The Ets domain transcription factor Spdef drives maturation of Goblet and Paneth cells in the intestinal epithelium

BACKGROUND & AIMS: Stems cells within the intestinal epithelium generate daughter cells which undergo lineage commitment and maturation through the concerted action of the Wnt and Notch signalling cascades. Both pathways, in turn, regulate transcription factor networks which further define differentiation towards either enterocytes or one of three secretory cell lineages (Paneth, goblet or enteroendocrine cells). In this manuscript, we identified the Ets domain transcription factor, Spdef, as a novel lineage maker of goblet and Paneth cells. METHODS: To address the function of Spdef in vivo, we inactivated the Spdef gene and analysed the intestinal phenotype using a range of histological techniques and DNA microarray profiling. RESULTS: In accordance with the expression data we found that loss of Spdef severely impaired the maturation of goblet and Paneth cells and conversely lead to an accumulation of immature secretory progenitors. Moreover, we provide evidence suggesting that Spdef positively and negatively regulates a specific subset of goblet and Paneth cell genes including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2. CONCLUSION: We propose a model whereby Spdef acts downstream of Math1 to promote terminal differentiation of a secretory progenitor pool towards Paneth and goblet cells. Keywords: expression profiling To compare wild-type versus Spdef-/- intestinal or colonic tissue samples, pooled total RNA from 2-3 littermates were utilized for each experiment. In addition, tissue samples from both adult (P30) or E18.5 fetal mice were compared. Overall, we carried out 3 dye swap experiments for E18.5 small intestine and P30 colon resulting in 6 arrays each, and 2 dye swap experiments for E18.5 colon and P30 small intestine resulting in 4 arrays each.背景与目的：肠上皮干细胞（intestinal epithelial stem cells）产生的子代细胞通过Wnt（Wnt）与Notch（Notch）信号级联通路的协同作用完成谱系定型与成熟。反过来，这两条通路均可调控转录因子网络，进一步决定细胞的分化方向：要么成为吸收性肠上皮细胞（enterocytes），要么分化为三种分泌细胞谱系之一——潘氏细胞（Paneth）、杯状细胞（goblet）或肠内分泌细胞（enteroendocrine cells）。本研究鉴定出Ets结构域转录因子（Ets domain transcription factor）Spdef（Spdef），可作为杯状细胞与潘氏细胞的新型谱系标志物。 方法：为探究Spdef在体内的功能，我们敲除了Spdef基因，并通过一系列组织学技术与DNA微阵列基因表达谱分析（DNA microarray profiling）对肠道表型进行分析。 结果：与表达谱数据相符，我们发现Spdef缺失会严重损害杯状细胞与潘氏细胞的成熟过程，反而造成未成熟分泌祖细胞的蓄积。此外，我们提供的证据表明，Spdef可正向及负向调控杯状细胞与潘氏细胞的特定基因子集，包括隐窝素（Cryptdins）、基质金属蛋白酶7（Mmp7）、血管生成素4（Ang4）、激肽释放酶（Kallikreins）以及黏蛋白2（Muc2）。 结论：我们提出了一个模型，即Spdef在Math1的下游发挥作用，促进分泌祖细胞库向潘氏细胞与杯状细胞方向完成终末分化。关键词：表达谱分析（expression profiling）。为比较野生型与Spdef基因敲除（Spdef-/-）小鼠的肠道或结肠组织样本，本研究为每组实验采用2~3只同窝小鼠的总RNA混合样本。此外，我们还比较了成年（出生后第30天，P30）与E18.5胎鼠的组织样本。总体而言，我们针对E18.5胎鼠小肠与P30结肠开展了3次荧光染料交换实验（dye swap experiments），每组各得到6张微阵列芯片；针对E18.5胎鼠结肠与P30小肠开展了2次荧光染料交换实验，每组各得到4张微阵列芯片。