1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

473 Poster Developmental, Neurogenesis, and ConsumerResearchMEDIATION OF CELL SIGNALING EVENTS INDEVELOPING OLFACTORY SYSTEM OF MANDUCA SEXTABY LIPID RAFTSGibson N.J. 1 , Hildebrand J.G. 1 , Tolbert L.P. 1 1 ARL Div. ofNeurobiology, University of Arizona, Tucson, AZDuring development of the adult olfactory system of the mothManduca sexta, axons of olfactory receptor neurons (ORNs) extendinginto the brain induce centrally derived glia to migrate and populate asorting zone; encountering these glia causes later-growing ORN axonsto sort and fasciculate according to their target glomerulus. Past workshowed that EGF receptor/neuroglian interactions in the sorting zonepromote axonal outgrowth and sorting, that FGF receptors present onglia are activated at critical times, and that ORN axons sort according toexpression of the IgCAM fasciclin II. Furthermore, ORN axons displayglycosphingolipids (GSLs) in patterns that change during development.In other systems GSLs are concentrated in membrane domains calledlipid rafts, which have been shown to be important in the function ofEGF and FGF receptors and IgCAMs. Current experiments explore therole played by lipid rafts in cell signaling events in the sorting zone.Sucrose gradient flotation reveals two low-density bands that mayrepresent different types of lipid rafts; Western blots reveal that onlyone contains GPI-linked fasciclin II, which previous studies showed isassociated with glial cells. General disruption of raft assembly withmethyl--cyclodextrin causes aberrant glial migration and abnormalglomerular microarchitecture. Ongoing experiments will furthercharacterize raft components and examine the functional organization ofthose rafts with respect to neuron-glia signaling in the sorting zone.Supported by NIH Grants DC004598 and P01-NS28495.475 Poster Developmental, Neurogenesis, and ConsumerResearchEXPRESSION OF GONADOTROPIN-RELEASING HORMONE(GNRH) AND GONADOTROPIN RELEASING HORMONERECEPTORS (GNRH-R) IN THE ZEBRAFISHTwomey S.L. 1 , Illing N. 1 , Brideau N. 1 , Smith K. 1 , Whitlock K. 11 Molecular Biology and Genetics, Cornell University, Ithaca, NYWe have shown that GnRH cells originate from precursors lyingoutside the olfactory placode: the region of the anterior pituitary givesrise to hypothalamic GnRH cells and the cranial neural crest gives riseto the GnRH cells of the terminal nerve and midbrain (1, 2). Ouranalysis of the molecular forms of GnRH expressed in these cellssuggests that zebrafish have a third form of GnRH as has been observedin other fishes. Concurrently with the examination of the GnRHdecapeptide expression, we are examining GnRH-R expression. Wecloned GnRH-Rs from the zebrafish and our analysis of these putativereceptors confirms that there are four receptors: two type I receptors(GnRH-R2, GnRH-R4) and two type II receptors (GnRH-R1, GnRH-R3). Using digoxygenin labeled mRNA probes generated against thesesequences we were able to detect signal for putative receptor GnRH-R3(Type II) and GnRH-R4 (Type I) at 56 hours post fertilization. Using anantibody recognizing the Type II receptor (ISPR3) we were able toidentify two cell types in the olfactory sensory system, large diametercells in the respiratory epithelium and smaller, apparently neuronal cellsin the sensory epithelium. This last observation suggests that GnRHmay affect the olfactory sensory epithelium via a Type II GnRHreceptor. Support: NIH/HD050820, NYS Hatch Grant 165047 (KEW).1. Whitlock KE, Wolf CD, Boyce ML (2003). Dev. Biol. 257(1):140-152. 