Browsing by Person "Breer, Heinz"

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Lokalisation von Pheromon-Rezeptoren und -Bindeproteinen in antennalen Sensillen von Insekten
(2007) Gohl, Thomas; Breer, Heinz
The remarkable reactivity of moth to specific pheromones is based on the extreme selectivity and sensitivity of sensory cells in the male antennae. This feature is supposed to be based on cells equipped with specific receptors. Only the sequencing of the genomes of Bombyx mori and Heliothis virescens provided the possibility to identify candidate genes of olfactory receptors in moths. Upon detailed inspection of candidate receptors it turned out that within the generally very heterogeneous group of receptor-genes a subfamily exists containing members of both species showing striking sequence homology. A conservation of the primary structure of receptors for pheromones has been postulated. For a continuing characterization in these studies different approaches were used to verify that these receptors are indeed expressed in cells of pheromone-sensitive sensilla (sensilla trichodea). By means of ?whole mount? in situ hybridization experiments the RNA of the receptor types BmOR1 and BmOR3 could be visualized in directly neighboring cells reflecting the topology of trichoid sensilla. Also some of the Heliothis receptor types (HR13, HR14, HR16) could be assigned to sensilla trichodea. In addition to the specific receptors, the pheromone binding proteins (PBPs) are expected to play an important role in the detection of hydrophobic pheromone molecules. PBPs are produced by glia-like cells surrounding the sensory neurons. In double in situ hybridization experiments it could be shown that HR13-cells are indeed surrounded by cells expressing HvirPBP1 and HvirPBP2. Analysis comparing the topology of different receptor-types showed that cells expressing HR13 can be assigned to sensilla trichodea type A, whereas HR14 and HR16 are expressed in cells of sensilla trichodea type C. This characteristic expression pattern is considered as a further indication that these candidate-receptors are indeed pheromone-receptors. The assignment of individual receptor-types to distinct sensilla-types provides the basis for investigating the functional implications of receptor-types for the registration of main or minor components of complex pheromone-blends. Further it turned out that HR13 shows coexpression with SNMP1 (sensory neuron membrane protein 1) which is considered as a ?marker?-protein for antennal sensory neurons. This is however not the case for receptor types HR14 and HR16. In search of further SNMP-types screening-experiments were carried out which led to the identification of a novel SNMP-type (SNMP2) of Heliothis virescens. Subsequent studies concerning the expression of SNMP2 showed that the topologic distribution of SNMP2-cells is comparable to SNMP1-cells, but they show a different morphology. Further experiments revealed that SNMP2 is in fact expressed in PBP-producing cells. These findings imply that the proposed putative function of SNMPs has to be reconsidered. One major goal of this study was the attempt, to identify receptor-relevant cells by visualization of mRNA via in situ hybridization but to visualize the localization of the receptor-protein via immunohistochemical approaches. Although the generation of antibodies for olfactory receptors is very difficult, it was possible to raise antibodies specific for receptor type HR13. Using these antibodies in immunohistochemical approaches allowed to also visualize HR13-receptor-protein. By means of double-staining experiments using HR13-specific antisense RNA-probes and anti-HR13 antibodies mRNA and protein were visualized in the same specific cells. Using confocal laserscanning microscopy, it was possible to document that receptor-protein was indeed located in the sensory dendrites. Further, the receptor-protein was also visualized in the axonal processes of sensory cells and the receptor-specific staining revealed that within the antennal nerve HR13-axons appear to be organized in fascicles. These HR13-immunolabeled fascicles were visible until they reach the ?sorting zone? of the antennal lobe; in contrast to mouse olfactory bulb, no receptor specific staining was visible in the antennal lobe.
