Expression profiling of palatal shelves in Tbx1 mutant mice

To assess the influence of Tbx1 on gene expression profile within the developing palate we performed a microarray screen using RNA isolated from dissected secondary palate shelves of E13.5 wild type, Tbx1+/- and Tbx1-/- mice. Significant differences were identified between genotypes, with a total of 67 genes demonstrating at least a 2-fold change (p<0.05) in expression. These were clustered into 5 groups, including those downregulated in mutant compared to wild type and heterozygote (n=36); those progressively downregulated from wild type to mutant (n=12); those upregulated in heterozygote and downregulated in mutant compared to wild type (n=2); those progressively upregulated from wild type to mutant (n=12) and those downregulated in heterozygote and upregulated in mutant compared to wild type (n=5). High-throughput real time quantitative RT-PCR confirmed a total of 18 genes significantly changed between wild type and mutant and 24 between heterozygote and mutant. Amongst these, 15 were present in both groups and all except 1 were downregulated in the mutant. There were no significant differences in gene expression between wild type and heterozygous palatal shelves. Secondary palatal shelf pairs were carefully microdissected from E13.5 Tbx1+/+; Tbx1+/- and Tbx1-/- embryos (3 embryos per genotype as biological replicates). RNA was extracted from each pooled shelf pair generating nine RNA samples in total, each one analysed using a single microarray.为探究Tbx1对发育中腭组织内基因表达谱的影响，我们采用从E13.5野生型、Tbx1+/-及Tbx1-/-小鼠的解剖次级腭突中分离的RNA开展了基因芯片（microarray）筛选实验。不同基因型样本间存在显著表达差异，共计67个基因的表达量至少发生2倍变化（p<0.05）。上述差异基因可划分为5个聚类组：与野生型及杂合子相比，在突变型中表达下调的基因（n=36）；从野生型到突变型表达逐步下调的基因（n=12）；相较于野生型，在杂合子中表达上调、在突变型中表达下调的基因（n=2）；从野生型到突变型表达逐步上调的基因（n=12）；以及相较于野生型，在杂合子中表达下调、在突变型中表达上调的基因（n=5）。高通量实时定量逆转录聚合酶链反应（High-throughput real time quantitative RT-PCR）验证结果显示，野生型与突变型间共有18个基因表达存在显著差异，杂合子与突变型间则有24个。其中15个基因在两组比较中均出现显著差异，且除1个基因外，其余均在突变型中表达下调。野生型与杂合子腭突之间的基因表达无显著差异。我们从E13.5的Tbx1+/+、Tbx1+/-及Tbx1-/-胚胎中精细显微解剖获取成对次级腭突（每个基因型取3个胚胎作为生物学重复）；从每一组混合的腭突对中提取RNA，共计获得9份RNA样本，每份样本均通过单次基因芯片实验完成分析。