Microarray-Based Discovery of Highly Expressed Olfactory Mucosal Genes: Potential Roles in the Various Functions of the Olfactory System
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Physiol Genomics 16: 67–81, 2003. First published October 21, 2003; 10.1152/physiolgenomics.00117.2003. Microarray-based discovery of highly expressed olfactory mucosal genes: potential roles in the various functions of the olfactory system Mary Beth Genter,1 Paul P. Van Veldhoven,2 Anil G. Jegga,3 Bhuvana Sakthivel,3 Sue Kong,3 Kristin Stanley,3 David P. Witte,3 Catherine L. Ebert,3 and Bruce J. Aronow3 1Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267-0056; 2Katholieke Universiteit Leuven, Department of Molecular and Cellular Biology, Pharmacology Division, B-3000 Leuven, Belgium; and 3Children’s Hospital Medical Center, Cincinnati, Ohio 45229 Submitted 17 June 2003; accepted in final form 8 October 2003 Genter, Mary Beth, Paul P. Van Veldhoven, Anil G. Jegga, nervous system, as it has a basal cell population that provides Bhuvana Sakthivel, Sue Kong, Kristin Stanley, David P. Witte, a continuous supply of new olfactory neurons. Thus neurons Catherine L. Ebert, and Bruce J. Aronow. Microarray-based discov- lost to injury or toxic insult can be replaced (13, 14, 27, 33). At ery of highly expressed olfactory mucosal genes: potential roles in the the present time, we lack an understanding of the overall various functions of the olfactory system. Physiol Genomics 16: 67–81, genomic expression patterns that allow for this to be accom- 2003. First published October 21, 2003; 10.1152/physiolgenomics.00117. plished. 2003.—We sought to gain a global view of tissue-specific gene expres- sion in the olfactory mucosa (OM), the major site of neurogenesis and We have used the power of microarray-based genomics to neuroregeneration in adult vertebrates, by examination of its overex- examine gene expression in the mouse OM relative to that of pressed genes relative to that in 81 other developing and adult mouse many other tissues. For example, other studies utilizing the tissues. We used a combination of statistical and fold-difference criteria University of Cincinnati-Children’s Hospital Medical Center to identify the top 269 cloned cDNAs from an array of 8,734 mouse (UC-CHMCC) Mouse Tissue Specific Gene Expression Data- cDNA elements on the Incyte Mouse GEM1 array. These clones, repre- base described gene expression in liver development and re- senting known and poorly characterized gene transcripts, were grouped generation (42) and in various anatomical segments of the according to their relative expression patterns across the other tissues and gastrointestinal tract (6). In the present study we are specifi- then further examined with respect to gene ontology categories. Approx- cally interested in understanding gene expression patterns that imately one-third of the 269 genes were also highly expressed in devel- distinguish the OM, with its capacity to support continuous oping and/or adult central nervous system tissues. Several of these have neurogenesis, from more quiescent regions of the brain, as well been suggested or demonstrated to play roles in neurogenesis, neuronal differentiation, and/or neuronal migration, further suggesting that many as from adjacent tissue in the nasal cavity, namely, the nasal of the unknown genes that share this expression pattern may play similar respiratory mucosa. We hypothesized that the molecular defi- roles. Highly OM-specific genes included a palate, lung, and nasal nition of these different, but closely related, tissues could be epithelium carcinoma-associated gene (Plunc); sphingosine phosphate performed by comparing the expression and function of large lyase (Sgpl1), and paraoxonase 1 (Pon1). Cell-type-specific expression numbers of genes in OM, nasal respiratory mucosa, and brain. within OM was established using in situ hybridization for several repre- This approach revealed a group of genes with expression sentative expression pattern clusters. Using the ENSEMBL-assembled highly restricted to the OM. Other clusters of genes were mouse genome and comparative genomics analyses to the human ge- highly expressed in OM and other tissues, including in 1)OM nome, we assigned many of the unknown expressed sequence tags and olfactory bulb; 2) OM and lung; 3) OM and liver; 4)OM (ESTs) and poorly characterized genes to either novel or known gene and respiratory mucosa; and 5) an OM and skin gene clusters. products and provided predictive classification. Further exploration of We also demonstrated the utility of this approach for gene this database will provide additional insights into genes and pathways discovery with the detection of novel genes likely to be critical for olfactory neurogenesis, neuronal differentiation, olfaction, and mucosal defense. relevant in the life cycle