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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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In situ hybridization analysis of Sox11, Pcdh8, Lepre1 and Trpv4 in embryonic mouse hindlimbs. Antisense probes were used to determine the level and distribution of mRNA for each gene. Expression of Sox11 is widespread throughout the limb bud and somites (arrowhead) at E11.5 but declines in level by E12.5 and becomes undetectable by E13.5. Pcdh8 is expressed within the pre-condensations of the tibia and fibula (arrowheads) at E11.5 but is downregulated by the condensation phase at E12.5. At this stage expression is detected in the tissue surrounding the femoral condensation (arrowhead) but declines to background levels by E13.5. Leprecan1 is not detectable at E11.5 but becomes visible in the femoral condensation at E12.5 (arrowhead) and is upregulated within the tibia and fibula at E13.5 (arrowheads). Trpv4 is not detectable within the limb buds at E11.5 or E12.5 but is strongly upregulated in the tibia and fibula at E13.5 (arrowheads). Distal is to the left in all images.
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Figure 4: In situ hybridization analysis of Sox11, Pcdh8, Lepre1 and Trpv4 in embryonic mouse hindlimbs. Antisense probes were used to determine the level and distribution of mRNA for each gene. Expression of Sox11 is widespread throughout the limb bud and somites (arrowhead) at E11.5 but declines in level by E12.5 and becomes undetectable by E13.5. Pcdh8 is expressed within the pre-condensations of the tibia and fibula (arrowheads) at E11.5 but is downregulated by the condensation phase at E12.5. At this stage expression is detected in the tissue surrounding the femoral condensation (arrowhead) but declines to background levels by E13.5. Leprecan1 is not detectable at E11.5 but becomes visible in the femoral condensation at E12.5 (arrowhead) and is upregulated within the tibia and fibula at E13.5 (arrowheads). Trpv4 is not detectable within the limb buds at E11.5 or E12.5 but is strongly upregulated in the tibia and fibula at E13.5 (arrowheads). Distal is to the left in all images.

Mentions: In order to validate the microarray data further with independent, biological replicates, in situ hybridization was performed on sagittal sections from 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hind limb buds using probes for Sox11 (Fig. 4A–C), Pcdh8 (Fig. 4D–F), Lepre1 (Fig. 4G–I), and Trpv4 (Fig. 4J–L). In each case, the gene expression detected by in situ hybridization corroborated the expression profiles generated by microarray and clarified the expression of these genes in the limb bud beyond the microdissected areas.


Global comparative transcriptome analysis of cartilage formation in vivo.

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

In situ hybridization analysis of Sox11, Pcdh8, Lepre1 and Trpv4 in embryonic mouse hindlimbs. Antisense probes were used to determine the level and distribution of mRNA for each gene. Expression of Sox11 is widespread throughout the limb bud and somites (arrowhead) at E11.5 but declines in level by E12.5 and becomes undetectable by E13.5. Pcdh8 is expressed within the pre-condensations of the tibia and fibula (arrowheads) at E11.5 but is downregulated by the condensation phase at E12.5. At this stage expression is detected in the tissue surrounding the femoral condensation (arrowhead) but declines to background levels by E13.5. Leprecan1 is not detectable at E11.5 but becomes visible in the femoral condensation at E12.5 (arrowhead) and is upregulated within the tibia and fibula at E13.5 (arrowheads). Trpv4 is not detectable within the limb buds at E11.5 or E12.5 but is strongly upregulated in the tibia and fibula at E13.5 (arrowheads). Distal is to the left in all images.
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Figure 4: In situ hybridization analysis of Sox11, Pcdh8, Lepre1 and Trpv4 in embryonic mouse hindlimbs. Antisense probes were used to determine the level and distribution of mRNA for each gene. Expression of Sox11 is widespread throughout the limb bud and somites (arrowhead) at E11.5 but declines in level by E12.5 and becomes undetectable by E13.5. Pcdh8 is expressed within the pre-condensations of the tibia and fibula (arrowheads) at E11.5 but is downregulated by the condensation phase at E12.5. At this stage expression is detected in the tissue surrounding the femoral condensation (arrowhead) but declines to background levels by E13.5. Leprecan1 is not detectable at E11.5 but becomes visible in the femoral condensation at E12.5 (arrowhead) and is upregulated within the tibia and fibula at E13.5 (arrowheads). Trpv4 is not detectable within the limb buds at E11.5 or E12.5 but is strongly upregulated in the tibia and fibula at E13.5 (arrowheads). Distal is to the left in all images.
Mentions: In order to validate the microarray data further with independent, biological replicates, in situ hybridization was performed on sagittal sections from 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hind limb buds using probes for Sox11 (Fig. 4A–C), Pcdh8 (Fig. 4D–F), Lepre1 (Fig. 4G–I), and Trpv4 (Fig. 4J–L). In each case, the gene expression detected by in situ hybridization corroborated the expression profiles generated by microarray and clarified the expression of these genes in the limb bud beyond the microdissected areas.

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