Transcription profiling of fetal lung from wild type and CREB knock-out mice

The cAMP response element binding protein (Creb) is a member of a leucine zipper transcription factor family that regulates gene expression primarily in response to the intracellular cAMP signalling pathway. Previous studies have shown Creb1-null mice suffer respiratory failure with lung atelectasis and a large reduction in Sftpd mRNA. Using a new line of Creb1-null mice we have further investigated Creb function in the developing mouse lung, focussing on differentiation of the airway epithelium. The lungs of Creb1-null fetal mice showed normal respiratory development until E17.5 when proximal and distal airways fail to inflate. Subsequent ultrastructural analysis of the lungs of E17.5 Creb1-null fetal mice revealed a defect in AEC differentiation with a reduction in proportions of type-II AECs and in particular, a very large reduction in type-I AECs. Furthermore, immunostaining for the proximal epithelial cell markers Scgb1a1 (also known as CC10) (Clara cells), Foxj1 (Ciliated cells), and CGRP (Neuroendocrine cells) showed delayed or defective proximal epithelial differentiation in Creb1-null fetal lungs. Quantitative real time PCR (qRT-PCR) analysis at E17.5 in Creb1-null fetal lungs showed differential expression of mRNAs for Creb/Atf1 subfamily members, surfactant-associated proteins, type-I AEC markers and proximal epithelial markers. Furthermore, whole-genome microarray analysis at E17.5 in Creb1-null fetal lungs has provided novel genes which will prove useful to further investigate Creb-mediated signalling in lung development. Together these results demonstrate that Creb plays a key role in determining cell lineages and differentiation of the developing lung epithelium.cAMP应答元件结合蛋白（cAMP response element binding protein, CREB）属于亮氨酸拉链转录因子家族，主要通过细胞内cAMP信号通路调控基因表达。既往研究显示，Creb1敲除小鼠会出现呼吸衰竭伴肺不张，且Sftpd mRNA水平大幅降低。本研究利用新构建的Creb1敲除小鼠品系，进一步探究了CREB在发育中小鼠肺中的功能，重点关注气道上皮细胞的分化过程。Creb1敲除胎鼠的肺部在胚胎期17.5天（E17.5）前呼吸发育均正常，而该时间点后近端与远端气道无法扩张。随后对E17.5天Creb1敲除胎鼠肺部开展超微结构分析，结果发现肺泡上皮细胞（Alveolar Epithelial Cell, AEC）分化存在缺陷：II型肺泡上皮细胞占比降低，尤其是I型肺泡上皮细胞出现大幅减少。进一步针对近端上皮细胞标志物开展免疫染色实验，结果显示，克拉拉细胞标志物Scgb1a1（又称CC10）、纤毛细胞标志物Foxj1以及神经内分泌细胞标志物降钙素基因相关肽（Calcitonin Gene-Related Peptide, CGRP）的表达均出现延迟或异常，提示Creb1敲除胎鼠肺部近端上皮分化存在缺陷。对E17.5天Creb1敲除胎鼠肺部进行实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）分析，发现CREB/ATF1亚家族成员、表面活性蛋白相关基因、I型肺泡上皮细胞标志物以及近端上皮标志物的mRNA表达均存在显著差异。此外，对该阶段胎鼠肺部开展全基因组微阵列分析，筛选出多个新的候选基因，可为后续研究CREB介导的肺发育信号通路提供新的研究靶点。综上，上述实验结果证实，CREB在决定发育中肺上皮细胞的细胞谱系及分化过程中发挥关键作用。