Introduction

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The coordination of cell proliferation and differentiation is a central theme in organogenesis. For the series of events in lung development that lead from the initial outpouching of the embryonic foregut to the appearance of a functional blood-gas interface of adequate surface area to maintain tissue oxygenation, endocrine, paracrine, and epithelial-mesenchymal interactions are brought into play (1, 2). One of the first hormonal influences recognized as altering the rate of pulmonary maturation was that of exogenous glucocorticoid administration (3). In vivo studies by Liggins demonstrated increased survival in premature sheep after precipitation of labor with glucocorticoids (4, 5). Subsequent biochemical and mechanical analyses of lung function in preterm rats, rabbits, mice, sheep, and primates have demonstrated increased synthesis of messenger RNA (mRNA) for surfactant protein-A (SP-A), SP-B, and SP-C, and of the corresponding proteins; increased fatty acid synthase (FAS) and cytidylyl transferase activity; and improved lung compliance independent of effects on surfactant production in animals treated in utero with glucocorticoids (6, 7). In addition to these biochemical and biophysical parameters, the thinning of alveolar septae, increased number of type I pneumocytes, and decreased cell division found after glucocorticoid administration is consistent with morphologic maturation (8).

The requirement for glucocorticoids during normal lung development has been rigorously demonstrated more recently by the phenotype of mice with targeted mutation of the corticotropin-releasing hormone (CRH) gene (11, 12). CRH deficiency results in adrenal atrophy with impaired glucocorticoid production, and abnormal pulmonary development in the setting of both maternal and fetal adrenal insufficiency. Other human (13) and animal models (14) of isolated maternal or fetal adrenal insufficiency still allow sufficient glucocorticoid exposure (15) for normal lung development. In contrast, glucocorticoid receptor-deficient (GR-KO) mice (16), which exhibit a defect in lung development similar to that of CRH-deficient (CRH-KO) mice, will not respond to exogenous administration of glucocorticoid. Studies using fetal lung of different gestational ages to determine the role of endogenous glucocorticoids in maturation are hindered by the inability to distinguish effects caused by the steroids from those caused by other developmental processes. Thus, the CRH-deficient mouse provides a unique model system for definition of the impact of glucocorticoid on pulmonary maturation.

The sites of glucocorticoid action in the fetal lung and the nature of the factors induced by glucocorticoid exposure to promote maturation remain uncertain. During the pseudoglandular phase of mouse lung development, radiolabeled dexamethasone binding occurs primarily in the interstitial mesenchyme, whereas during late gestation, increasing GR content is noted in the distal airway epithelium (17). The existence of a "fibroblast pneumonocyte factor," synthesized by mesenchymal stroma in response to glucocorticoids and which induces epithelial maturation, has been postulated (18), though the isolation of a molecule with such activity remains elusive. To understand better the cell-type spectrum and the timing of glucocorticoid action during lung development, we investigated the ontogeny of biochemical and histologic lung maturation resulting from CRH deficiency and concomitant glucocorticoid deficiency.

	Materials and Methods

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Animal Husbandry

CRH-KO and wild-type (WT) mice of 129 × C57BL/6 or outbred genetic background were maintained on a 12:12 light:dark cycle with ad libitum access to rodent chow. All mouse protocols were in accordance with National Institutes of Health (NIH) guidelines, and were approved by the Animal Care and Use Committees of the Children's Hospital of Boston and Washington University School of Medicine in St. Louis. Natural mating of estrous females with stud males was confirmed by detection of a copulation plug on the morning after introduction of the female into the male cage. To ensure accurate gestational timing, females were subsequently isolated until the time of tissue harvest, with the morning of copulation-plug detection designated as 0.5 d gestation. Gravid females were killed by carbon dioxide inhalation, after which fetuses were dissected from the uterus for analysis.

Histology

Thoracic cavities from embryos of 14.5 d gestation, or isolated lung from embryos of 16.5 to 18.5 d gestation, were fixed by immersion in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h at 4°C, followed by paraffin embedding for generation of 10-µm-thick sections. Lung sections were subjected to hematoxylin and eosin (H&E) staining for assessment of overall histopathology and architecture, and to quantify of apoptosis with an ApopTag Peroxidase Kit (Oncor, Gaithersburg, MD) according to the manufacturer's specifications. Cell proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA)-polymerase delta accessory protein, using mouse monoclonal anti-PCNA/horseradish peroxidase (HRP; Dako, Carpinteria, CA). Immunoperoxidase detection of Clara cell secretory protein (CC10) and the neuroendocrine cell marker protein gene product 9.5 (PGP9.5) was done as described previously (19), with visualization through formation of avidin-biotin-HRP complex (Vector Laboratories, Burlingame, CA).

