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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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Mouse hindlimb microdissections. 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hindlimb cryosections stained with Toluidine Blue. Upper panels (A-C) show intact tissue; lower panels (D-F) show cryosections following microdissection of tibial and fibular tissues. Staining of tissues shown in lower panels was performed following microdissection. T = Tibia, F = Fibula. Scale bar = 500 μm.
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Figure 6: Mouse hindlimb microdissections. 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hindlimb cryosections stained with Toluidine Blue. Upper panels (A-C) show intact tissue; lower panels (D-F) show cryosections following microdissection of tibial and fibular tissues. Staining of tissues shown in lower panels was performed following microdissection. T = Tibia, F = Fibula. Scale bar = 500 μm.

Mentions: We have previously reported global differential expression profiles corresponding to zones of the growth plate generated by microarray analysis of RNA derived from tissue microdissected from long bones of Swiss white mice [66]. So that these datasets could be compared with those of the present study, Swiss white mice were used here also. Accordingly, pregnant female Swiss white mice were sacrificed in accordance with Institutional Animal Ethics guidelines and embryos were harvested at 11.5 dpc, 12.5 dpc and 13.5 dpc and transferred to PBS at 4°C. The hind limb buds were promptly dissected and embedded in Tissue-Tek OCT (Sakura Fine Technical), snap-frozen in isopentane and stored at -80°C. 6 μm sections of limb tissue corresponding to the regions of tibial and fibular development were prepared using a cryostat (Leica CM1850), mounted on RNAse-free SuperFrost Plus slides (Biolab Scientific), fixed in 70% ethanol, washed in RNAse-free water, and dehydrated in 70%, 95%, and 100% ethanol for thirty seconds each, and air-dried. Regions corresponding to the mesenchymal condensations of developing tibiae and fibulae were microdissected from slides immobilized on the stage of an inverted light microscope (Leica DM IL) using an ophthalmic scalpel (Feather) fixed to the scanning xy-object guide. Mesenchymal condensations and cartilage anlagen at 12.5 dpc and 13.5 dpc were readily visible in untreated (ie fixed and dehydrated, but not stained) sections by light microscopy, allowing precise microdissection of these tissues. In 12.5 dpc hind limb sections, a prominent blood vessel was found to run between, and perpendicular to the developing tibia and fibula, thus appearing in the transverse plane as the tibia and fibula came into view (Fig. 6). The appearance of this blood vessel was used as a guide for locating the precondensed mesenchymal cells in 11.5 dpc hind limb sections. 231 sections from a total of four 11.5 dpc mouse hind limbs, three 12.5 dpc mouse hind limbs, and four 13.5 dpc mouse hind limbs were microdissected (Fig. 6). The tissue from each time point was pooled into RNase-free Eppendorf tubes and total RNA was extracted using the RNeasy Micro Kit (Qiagen). To assess RNA yield, purity, and integrity, total RNA samples were interrogated by capillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies), using a Series II RNA 6000 Pico Kit, according to the manufacturer's specifications (Agilent Technologies). All total RNA samples were confirmed to be of high quality, with a RIN of >7.7 (Agilent). Total RNA from tibial and fibular tissue microdissected from each time point was linearly amplified [67] in two rounds using the MessageAmp aRNA kit (Ambion) following the manufacturer's instructions. 100–150 ng of first-round amplified cRNA was used as template for each of the second round amplifications. Quality and yield was assessed by capillary electrophoresis. Amplified RNA samples (1.25 μg) were fluorescently labelled with Cy3 or Cy5 fluorophores (Amersham) according to the manufacturer's instructions, analysed by 1.5% agarose gel electrophoresis, and visualized using a Typhoon fluorescence scanner (Amersham) (see Additional file 5). These data demonstrated that the majority of transcripts in each sample were approximately 150 nt – 400 nt in length, and the lack of low molecular weight fluorescent signal confirmed that all unincorporated dyes were successfully removed during the purification procedure.


Global comparative transcriptome analysis of cartilage formation in vivo.

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

Mouse hindlimb microdissections. 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hindlimb cryosections stained with Toluidine Blue. Upper panels (A-C) show intact tissue; lower panels (D-F) show cryosections following microdissection of tibial and fibular tissues. Staining of tissues shown in lower panels was performed following microdissection. T = Tibia, F = Fibula. Scale bar = 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Mouse hindlimb microdissections. 11.5 dpc, 12.5 dpc, and 13.5 dpc mouse hindlimb cryosections stained with Toluidine Blue. Upper panels (A-C) show intact tissue; lower panels (D-F) show cryosections following microdissection of tibial and fibular tissues. Staining of tissues shown in lower panels was performed following microdissection. T = Tibia, F = Fibula. Scale bar = 500 μm.
Mentions: We have previously reported global differential expression profiles corresponding to zones of the growth plate generated by microarray analysis of RNA derived from tissue microdissected from long bones of Swiss white mice [66]. So that these datasets could be compared with those of the present study, Swiss white mice were used here also. Accordingly, pregnant female Swiss white mice were sacrificed in accordance with Institutional Animal Ethics guidelines and embryos were harvested at 11.5 dpc, 12.5 dpc and 13.5 dpc and transferred to PBS at 4°C. The hind limb buds were promptly dissected and embedded in Tissue-Tek OCT (Sakura Fine Technical), snap-frozen in isopentane and stored at -80°C. 6 μm sections of limb tissue corresponding to the regions of tibial and fibular development were prepared using a cryostat (Leica CM1850), mounted on RNAse-free SuperFrost Plus slides (Biolab Scientific), fixed in 70% ethanol, washed in RNAse-free water, and dehydrated in 70%, 95%, and 100% ethanol for thirty seconds each, and air-dried. Regions corresponding to the mesenchymal condensations of developing tibiae and fibulae were microdissected from slides immobilized on the stage of an inverted light microscope (Leica DM IL) using an ophthalmic scalpel (Feather) fixed to the scanning xy-object guide. Mesenchymal condensations and cartilage anlagen at 12.5 dpc and 13.5 dpc were readily visible in untreated (ie fixed and dehydrated, but not stained) sections by light microscopy, allowing precise microdissection of these tissues. In 12.5 dpc hind limb sections, a prominent blood vessel was found to run between, and perpendicular to the developing tibia and fibula, thus appearing in the transverse plane as the tibia and fibula came into view (Fig. 6). The appearance of this blood vessel was used as a guide for locating the precondensed mesenchymal cells in 11.5 dpc hind limb sections. 231 sections from a total of four 11.5 dpc mouse hind limbs, three 12.5 dpc mouse hind limbs, and four 13.5 dpc mouse hind limbs were microdissected (Fig. 6). The tissue from each time point was pooled into RNase-free Eppendorf tubes and total RNA was extracted using the RNeasy Micro Kit (Qiagen). To assess RNA yield, purity, and integrity, total RNA samples were interrogated by capillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies), using a Series II RNA 6000 Pico Kit, according to the manufacturer's specifications (Agilent Technologies). All total RNA samples were confirmed to be of high quality, with a RIN of >7.7 (Agilent). Total RNA from tibial and fibular tissue microdissected from each time point was linearly amplified [67] in two rounds using the MessageAmp aRNA kit (Ambion) following the manufacturer's instructions. 100–150 ng of first-round amplified cRNA was used as template for each of the second round amplifications. Quality and yield was assessed by capillary electrophoresis. Amplified RNA samples (1.25 μg) were fluorescently labelled with Cy3 or Cy5 fluorophores (Amersham) according to the manufacturer's instructions, analysed by 1.5% agarose gel electrophoresis, and visualized using a Typhoon fluorescence scanner (Amersham) (see Additional file 5). These data demonstrated that the majority of transcripts in each sample were approximately 150 nt – 400 nt in length, and the lack of low molecular weight fluorescent signal confirmed that all unincorporated dyes were successfully removed during the purification procedure.

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