Anne-Karina T. Perl

Early restriction of peripheral and proximal cell lineages during formation of the lung.

To establish the timing of lineage restriction among endodermal derivatives, we developed a method to label permanently subsets of lung precursor cells at defined times during development by using Cre recombinase to activate floxed alkaline phosphatase or green fluorescent protein genes under control of doxycycline-dependent surfactant protein C promoter. Extensive or complete labeling of peripheral lung, thyroid, and thymic epithelia, but not trachea, bronchi, or gastrointestinal tract occurred when mice were exposed to doxycycline from embryonic day (E) 4.5 to E6.5. Nonoverlapping cell lineages of conducting airways (trachea and bronchi), as distinct from those of peripheral airways (bronchioles, acini, and alveoli), were established well before formation of the definitive lung buds at E9–9.5. At E11.5, the labeled precursors of peripheral lung were restricted to relatively few cells along the bronchial tubes and clusters in bronchial tips and lateral buds. Thereafter, these cells underwent marked expansion to form the entire gas-exchange region in the lung. This study demonstrates early restriction of endodermal progenitor cells forming peripheral as compared with proximal airways, identifies distinct cell lineages in conducting airways, and distinguishes neuroepithelial and tracheal–bronchial gland cell lineages from those lining peripheral regions of the lung. This system for conditional gene addition or deletion is useful for the study of lung morphogenesis and gene function in vivo, and identifies progenitor cells that may serve as useful targets for cell or gene replacement for pulmonary disorders.

Role of Sonic hedgehog in Patterning of Tracheal- Bronchial Cartilage and the Peripheral Lung

Sonic hedgehog (Shh) was conditionally deleted in respiratory epithelial cells of the embryonic lung in vivo. Deletion of Shh before embryonic day (E) 13.5 resulted in respiratory failure at birth. While lobulation was not perturbed, the lungs were hypoplastic, with reduced branching of peripheral lung tubules, evident from E13.5. Smooth muscle and endothelial cells were absent or reduced, the latter in relationship to the loss of peripheral lung parenchyma. Tracheal–bronchial ring abnormalities occurred when Shh was deleted between E8.5 and E12.5. Deletion of Shh later in gestation (after E13.5) caused mild abrogation of peripheral branching morphogenesis but did not disrupt tracheal‐bronchial development. Defects in branching morphogenesis and vascularization seen in Shh null mutant (Shh‐/‐) mice were substantially corrected when SHH was ectopically expressed in the respiratory epithelium; however, peripheral expression of SHH failed to correct cartilage abnormalities in the trachea and bronchi, indicating a spatial requirement for SHH expression near sites of cartilage formation. Expression of SHH by the respiratory epithelium plays an important role in the patterning of tracheal–bronchial mesenchyme required for formation of cartilage rings in conducting airways. SHH regulates branching morphogenesis and influences differentiation of the peripheral lung mesenchyme required for formation of bronchial and vascular smooth muscle. Developmental Dynamics 231:57–71, 2004.

C/EBPα is required for lung maturation at birth

Epithelial cells lining the peripheral lung synthesize pulmonary surfactant that reduces surface tension at the air-liquid interface. Lack of surfactant lipids and proteins in the lungs causes respiratory distress syndrome, a common cause of morbidity and mortality in preterm infants. We show that C/EBPα plays a crucial role in the maturation of the respiratory epithelium in late gestation, being required for the production of surfactant lipids and proteins necessary for lung function. Deletion of the Cebpa gene in respiratory epithelial cells in fetal mice caused respiratory failure at birth. Structural and biochemical maturation of the lung was delayed. Normal synthesis of surfactant lipids and proteins, including SP-A, SP-B, SP-C, SP-D, ABCA3 (a lamellar body associated protein) and FAS (precursor of fatty acid synthesis) were dependent upon expression of the C/EBPα in respiratory epithelial cells. Deletion of the Cebpa gene caused increased expression of Tgfb2, a growth factor that inhibits lung epithelial cell proliferation and differentiation. Normal expression of C/EBPα required Titf1 and Foxa2, transcription factors that also play an important role in perinatal lung differentiation. C/EBPα participates in a transcriptional network that is required for the regulation of genes mediating perinatal lung maturation and surfactant homeostasis that is necessary for adaptation to air breathing at birth.

Stat-3 is required for pulmonary homeostasis during hyperoxia

Acute lung injury syndromes remain common causes of morbidity and mortality in adults and children. Cellular and physiologic mechanisms maintaining pulmonary homeostasis during lung injury remain poorly understood. In the present study, the Stat-3 gene was selectively deleted in respiratory epithelial cells by conditional expression of Cre-recombinase under control of the surfactant protein C gene promoter. Cell-selective deletion of Stat-3 in respiratory epithelial cells did not alter prenatal lung morphogenesis or postnatal lung function. However, exposure of adult Stat-3-deleted mice to 95% oxygen caused a more rapidly progressive lung injury associated with alveolar capillary leak and acute respiratory distress. Epithelial cell injury and inflammatory responses were increased in the Stat-3-deleted mice. Surfactant proteins and lipids were decreased or absent in alveolar lavage material. Intratracheal treatment with exogenous surfactant protein B improved survival and lung histology in Stat-3-deleted mice during hyperoxia. Expression of Stat-3 in respiratory epithelial cells is not required for lung formation, but plays a critical role in maintenance of surfactant homeostasis and lung function during oxygen injury.

Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.

Conditional expression of genes in the respiratory epithelium in transgenic mice: cautionary notes and toward building a better mouse trap.

In vitro and in vivo studies have repeatedly demonstrated the limitations and potential toxicity of various genes (proteins) used for both labeling cells (e.g., with green fluorescent protein [GFP], β-galactosidase, and luciferase) or for the deletion/addition of mutation of genes (e.g., reverse tetracycline transactivator protein [rtTA], tetracycline transactivator protein [tTA], Cre-recombinase [Cre], or CreER). High levels of the introduced protein can cause endoplasmic reticulum stress, genetic instability, immunologic recognition, and/or disrupt cellular homeostasis. Resultant cell injury, death, or other off-target effects on gene expression may be caused by expression of the transgene. A number of systems for gene addition and deletion in the respiratory epithelium have been developed and widely used for the study of gene function, lung morphogenesis, and function. The Scgb1a1 (Clara Cell Secretory Protein or CCSP) and SFTPC (Surfactant Protein C or SP-C) promoters have been used by our laboratory and others (1, 2). These promoters are highly cell specific and generate robust levels of gene expression. To conditionally express genes in the lung, transgenic mice were produced expressing the reverse tetracycline transactivator that is active when doxycycline is provided to the mouse (3, 4). To target proximal airways, the 2.3-kb rat Scgb1a1 promoter was used to drive the reverse tetracycline activator (CCSP-rtTA transgenic mice). To target distal lung structures, we used the 3.7-kb human SFTPC promoter (SP-C-rtTA transgenic mice). Airspace enlargement unrelated to the effects of the transgene were observed in various mouse strains bearing the CCSP-rtTA transgene (line 1) (5–7). The potential for toxicity related to the reverse tetracycline activator, doxycycline, and Cre-recombinase was reviewed previously (7). Such overt rtTA toxicity was not seen in many experiments with the initial 3.7-kb human SP-C-rtTA transgenic mice that have been used in numerous studies. Subsequently, both CCSP-rtTA and SP-C-rtTA mice were bred to a line of (otet)7CMV-Cre mice that enables doxycycline-regulated expression of Cre-recombinase in the respiratory epithelium in vivo (6, 8). When mated to mice bearing floxed alleles, the addition of doxycycline to chow or drinking water (3) causes excision and recombination of DNA sequences located between engineered loxP recognition sites, causing deletion, mutation, or activation of the appropriately engineered floxed gene in the mouse lung (6–8). This system has been useful in lineage analysis, the study of gene function, and lung development. Initial studies failed to reveal significant misexpression of Cre-recombinase or overt histologic or biological toxicity. As our laboratory has bred the SP-C-rtTA, (otet)7CMV-Cre mice (primarily maintained in FVBN strain) with other strains bearing floxed alleles and other mouse genetic backgrounds, we have observed off-target effects influencing both lung morphogenesis and perinatal survival. In generating mice in which GP130 receptor, mediating the activation of STAT3, was deleted (9), initial F1 mice were bred, in line, with resultant production of two distinct strains that (1) lacked discernable phenotype, unless injured (9), or (2) died of severe lung pathology at birth. Severe morphologic effects were observed when doxycycline was provided to the dam from approximately Embryonic Day (E)6 to birth. Toxicity was not dependent on the presence of the floxed allele, but on the presence of both SP-C-rtTA and (otet)7CMV-Cre, indicating that toxicity was likely dependent on the expression of Cre-recombinase or the combined expression of rtTA and Cre-recombinase in this strain. Changes in lung morphogenesis and perinatal survival have not been seen if doxycycline was limited to E6.5–14.5 in a number of experiments with SP-C-rtTA, (otet)7CMV-Cre mice bred to a number of mouse strains in our laboratory. The distinct mouse lines produced from the initial SP-C-rtTA, (otet)7CMV-Cre, GP130 loxP founders maintained these distinct features, indicating the potential for the presence of inherited genes that modify susceptibility or resistance to this pulmonary toxicity. As previously noted (4), SP-C-rtTA mice (line 1) cannot be maintained in homozygous state, indicating the potential for a gene dose effect on survival. SP-C-rtTA line 1 mice express high levels of rtTA, and the activity increases in the perinatal period, consistent with the expression of endogenous Sftpc gene expression in the lung (3). Taken together, these observations demonstrate the potential for variable and strain-dependent off-target toxicity of rtTA, Cre-recombinase, and doxycycline. Because doxycycline is stored in tissues, prolonged exposure to doxycycline can result in its continued release from tissue pools. To avoid repeated or prolonged doxycycline exposure to the dams or pups, we now routinely limit the period of doxycycline treatment. The timing and duration of treatment needed to target subsets of epithelial cells have been described (6, 8). It is sufficient to expose dams to doxycycline from E8.5 to E14.5 (SP-C-rtTA mice) and E14.5 to E18.5 (CCSP-rtTA mice) to permanently target floxed genes. To control for phenotypes not related to the activation/inactivation of the gene of interest, compound mutant mice should be tested in the absence of doxycycline. Similarly, heterozygous compound mutant mice bearing the floxed allele should be tested after doxycycline treatment. Littermate controls are used when possible to minimize strain or age related variability.