Lung Weight and DNA Content Determination

Fetuses (n = 7 WT, n = 6 CRH-KO) were isolated from pregnancies arising from either WT × WT or CRH-KO × CRH-KO matings at 18.5 d gestation. Each lung was removed intact from the thoracic cavity, blotted free of excess fluid, and weighed (wet weight). Dry weight was determined after baking at 80°C in a vacuum oven until no further changes in weight were seen on daily weighing. Following dry-weight determination, lung DNA was purified by standard methods (20). DNA concentration was determined by solution absorbance at 260 nm, and was confirmed by agarose gel electrophoresis to ensure that contaminating RNA was insignificant.

PCNA Morphometry

Multiple sections of fetal murine lung immunostained for PCNA were analyzed to determine the percentage of total cells labeled for PCNA in distinct anatomic compartments. The anatomic compartments were defined as follows: (1) proximal airway epithelium: both cartilaginous and noncartilaginous airways lined by cuboidal epithelial cells, including predominantly ciliated cells and Clara cell-like nonciliated cells; (2) distal (primitive alveolar) epithelium: undifferentiated, near-subpleural cuboidal epithelial cells lining acinar structures and facing a lumen; (3) proximal mesenchyme: predominantly spindle-shaped cells surrounding proximal airways and blood vessels, clearly separate from the epithelium, with up to four layers of nuclei counted surrounding a given airway or blood vessel; and (4) distal mesenchyme: predominantly spindle-shaped cells located in the pulmonary interstitium either subpleurally or between adjacent primitive alveoli, with only nonambiguous cells scored. Total cell number was determined in each ×400 high-power field (hpf), and included both PCNA-positive and PCNA-negative (methyl green-positive) nuclei. The percentage of PCNA-positive cells was expressed as a subset of the whole, with at least 3 hpf counted per cellular compartment.

RNA Preparation and Analysis

RNA was prepared from lung tissue of WT and CRH-KO mice of 16.5 to 18.5 d gestation using the guanidinium thiocyanate-cesium chloride method (21). Five or 10 µg of total RNA from three separate embryos of a given gestational age and genotype were subjected to electrophoresis through 1.2% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Random-primed, ³²P-labeled DNA fragments of rat SP-A (22), SP-B (23), SP-C (24), and FAS complementary DNAs (cDNAs) (25) were hybridized to RNA immobilized on the membranes in 5× standard saline citrate (0.75 M NaCl, 0.075 M sodium citrate); 5× Denhardt's solution (0.1% polyvinylpyrrolidine, 0.1% bovine serum albumin, 0.1% Ficoll type 400); 0.5% sodium dodecyl sulfate; 100 µg/ml sheared, denatured salmon sperm DNA; and 16 µg/ml yeast transfer RNA (tRNA) at 65°C (26). After washing of the membranes, hybridizing probes were quantitated with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Each mRNA hybridization signal was corrected for loading and recovery by normalization to -actin or cyclophilin A hybridization on the same filter.

Amniotic Fluid Analysis for Dipalmitoyl Phosphatidyl Choline

Amniotic fluid was aspirated by needle through intact fetal membranes after removal of the fetoplacental unit from the uterus. Aliquots of 25 µl were hydrolyzed with Bacillus cereus phospholipase C, and the resulting diglycerides were analyzed with AgNO₃-modified high-performance thin-layer chromatography (HPTLC)-reflectance spectrodensitometry as described (27).

Statistical Analysis

All comparisons were made within a given category (e.g., lung weight) and between the two genotypes of mice with analysis of variance. Significance was accepted at P < 0.05. All data are expressed as means ± SEM.

