Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling of ICAM-positive and -negative cells from the mouse olfactory epithelium

ABSTRACT: We used trasncriptional profiling of fluorescent activated cell sorting (FACS) purified ICAM1-positive and negative cells from the olfactory epithelium (OE) of three-week old mice to identify genes enriched in the horizontal basal cells. The olfactory epithelia from P21-P25 CD1 mice (Charles River Laboratories, Wilmington, MA), were isolated, dissociated, and then purified by FACS using a FITC-conjugated antibody for ICAM1 (CD54). ICAM1-positive and negative cell populations were collected and compared. RNA was purified with TRIzol reagent (Invitrogen, Carlsbad, CA), subjected to two rounds of amplification, labeled, and hybridized to Affymetrix Mouse Genome 430.2 GeneChip microarrays (Affymetrix Inc., Santa Clara, CA, USA) using Affymetrix reagents and protocols (http://www.affymetrix.com). There were three experimental replicates, each consisting of the ICAM1 (+) and ICAM1 (-) FACS purified cell populations from individual litters of mice that were dissected/dissociated/FACS-purified using an ICAM1-FITC conjugated antibody; one microarray was used for each ICAM1 (+) and ICAM1 (-) sample for a total of six microarrays. Microarray data were normalized using GCRMA.

ORGANISM(S): Mus musculus

SUBMITTER: Russell Fletcher

PROVIDER: E-GEOD-31972 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of GFP and cell surface markers to FACS-isolate Sox2-GFP(+) GBCs, Neurog1-GFP(+) GBCs and immature neurons, and OMP-GFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2100 genes that are upregulated compared to sustentacular cells. A further cohort of >1200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-GFP(+) population. In contrast, the abbreviated lifespan of OMP-GFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream Sox2-GFP(+) GBCs and Neurog1-GFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion. Total RNA was isolated from dissected, dissociated and FACS-purified olfactory mucosal cells from normal adult mice or mice 3 weeks after unilateral bulbectomy. Cells were purified with FACS using endogenous GFP fluorescence from various transgenic lines and cell surface labeling. Two to seven adult mice were used per replicate and three replicates per condition were performed using Illumina bead arrays. 21 samples from olfactory mucosa were analyzed in this series and 3 samples were from a commercially available reference RNA sample. Universal mouse reference RNA from Stratagene was used as a general control and Normal olfactory mucosa was used as a tissue specific control.

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Transcriptional profiling of ICAM-positive and -negative cells from the mouse olfactory epithelium

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