A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity

Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, frizzled, and transforming growth factor-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.     

Identification of novel mRNAs expressed in oligodendrocytes

To identify new proteins, which are expressed in oligodendrocytes and which may have a functional role in myelination, a rat oligodendrocyte cDNA library was screened using differential and subtractive screening techniques. Ten clones that have elevated levels of expression in brain were isolated. Two of these clones were characterized further and one clone, pC26.H2, was found to be closely related to mouse stearoyl-CoA desaturase 2 (SCD2), which catalyzes the synthesis of unsaturated fatty acid. From Northern blot and in situ hybridization studies, SCD2 mRNA was expressed primarily in brain with lower levels found in lung and spleen. In brain sections, SCD2 mRNA was found primarily in oligodendrocytes, although mRNA was detected at a low level in neurons, in particular in Purkinje cells in the cerebellum. Northern blot analysis of the other clone, p973.HB, indicated that it was expressed more selectively in brain. In mixed glial cultures oligodendrocytes were the only cells that expressed this mRNA, whereas in brain, neurons expressed this mRNA at a higher level than in oligodendrocytes. This clone is being actively pursued because of its unique expression exclusively in oligodendrocytes and neurons.Special issue dedicated to Dr. Marjorie B. Lees.     

A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.     

Identification of members of the Bex gene family as olfactory marker protein (OMP) binding partners

Olfactory marker protein (OMP) expression is a hallmark of mature vertebrate olfactory receptor neurons (ORNs). Evidence for OMP function derives from altered behavioral and electrophysiological activities of OMP-KO mice. The molecular basis for the altered phenotype following the deletion of OMP is still unclear. Recent structural studies predict the involvement of OMP in protein-protein interaction. Here we report the identification of an OMP partner, Bex2, by phage-display screening of an olfactory mucosal cDNA-library. In situ hybridization demonstrates cellular co-localization of OMP mRNA with mRNAs for Bex1, Bex2, and Bex3 in ORNs of olfactory tissue of the mouse. The OMP/Bex interaction has been confirmed by demonstrating the chemical cross-linking of recombinant rat OMP with a synthetic peptide derived from the Bex amino acid sequence. The subcellular localization of Bex and OMP proteins was evaluated in transfected HEK293 cells. Bex is visualized in the nucleus and cytoplasm. Following co-transfection we observed the unexpected presence of some OMP in the nucleus along with Bex. Together, these data argue convincingly that we have identified Bex as an OMP partner whose further characterization will provide insight to the role of OMP and to the mechanism of the OMP/Bex interaction in ORN differentiation and function.     

Hybridization on squashed flies: A method to detect gene sequences in individual Drosophila

Specific gene sequences can be detected by DNA hybridization to individual Drosophila squashed on cellulose or nylon filters. This “squash-blot” method permits the rapid survey of DNA polymorphism in large Drosophila population samples. It could also be useful for studying chromosome aberrations, departure from diploidy, and detection of pathogenic agents in vector insects.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.     

Vege and Mar: two novel hAT MITE families from Drosophila willistoni

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.     

A mouse homolog of dunce,a gene important for learning and memory in drosophila,is preferentially expressed in olfactory receptor neurons

The dunce (dnc) gene in Drosophila codes for a cyclic adenosine monophosphate-specific phosphodiesterase (PDE). Flies with a mutation at this locus exhibit severe deficits in learning and memory. We have begun to analyze the neural distribution of mammalian homologs of dnc in the mouse. Surprisingly, in situ hybridization and northern blotting using a probe specific for one of the four mammalian dnc homologs (mPDE2) reveals high levels of expression in the olfactory neuroepithelium. Anti mPDE2 antibody confirms that this PDE protein is abundant in the axons and dendrites of the olfactory receptor neurons but is conspicuosly absent from the cilia, where the initial events in olfactory signal transduction occur. Lower levels of mPDE2 were also detected throughout the brain and in the testis. These findings suggest an important modulatory role for mPDE2 in mammalian olfaction. © 1995 John Wiley & Sons, Inc.     

标化消减杂交:提高差异表达cDNA筛选效率的新策略

在回顾分析了消减杂交技术改进历程的基础上,我们提出了“标化消碱杂交策略”,此策略的核心是“用标化cDNA制备消碱杂交反应的单链驱动子和单链示踪子,分离回收消减杂交后的单链示踪子用于制备差异表达cDNA库,并从标化cDNA库制备探讨来对差异表达cDNA库进行检测”将主要用于筛选与细胞特殊表型密切相关的（或与不同发育阶段等密切相关的）呈“全或无”差异形式表达的低丰度全长cDNA。此策略有望能较以往的消减杂交策略在降低假阳性背景、提高低丰度差异表达cDNA获得率、得到较长甚至全长差异表达cDNA等方面有较好表现,但尚待检验。     

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.     

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana⁹ mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana⁹ mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana⁹ mutants. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 199–211, 1997     

Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons

Evidence has accumulated to support a model for odorant detection in which individual olfactory receptor neurons (ORNs) express one of a large family of G protein-coupled receptor proteins that are activated by a small number of closely related volatile chemicals. However, the issue of whether an individual ORN expresses one or multiple types of receptor proteins has yet to be definitively addressed. Physiological data indicate that some individual ORNs can be activated by odorants differing substantially in structure and/or perceived quality, suggesting multiple receptors or one nonspecific receptor per cell. In contrast, molecular biological studies favor a scheme with a single, fairly selective receptor per cell. The present studies directly assessed whether individual rat ORNs can express multiple receptors using single-cell PCR techniques with degenerate primers designed to amplify a wide variety of receptor sequences. We found that whereas only a single OR sequence was obtained from most ORNs examined, one ORN produced two distinct receptor sequences that represented different receptor gene families. Double-label in situ hybridization studies indicated that a subset of ORNs co-express two distinct receptor mRNAs. A laminar segregation analysis of the cell nuclei of ORNs labeled with the two OR mRNA probes showed that for one probe, the histogram of the distribution of the cell nuclei along the depth of the epithelium was bimodal, with one peak overlapping the (unimodal) histogram for the other probe. These results are consistent with co-expression of two OR mRNAs in a population of single ORNs.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina

In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems.     

A novel thermosensitive escape behavior in Drosophila larvae

We describe a novel thermosensitive escape behavior in Drosophila larvae and a simple assay to accurately define the response temperature. When a larva is placed in a droplet of water that is subsequently heated, a stereotypical escape response is robustly elicited at 29°C. Larvae defective for the painless TRP receptor, or blocked in the function of class IV multi-dendritic sensory dendrites respond to this stimulus at reproducibly higher temperature (34°C). The escape response has novel behavioral components and a lower temperature threshold in comparison with the responses to touch with a hot needle. Furthermore the assay minimizes operator bias that is present in current tests of thermosensitive nociception and generates a precise determination of temperature at the point of response. This response is highly reproducible and directly applicable to genetic and neural circuit analysis of a simple escape behavior.     

DMAP-85: A τ-Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-βII-(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the βII-(422–434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-τ antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on τ, MAP-2, and MAP-4 and specific N-terminal sequences of τ, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that τ and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP. The finding on a Drosophila protein with functional homology and structural similarities to mammalian τ opens new perspectives to understand the cellular roles of MAPs.