	Results

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Histologic Evaluation of Lung Development

CRH-KO pups arising from pregnancies of CRH-KO parents develop cyanosis and die during the 24-h period following delivery. We have previously reported hypercellular, immature-appearing lungs in these mice on the first day of life, with restoration of lung architecture and postnatal viability following in utero administration of glucocorticoid (12). To define the critical window for the glucocorticoid effect, we established timed pregnancies of WT or CRH-KO matings. Histologic analysis, with H&E staining of fetal lungs from homozygous WT or CRH-KO pregnancies isolated at 14.5, 16.5, 17.5, and 18.5 d gestation, revealed equivalent morphology through 16.5 d gestation (Figure 1). A clear divergence in architecture was visible at 17.5 d, with the CRH-KO lung maintaining a dense pseudoglandular appearance, in contrast to WT lung, which showed increased airspace formation. An even more striking difference in architecture was apparent at 18.5 d, with essentially no additional airspace formation or alveolar thinning in the CRH-KO lung occurring as gestation proceeded. The WT lung at 18.5 d showed thinning of alveolar septae, a maturing blood-gas interface, and prominent air spaces. Thus, the maturation profiles of CRH-KO and WT lung diverged between 16.5 and 17.5 d gestation, with little continued differentiation apparent after 16.5 d in the CRH-KO lung.

View larger version (118K): [in this window] [in a new window]   Figure 1.   Developmental analysis of lung histology in CRH-KO and WT mice. H&E-stained sections from fetuses of 14.5, 16.5, 17.5, and 18.5 d gestation photographed at magnification of ×200 are shown. (a) WT 14.5 d; (b) CRH-KO 14.5 d; (c) WT 16.5 d; (d) CRH-KO 16.5 d; (e) WT 17.5 d; ( f ) CRH-KO 17.5 d; (g) WT 18.5 d; (h) CRH-KO 18.5 d.

Evaluation of Cell Proliferation and Cell Death

To differentiate true hypercellularity versus atelectasis as resulting in the dense appearance of the CRH-KO lung in late gestation, we measured lung weight and DNA content of WT and CRH-KO fetuses at 18.5 d gestation (Table 1). Confirming excess cell proliferation in the CRH-KO fetus, lung weight wet, dry weight, and total DNA content were all significantly increased in comparison with those of WT fetuses. Additionally, the lung wet/dry-weight ratio was significantly lower in the CRH-KO fetuses, in accord with the increased weight being due to hypercellularity rather than to increased lung water.

This hypercellularity in the late-gestation fetal CRH-KO lung could result either from failure of differentiation-related cell death or from continued cell proliferation during gestation. One common mechanism of glucocorticoid-mediated developmental cell death is apoptosis. We performed terminal deoxynucleotidyl transferase (TdT) nick end-labeling with incorporation of digoxigenin-conjugated nucleotides in fetal lung from 14.5 through 18.5 d gestation to assess nuclei for the presence of multiple endonuclease cleavages characteristic of apoptosis. No significant difference in staining between CRH-KO and WT lung samples was apparent, with very few cells noted to be undergoing apoptosis in lung tissue from either genotype (data not shown). In contrast, evaluation of continued cell proliferation by immunocytochemical detection of PCNA did show differences between CRH-KO and WT litters. In accord with the H&E-stained lung developmental series, PCNA immunohistochemistry was similar in WT and CRH-KO lung at 14.5 d gestation, with proliferating cells of both mesenchymal and epithelial origin present (data not shown). At 16.5 d gestation, morphometric analyses revealed a small but statistically significant increase in epithelial cells per hpf in CRH-KO lung, without a significant difference in mesenchymal cells (Figure 2a). The total PCNA immunostaining of nuclei in the lung was slightly reduced in the CRH-KO fetuses, however. When the different cell compartments were evaluated at this time point, we found a significant reduction in PCNA immunostaining in both proximal (approximately 30% reduction, P < 0.05) and distal mesenchyme (over 50% reduction, P < 0.005) in CRH-KO fetuses as compared with WT fetuses, with no change in epithelial PCNA immunoreactivity (Figure 2b). Between gestational ages 17.5 and 18.5 d, decreasing PCNA immunoreactivity was apparent in WT lung (Figure 3). At 18.5 d gestation the total number of cells per hpf was significantly increased in proximal and distal airways in CRH-KO as compared with WT mice (Figure 4a), in accord with the total lung DNA and dry weight data. When the epithelial cell compartments were compared, there was a highly significant increase in PCNA labeling of all CRH-KO epithelial cells, with more than a 4-fold increase in the proximal airways and a 2- to 3-fold increase in the distal primitive alveoli (Figure 4b). In contrast to Day 16.5 lung, there was also an increase in PCNA labeling of proximal mesenchymal cells (Figure 4b) in the CRH-KO mice, predominantly reflecting increased labeling around developing blood vessels (more than a fourfold increase), although there was also a significant (twofold) increase in mesenchymal cell proliferation around proximal developing airways (data not shown). In contrast, there was no difference in the PCNA labeling of distal mesenchymal cells at Day 18.5 of gestation.

View larger version (13K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 2.   Morphometric analysis of pulmonary development at 16.5 d gestation. (a) Evaluation of total epithelial (Epi.) and mesenchymal (Mes.) cells per hpf; *P < 0.05. (b) PCNA immunoreactivity in proximal (Prox.) and distal airways, expressed as a percentage of epithelial cells and mesenchymal cells per hpf; *P < 0.05, **P < 0.005.

View larger version (129K): [in this window] [in a new window]   Figure 3.   Immunohistochemical analysis of PCNA in CRH-KO and WT mice. PCNA-positive cells are visualized by peroxidase staining resulting in formation of a brown nuclear precipitate. Sections are counterstained with methyl green for demonstration of architecture and photographed at a magnification of ×200. (a) WT 17.5 d; (b) CRH-KO 17.5 d; (c) WT 18.5 d; (d) CRH-KO 18.5 d.

View larger version (14K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 4.   Morphometric analysis of pulmonary development at 18.5 d gestation. (a) Evaluation of total cellularity in proximal (Prox.) and distal airways; *P < 0.05. (b) Prevalence of PCNA immunoreactivity in epithelial (Epi.) and mesenchymal (Mes.) cells in proximal and distal airways; **P < 0.001.

Evaluation of Type II Pneumocyte Maturation

The ontogeny of expression of the surfactant proteins in WT mice is characterized by increased mRNA and protein abundance as gestation nears term, which is thought to be reflective of type II pneumocyte maturation (6). This increase in expression can be accelerated by exogenous administration of glucocorticoids. We have previously demonstrated impaired SP-B mRNA synthesis near term in CRH-KO mice (12). To characterize more precisely the expression profile of surfactant protein mRNAs in the setting of glucocorticoid deficiency, we prepared RNA from lungs of WT and CRH-KO mice at 16.5 through 18.5 d gestation, and performed Northern blot analysis. SP-A and SP-B mRNA levels were most significantly reduced at 17.5 d gestation in the CRH-KO mice (44% and 45% of the levels in WT mice, respectively, P  0.01; Figures 5a and 5b), but did not differ significantly from the levels in WT mice by 18.5 d gestation. In contrast, the concentration of mRNA for SP-C did not differ significantly from that of WT mice at any time between 16.5 and 18.5 d gestation (Figure 5c).

View larger version (24K): [in this window] [in a new window]   Figure 5.   Quantitation of parameters of type II pneumocyte maturation. Northern blot analysis at 16.5 through 18.5 d gestation for (a) SP-A mRNA; (b) SP-B mRNA; (c) SP-C mRNA; and (d) FAS mRNA. Average hybridization intensity as fraction of the Day-18.5 average for the indicated probe, with SEM, is shown; *P  0.01. (e) Amniotic fluid concentration of DPPC during late gestation in CRH-KO and WT mice determined by HPTLC.

The appearance of the surfactant lipid dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid is used during human pregnancy as a clinical indicator of lung maturity and probable functional capacity, although both lipid and apoprotein components of surfactant are required for adequate reduction of surface tension. Northern blot analysis of expression of mRNA for FAS, an enzyme exhibiting glucocorticoid modulation in the DPPC synthetic pathway in in vitro studies (28), revealed a developmental profile similar to that for SP-A and SP-B in CRH-KO mice (54% of that of WT mice at 17.5 d gestation, P < 0.01; Figure 5d). To assess the consequences of reduced FAS expression on production of surfactant lipid, we measured the DPPC concentration in amniotic fluid with HPTLC (27). At Day 17.5 of gestation, both WT and CRH-KO mice demonstrated a DPPC concentration of < 5 µg/ml, a level incompatible with mature lung function (29) (Figure 5e). Surprisingly, at 18.5 d gestation both the CRH-KO and WT mice showed a marked increase in DPPC concentration, with no significant difference between the two genotypes. In accord with the similar amniotic fluid concentration of DPPC in WT and CRH-KO mice, the time of appearance of lamellar bodies within type II pneumocytes, at 17.5 d gestation, also did not differ (data not shown).

