Abstract
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.
Highlights
During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations
These data provided clear evidence that the microdissected regions of the limb buds represented mesenchymal condensations undergoing the in vivo transition from undifferentiated chondroprogenitor cells to differentiated chondrocytes
Whole genome expression profiling As an overall representation of the microarray hybridizations, M/A scatter plots were generated for each time point comparison in which the mean signal intensity (Average Log2 Intensity; A) for each microarray probe was plotted against the relative fold difference for that probe (Fig. 2)
Summary
During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Cells within the pre-chondrogenic condensation up-regulate cell adhesion mechanisms and begin to synthesize specific extracellular matrix molecules, and the condensations expand through a combination of proliferation and recruitment of surrounding mesenchyme. The generation of these condensations creates an environment which is conducive to chondrogenic differentiation [1,2]. The hypertrophic cartilage is first calcified and following vascular invasion, replaced by primary bone that is subsequently remodelled to form secondary bone This process radiates outwards from the centre of the anlage with the development of highly ordered growth plates that separate the cartilaginous epiphyses from the bony diaphysis. Secondary centres of ossification develop within the epiphyses and, with subsequent fusion of the ossification centres during puberty, endochondral ossification and bone growth ceases
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