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Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-wide RNASeq Analysis

ABSTRACT: We report the application of RNA sequencing technology for high-throughput profiling of the articular and mature zones of the mandibular condyle in postnatal 7-day-old mice. Briefly, mandibular condyles of postnatal 7-day-old mice were frozen-sectioned with articular, mature zones idintified under a dissection microscope. Cells from the articular zone (az), mature zone (mz) were isolated by laser capture microscopy. Total RNAs from each sample were polyA selected and single-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit (Illumina). The samples were then sequenced using the Illumina HiSeq sequencer. Total sequencing reads representing RNA transcripts were generated and were mapped to the NCBI mouse reference genome. Differential expressed genes were identified using DESeq, with <0.05 false discovery rate (FDR). Fragments Per Kilobase of exon model per Million Mapped fragments (FPKM) were estimated for each gene in each sample. Hierarchical clustering was also performed on log2FPKM values with Cluster 3.0. Only genes with FDR<0.05 and ≥0.80 standard deviation were included. RNA comparison between articular and mature zones revealed distinctive gene expression profiles.

ORGANISM(S): Mus musculus

PROVIDER: GSE113116 | GEO | 2018/07/19

REPOSITORIES: GEO

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Project description:Articular and growth plate cartilage have comparable structures consisting of three distinct layers of chondrocytes, suggesting similar differentiation programs and therefore similar gene expression profiles. To address this hypothesis and to explore transcriptional changes that occur during the onset of articular and growth plate cartilage divergence, we used microdissection of 10-day-old rat proximal tibial epiphyses, microarray analysis, and bioinformatics to compare gene expression profiles in individual layers of articular and growth plate cartilage. We found that many genes that were spatially upregulated in intermediate/deep zone of articular cartilage were also spatially upregulated in resting zone of growth plate cartilage (overlap greater than expected by chance, P < 0.001). Interestingly, superficial zone of articular cartilage showed an expression profile with similarities to both proliferative and hypertrophic zones of growth plate cartilage (P < 0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in superficial zone compared to intermediate/deep zone (more than expected by chance, P < 0.001 each). In conclusion, we provide evidence that intermediate/deep zone of articular cartilage has a gene expression profile more similar to resting zone of growth plate cartilage, whereas superficial zone has a gene expression profile more similar to proliferative and hypertrophic zones. 10-day-old rat proximal tibial epiphyses were manually microdissected into articular cartilage superficial (SZ) and intermediate/deep (IDZ) zones and growth plate cartilage resting zone (RZ) for total RNA extraction and hybridization on Affymetrix microarrays. We used 10-day-old animals because, at this age, the secondary ossification center has recently begun to form and divides the epiphysis into articular cartilage distally and growth plate cartilage more centrally. The 4 SZ samples were taken from animals 5-8, respectively, whereas the 4 IDZ and 4 RZ samples were each taken from animals 1-2, 3-4, 5-6, and 7-8, respectively.

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Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-wide RNASeq Analysis

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