Evaluation of Clara and Pulmonary Neuroendocrine Cells

To determine whether cell types other than type II pneumocytes are affected by inadequate glucocorticoid exposure, we analyzed Clara cell and pulmonary neuroendocrine cell differentiation. Immunohistochemical analysis of CC10 induction showed no expression in CRH-KO or WT mice until 18.5 d gestation. At 18.5 d gestation, WT mice displayed prominent CC10 immunoreactivity in bronchial airway epithelium, whereas CC10 remained virtually undetectable in the CRH-KO mice (Figures 6a and 6b). In contrast to the induction of CC10 in normal lung development is the restriction of expression of the marker PGP9.5 as pulmonary differentiation proceeds (30). We found that immature pulmonary epithelial cells were diffusely positive for PGP9.5 immunoreactivity, as demonstrated in histologic specimens from Day 17.5 WT and CRH-KO mice (Figures 6c and 6d). At Day 18.5, however, the PGP9.5 immunoreactivity in WT mice was confined to the neuroendocrine cells populating the pulmonary epithelium, to peribronchial nerves, and to smooth-muscle bundles surrounding pulmonary vasculature. CRH-KO mice failed to restrict PGP9.5 expression to these cell types, continuing to express this protein in the majority of epithelial cells in a pattern similar to the earlier-gestation WT lung (Figures 6e and 6f). The neuroendocrine cells per se appeared normal in number and distribution in CRH-KO mice at Day 18.5. 

View larger version (191K): [in this window] [in a new window]   Figure 6.   Evaluation of Clara and pulmonary epithelial cell maturation. Immunoperoxidase staining for CC10 in bronchial epithelium from (a) WT or (b) CRH-KO lung at 18.5 d gestation. Immunoperoxidase staining for PGP9.5 in pulmonary epithelium from lung sections of (c) 17.5 d WT, (d) 17.5 d CRH-KO, (e) 18.5 d WT, and ( f ) 18.5 d CRH-KO mice. All panels were photographed at a magnification of ×200.

	Discussion

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The CRH-KO mouse provides a unique system of reversible glucocorticoid deficiency with which to study lung development, and establishes an essential glucocorticoid requirement in the mouse. To begin dissection of the mechanism of glucocorticoid action, we conducted a developmental analysis of the consequences of combined maternal and fetal glucocorticoid deficiency. Our results reveal pleiotropic effects of glucocorticoid deficiency manifest late in gestation. This timing of deviation from normal development coincides well with the known time course of the glucocorticoid surge during late mouse gestation, which peaks at approximately Day 16 (31). Thus, one probable consequence of this surge is to prepare the fetus for respiration outside the uterus as pregnancy nears term.

Histologic analysis of lungs from timed CRH-KO pregnancies most strikingly revealed a failure of overall morphologic maturation after Day 16.5 of mouse gestation, corresponding to progression from the early to the late canalicular phase. One prominent feature of the CRH-KO lung is its overall hypercellularity, confirmed by quantitative analyses of wet weight, dry weight, and DNA content at 18.5 d gestation. Although increase in plasma glucocorticoid concentration causes apoptosis in many cell types, we did not find apoptotic cell death to be a frequent event in the pulmonary development either of normal mice, in agreement with the work of others (32), or of CRH-KO mice. Cell proliferation in the CRH-KO lung in excess of that in the WT lung at 18.5 d gestation, however, was demonstrated by assessing immunoreactivity for PCNA. The greatest difference in PCNA immunoreactivity occurred in pulmonary epithelium both proximally and distally, with a significant increase in evidence of proliferation also apparent in proximal mesenchymal cells. The reduced PCNA reactivity in pulmonary mesenchyme at 16.5 d gestation in the CRH-KO mice suggests a defect in mesenchymal cell expansion during the period preceding type II cell differentiation and alveolarization. Subsequently, at 18.5 d gestation, the CRH-KO mice showed sustained epithelial hyperproliferation similar to that in more primitive stages of lung development, in both proximal and distal alveolar regions. Exogenous glucocorticoid administration during normal gestation leads to diminished lung size (9) as well as to pulmonary epithelial maturation, and our studies indicate that endogenous glucocorticoids likewise normally serve this purpose. The implication for administration of pharmacologic doses of glucocorticoid in the preterm human infant is one of caution in balancing increasing production of factors promoting a reduction in surface tension, and a more mechanically favorable architecture, with inhibition of lung proliferation resulting in overall lung hypoplasia.

