Attenuation of pulmonary neuroendocrine differentiation in mice lacking Clara cell secretory protein

During development and injury, pulmonary neuroendocrine (NE) cells may tran siently express Clara cell 10 kD protein (CC10), a major product of the non ciliated progenitor cells for normal and neoplastic airway epithelia sugges ting a close relationship between the cells. To assess the role of CC10 dur ing NE differentiation, we studied CC10-deficient mouse lungs by immunohist ochemistry and digital imaging. The knockout model revealed a lack of the d isrupted gene product in the lung. Because NE cells, which occur as solitar y cells or in neuroepithelial bodies (NEBS), comprise less than 1% of airwa y epithelia, we counted foci positive for each of the three NE markers-syna ptophysin, calcitonin gene-related peptide (CGRP), and protein gene product (PGP) 9.5-and developed a method to analyze numerous airways in serial sec tions. Digitized images of slides were segmented with Photoshop imaging sof tware. The length of airway epithelium and total section areas were then me asured using MetaMorph image analysis software. A comparable range of NE fo ci was observed regardless of CC10 expression patterns with all three marke rs, suggesting that CC10 is not critical for NE ontogenesis. However, discr imination according to size revealed that wild-type lungs harbored 30% to 4 0% greater synaptophysin- and CGRP-containing NEBs relative to CC10 deficie nt lungs. We posit that an attenuation of pulmonary NE differentiation affl icts the CC10-deficient state. Our imaging application greatly facilitates the acquisition and analysis of complex structures such as the lung and pro mises to be a widely applicable technique for assessments of tissue section s.