The in vivo function of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated in mice, carrying a null allele of the GM-CSF gene, that were generated by gene targeting techniques in embryonic stem cells. Although steady-state hematopoiesis was unimpaired in homozygous mutant animals, all animals developed the progressive accumulation of surfactant lipids and proteins in the alveolar space, the defining characteristic of the idiopathic human disorder pulmonary alveolar proteinosis. Extensive lymphoid hyperplasia associated with lung airways and blood vessels was also found, yet no infectious agents could be detected. These results demonstrate that GM-CSF is not an essential growth factor for basal hematopoiesis and reveal an unexpected, critical role for GM-CSF in pulmonary homeostasis.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/-offspring died of respiratory failure immediately after birth. Lungs of SP-B

The phylogenetically conserved nuclear factor I (NFI) gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects, whereas Nfic-deficient mice have agenesis of molar tooth roots and severe incisor defects. Here we show that Nfib-deficient mice possess unique defects in lung maturation and exhibit callosal agenesis and forebrain defects that are similar to, but more severe than, those seen in Nfia-deficient animals. In addition, loss of Nfib results in defects in basilar pons formation and hippocampus development that are not seen in Nfia-deficient mice. Heterozygous Nfib-deficient animals also exhibit callosal agenesis and delayed lung maturation, indicating haploinsufficiency at the Nfib locus. The similarity in brain defects in Nfia-and Nfib-deficient animals suggests that these two genes may cooperate in late fetal forebrain development, while Nfib is essential for late fetal lung maturation and development of the pons.Nuclear factor I (NFI) transcription and replication proteins function both in adenoviral DNA replication (12,43,44) and in the regulation of transcription throughout development (21). There are four NFI genes in mammals (Nfia, Nfib, Nfic, and Nfix) and single NFI genes in Drosophila melanogaster, Caenorhabditis elegans, Anopheles spp., and other simple animals (21,30,50). No NFI genes have been found in plants, bacteria, or single-cell eukaryotes. In mammals, NFI proteins function as homo-or heterodimers and are expressed in complex, overlapping patterns during embryogenesis (6, 31). NFI proteins bind to a dyad-symmetric binding site (TTGGCN 5 GCCAA) with high affinity (20,40), and NFI proteins have been shown to either activate or repress gene expression depending on the promoter and cellular context (21, 42). The presence of four NFI genes in mammals with possibly overlapping functions makes it a challenge to identify in vivo targets of individual NFI proteins and the roles of NFI genes in development.We showed previously that disruption of Nfia causes late gestation neuroanatomical defects, including agenesis of the corpus callosum, size reductions in other forebrain commissures, and loss of specific midline glial populations (11, 56). In contrast, disruption of Nfic results in early postnatal defects in tooth formation, including the loss of molar roots and aberrant incisor development (61). In a previous study, insertion of a lacZ reporter gene into the Nfib locus resulted in defects in lung maturation but no apparent defects in brain development (22). Here we report the replacement of the essential exon 2 of the Nfib gene with a lacZ reporter gene and show that mice homozygous for our replacement mutation have major neuroanatomical defects similar to, but more severe than, those of Nfia Ϫ/Ϫ mice. These defects include callosal agenesis, aberrant hippocampus and pons formation, and loss of specific midline glial populatio...

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs

Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack ofcontact inhibition, and an epithelial cel morphology for 30-40 passages in culture. MicroviUi, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression ofsurfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in al of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as wel as lung development and tumorigenesis. (4), and synthesis of SPs (5) in culture.Clara cells and type II epithelial cells also serve as progenitor cells of the distal respiratory epithelium in the adult lung (6, 7) and are believed to be the cell of origin forThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. pulmonary adenocarcinomas, a subtype of non-small-cell lung cancer (8). We have previously produced transgenic mice harboring the simian virus 40 (SV40) large tumor antigen (TAg) under the transcriptional control of regulatory sequences derived from the human SP-C promoter region to study the development of pulmonary adenocarcinomas in vivo (9). Transgenic mice bearing the exogenous SP-C/TAg chimeric gene developed pulmonary tumors within 4-6 months of age. The presence ofmicrovilli and lamellar bodies by electron microscopy, as well as the presence of respiratory epithelial cell markers by in situ hybridization, were consistent with the identification of the tumor cells as both bronchiolar and alveolar subtypes in vivo. While cells of the proximal respiratory epithelium and lung epithelial cells of fetal origin (10) have been established in culture, distal respiratory epithelial cell lines that maintain a differentiated phenotype in culture have not been previously produced. In the current study, we describe the production and characterization of distal respiratory epithelial cell lines derived from the SP-C/TAg transgenic mice.