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Global comparative transcriptome analysis of cartilage formation in vivo.

Cameron TL, Belluoccio D, Farlie PG, Brachvogel B, Bateman JF - BMC Dev. Biol. (2009)

Bottom Line: We found significant differential expression of 931 genes during these early stages of chondrogenesis.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. trevor.cameron@mcri.edu.au

ABSTRACT

Background: During 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.

Results: Tibial 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.

Conclusion: These 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.

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Comparison of differential expression profiles of selected genes analysed by quantitative real time PCR or microarrays. Expression was analyzed in amplified mRNA by quantitative real time PCR analysis (qPCR; yellow bars) or microarray analysis (blue bars) for A) aggrecan (Agc1); B), Bmp8a; C), Col2a1; D), Fgfr4; E), Foxa3, F), Gdf5; G), Klf2, H), Klf4; I), Lepre1; J), Ncad; K), Sox11; L) Trpv4. qPCR experiments were performed in triplicate.
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Figure 3: Comparison of differential expression profiles of selected genes analysed by quantitative real time PCR or microarrays. Expression was analyzed in amplified mRNA by quantitative real time PCR analysis (qPCR; yellow bars) or microarray analysis (blue bars) for A) aggrecan (Agc1); B), Bmp8a; C), Col2a1; D), Fgfr4; E), Foxa3, F), Gdf5; G), Klf2, H), Klf4; I), Lepre1; J), Ncad; K), Sox11; L) Trpv4. qPCR experiments were performed in triplicate.

Mentions: Using unmodified cRNA samples amplified in parallel to those cRNA samples generated for the microarray analyses, qPCR was performed on selected genes as a technical validation for the microarray data. qPCR was performed on Agc1 (Fig. 3A), Bmp8a (Fig. 3B), Col2a1 (Fig. 3C), Fgfr4 (Fig. 3D), Foxa3 (Fig. 3E), Gdf5 (Fig. 3F), Klf2 (Fig. 3G), Klf4 (Fig. 3H), Lepre1 (Fig. 3I), Ncad (Fig. 3J), Sox11 (Fig. 3K), and Trpv4 (Fig. 3L). For each marker, a close correlation was observed between the pattern of differential gene expression determined using either technique, with highest expression for markers of mesenchymal condensation seen between 11.5 dpc and 12.5 dpc, and highest expression for markers of chondrogenesis seen at 13.5 dpc.


Global comparative transcriptome analysis of cartilage formation in vivo.

Cameron TL, Belluoccio D, Farlie PG, Brachvogel B, Bateman JF - BMC Dev. Biol. (2009)

Comparison of differential expression profiles of selected genes analysed by quantitative real time PCR or microarrays. Expression was analyzed in amplified mRNA by quantitative real time PCR analysis (qPCR; yellow bars) or microarray analysis (blue bars) for A) aggrecan (Agc1); B), Bmp8a; C), Col2a1; D), Fgfr4; E), Foxa3, F), Gdf5; G), Klf2, H), Klf4; I), Lepre1; J), Ncad; K), Sox11; L) Trpv4. qPCR experiments were performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662817&req=5

Figure 3: Comparison of differential expression profiles of selected genes analysed by quantitative real time PCR or microarrays. Expression was analyzed in amplified mRNA by quantitative real time PCR analysis (qPCR; yellow bars) or microarray analysis (blue bars) for A) aggrecan (Agc1); B), Bmp8a; C), Col2a1; D), Fgfr4; E), Foxa3, F), Gdf5; G), Klf2, H), Klf4; I), Lepre1; J), Ncad; K), Sox11; L) Trpv4. qPCR experiments were performed in triplicate.
Mentions: Using unmodified cRNA samples amplified in parallel to those cRNA samples generated for the microarray analyses, qPCR was performed on selected genes as a technical validation for the microarray data. qPCR was performed on Agc1 (Fig. 3A), Bmp8a (Fig. 3B), Col2a1 (Fig. 3C), Fgfr4 (Fig. 3D), Foxa3 (Fig. 3E), Gdf5 (Fig. 3F), Klf2 (Fig. 3G), Klf4 (Fig. 3H), Lepre1 (Fig. 3I), Ncad (Fig. 3J), Sox11 (Fig. 3K), and Trpv4 (Fig. 3L). For each marker, a close correlation was observed between the pattern of differential gene expression determined using either technique, with highest expression for markers of mesenchymal condensation seen between 11.5 dpc and 12.5 dpc, and highest expression for markers of chondrogenesis seen at 13.5 dpc.

Bottom Line: We found significant differential expression of 931 genes during these early stages of chondrogenesis.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. trevor.cameron@mcri.edu.au

ABSTRACT

Background: During 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.

Results: Tibial 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus