1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

465 Poster Developmental, Neurogenesis, and ConsumerResearchPROTOCADHERIN 20 EXPRESSION IS RESTRICTED IN THENEWLY DIFFERENTIATED OLFACTORY SENSORYNEURONSLee W. 1 , Gong Q. 1 1 Cell Biology and Human Anatomy, University ofCalifornia, Davis, CAOlfactory sensory neurons (OSNs) expressing the same odorantreceptor project their axons to a predicted region of the olfactory bulb(OB) and converge into the same foci, the glomeruli. Guidancemechanisms for olfactory axons during development have beeninvestigated intensely. In an effort to study this question, we haveexamined and detected the expression of protocadherin 20 (pcdh20)transcripts in the nasal epithelium. Pcdh20 is a novel non-clusteredprotocadherin. 3T3 fibroblasts transfected with pcdh20 expressionconstructs demonstrated Ca 2+ -dependent homophilic adhesion by cellaggregation assay. An antibody against the C-terminus region ofPcdh20 was developed. Pcdh20 immunostaining is restricted in theOSNs at embryonic and early postnatal stages. Pcdh20 was not detectedin other regions of the brain. At P6, Pcdh20 is highly expressed in theolfactory nerve fascicles and continues in the olfactory nerve layer andin all glomeruli of the OB. In the adult, however, Pcdh20 isdramatically down regulated. Although most of the glomeruli aredevoid of Pcdh20, a few glomeruli are found to have high Pcdh20expression in adult OB. To examine whether Pcdh20 is expressed innewly generated OSNs in adult, we have performed diphtheria toxinmediated OSN specific ablation using OMP-DTR transgenic mice. At17 days after OSN ablation, Pcdh20 is up-regulated in the olfactoryaxon fascicles and more Pcdh20 positive glomeruli are observed in theadult OB. These data indicate that pcdh20 is an OSN specific adhesionmolecule which is expressed exclusively in newly generated OSNs.Supported by NIH DC006015 NSF0324769466 Poster Developmental, Neurogenesis, and ConsumerResearchLOCALIZATION OF NUCLEAR RETINOIC ACIDRECEPTORS, RAR AND RXR IN POSTNATAL RODENTOLFACTORY EPITHELIUMAsson-Batres M. 1 , Smith W. 1 , Ahmad O. 1 , Zeng M. 2 1 BiologicalSciences, Tennessee State University, Nashville, TN; 2 Sun Yat-senUniversity, Guangzhou, ChinaRetinoic acid (RA) is a derivative of vitamin A (VA) that is known toaffect developmental processes, including cell differentiation. RA issynthesized from VA, all-trans-retinol, via a biosynthetic pathway thatincludes a terminal oxidation step catalyzed by a retinaldehydedehydrogenase (RALDH). We have recently published that RALDHsare present and relatively abundant in the postnatal rodent olfactoryepithelium (OE) and underlying lamina propria, an indication that RAis produced by this tissue. RA is thought to facilitate gene transcriptionby activating nuclear RA receptors. Our work has demonstrated that VAdeficiency (VAD) leads to a significant loss of mature olfactory neuronsin postnatal rat OE, a continuously differentiating neural tissue. Ourinterpretation is that neuron development is impeded when RA isunavailable. An assumption of this hypothesis is that nuclear receptorsare present. Using immunohistochemical methods, we show here thatRARβ and RXRβ are present in cell bodies in the the central andsupranuclear region of postnatal rodent OE. Positive staining of thesecells colocalizes with markers for nuclei. The RARβ gene has anupstream RA response element that regulates transcription of the gene.Using quantitative RT-PCR, we find that RARβ transcript expression issignificantly reduced in VAD olfactory tissue, an indication that RAlevels are also reduced. These results and our previous findings suggestthat RA is synthesized locally in postnatal rodent OE, where it interactswith nuclear RA receptors that appear to be expressed in sustentacularcells and cells of neuronal origin. Supported by NIH/NIDCD 1 K02DC180-01 and NIH/NIGMS/MBRS/SCORE 3 S06 GM008092-28S1.467 Poster Developmental, Neurogenesis, and ConsumerResearchMETHYL BINDING DOMAIN PROTEINS IN THE STAGE-SPECIFIC DIFFERENTIATION OF OLFACTORY RECEPTORNEURONSMacDonald J.L. 1 , Roskams J. 2 1 Neuroscience Graduate Program,University of British Columbia, Vancouver, British Columbia, Canada;2 Zoology, University of British Columbia, Vancouver, British Columbia,CanadaMethylation of cytosine residues is associated with epigenetic genesilencing and is critical for mammalian development. De novo DNAmethyltransferases (DNMTs) catalyze the methylation, producing siteswhich may then be bound by methyl-CpG binding domain proteins(MBDs), forming repressor complexes that modify chromatin structurevia recruitment of histone deacetylases. Disruptions in this process havebeen implicated in developmental disorders. The olfactory epithelium,where neurogenesis is ongoing, is an ideal system in which to studyDNA methylation-dependent gene silencing during neuronaldifferentiation. The DNMTs are expressed in a stage-specific,sequential pattern during olfactory neurogenesis. DNMT3b is expressedin cycling progenitors, as they commit to the neuronal lineage, and islikely necessary to mediate this transition. DNMT3a is expressed inimmature receptor neurons, and is down-regulated as they functionallymature, suggesting that it initiates gene silencing necessary to transitionfrom an immature to a mature neuron. The MBD proteins MBD2 andMeCP2 are similarly expressed at distinct stages of olfactorydevelopment. Expression of MBD2 is initiated in the progenitors of theOE, and is maintained throughout differentiation. MeCP2 is expressedin immature receptor neurons as they functionally mature. Furthermore,MBD2 and MeCP2 knockout mice display stage-specific defects inolfactory neurogenesis corresponding to their observed expressionpatterns. Our findings indicate that the sequential recruitment ofepigenetic modifiers may be essential for the successful stage-specificdifferentiation of olfactory receptor neurons. Funding was provided byNSERC and CIHR (JM) and NIDCD (JR)468 Poster Developmental, Neurogenesis, and ConsumerResearchEXPRESSION OF TRANSCRIPTIONAL REGULATORS INOLFACTORY SENSORY NEURONSSammeta N. 1 , McClintock T. 2 1 Basic Science: Physiology, University ofKentucky, Lexington, KY; 2 Physiology, University of Kentucky,Lexington, KYMicroarray comparison of a purified population of mature olfactorysensory neurons (OSNs) against all other cells in the olfactoryepithelium predicts that OSNs express 238 mRNAs whose genes areannotated as regulators of transcription. Signal intensity distributionspredict which of these mRNAs are expressed in mature OSNs versusother cell types in the epithelum. We attempted in situ hybridization on63 of these mRNAs. Of the 36 that gave insitu hybridization signals, 28were detected in the OSN layers of the olfactory epithelium, with somebeing restricted to either the mature or immature OSN layer. ThosemRNAs encoding transcriptional regulators involved in differentiationand maturation should show preferential expression in immatureneurons. Six1, Sox6, Msx1, Tle1, Tle3 and Mef2b fit this pattern, andthese genes are known to be associated with developmental events inother tissues. In contrast, mRNAs associated with mature neurons arelikely to be associated with homeostasis or the final stages ofmaturation of OSNs. Nfat5, Crebl1, Xbp1, Nfatc1, Nfe2l2, Ches1 andUsf2 fit this pattern. These genes share links to several types of stressresponses. The type of transcriptional regulators expressed in OSNsgives an insight into the development and homeostasis of the tworecognized OSN phenotypes. Supported by R01DC002736117