FGF signaling is required for myofibroblast differentiation during alveolar regeneration

Normal alveolarization has been studied in rodents using detailed morphometric techniques and loss of function approaches for growth factors and their receptors. However, it remains unclear how these growth factors direct the formation of secondary septae. We have previously developed a transgenic mouse model in which expression of a soluble dominant-negative FGF receptor (dnFGFR) in the prenatal period results in reduced alveolar septae formation and subsequent alveolar simplification. Retinoic acid (RA), a biologically active derivative of vitamin A, can induce regeneration of alveoli in adult rodents. In this study, we demonstrate that RA induces alveolar reseptation in this transgenic mouse model and that realveolarization in adult mice is FGF dependent. Proliferation in the lung parenchyma, an essential prerequisite for lung regrowth was enhanced after 14 days of RA treatment and was not influenced by dnFGFR expression. During normal lung development, formation of secondary septae is associated with the transient presence of alpha-smooth muscle actin (alphaSMA)-positive interstitial myofibroblasts. One week after completion of RA treatment, alphaSMA expression was detected in interstitial fibroblasts, supporting the concept that RA-initiated realveolarization recapitulates aspects of septation that occur during normal lung development. Expression of dnFGFR blocked realveolarization with increased PDGF receptor-alpha (PDGFRalpha)-positive cells and decreased alphaSMA-positive cells. Taken together, our data demonstrate that FGF signaling is required for the induction of alphaSMA in the PDGFRalpha-positive myofibroblast progenitor and the progression of alveolar regeneration.

Temporal effects of Sprouty on lung morphogenesis

Paracrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5-E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduced lung growth and resulted in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, caused mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma.

Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis

RATIONALE The respiratory epithelium has a remarkable capacity to respond to acute injury. In contrast, repeated epithelial injury is often associated with abnormal repair, inflammation, and fibrosis. There is increasing evidence that nonciliated epithelial cells play important roles in the repair of the bronchiolar epithelium after acute injury. Cellular processes underlying the repair and remodeling of the lung after chronic epithelial injury are poorly understood. OBJECTIVES To identify cell processes mediating epithelial regeneration and remodeling after acute and chronic Clara cell depletion. METHODS A transgenic mouse model was generated to conditionally express diphtheria toxin A to ablate Clara cells in the adult lung. Epithelial regeneration and peribronchiolar fibrosis were assessed after acute and chronic Clara cell depletion. MEASUREMENTS AND MAIN RESULTS Acute Clara cell ablation caused squamous metaplasia of ciliated cells and induced proliferation of residual progenitor cells. Ciliated cells in the bronchioles and pro-surfactant protein C-expressing cells in the bronchiolar alveolar duct junctions did not proliferate. Epithelial cell proliferation occurred at multiple sites along the airways and was not selectively associated with regions around neuroepithelial bodies. Chronic Clara cell depletion resulted in ineffective repair and caused peribronchiolar fibrosis. CONCLUSIONS Colocalization of proliferation and cell type-specific markers demonstrate that Clara cells are critical airway progenitor cells. Continuous depletion of Clara cells resulted in persistent squamous metaplasia, lack of normal reepithelialization, and peribronchiolar fibrosis. Induction of proliferation in subepithelial fibroblasts supports the concept that chronic epithelial depletion caused peribronchiolar fibrosis.

Signaling pathways in the epithelial origins of pulmonary fibrosis

Pulmonary fibrosis complicates a number of disease processes and leads to substantial morbidity and mortality. Idiopathic pulmonary fibrosis (IPF) is perhaps the most pernicious and enigmatic form of the greater problem of lung fibrogenesis with a median survival of three years from diagnosis in affected patients. In this review, we will focus on the pathology of IPF as a model of pulmonary fibrotic processes, review possible cellular mechanisms, review current treatment approaches and review two transgenic mouse models of lung fibrosis to provide insight into processes that cause lung fibrosis. We will also summarize the potential utility of signaling pathway inhibitors as a future treatment in pulmonary fibrosis. Finally, we will present data demonstrating a minimal contribution of epithelial-mesenchymal transition in the development of fibrotic lesions in the transforming growth factor-alpha transgenic model of lung fibrosis.