Carol Wuenschell

Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.

Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia

Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of α-smooth muscle actin (α-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and α-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of α-SMA mRNA, fewer and more muscular arterioles (α-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of alpha-smooth muscle actin (alpha-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and alpha-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of alpha-SMA mRNA, fewer and more muscular arterioles (alpha-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.

Smad7 is a TGF-β-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis

Smad7 was recently shown to antagonize TGF-beta-induced activation of signal-transducing Smad2 and Smad3 proteins. However, the biological function of Smad7 in the process of lung organogenesis is not known. Since Smad2/3-mediated TGF-beta signaling is known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad7 regulates early lung development by modulating TGF-beta signal transduction. An antisense oligodeoxynucleotide (ODN) was designed to specifically block endogenous Smad7 gene expression at both transcriptional and translational levels in embryonic mouse lungs in culture. TGF-beta-mediated inhibition of lung branching morphogenesis was significantly potentiated in cultured embryonic lungs in the absence of Smad7 gene expression: abrogation of Smad7 potentiated TGF-beta-mediated inhibition of lung branching morphogenesis from 76 to 52% of the basal level in lungs cultured in the presence of 5 ng/ml TGF-beta1 ligand. Likewise, TGF-beta1 EC(50) (concentration of TGF-beta1 that induced half maximal branching inhibition) was reduced from 5 to 1 ng/ml when Smad7 gene expression was abrogated in lung culture, indicating an enhanced level of TGF-beta signaling in lung tissue with abolished Smad7 gene expression. By immunocytochemistry, Smad7 protein was co-localized with both Smad2 and Smad3 in distal bronchial epithelial cells, supporting the concept that Smad7 inhibits TGF-beta signaling by competing locally with Smad2 and Smad3 for TGF-beta receptor complex binding during lung morphogenesis. Furthermore, antisense Smad7 ODN increased the negative effect of TGF-beta1 on epithelial cell growth in developing lungs in culture. We also demonstrated that Smad7 mRNA levels were rapidly and potently induced upon TGF-beta1 stimulation of lungs in culture, suggesting that Smad7 regulates TGF-beta responses in a negative feedback loop. These studies define a novel function for Smad7 as an intracellular antagonist of TGF-beta-induced, Smad2/3-mediated inhibition of murine embryonic lung growth and branching morphogenesis in culture. The optimization of TGF-beta signaling during early lung development therefore requires a finely-regulated competitive balance between both permissive and inhibitory members of the Smad family.

Nicotine stimulates branching and expression of SP-A and SP-C mRNAs in embryonic mouse lung culture

Although the effects of maternal smoking on fetal growth and viability are overwhelmingly negative, there is a paradoxical enhancement of lung maturation as evidenced, in part, by a lower incidence of respiratory distress syndrome in infants of smoking mothers. Other epidemiologic and experimental evidence further support the view that a tobacco smoke constituent, possibly nicotine, affects the development of the lung in utero. We are studying the direct effects of nicotine on murine lung development using a serumless organ culture system. We have found that embryonic lungs explanted at 11 days gestation showed a 32% increase in branching after 4 days in culture in the presence of 1 μM nicotine and 7- to 15-fold increases in mRNAs encoding surfactant proteins A and C after 11 days. The effect of nicotine exposure on surfactant gene expression is apparently mediated by nicotinic acetylcholine receptors because it was blocked byd-tubocurarine. The nicotine-induced stimulation of surfactant gene expression could, in part, account for the effect of maternal smoking on the incidence of respiratory distress syndrome.