Levesque Bernadette M, Zhou Shutang, Shan Lin, Johnston Pamela, Kong Yanping, Degan Simone, Sunday Mary E
Department of Pathology, Duke University Medical Center, Research Drive, Carl Building, Room 0043, Durham, NC 27710, USA.
Am J Respir Cell Mol Biol. 2007 Apr;36(4):427-34. doi: 10.1165/rcmb.2006-0314OC. Epub 2006 Nov 16.
Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for alpha-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5-E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: beta-ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.
果蝇无气管基因（Trl）是气管生成的主要调节因子，目前尚无已知的功能性哺乳动物同源物。我们推测，与无气管基因相似的基因可调节肺的发育。采用定量（Q）RT-PCR和免疫染色法来确定npas1基因在发育中的小鼠肺内的时空表达模式。α-平滑肌肌动蛋白免疫染色可显示肌成纤维细胞，蛋白基因产物（PGP）9.5可识别神经内分泌细胞。在反义或正义寡脱氧核苷酸（ODN）存在的情况下，分析胚胎肺芽的分支形态发生。进行微阵列分析以筛选反义处理的肺中基因表达的变化。使用QRT-PCR验证微阵列上鉴定出的关键基因的表达改变。我们证明npas1在小鼠胚胎肺中表达。npas1 mRNA在胚胎第（E）10.5 - E11.5天早期达到峰值，然后降至低水平。测序验证了胚胎肺中npas1转录本的身份。NPAS1免疫染色出现在副支气管间充质细胞的细胞核中，尤其是在气管分叉处。Arnt是Tango（Trl的异二聚体化伴侣）的小鼠同源物，也在发育中的肺中表达，但水平恒定。npas1或arnt反义ODN抑制肺分支形态发生，伴有肌成纤维细胞发育改变和肺神经内分泌细胞增加。在微阵列上，我们鉴定出>50个被npas1反义下调的已知基因，包括多个调节细胞迁移和细胞分化的基因。QRT-PCR证实神经源性基因RBP-Jk和Tle以及三个参与肌肉发育的基因：β-ig-h3、claudin-11和心肌素的表达显著降低。Npas1可调节小鼠胚胎肺中肌成纤维细胞的分布、分支形态发生和神经内分泌细胞分化。
