Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.

Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2 during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud epithelium in transgenic mice. Newborn mice expressing the transgene were completely normal except that instead of normally developed lungs they had two undifferentiated epithelial tubes that extended from the bifurcation of the trachea down to the diaphragm, a defect that resulted in perinatal death. Thus, the dominant negative FGF receptor completely blocked airway branching and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in branching morphogenesis of the mammalian lung.     

Alpha smooth muscle actin expression in developing and adult human lung

Abstract. Myofibroblast-like cells containing smooth muscle actin have been identified in lung injury and repair. These cells differ from typical smooth muscle cells by architectural configuration, location and lack of smooth muscle myosin. Their progenitors are unknown. We hypothesized that these cells might have a developmental analog critical to lung morphogenesis. Lung tissue from developing and adult human lungs was studied using a highly specific monoclonal antibody directed against alpha smooth muscle actin (ASMA). Cells im-munoreactive for ASMA (ASMA cells) were identified prenatally in the form of smooth muscle investing the developing vasculature and airway structures. ASMA was not expressed in undifferentiated mesenchymal cells at any prenatal stage. Late in development, ASMA cells within the lung acinus increased proportionally to terminal airway and vascular complexity. In the early postnatal period, the specific distribution of ASMA cells within inflated lung became clearer, and three populations were identified: (1) typical smooth muscle investing the large airways and blood vessels; (2) small clusters of cells with in the acinus distributed at the tips of septa protruding into the alveolar duct; (3) individual cells within the alveolar sac sparsely distributed near the junctions of individual alveoli, frequently in association with small blood vessels. We conclude that ASMA cells appear only in developing small and large airways and pulmonary vessels and that they may play a critical role in branching morphogenesis during development.     

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

Distribution of human PLUNC/BPI fold-containing (BPIF) proteins

Although gene expression studies have shown that human PLUNC (palate, lung and nasal epithelium clone) proteins are predominantly expressed in the upper airways, nose and mouth, and proteomic studies have indicated they are secreted into airway and nasal lining fluids and saliva, there is currently little information concerning the localization of human PLUNC proteins. Our studies have focused on the localization of three members of this protein family, namely SPLUNC1 (short PLUNC1), SPLUNC2 and LPLUNC1 (long PLUNC1). Western blotting has indicated that PLUNC proteins are highly glycosylated, whereas immunohistochemical analysis demonstrated distinct patterns of expression. For example, SPLUNC2 is expressed in serous cells of the major salivary glands and in minor mucosal glands, whereas SPLUNC1 is expressed in the mucous cells of these glands. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and expressed in airway submucosal glands and minor glands of the oral and nasal cavities. SPLUNC1 is also found in the epithelium of the upper airways and nasal passages and in airway submucosal glands, but is not co-expressed with LPLUNC1. We suggest that this differential expression may be reflected in the function of individual PLUNC proteins.     

Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.     

Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation

The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56gamma is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56gamma was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56gamma was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56gamma using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56gamma transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, beta-catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56gamma transgenic fetuses. We propose that B56gamma is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/beta-catenin signaling pathway during lung airway morphogenesis.     

Cell type specific expression of Follistatin-like 1 (Fstl1) in mouse embryonic lung development

Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-β1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα⁺ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.     

Notch-dependent differentiation of adult airway basal stem cells

The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily toward secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways.     

Evidence That SOX2 Overexpression Is Oncogenic in the Lung

Background

SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung.

Methodology/Principal Findings

We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12–34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63).

Conclusions

These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.     

Fibroblast growth factor 18 influences proximal programming during lung morphogenesis

The structure and functions of the airways of the lung change dramatically along their lengths. Large-diameter conducting airways are supported by cartilaginous rings and smooth muscle tissue and are lined by ciliated and secretory epithelial cells that are involved in mucociliary clearance. Smaller peripheral airways formed during branching morphogenesis are lined by cuboidal and squamous cells that facilitate gas exchange to a network of fine capillaries. The factors that mediate formation of these changing cell types and structures along the length of the airways are unknown. We report here that conditional expression of fibroblast growth factor (FGF)-18 in epithelial cells of the developing lung caused the airway to adopt structural features of proximal airways. Peripheral lung tubules were markedly diminished in numbers, whereas the size and extent of conducting airways were increased. Abnormal smooth muscle and cartilage were found in the walls of expanded distal airways, which were accompanied by atypically large pulmonary blood vessels. Expression of proteins normally expressed in peripheral lung tubules, including SP-B and pro-SP-C, was inhibited. FGF-18 mRNA was detected in normal mouse lung in stromal cells surrounding proximal airway cartilage and in peripheral lung mesenchyme. Effects were unique to FGF-18 because expression of other members of the FGF family had different consequences. These data show that FGF-18 is capable of enhancing proximal and inhibiting peripheral programs during lung morphogenesis.