Identification of endoderm-specific genes in mouse embryos

The foregut definitive endoderm is the precursor of many tissues including the liver, pancreas, thyroid, lungs, trachea and oesophagus. However, networks and pathways involved in the early development of the definitive endoderm of mammals are not well studied. To identify genes with potential roles in the early development of the foregut definitive endoderm in mouse embryos, we performed microarray analysis to compare the gene expression profile of foregut endoderm and non-endodermal tissues from early somite-stage mouse embryos. ARC/s embryos at 4- to 5-somite stages were collected and the anterior intestinal portal was isolated by digestion with trypsin and pancreatin and mechanical dissection. Ectoderm and mesoderm tissues were dissected from the head folds and the heart, and combined as the non-endoderm tissues for comparison of gene expression profiles. RNA was extracted from 15-20 pooled samples of endoderm or ectoderm plus mesoderm cells and subjected to two rounds of linear amplification and labelling according to standard methods (Affymetrix). The amplified products were hybridized to Affymetrix mouse MOE430 GeneChips. The experiment was performed twice. RNA amplification labelling, hybridization and scanning were performed by the Australian Genome Research Facility. GC-RMA data normalization and further analysis were performed using GenePattern software.

前肠定型内胚层（foregut definitive endoderm）是肝脏、胰腺、甲状腺、肺、气管及食管等多种组织的前体。然而，哺乳动物定型内胚层早期发育所涉及的调控网络与信号通路尚未得到充分研究。为鉴定在小鼠胚胎前肠定型内胚层早期发育中潜在发挥功能的基因，我们开展了微阵列分析（microarray analysis），对比早期体节期（somite-stage）小鼠胚胎的前肠内胚层与非内胚层组织的基因表达谱（gene expression profile）。我们收集了4至5体节期的ARC/s品系小鼠胚胎，通过胰蛋白酶（trypsin）与胰酶（pancreatin）消化结合机械解剖（mechanical dissection）分离前肠门（anterior intestinal portal）。从头部褶皱与心脏中分离外胚层（ectoderm）与中胚层（mesoderm）组织，将其合并作为非内胚层组织以用于基因表达谱的对比分析。从15至20份混合的内胚层样本，或外胚层+中胚层细胞样本中提取RNA，依照标准方法（Affymetrix）进行两轮线性扩增与标记。将扩增产物与Affymetrix小鼠MOE430基因芯片（GeneChips）进行杂交。本实验重复开展两次。RNA扩增标记、杂交及扫描工作均由澳大利亚基因组研究设施（Australian Genome Research Facility）完成。使用GenePattern软件进行GC-RMA数据标准化（GC-RMA data normalization）与后续分析。