1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

469 Poster Developmental, Neurogenesis, and ConsumerResearchWNT AND FZ IN THE DEVELOPING MOUSE OLFACTORYSYSTEMRodriguez Gil D.J. 1 , Greer C.A. 2 1 Neurosurgery, Yale University, NewHaven, CT; 2 Neurobiology, Yale University, New Haven, CTThe general principles of axon extension and regeneration can beeffectively studied in the olfactory system. Olfactory sensory neurons(OSNs) are broadly distributed in the olfactory epithelium (OE) yettheir axons target restricted areas of olfactory bulb (OB) neuropil,glomeruli, with exceptional precision. The odor receptors have beenstrongly implicated in the targeting of these axons. Nevertheless, howOSN axons navigate from the OE up to the OB is still not known.Formerly known as morphogens, there is increasing evidence thatWingless-Int (Wnt) molecules, signaling through Frizzled receptors (Fz)contribute in a variety of processes, including development of neuronalcircuits. In this context, the aim of the present work was to study theexpression of several Wnt, Fz and secreted Fz-related protein (sFrp) inthe developing OB and OE. RT-PCR was performed from mouseembryonic (E13, E17) and early postnatal (P0, P4) tissues. Wnt-1mRNA was the only one not found either in the OB or OE at any age.All Fz and sFrp studied were expressed at all analyzed ages.Immunohistochemical characterization showed that Wnt-5a is expressedby OMP positive cell bodies throughout the OE while, the expressionFz-7 is restricted to cells as yet unidentified in zones 2 to 4. In the OBFz-7 was present in olfactory ensheathing cells, olfactory nerve layerand glomeruli, while Wnt-5a showed a relatively uniform stainingthroughout the OB. Taking into account that during the period of studyOSN axons are growing towards the OB and starting to synapse in theglomeruli, it could be suggested that Wnt and Fz are involved in thedevelopment of the primary olfactory pathway and in synaptogenesis inthe OB. Support Contributed By: NIH DC00210, DC006792 andDC006291 to CAG.470 Poster Developmental, Neurogenesis, and ConsumerResearchEXPRESSION OF AXON GROWTH AND GUIDANCE GENESIN IMMATURE OSNSMcIntyre J.C. 1 , McClintock T.S. 1 1 Basic Science: Physiology,University of Kentucky, Lexington, KYNewly differentiated olfactory sensory neurons (OSNs) grow axonsinto glomeruli in the olfactory bulb. The identity of the single odorantreceptor expressed in each OSN determines which axons convergetogether. However, extrinsic guidance cues and neuronal activity mustalso be involved in guiding and targeting OSN axons. Differentiallyabundant mRNAs from two microarray experiments have identifiedmouse genes that may be involved in OSN axon growth and guidance.In situ hybridization revealed that several of these mRNAs are presentonly in immature OSNs, or in immature OSNs and basal cells. ThesemRNAs include Cxcr4, Dpysl3, Mlp, Ppp2cb, and Dbn1. The proteinsencoded by these mRNAs are known to regulate axon growth in otherparts of the nervous system. For example, Cxcr4 and its ligand Cxcl12have been shown to direct the ventral trajectory of ventral motorneurons (Lieberam et al., 2005, Neuron. 47:667). We find that Cxcl12 isexpressed in the stroma of postnatal day 0 mice while Cxcr4 isexpressed in immature OSNs, suggesting that Cxcl12 could direct thetrajectory of nascent OSN axons. At postnatal day 21, expression ofCxcl12 is absent from the stroma and restricted to underlying bonewhile Cxcr4 is detectable only in a subset of the immature OSNs thatexpress Gap43. These data suggest that if Cxcr4/Cxcl12 signalingorients OSN axons, it is most important during development and theinitial outgrowth of the axon. Supported by R01 DC002736.471 Poster Developmental, Neurogenesis, and ConsumerResearchGENE-TARGETED DELETION OF KV1.3 CHANNEL ALTERSOLFACTORY RECEPTOR GENE EXPRESSION ANDMODIFIES PRIMARY OLFACTORY PROJECTIONSBiju K.C. 1 , Walker D.W. 1 , Fadool D.A. 1 1 Biological Science, Programsin Neuroscience and Molecular Biophysics, The Florida StateUniversity, Tallahassee, FLKv1.3, a member of the Shaker family of potassium channels, plays akey role in the excitability of olfactory bulb neurons. Previously wehave demonstrated that gene-targeted deletion of Kv1.3 created a “super-smeller” phenotype with an increased ability to discriminate anddetect odors, an increased firing frequency of mitral cells, and anincreased number of glomeruli. Since sensory activity plays a key rolein axonal targeting, we asked whether deletion of Kv1.3 affects axonaltargeting during development. We generated a mouse line that carriesM72-IRES-taulacZ in a Kv1.3-null background (double-mutant) andcompared its projection pattern with that of mice expressing M72-IRES-taulacZ (wildtype) at P20. The number of olfactory sensoryneurons (OSNs) expressing M72 receptors was dramatically reduced inthe double-mutant mice. The morphology of M72 OSNs was alsoaltered in the double-mutant mice; OSNs were comparatively slenderwith an elongated dendrite. In the olfactory bulb, M72 positive axonswere less numerous and their axons coalesced into two or three novelglomeruli at positions different from that observed in wildtype mice.We are currently analyzing M72 double-mutant mice over postnataldevelopment and comparing with patterns of P2 expressing OSNs in theKv1.3-null background. These data indicate that Kv1.3 influences theguidance of primary sensory axons and the targeting to specificglomeruli that may be receptor-type dependent. This work wassupported by NIH DC03387 (NIDCD).472 Poster Developmental, Neurogenesis, and ConsumerResearchSIGNALING MOLECULES INVOLVED IN REGULATINGMOUSE OLFACTORY AXON OUTGROWTHChen H. 1 , Gong Q. 1 1 Cell Biology and Human Anatomy, University ofCalifornia, Davis, Davis, CAOlfactory sensory neurons (OSNs) in the olfactory epithelium projecttheir axons into defined glomeruli in the olfactory bulb. Althoughseveral cell surface molecules have been shown to play a role inolfactory axon growth and targeting, the signaling pathway in the OSNsinvolved in this process is largely unknown. To investigate the signalingmechanisms for OSN axon outgrowth and guidance, we established adissociated OSN culture system, which allows the visualization of axongrowth and manipulation of single OSN. Pharmacological studies wereconducted to investigate the involvement of PKC, PKA, PKG, and RhoGTPase pathways in OSN axon elongation. We observed that OSNaxons were dramatically shortened at 30 hrs after the treatment ofRottlerin (20 nM), a PKCδ specific inhibitor. However, increasedelongation of OSN axons was observed after the treatment of Go6976(100 nM), a specific inhibitor for PKCα and βI. These results suggestthat isoforms of PKC play antagonistic roles in the OSN axonoutgrowth and targeting. Small GTPases are also involved in the OSNaxon outgrowth. Rac1 promotes the elongation of OSN axons indicatedby Rac1 inhibitor (NSC23766, 30 µM) result. However, downstreamregulator of RhoA, Rho kinase, negatively regulates the outgrowth ofOSN axons, suggested by Rho kinase inhibitor (Y27632,10 µM) results.Unexpectedly, PKA activator, Sp-cAMPS; PKA inhibitors, H89 andRp-cAMPs; PKG activator, 8-Bromo-cGMP; PKG inhibitors, KT5823and Rp-8-pCPT-cGMPs, did not affect the axonal outgrowth of OSNsunder our culture condition. Supported by NSF0324769118