Browsing by Subject "Odorant receptor"

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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.