Abstract
We standardized conditions for ex vivo mouse limb culture to study cartilage maturation and joint formation. We compared 12.5 d.p.c. mouse forelimbs that were cultured either mounted or freely rotating for up to 72 h. Limb outgrowth progressed ex vivo at a variable rate as compared to its development in vivo, spanning approximately 48 h. Although cartilage maturation and joint formation developed grossly normal, aberrant expression of skeletal marker genes was seen. Interestingly, no regression of the interdigital webs took place in mounted cultures, in contrast to limited webbing under freely rotating conditions. Caspase inhibition, by addition of zVAD-fmk to the culture medium of freely rotating limbs, supported proper gene expression associated with skeletal development, and prevented interdigital web regression. Taken together, a freely rotating ex vivo culture for mouse limb outgrowth that is combined with caspase inhibition provides a good model to study cartilage maturation and joint formation. Cell Death and Differentiation (2001) 8, 985–994
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Abbreviations
- abc1 :
-
ATP binding cassette transporter-1
- ALP:
-
alkaline phosphatase
- BMP:
-
bone morphogenetic protein
- c1qa :
-
complement component 1qa
- caspase:
-
cysteine aspartic acid specific protease
- col2a1 :
-
collagen type 2a1
- d.p.c.:
-
days post coitum
- gdf5 :
-
growth and differentiation factor-5
- TGF-β:
-
transforming growth factor-β
- TUNEL:
-
terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling
- zVAD-fmk:
-
benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone
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Acknowledgements
The authors thank Dr. Suneel Apte (Cleveland Clinic Foundation, Cleveland, OH, USA) and Dr. Paul Martin (University College London, London, UK) for the generous gifts of col2a1, abc1 and c1qa cDNAs (courtesy of Dr. Giovanna Chimini and Dr. Marina Botto). Dr. An Zwijsen (Laboratory for Molecular Biology and Flanders Interuniversity Institute for Biotechnology) is acknowledged for laboratory animal facility service and assistance to the procedure of whole mount in situ hybridization. We thank Jenny Peeters and Giselle De Geest (Center for Human Genetics and Flanders Interuniversity Institute for Biotechnology) for technical advice on tissue sectioning. Dirk De Valck is a postdoctoral research assistant with the Fund for Scientific Research-Flanders. Research was supported by the Fund for Scientific Research-Flanders (grant nr G.0200.99) and the National Lottery (grant nr 9.0200.99).
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De Valck, D., Luyten, F. Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures. Cell Death Differ 8, 985–994 (2001). https://doi.org/10.1038/sj.cdd.4400912
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DOI: https://doi.org/10.1038/sj.cdd.4400912
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