Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes.

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.

Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells.

Pulmonary surfactant is a complex of lipids and proteins produced and secreted by alveolar type II cells that provides the low surface tension at the air-liquid interface. The phospholipid most responsible for providing the low surface tension in the lung is dipalmitoylphosphatidylcholine. Dipalmitoylphosphatidylcholine is synthesized in large part by phosphatidylcholine (PC) remodeling, and a lysophosphatidylcholine (lysoPC) acyltransferase is thought to play a critical role in its synthesis. However, this acyltransferase has not yet been identified. We have cloned full-length rat and mouse cDNAs coding for a lysoPC acyltransferase (LPCAT). LPCAT encodes a 535-aa protein of approximately 59 kDa that contains a transmembrane domain and a putative acyltransferase domain. When transfected into COS-7 cells and HEK293 cells, LPCAT significantly increased lysoPC acyltransferase activity. LPCAT preferred lysoPC as a substrate over lysoPA, lysoPI, lysoPS, lysoPE, or lysoPG and prefers palmitoyl-CoA to oleoyl-CoA as the acyl donor. This LPCAT was preferentially expressed in the lung, specifically within alveolar type II cells. Expression in the fetal lung and in rat type II cells correlated with the expression of the surfactant proteins. LPCAT expression in fetal lung explants was sensitive to dexamethasone and FGFs. KGF was a potent stimulator of LPCAT expression in cultured adult type II cells. We hypothesize that LPCAT plays a critical role in regulating surfactant phospholipid biosynthesis and suggest that understanding the regulation of LPCAT will offer important insight into surfactant phospholipid biosynthesis.

Lung development is not necessary for diaphragm development in mice.

BACKGROUND/PURPOSE: Congenital diaphragmatic hernia affects approximately 1 in every 2000 live births. The etiology of these diaphragmatic defects is unknown. Using mice with a targeted deletion of fibroblast growth factor 10 (FGF10), which display a complete lack of lung tissue, we have examined the relationship between lung hypoplasia and diaphragmatic development. METHODS: The diaphragms of FGF10 null mice were examined at 2 embryonic time-points and compared with their heterozygous and wild-type littermates. RESULTS: FGF10 null mice had phenotypically normal diaphragms when compared with wild-type littermates at both time-points studied. CONCLUSION: Normal diaphragm development appears to occur independent of lung development in mice.

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium.

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.

Epithelial-mesenchymal interactions in the developing lung.

Classical experiments in embryology have shown that normal growth, morphogenetic patterning, and cellular differentiation in the developing lung depend on interactive signaling between the endodermal epithelium and mesenchyme derived from splanchnic mesoderm. These interactions are mediated by a myriad of diffusible factors that are precisely regulated in their temporal and spatial expression. In this review we first describe factors regulating formation of the embryonic foregut. We then discuss the experiments demonstrating the importance of tissue interactions in lung patterning and differentiation. Finally, we detail the roles that a few key signaling systems-fibroblast growth factors and their receptors, sonic hedgehog and Gli genes, Wnt genes and beta-catenin, and BMP4-play as mediators of epithelial-mesenchymal interactions in the developing lung.

Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro.

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene (SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein (CC10), sonic hedgehog (Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.

BMP4 modulates fibroblast growth factor-mediated induction of proximal and distal lung differentiation in mouse embryonic tracheal epithelium in mesenchyme-free culture.

Lung morphogenesis and differentiation require interaction between the epithelium and mesenchyme, which is mediated by diffusible molecules such as fibroblast growth factors (FGFs), bone morphogenetic protein 4 (BMP4), and Shh. We have used mesenchyme-free culture to study the effects of these molecules on lung epithelial differentiation. We have tested the individual abilities of FGF1, FGF2, FGF7, FGF9, FGF10, and FGF18, as well as BMP4 and Shh to promote growth and specify distal lung differentiation in mouse tracheal epithelium. The different FGFs exhibited distinct abilities to induce epithelial growth and the expression of the distal lung epithelial marker, surfactant protein C (SP-C), although all FGFs were able to induce expression of BMP4. Tracheal epithelium treated with FGF10 showed little growth and failed to express SP-C as measured by whole-mount in situ hybridization and quantitative real-time polymerase chain reaction. FGF1 treatment resulted in the strongest induction of SP-C. Treatment with BMP4 inhibited epithelial growth and differentiation and antagonized the stimulatory effects of FGF1. In contrast, inhibition of endogenous BMP4 signaling with Noggin protein did not inhibit growth or expression of SP-C but did increase the expression of the proximal lung markers CCSP and HFH4. Expression of Shh was not affected by any of the conditions tested. These results suggest that BMP4 does not signal epithelial cells to adopt a distal fate but may regulate the expansion of proximal epithelial cells in the lung.