Airway epithelial cell differentiation during lung organogenesis requires C/EBPa and C/EBP ss
Authors: Roos, Abraham B.; Berg, Tove; Barton, Jenny L.; Didon, Lukas; Nord, Magnus
Background: CCAAT/enhancer-binding protein (C/EBP)a is crucial for lung development and differentiation of the pulmonary epithelium. Conversely, no lung defects have been observed in C/EBP beta-deficient mice, although C/EBP beta trans-activate pulmonary genes by binding to virtually identical DNA-sequences as C/EBPa. Thus, the pulmonary phenotype of mice lacking C/EBP beta could be explained by functional replacement with C/EBPa. We investigated whether C/EBPa and C/EBP beta have overlapping functions in regulating lung epithelial differentiation during organogenesis. Epithelial differentiation was assessed in mice with a lung epithelialspecific (SFTPC-Cre-mediated) deletion of C/EBPa (Cebpa?LE), C/EBP beta (Cebpb?LE), or both genes (Cebpa?LE; Cebpb?LE). Results: Both Cebpa?LE mice and Cebpa?LE; Cebpb?LE mice demonstrated severe pulmonary immaturity compared to wild-type littermates, while no differences in lung histology or epithelial differentiation were observed in Cebpb?LE mice. In contrast to Cebpa?LE mice, Cebpa?LE; Cebpb?LE mice also displayed undifferentiated Clara cells with markedly impaired protein and mRNA expression of Clara cell secretory protein (SCGB1A1), compared to wild-type littermates. In addition, ectopic mucus-producing cells were observed in the conducting airways of Cebpa?LE; Cebpb?LE mice. Conclusions: Our findings demonstrate that C/EBPa and C/EBP beta play pivotal, and partly overlapping roles in determining airway epithelial differentiation, with possible implications for tissue regeneration in lung homeostasis and disease. Developmental Dynamics 241:911923, 2012. (c) 2012 Wiley Periodicals, Inc.
Imaging lung regeneration by light sheet microscopy
HISTOCHEMISTRY AND CELL BIOLOGY
Authors: Salwig, Isabelle; Spitznagel, Birgit; Wiesnet, Marion; Braun, Thomas
Optical clearing combined with deep imaging of large biological specimen allows organ-wide visualization of cells in three dimensions (3D) to explore regenerative processes in a spatial context. Here, we investigate the dynamics of airway regeneration following toxin-mediated epithelial injury in cleared whole lung preparations by light sheet microscopy. We use a recently developed knock-in mouse strain labeling bronchiolar Club cells (Scgb1a1-mCherry) to define an optimal clearing procedure that efficiently preserves genetically encoded fluorophores. Dehydration in pH-adjusted tert-butanol followed by clearing in ethyl cinnamate maintained maximum mCherry fluorescence while preventing unfavorable background fluorescence. We apply this technique to depict the course of bronchiolar epithelial renewal from an acute injury phase to early and late recovery stages. 3D reconstructions of whole lungs demonstrate near-complete loss of secretory Club cells throughout the entire respiratory tract 3 days post naphthalene (dpn). Multiple foci of regenerating Club cells emerge at 7 dpn, predominantly at airway bifurcations and in distal terminal bronchioles-anatomical regions assumed to harbor distinct stem/progenitor cells subsets. At 21 dpn, clusters of newly formed Club cells have largely expanded, although the bronchiolar epithelial lining continues to regenerate. This study identifies regional stem cell niches as starting points for epithelial recovery, underscores the enormous regenerative capacity of the respiratory epithelium and demonstrates the power of whole lung 3D imaging for evaluating the extent of pulmonary damage and subsequent repair processes.