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RNASeq of OMP and TRPC2 neuronal population of zebrafish olfactory sensory neurons

ABSTRACT: RNASeq was performed on mRNA obtained from OMP expressing and TRPC2 expressing olfactory sensory neurons from 48hpf zebrafish embryos. Single-cell suspensions of 48hpf olfactory epithelia were FAC-sorted to obtain RFP expressing or venus expressing cells (RFP sample1: 3500 cells, RFP sample2: 5500 cells. Venus sample1: 676 cells, venus sample2:201 cells. RNA was extracted using QIAGEN RNEasy kit and cDNA was synthesized using a custom oligo-dT primer containing T7 promoter sequence courtesy of Jim Eberwine, University of Pennsylvanina. In vitro RNA amplification was carried out through one round for OMP:RFP neurons and through two rounds for TRPC2:venus neurons as per the protocol in Morris et al, 2011. Adapter-tagged libraries were synthesized using Illumina TruSeq v2.0 and deep-sequenced on HiSeq2500 to obtain ~100million reads per sample.

ORGANISM(S): Danio rerio

PROVIDER: GSE86023 | GEO | 2018/03/03

REPOSITORIES: GEO

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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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RNASeq of OMP and TRPC2 neuronal population of zebrafish olfactory sensory neurons

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