Molecular elements involved in locust olfaction : gene families in the desert locust Schistocerca gregaria
(2018) Jiang, Xingcong; Breer, Heinz
Locusts are remarkable insects due to their unique and potentially devastating phenotypic plasticity based on the local population density. While “solitarious” phase locusts avoid one another, “gregarious” locusts can form dense and highly mobile swarms, which have been feared as agricultural pests since ancient history. For this reason alone, locust biology has long been the object of intense scientific studies; moreover, from a purely scientific perspective it is of great interest to unravel the mystery underlying the phenotypic plasticity. The unique phase transition including the behavioral plasticity heavily relies on chemical communication by means of critical volatiles. It is therefore important to elucidate the mechanisms underlying locust chemosensory communication, including the identification of molecular elements involved in recognizing odorous compounds. Towards this goal, the desert locust Schistocerca gregaria, as a representative locust species, was investigated in this study. One of the key elements for recognizing odorous compounds are odorant binding proteins (OBPs). To gain insight into the repertoire of locust OBPs, genomic sequences encoding candidate OBPs from Schistocerca gregaria together with those from three other locust species were subjected to thorough comparative analyses. The results indicated that locust OBPs could be classified into several categories, namely, “classic OBPs”, “plus-C OBPs”, “minus-C OBPs” and “atypical OBPs” which reside in four major phylogenetic families (I to IV). With the aim to uncover distinct features of the various OBP types, the initial studies were concentrating on the conserved subfamilies I-A and II-A which comprise “classic OBPs”. The sequence analyses provided evidence for both common and subfamily-specific motifs as well as evolutionary clues based on the calculation of coden substitution rates, which suggested the effect of purifying selection pressure. The subfamily I-A comprised a much higher number of orthologous OBPs than subfamily II-A, which resulted in a distinct re-clustering patterns for subfamily I-A and subfamily II-A. Exploring the topographic expression pattern on the antennae revealed that OBPs of subfamily I-A were selectively expressed in sensilla basiconica and sensilla trichodea, whereas OBPs of subfamily II-A were restricted to sensilla coeloconica. Furthermore, cells expressing the subtype OBP1 were present in almost all sensilla basiconica and trichodea, whereas other subtypes were only present in subpopulations. The OBPs of subfamily II-A, were expressed in distinct subpopulations of sensilla coeloconica. Analyses of representative OBPs from the remaining phylogenetic subfamilies revealed that representative subtypes from subfamily III-A and III-B were expressed in sensilla chaetica, similarly the two representatives of subfamily I-B were also expressed in this sensillum type. The selective expression of these OBPs in sensilla chaetica was substantiated by analyzing the antennal tip, which comprises numerous sensilla chaetica. The “atypical OBP” OBP12, a representative of subfamily IV-A was found to be selectively expressed in a distinct subpopulation of sensilla coeloconica, while “plus-C OBP” OBP9, from subfamily IV-B, showed a unique expression pattern and seemed to be associate with all four sensillum types. The diversity and complex sensilla- and cellular-specific distribution implies distinct functional implications of OBP subtypes in the process of chemoreception.
Odorantrezeptoren in Axonen olfaktorischer Sinneszellen : in vitro Studien an Explantatkulturen
(2008) Luxenhofer, Georg; Breer, Heinz
Olfactory sensory neurons (OSN) expressing a particular odorant receptor (OR) are widely scattered throughout the olfactory epithelium (OE) of the nose and send their axon into a small number of common glomeruli in the olfactory bulb (OB). In a spatially well conserved pattern these axons establish synaptic contacts to second order neurons. The molecular mechanisms underlying the precise wiring are still not well understood. To generate a system which may facilitate the investigation of distinct aspects of this complex process, an in vitro culture with tissue explants from the olfactory system was established in the present work. The use of tissues from transgenic mice which enabled the visualisation of OSN and their processes by intrinsic fluorescence allowed a continuous observation of distinct axonal populations under defined and manipulable conditions. Cells within an explant from the OE harvested at the embryonic stage 14 (E14) extended numerous axonal processes within a few days which grew out radially and without fasciculation. During the initial culture period the explants contained mainly progenitor cells; after several days in culture cells differentiated to OMP-positive, thus mature OSN. Using receptor specific transgenic mouse lines the expression of distinct OR genes in a subpopulation of OSN could be detected. Altogether, the culture conditions thus allowed the differentiation of progenitor cells into OSN with characteristic gene expression. Concerning the key question of how axons of OSN interact with their target tissue, co-culture experiments with OB tissue were performed; they showed that axons were initially repelled by their target. A precultivation of OB tissue, however, resulted in an attraction of axons even from larger distances. Moreover, the bulb tissue exerted a positive effect on the