Gene expression of the intermediate and outer interzone of murine embryos at 15.5 days of gestation

Nascent embryonic joints, interzones, contain a distinct cohort of progenitor cells responsible for the formation of the majority of articular tissues. However, to date the interzone has largely been studied using in situ analysis for candidate genes in the context of the embryo rather than using an unbiased genome wide expression analysis on isolated interzone cells, leaving significant controversy regarding the exact role of the intermediate and outer interzone layers in joint formation. Therefore, in this study, using laser capture microdissection (three biological replicates), we selectively harvested the intermediate and outer interzones of mouse embryos at gestational age 15.5 days, just prior to cavitation, when the differences between the layers should be most profound. Microarray analysis (Agilent Whole Mouse Genome Oligo Microarrays) was performed and the differential gene expression between the intermediate interzone cells and outer interzone cells was examined by performing a 2-sided paired student t-test and pathway analysis. 197 genes were differentially expressed (+/- 2-fold) between the intermediate interzone and the outer interzone with a p-value >= 0.01. Of these, 91 genes showed higher expression levels in the intermediate interzone and 106 were expressed higher in the outer interzone. Pathway analysis of differentially expressed genes suggests an important role for inflammatory processes in the interzone layers, especially in the intermediate interzone, and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that it undergoes endochondral ossification. 2 study groups (intermediate and outer interzone) with three biological replicates (1 biological replicate = 1 embryo)新生胚胎关节的间区（interzone）拥有独特的祖细胞群，负责大多数关节组织的形成。然而迄今为止，针对间区的研究大多是在胚胎背景下针对候选基因开展原位分析，而非对分离获取的间区细胞进行无偏全基因组表达检测，这使得学界对于中间层与外层间区在关节形成中的确切功能仍存在显著争议。为此，本研究采用激光捕获显微切割（laser capture microdissection）技术，设置3次生物学重复，于关节腔形成前夕的妊娠15.5天小鼠胚胎中，选择性采集中间层与外层间区组织——此时两层间的差异最为显著。本研究采用安捷伦全小鼠基因组寡核苷酸芯片（Agilent Whole Mouse Genome Oligo Microarrays）进行基因芯片分析，并通过双侧配对t检验与通路分析，探究了中间层间区细胞与外层间区细胞的差异基因表达情况。在p值≥0.01的筛选标准下，中间层与外层间区共有197个基因呈现±2倍的差异表达，其中91个基因在中间层间区中高表达，106个基因在外层间区中高表达。对差异表达基因的通路分析结果显示，炎症过程在间区各层（尤其是中间层间区）乃至关节与关节软骨的发育过程中均发挥重要作用。外层间区中富集大量与软骨细胞肥大及软骨内骨化相关的基因，提示该层可能发生软骨内骨化进程。本研究设置2个实验组（中间层间区与外层间区），每组包含3次生物学重复（1次生物学重复对应1枚胚胎）