Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures.
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.
De Valck
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Laboratory of Skeletal Development and Joint Disorders, Department of Rheumatology, University Hospitals, K.U. Leuven, Belgium.
Luyten
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FP
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D002356:Cartilage / Q000033:anatomy & histology / Q000196:embryology*; D061945:Caspase Inhibitors; D046508:Culture Techniques / Q000379:methods*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005121:Extremities / Q000033:anatomy & histology / Q000196:embryology*; D017403:In Situ Hybridization; D007596:Joints / Q000033:anatomy & histology / Q000196:embryology* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D014158:Transcription, Genetic
D000590:Amino Acid Chloromethyl Ketones; D015415:Biomarkers; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone