Sox17 and Lung Progenitor Cell Figure 1. Sox17-induced cell clusters are reversible. Adult CCSPrtTA (A,D,G) and CCSPrtTA/tetO-Sox17 (B,C,E,F,H,I) mice were maintained on Dox, and lungs were harvested after 4 weeks (wks) (A,B,D,E,G,H) or 1 week after discontinuing Dox (C,F,I). Immunostaining for Sox17 (A–C), CCSP (D– F), and Foxj1 (G–I) was performed on lung sections. (A) Sox17 staining was not observed in the airway epithelium in the absence of Dox. (B,E,H) Hyperplastic clusters of cells were observed in the alveolar region following Sox17 expression (arrows and insets). CCSP and Foxj1 staining was detected in a subset of the Sox17-induced alveolar cell clusters (arrows and insets; E,H). (C,F,I) Neither Sox17 transgene expression nor hyperplastic cell clusters were detected 1 week after removal from Dox. Scale bar, 50 mm. doi:10.1371/journal.pone.0005711.g001 Sox17 induces proliferation in adult respiratory epithelial cells To assess the molecular mechanisms by which Sox17 initiates cell cluster formation in respiratory epithelial progenitor cells, Sox17 was conditionally expressed in adult CCSPrtTA/tetO-Sox17 mice for 1, 3, or 5 days. Endogenous expression of Sox17 was detected in endothelial cells in the peripheral lung but not in airway epithelial cells. In contrast, Sox17 staining was readily detected in bronchioles and alveolar type II cells of CCSPrtTA/ tetO-Sox17 mice, consistent with sites of rtTA-directed gene expression in this mouse line (Fig. 3A–F) [33]. The formation of cell clusters in the alveolar regions was evident 5 days after the induction of Sox17 expression (Fig. 3F). Immunostaining for phospho-histone H3 revealed the presence of mitotic cells in the both the bronchioles and alveoli as early as 3 days following Sox17 expression (Fig. 3G–L), with a 4.7-fold increase in the number of proliferative cells compared to control lungs (Fig. S1). Phospho- histone H3 staining was also observed in a subset cells at the bronchoalveolar duct junctions in the lungs of CCSPrtTA/tetO- Sox17 mice (Fig. 3L; arrowhead). Although Sox17 expression and cell proliferation were detected in the bronchioles and broncho- alveolar duct junctions of CCSPrtTA/tetO-Sox17 mice, hyperplastic foci within these regions were not evident. Dual immunofluores- cence revealed colocalized expression of phospho-histone H3 with Sox17, demonstrating that proliferation occurs within the Sox17- expressing population of cells in CCSPrtTA/tetO-Sox17 mice (Fig. 3M–O). Of the respiratory epithelial cells expressing Sox17, 28% coexpressed phospho-histone H3 (Fig. S1) and only a subset of alveolar type II cells expressing Sox17 formed cell clusters. Thus, Sox17 expression induces a subset of mature respiratory epithelial cells in the adult mouse lung to reenter the cell cycle, resulting in the formation of atypical cell clusters in the alveolar region within 5 days. Sox17 increases a Sca-1-positive cell population. Since Sox17 induced proliferation of alveolar type II cells and bronchiolar cells near the bronchoalveolar duct junctions, both considered lung stem/progenitor cell niches, expression of proposed pulmonary stem/progenitor cell markers was examined in CCSPrtTA/tetO-Sox17 mice. Increased expression of Sca-1 was detected in the alveolar cells as well as in cells near the bronchoalveolar duct junction after 5 days of conditional Sox17 expression in CCSPrtTA/tetO-Sox17 mice (Fig. 4A–B). Further, immunofluorescent double labeling demonstrated that the Sca-1 positive cells colocalized with Sox17-expressing cells in lungs of CCSPrtTA/tetO-Sox17 mice (Fig. 4C–E). While the majority of the Sca-1-expressing cells did not show evidence of active proliferation, a rare subset of the Sca-1 positive cells coexpressed phospho-histone PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5711