform layer in all three species. Binding sites also were present in the nerve fiber layer, the ganglion cell layer, and in portions of the inner nuclear layer. Peripheral benzodiazepine binding sites, visualized with 3H-PK-11195, were found in the corneal epithelium and endothelium, iris, ciliary epithelium, trabecular meshwork, and throughout the retina. Binding sites for 3H-PK-11195 also were present in the retinal pigment epithelium and choriocapillaris areas and retinal vascular structures.