2. Whitlock KE, Smith, K, Kim, H, Harden MV (2005).Development 132(24): 5491-5502.474 Poster Developmental, Neurogenesis, and ConsumerResearchDISRUPTION OF KALLMANN AND FGFR1 GENE FUNCTIONIN ZEBRAFISH DIFFERENTIALLY AFFECTS GNRH ANDOLFACTORY CELL DEVELOPMENTKim H.K. 1 , Smith K.M. 1 , Whitlock K.E. 1 1 Molecular Biology andGenetics, Cornell University, Ithaca, NYHuman Kallmann syndrome is characterized by hypogonadichypogonadism (deficits in GnRH) and anosmia (loss of sense of smell).Two of the genes known to underlie Kallmann syndrome are KAL1(anosmin1) and KAL2 (fibroblast growth factor receptor 1, fgfr1).Though KAL1 has not been found in mouse, zebrafish have two KAL1homologues, kallmann1.1 (kal1.1) and kallmann1.2 (kal1.2) (Ardouin etal., 2000), and also have one KAL2 homologue, fibroblast growthfactor receptor 1 (fgfr1) (Scholpp et al., 2004). To identify mechanismscontrolling GnRH and olfactory sensory cell development, we disruptedkallman and fgfr1 gene function in the developing zebrafish. We usedmorpholinos (modified oligonucleotides; MO) to block proteintranslation of kal1.1, kal1.2, and fgfr1. “Knockdown” of gene functioncaused reduction of endocrine GnRH cells, but had no effect onneuromodulatory midbrain or nervus terminalis GnRH cells (Whitlocket al., 2005). The olfactory nerves of these animals were disrupted butnot absent. Our data indicate that knockdown of kal1.1, kal1.2, andfgfr1 results in a different GnRH cell and olfactory sensory systemphenotype for each gene. Also, we studied the developmental origins ofendocrine GnRH cells in relation to anterior pituitary and hypothalamicdevelopment. The anterior pituitary development was not greatlydisrupted in kal1.1 MO injected fish, yet a hypothalamic marker wascompletely absent in many injected fish. Our data suggest kal1.1 isinvolved in both endocrine GnRH neuron and hypothalamicdevelopment. Support: NIH/HD050820 (KEW); NYS Hatch Grant165047.476 Poster Developmental, Neurogenesis, and ConsumerResearchODORANT MODULATION OF IMMEDIATE EARLY GENEEXPRESSION IN THE ZEBRAFISH OLFACTORY EPITHELIAMcKenzie M.G. 1 , Harden M.V. 1 , Whitlock K.E. 1 1 Dept. of MolecularBiology and Genetics, Cornell University, Ithaca, NYImmediate early genes (IEGs) are transcription factors that arerapidly up-regulated in response to sensory stimuli. Our previous workhas shown that one IEG, c-fos is expressed in the developing olfactoryepithelia as early as 24 hours post fertilization (hpf) and is retained in asmall and variable number of cells up to 72hpf. To investigate whetherodorant exposure modulates the expression of c-fos in the olfactoryepithelia, we have used in situ hybridization to compare c-fosexpression in the olfactory epithelia of odor-exposed and controlembryos. Two odorants shown to be environmentally significant inadult goldfish, prostaglandin (PGF2α) and Progesterone (17α,20βdihydroxyprogesteronesodiumsulphate) (Sorensen et al., 1988),have an effect on olfactory epithelial c-fos expression in the zebrafish.After 2 days of chronic exposure to these odorants [10-8 M], thefrequency of embryos displaying high numbers of c-fos expressing cellsincreased relative to wild-type control siblings. The difference wasfound to be statistically significant by a Mann-Whitney rank-sum test (P< 0.005) in both cases. Our results suggest a possible role of odorenvironment in influencing gene expression during the development ofthe olfactory epithelium. Support: Cornell Irving Tanner Dean´s Grant(MGM), NIHDC0421801 (KEW) 1. Sorensen, P.W., Hara, T.J., Stacy,N.E., Goetz, F.W.M. (1988). Biology of Reproduction 39, 1039-1050.119