Global comparative transcriptome analysis of cartilage formation in vivo

We set out to characterize the gene expression changes which take place during chondrogenesis in the developing mouse limb. RNA derived from pre-condensed mesenchyme, mesenchymal condensations, and cartilage anlagen representing the earliest stages of tibial and fibular development was analysed by whole genome microarray analysis, and revealed 931 genes differentially expressed in these tissues. Among them were 892 genes not previously identified during the initation of chondrogenesis, including members of the Bmp, Wnt, Gdf, Sox, and Fox gene families. These microarray data were validated by qPCR, in situ hybridisation, and analysis of numerous genes already implicated in chondrogenesis in the scientific literature. 231 sections from a total of four 11.5dpc mouse hind limbs, three 12.5dpc mouse hind limbs, and four 13.5dpc mouse hind limbs (all from separate mice) were microdissected, and tissues from each time point were pooled. Thus, this experiment consisted of one replicate only.本研究旨在表征发育中小鼠肢体软骨发生过程中发生的基因表达变化。研究针对代表胫骨与腓骨发育最早阶段的预凝聚间充质、间充质凝聚体及软骨原基提取的RNA开展全基因组微阵列分析，共鉴定出931个在上述组织中差异表达的基因。其中892个基因为此前软骨发生启动阶段未被报道的新基因，涵盖骨形态发生蛋白（Bmp）、Wnt、生长分化因子（Gdf）、Sox及Fox基因家族成员。本研究通过实时定量聚合酶链式反应（qPCR）、原位杂交，以及对已有学术文献中已被证实与软骨发生相关的大量基因进行分析，对微阵列数据进行了验证。实验团队从4只11.5dpc小鼠后肢、3只12.5dpc小鼠后肢及4只13.5dpc小鼠后肢（均来自不同个体）中共获取231份切片并进行显微解剖，随后将每个时间点的组织进行混合。因此，本实验仅设置一次生物学重复。