of olfactory neurons. cDNA microarray; gene expression profiles; gene discovery; bioin- MATERIALS AND METHODS formatics; neurodevelopment; Plunc; sphingosine phosphate lyase; paraoxonase Animals, tissue samples, and RNA preparation. Adult OM, olfac- tory bulb, and nasal respiratory mucosa were obtained from 6- to 8-wk-old male C57BL/6 mice (Jackson Laboratories) as part of an THE OLFACTORY MUCOSA (OM) is positioned at the junction of the institutional consortium effort to develop a generalized mouse gene respiratory tract and nervous system and performs functions expression database. This database includes a wide range of develop- integral to both organ systems. Olfactory sensory neurons are ing, normal adult, and diseased adult mouse tissues, including multi- responsible for odorant transduction. In addition, inhaled air ple brain subregions, dorsal root ganglion, heart, lung, kidney, liver, can be detoxified of toxic agents by mucociliary mechanisms reproductive and endocrine organs, immune organs, muscle, and skin. and metabolic enzymes in the nasal respiratory and olfactory The ethmoid turbinates and caudal one-third of the nasal septum served as the source of OM RNA, and the nasoturbinates, maxillo- tissues. The OM is unique with respect to the rest of the turbinates, and anterior two-thirds of the nasal septum were the source of respiratory mucosal RNA (for anatomy, see Ref. 87). Mice were Article published online before print. See web site for date of publication killed by carbon dioxide inhalation according to institutional and (http://physiolgenomics.physiology.org). national animal care guidelines, and tissues were rapidly dissected and Address for reprint requests and other correspondence: M. B. Genter, Dept. snap frozen in liquid nitrogen. Frozen tissues, pooled from eight mice, of Environmental Health, Univ. of Cincinnati, Cincinnati, OH 45267-0056 were homogenized on ice with a stainless steel tissue grinder, and total (E-mail: [email protected]). RNA was prepared using TRI Reagent (Molecular Research Center, 1094-8341/03 $5.00 Copyright © 2003 the American Physiological Society 67 68 GENE EXPRESSION IN OLFACTORY MUCOSA Cincinnati, OH), following the manufacturer’s protocol. Total RNA sample channel was divided by the whole mouse reference channel was reprecipitated with ethanol/sodium acetate and resuspended in followed by a LOWESS (“locally weighted scatter plot smoothing”) DEPC-treated water. Polyadenylated [poly(A)ϩ] RNA was prepared intensity-dependent normalization, and then per gene across the series from total RNA using Oligotex (Qiagen, Valencia, CA). Poly(A)ϩ of probe arrays using the median expression ratio observed for the RNA was quantitated using RiboGreen dye (Molecular Probes, Eu- gene across the entire University of Cincinnati-Children’s Hospital gene, OR) and checked for RNase degradation using an Agilent Medical Center (UC-CHMCC) Mouse Tissue Specific Gene Expres- Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). A total of sion Database. 600 ng of poly(A)ϩ RNA (50 ng/l) for each tissue was submitted for The measured intensity of each gene was divided by its control cDNA labeling and microarray hybridization. channel value in each sample. When the control channel value was Fluorescent probe preparation and microarray hybridization. below 10.0, the data point was considered invalid. Intensity-dependent Probe preparation and microarray hybridization were performed by normalization was also applied, where the ratio was reduced to the Incyte Genomics (Palo Alto, CA) using the GEMBright random residual of the LOWESS fit of the intensity vs. ratio curve as primer reverse-transcription labeling kit (Incyte Genomics). Labeled implemented in GeneSpring 4.2.1. The 50th percentile of all measure- cDNA was prepared from each poly(A)ϩ RNA sample using nucle- ments was used as a positive control for each sample; each measure- otides labeled with the fluorescent dye Cy5. Labeled cDNAs were ment for each gene was divided by this synthetic positive control, then subjected to competitive hybridization to mouse GEM1 microar- assuming that this was at least 0.01. Each gene was normalized to rays, composed of spotted cDNAs corresponding to 8,638 sequence- itself by making a synthetic positive control for that gene and dividing verified IMAGE expressed sequence tag (EST) clones (500–5,000 all measurements for that gene by this positive control, assuming it nucleotides in length) with a low redundancy rate with respect to their was at least 0.01. This synthetic control was the median of the gene’s corresponding gene products (a complete list of the genes represented expression values over all the samples. on the GEM1 microarray can be found at http://microarray.uc.edu/ Hierarchical tree clustering allowed grouping of genes based on DataBases/Incyte_Mouse_GEM1.xls). For all samples