growth rate of OSN axons. During their growth these axons formed bundles which defasciculated in the vicinity of the OB explants. These results showed that characteristic parameters in the generation of OSN, their axonal growth and interactions with the target tissue were recapitulated by the in vitro culture system, thus, providing optimal conditions for the examination of key questions regarding the molecular mechanisms involved in establishing the unique projection pattern. Subsequently, the explant culture system was used to investigate the role of the odorant receptor protein in the process of path finding. Expression of genetically modified receptor variants in the explants revealed novel insights into the subcellular localisation of the odorant receptor mOR256-17. An mOR256-17-EGFP fusion protein could be detected in vesicles transported into the dendrite of OSN, resulting in an accumulation of the OR in the cilia. Using this technique it was possible to observe for the first time OR proteins in vesicles which were transported anterogradely and retrogradely along the entire axon. The OR could be visualised within the growth cones and the attached filopodia. Taking advantage of a novel detection method in which proteins integrated into the plasma membrane were selectively marked, retrogadely transported vesicles containing internalised mOR256-17 protein could be observed. The generation of an OR variant, in which the G-protein binding domain was mutated resulted in a disturbed localisation of the OR protein within OSN. Hence, by developing an improved in vitro explant system, an important tool was generated that allowed novel insights into the function of distinct molecular components and should be valuable for future studies aimed at understanding the complex processes that lead to the precise connection of OSN with their target.
Olfaktorische Rezeptoren mit speziellen topographischen Expressionsmustern
(2007) Feistel, Torben; Breer, Heinz
In the present study, olfactory receptors (ORs) featuring special expression patterns in chemosensory subsystems were analyzed. The data showed that out of a repertoire of about 1000 ORs a restricted group (about 50) was not only expressed in the olfactory epithelium but also in the vomeronasal organ, which is generally defined by the expression of characteristic V1R and V2R receptors. The ?ectopically? expressed OR genes represent different receptor families including genes from gene-clusters located on different chromosomes. However, in all individuals the same set of genes seemed to be expressed. The majority of the expressed ORs was present in only a small number of VNO cells, however some were found to be expressed in more than 100 cells. One distinct OR gene (mOR261-6) was expressed in many more cells of female VNOs than in males. The highest number of OR expressing cells was observed in a short postnatal, pre-pubertal period. Cells with mOR18-2-receptors were also found to express a V1R gene. In these cells the OR18-2 gene did not show a mono-allelic expression as compared to expression in the main olfactory system but rather was transcribed from both alleles (bi-allelic) in the vomeronasal neurons. The functional implication of receptor coexpression and bi-allelic OR-expression are unknown. Receptors of the OR37 gene family are exclusively expressed in the olfactory epithelium, in which they are expressed in a special pattern in a central area of the nasal turbinate. Detailed analysis of the spatial distribution of cells equipped with distinct OR37 subtypes revealed that these cells were positioned in specific sub-compartments within this central region. The high number of OR37 expressing cells resulted in the lower number of cells with receptors which are zonally distributed. This implies that cells in a small circumscribed area preferentially expressed OR37 genes. The mechanisms underlying this unique spatial expression pattern of OR37 genes in the olfactory epithelium are currently unknown. A newly generated mouse line was arranged in which even very transient transcription of a OR37 gene is visualized by permanent label in the expressing cells. The examination showed that cells outside of the OR37 cluster did express a OR 37 family member, yet transcription in these cells was rapidly terminated. These results suggest a mechanism which allows an initial transcription of these genes in different areas of the epithelium, but a sustained expression of OR37 genes is restricted exclusively to the central recess of the turbinate. The special features of the OR37 subtypes have led to the hypothesis that these receptors possibly react to chemical compounds relevant for mammalian species. Thus complex mixtures of volatile compounds from the habitat of mice were examined. Electroolfactograms from different regions of the olfactory epithelium showed that the ?head-space? of the embedding from mice cages elicited a stronger response in the central area of the turbinate IIb than in areas outside of this region. Single substances out of this complex mixture induced similar responses in different epithelial recesses. However, 6-hydro-6-methyl-3-heptanon elicited stronger responses within the central OR37 expressing region. This substance is considered to be a mouse pheromone. Taken together, these data on expression and ligand specificity of distinct olfactory receptors suggest, that the vomeronasal organ and the olfactory epithelium although structurally separated chemosensory systems, overlap in their response, spectrum and in their functions, particularly in detecting compounds with biological relevance.
Regulatory elements controlling the expression of OR37 genes
(2007) Zhang, Yongquan; Breer, Heinz
The genes of the OR37 family are clustered in two loci (cluster I and cluster II) on mouse chromosome 4. These genes encode distinct olfactory receptors (ORs) which are characterised by an insertion of six amino acids in the third extracellular loop and moreover, these receptor types are only expressed in cells which are segregated in a small patch on the central nasal turbinate. As first steps to unravel the molecular basis of this unique topographic expression pattern previous studies have led to the identification of highly conserved sequence motifs including an olf-1 site in the putative promoter region of these genes and subsequently several transcription factors were identified which did bind to these sites. However, it remained elusive if an interaction between the transcription factors and the putative promoter sites may have functional implications. Therefore, a heterologous system was employed to assess the consequence of an interaction between the putative promoters and the transcription factors. HEK 293 cells were cotransfected with a reporter gene under the control of putative mOR37 promoter regions and an expression vector based gene encoding the transcription factor. The expression rate of the reporter gene was monitored by measuring luciferase activity. It was found that the three O/E transcription factors (O/E-1, O/E-2 and O/E-4) induced significant activation of the mOR37 promoters; in addition, it was observed that the putative promoters of other OR genes were also activated, suggesting that the O/E proteins may play a general role in the regulation of OR gene expression. Mutagenesis experiments revealed that the effects of O/E proteins were dependent on the presence of an olf-1 site within the promoter region. For the transcription factor Lhx-2 it was found that not all but only promoters of distinct OR-genes were affected. For the mOR37 promoters a simultaneous action of O/E protein and Lhx-2 elicited an increase of reporter gene expression. The data indicate that the putative mOR37 promoters could drive gene expression in the presence of the crucial transcription factors in this heterologous system. In order to explore to what extent the promoter may contribute to the characteristic topographic expression pattern of the mOR37 genes in vivo, a mOR37C transgene which included the coding exon and the putative promoter, was randomly inserted into the mouse genome. Seven lines were obtained; in all lines the transgene was specifically expressed in olfactory sensory neurons (OSN). In six lines the transgene expression was restricted to the central patch of the olfactory turbinates, typical for the OR37 genes. In one line (line 7) the transgene was also expressed in OSNs ectopically positioned outside the patch within the medial zone. It was found that the transgene was expressed in a mutually exclusive manner and from only one allele. The axons of OSNs expressing the transgene co-converged in the same glomerulus with the axons from neurons expressing the endogenous gene. In line #7 the formation of ectopic glomeruli was observed. The number of OSNs expressing the transgene varied considerably among lines; these differences were independent from the copy number of the transgene. The data indicate that the short putative promoters, most likely the conserved motifs, were sufficient to drive the OR37 gene expression in a tissue specific way and most aspects of the OR37 gene expression were mimicked by the transgene; however, considerable differences between certain lines suggested additional regulatory elements, such as a locus control region (LCR). Since regulatory elements for gene transcription, such as promoters, enhancers and LCRs, appear to be conserved across species, a comparative approach was utilized to search for the LCR-like element for the OR37 locus by sequence alignment across distantly related mammals. A segment of 270 base pairs located 137 Kb upstream of OR37 cluster I was found to be highly conserved between mouse, human, dog and opossum. It was not associated with an exon of any known gene and was highly correlated with OR37 cluster I rather than with the neighboring genes, since the flanking genes did not show syntenic conservation in the opossum genome. A homologous counterpart for this segment was found downstream of the OR37 cluster II locus; an alignment of the cluster II sequence across species identified the conservation of this counterpart. Examination for relevant motifs in this segment and comparison with the conserved H element revealed two common transcription factor binding sites, at least one of them is known to be essential for generating DNase I hypersensitive sites in the LCR of the beta globin gene locus. Further studies are required to evaluate a possible role of this conserved segment in the regulation of the OR37 gene expression.