Global comparative transcriptome analysis of cartilage formation in vivo
BackgroundDuring vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of in vivo gene expression.ResultsTibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for Agc1, Bmp8a, Col2a1, Fgfr4, Foxa3, Gdf5, Klf2, Klf4, Lepre1, Ncad, Sox11, and Trpv4. Further, independent validation of the microarray data was achieved by in situ hybridization to analyse the expression of Lepre1, Pcdh8, Sox11, and Trpv4 from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several Bmp, Gdf, Wnt, Sox and Fox family members.ConclusionThese data represent the first global gene expression profiling analysis of chondrogenic tissues during in vivo development. They identify genes for further study on their functional roles in chondrogenesis, and provide a comprehensive and important resource for future studies on cartilage development and disease.
[…] Interrogation of the 11.5 dpc, 12.5 dpc, and 13.5 dpc cRNA samples by microarray analysis involved a saturated hybridization strategy with dye swaps. Thus, cRNA samples from each time point were labelled with both Cy3 and Cy5 fluorophores, and six hybridizations were performed such that each time point was compared with each other time point using both dye combinations (to normalize against dye biases). 44 K whole mouse genome microarrays (G4122F, ID014868, Agilent) were hybridized according to the manufacturer's instructions. The arrays were then scanned at 10 μm resolution on an Axon 4000B scanner and the features acquired with the GenePix Pro 4.1 software (Axon Instruments). The raw data was then processed using a print-tip loess normalization  using limmaGUI [,] which is an implemented package of R used to fit linear models to microarray data . Genes were ranked according to their differential expression and gene lists were generated based on biological process and function (GOStat and OntoExpress). […]
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