On the basis of previous in vivo and in vitro studies using administration of exogenous glucocorticoids, decreased surfactant apoprotein gene expression would be anticipated in the context of in utero glucocorticoid deficiency as a result of CRH deficiency. Our findings are consistent with this hypothesis, and further show variable sensitivity to inadequate in utero steroid exposure among the various surfactant components. SP-C, which appears the earliest in gestation and in significant amount before the glucocorticoid surge, is least affected, whereas more significant reductions in both SP-A and SP-B are found on Day 17.5 of gestation. The increases in SP-A and SP-B mRNA concentrations on Day 18.5, to values near those found in WT mouse lungs, show that endogenous glucocorticoids modulate the timing of the increase in expression of the genes for these proteins. Because the epithelial cell compartment is relatively increased in the CRH-KO lung at 18.5 d gestation, the equivalent overall surfactant apoprotein mRNA concentrations, as normalized to total lung RNA, also suggest a somewhat decreased synthesis rate per epithelial cell. The pattern of induction of surfactant protein mRNAs in the CRH-KO mouse is consistent with normal levels of these mRNAs in the GR-KO mouse at birth (16), and argues against an absolute requirement of glucocorticoid for surfactant protein gene expression. The normal production of surfactant lipid in the CRH-KO mouse suggests that FAS expression, though reduced, is not limiting in the setting of glucocorticoid insufficiency. Overall, our data suggest that during lung maturation, glucocorticoid has more marked effects on cell proliferation and architectural maturation than on surfactant expression.

These modest reductions in SP-A and SP-B would not be anticipated to be the primary cause of demise in CRH-KO mice. Mice with a targeted mutation in the gene for SP-A, resulting in complete deficiency of this protein without other abnormalities in lung morphologic differentiation, do not develop respiratory distress in the neonatal period (33). Mice with complete deficiency of SP-B (34), and as a consequence impaired secretion of SP-C, die in the neonatal period, although heterozygotes are normal. The generalized parenchymal defects we found, such as the lack of induction of CC10 in Clara cells, the failure to restrict PGP9.5 expression, and the continued proliferation of epithelial and mesenchymal cells, suggest that the fatality observed in CRH-KO mice as a result of glucocorticoid insufficiency stems from an overall delay in the timing of pulmonary maturation. This diversity in manifestations of glucocorticoid action on in utero lung development is analogous to the variety of postnatal effects of glucocorticoids on gastrointestinal maturation. Classic studies by Moog (35) and others (36, 37) on induction of intestinal enzymes initially defined a role for glucocorticoids in biochemical differentiation. The role for glucocorticoids was later shown to include alteration in gut epithelial proliferation and morphogenesis of the crypts and villi (38). In contrast to the acceleration of epithelial proliferation in the gut that is promoted by the postnatal glucocorticoid surge, the prenatal surge results in an overall decrease in pulmonary cell division. We speculate that the increment in pulmonary function observed when glucocorticoids are given to premature human infants is due to morphologic lung maturation and remodeling through effects on cell proliferation, in addition to the acceleration of surfactant synthesis.

In summary, the CRH-KO mouse is a novel model with which to analyze the effects of combined maternal and fetal glucocorticoid deficiency on lung development. This model, in which glucocorticoid deficiency is separable from other developmental processes, clearly shows that glucocorticoid broadly affects pulmonary epithelial and mesenchymal maturation, leading to alterations in cell proliferation and Clara cell differentiation, with surprisingly minimal effects on the induction of surfactant proteins and lipids. This system should prove valuable for continued dissection of the mechanism of glucocorticoid action and delineation of the program of cell differentiation in the lung.