1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

229 Slide Molecular Genetic Approaches toChemoreceptionNEURON-SPECIFIC ODOR RECEPTOR GENE CHOICE INDROSOPHILARay A. 1 , Van Der Goes Van Naters W. 1 , Carlson J. 1 1 MCDB, YaleUniversity, New Haven, CTWe have uncovered a combinatorial code of cis-regulatory elementscritical to the process of odor receptor (Or) gene choice in Drosophila.The Or gene family in Drosophila consists of 60 members. Individualmembers are expressed in specific functional classes of olfactoryreceptor neurons (ORNs) of either the antenna or the maxillary palp.We have identified both positive and negative cis-regulatory elementsthat act together to dictate the organ-specific expression of individualreceptor genes in the maxillary palp. In Drosophila a special subset ofORNs expresses not one but two Or genes. We have identified twodistinct mechanisms by which such co-expression can be achieved.First, we demonstrate that some co-expressed Or genes share specificsequence motifs in their flanking DNA that can direct expression in acertain neuronal type. Second, for some of the tightly linked Or genes,we have found evidence that the downstream gene can share the samepromoter as the upstream one by virtue of alternative splicing of themRNA. The majority of ORNs, however, express a single Or gene. Wehave used comparative genomics to identify evolutionarily conservedcis-acting neuron-specific elements that act in specifying the expressionof these classes of palp ORNs. In vivo analysis of these elements hasled to a model for Or gene choice. We find that within an olfactoryorgan a given Or gene has the ability to express in a few different ORNclasses; however, transcriptional repressors restrict expression to theappropriate neuronal class. Finally, we have identified transcriptionfactors that are required for this highly regulated process of Or genechoice.230 Slide Molecular Genetic Approaches toChemoreceptionALTERING OLFACTORY NEURON IDENTITY WITHECTOPIC EXPRESSION OF G-PROTEIN COUPLEDRECEPTORSChesler A. 1 , Le Pichon C. 2 , Peterlin Z.A. 3 , Matthews G. 4 , Zou D. 2 ,Firestein S. 2 1 Biology, Columbia University, New York, NY; 2 ColumbiaUniversity, New York, NY; 3 Biological Sciences, Columbia University,New York, NY; 4 Neurobiology and Behavior, Columbia University, NewYork, NYIn the mammalian olfactory epithelium (OE), each olfactory sensoryneuron (OSN) is thought to express only one allele of a single olfactoryreceptor (OR) gene. It has been proposed that the expressed OR proteinrepresses activation of additional OR genes through feedback inhibition.The resulting singular OR expression effectively determines the identityof the OSN by defining its odorant response properties and influencingits axonal projection. The mechanisms by which ORs influence OSNidentity are unknown. We have taken a gain-of-function approachusing ultrasound-guided injection of the embryonic OE with retroviralvectors to study the role of ORs in the development of OSN identity.We find that the ectopic expression of functional GPCRs, either ORI7or beta-2-Adrenergic Receptor, perturb odorant response profiles andaxonal sorting without a noticeable effect on endogenous ORexpression. Additionally, expression of an amino-terminally mutatedORI7 that encodes a full-length and properly trafficked OR does notconfer octanal responsiveness or perturb axonal sorting. Our worksupports a model wherein modulation of G-protein signaling, at least inpart, encodes OSN identity. In conclusion, embryonic expression ofGPCRs using retroviral vectors offers a new gain-of-function approachfor studying the role of ORs in olfactory development. SupportContributed By: NIDCD.231 Slide Molecular Genetic Approaches toChemoreceptionUBIQUITOUS EXPRESSION OF AN ODORANT RECEPTORIMPAIRS PROPER AXON TARGETING TO THE OLFACTORYBULBVidaltamayo R. 1 , Reed R.R. 1 1 Molecular Biology and Genetics,Howard Hughes Medical Institute, Baltimore, MDIn order to study the effects of widespread expression of an odorantreceptor (OR) in the olfactory epithelium, we have generated atransgenic mouse line in which we placed the coding region of the mI7OR gene into the transcription factor O/E3 locus. Expression of thistranscription factor occurs early in olfactory neuron development andpersists in mature olfactory neurons in the adult mouse. In the O/E3-mI7 line, we detect widespread expression of mI7 OR, encompassing allfour zones of the olfactory epithelium, from birth (postnatal day 0) up toadulthood (8 week-old mice), both at the mRNA, by in situhybridization, and protein level, by immunofluorescence.Electroolfactogram recordings show a 100-fold increase in the doseresponse to the mI7 ligand, heptaldehyde (-4.59 ± 1.17 mV at 1 x 10 -6M 7-al in O/E3-mI7 and -5.35 mV ± 0.25 mV at 1 X 10 -4 M 7-al in wtmice, P < 0.05, Student´s T-test, n = 4). The olfactory neurons fromthese animals express high levels of mI7 protein and provide a usefulmodel for examining adaptation and OR protein biochemistry. Althoughubiquitous expression of mI7 does not abolish the expression ofendogenous ORs (M72, mOR28), it dramatically affects the targeting ofM72-expressing axons. Moreover, stereotyped alterations in theposition of convergent axons to generate glomeruli suggest a model forsorting axons and glomerular formation. Supported by Howard HughesMedical Institute and NIH232 Symposium Changing the Development of Tasteand OlfactionFLAVOR PROGRAMMING DURING INFANCYMennella J.A. 1 1 Monell Chemical Senses Center, Philadelphia, PAA major factor that has inhibited progress in understanding the originof human flavor preferences is the absence of a robust, experimentalparadigm. In addition to demonstrating that experience with flavors inamniotic fluid, mothers´ milk and formulas contribute to individualdifferences in flavor preferences, we have identified a naturallyoccurring flavor variation that can be exploited to study this important,yet under-investigated, research area. Using as a model system a classof infant formulas that are hydrolyzed-protein based and havedistinctive flavors which are unpalatable to older-aged infants andadults, we found that early exposure to this formula (Nutramigen)resulted in a complete shift in hedonic tone to the flavor of this formulafrom one of absolute distaste to eager acceptance. The effects of earlyexposure were particularly persistent, leading to heightened preferencesfor sour tastes, as well as for the taste and aroma of the formula andsimilarly flavored foods, several years after the child´s last exposure.These finding provided evidence of the clearest example of a sensitiveperiod in the development of responses to flavors in humans thus faridentified. This research is supported by NIH Grant HD37119.58