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Apr 22, 2004 - expression in isolated developing lung is sufficient to disrupt normal development of the alveolar ducts and the centriacinar regions.
DEVELOPMENTAL DYNAMICS 230:278 –289, 2004

RESEARCH ARTICLE

Arrested Pulmonary Alveolar Cytodifferentiation and Defective Surfactant Synthesis in Mice Missing the Gene for Parathyroid Hormone-Related Protein Lewis P. Rubin,1,2* Christopher S. Kovacs,3 Monique E. De Paepe,2,4 Shu-Whei Tsai,1 John S. Torday,5 and Henry M. Kronenberg6

Parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor expression are developmentally regulated in lung epithelium and adepithelial mesenchyme, respectively. To test the hypothesis that PTHrP is a developmental regulator of terminal airway development, we investigated in vivo and in vitro models of alveolar cytodifferentiation using mice in which the gene encoding PTHrP was ablated by homologous recombination. We have determined that fetal and newborn PTHrP(ⴚ/ⴚ) lungs showed delayed mesenchymal– epithelial interactions, arrested type II cell differentiation, and reduced surfactant lamellar body formation and pulmonary surfactant production. Embryonic PTHrP(ⴚ/ⴚ) lung buds cultured in the absence of skeletal constriction or systemic compensating factors also exhibited delayed alveolar epithelial (type II cell) and mesenchymal cytodifferentiation, as well as a >40% inhibition of surfactant phospholipid production (n ⴝ 3–5). Addition of exogenous PTHrP to embryonic PTHrP(ⴚ/ⴚ) lung cultures normalized interstitial cell morphology and surfactant phospholipid production. The importance of PTHrP as an endogenous regulatory molecule in mammalian lung development is supported by the findings that ablation of PTHrP expression in isolated developing lung is sufficient to disrupt normal development of the alveolar ducts and the centriacinar regions. Developmental Dynamics 230:278 –289, 2004. © 2004 Wiley-Liss, Inc. Key words: PTHrP; alveolarization; surfactant; lung development; gene ablation Received 14 November 2003; Revised 2 January 2004; Accepted 22 January 2004

INTRODUCTION Parathyroid hormone-related protein (PTHrP) is an autocrine and paracrine factor initially identified as the product of human tumors, including all major lung cancer cell types (Brandt et al., 1991). Excessive production of PTHrP by tumors commonly results in the syndrome of humoral hypercalcemia of malignancy (reviewed by Dunbar et al.,

1996). PTHrP is also produced by a wide range of normal fetal and adult tissues (Wysolmerski and Stewart, 1998; Rubin and Torday, 2000) and is widely expressed during embryogenesis (van de Stolpe et al., 1993; Karperien et al., 1994). The phenotypes of mice overexpressing a PTHrP transgene (Wysolmerski and Stewart, 1998) or homozygous for a disrupted PTHrP allele (Karaplis et al.,

1

1994; Amizuka et al., 1994; Kovacs et al., 1996) provide further evidence that PTHrP is an important developmental regulatory protein in skin, breast, placenta, bone, and tooth. Homology of the amino-terminal region of PTHrP with the corresponding domain of parathyroid hormone (PTH) permits both molecules to interact with a common receptor (Ju¨ppner et al., 1991) equipotently on a

Department of Pediatrics, Brown Medical School and Women and Infants Hospital, Providence, Rhode Island Program in Fetal Medicine, Brown Medical School and Women and Infants Hospital, Providence, Rhode Island Faculty of Medicine-Endocrinology, Health Sciences Centre, Memorial University of Newfoundland, St. Johns, NF, Canada 4 Department of Pathology and Laboratory Medicine, Brown Medical School and Women and Infants Hospital, Providence, Rhode Island 5 Departments of Pediatrics and Obstetrics and Gynecology, Harbor-UCLA Research and Education Institute, Torrance, California 6 Endocrine Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts Grant sponsor: NIH; Grant numbers: HL55268; DK47038; Grant sponsors: the American Heart Association; the Rhode Island Foundation; the Medical Research Council of Canada. *Correspondence to: Lewis P. Rubin, M.D., Department of Pediatrics, Women and Infants’ Hospital of Rhode Island, 101 Dudley Street, Providence, RI 02905-2499. E-mail: [email protected] 2 3

DOI 10.1002/dvdy.20058 Published online 22 April 2004 in Wiley InterScience (www.interscience.wiley.com).

© 2004 Wiley-Liss, Inc.

PTHRP AND PULMONARY ALVEOLAR DEVELOPMENT 279

molar basis, e.g., in pulmonary fibroblasts (Rubin et al., 1994). This PTH/ PTHrP receptor is also expressed widely, principally in mesenchymal cells of many developing organs, often adjacent to cells producing PTHrP (Karperien et al., 1994; Wysolmerski et al., 1998). The similar effects in PTHrP(⫺/⫺) and PTH/PTHrP receptor(⫺/⫺) mice on bone growth plate (Lanske et al., 1996) and developing breast tissue (Wysolmerski et al., 1998) suggest that this PTH/PTHrP receptor mediates many actions of PTHrP. In the developing lung, paracrine signals between the rudimentary epithelial cells and surrounding mesenchymal cells are crucial for airway branching and air sac cytodifferentiation (Adamson, 1992; Shannon, 1994). By the end of mammalian gestation, pulmonary saccules (the presumptive alveoli) become lined by a continuous layer of epithelium consisting of flattened, gas exchanging squamous cells (alveolar type I cells) interspersed in the saccular niches with cuboidal, surfactant-producing alveolar type II cells. After birth, pulmonary surfactant confers mechanical stability to the alveoli at low lung volumes and prevents transudation of fluid into the air spaces. Pulmonary PTHrP expression is highly cell type- and developmental stage-specific (Lee et al., 1995), observed first in the undifferentiated columnar epithelium and, later in development, in the epithelial progenitor cells of the alveoli, i.e., type II cells (Hastings et al., 1994), and bronchioles, i.e., Clara cells (Rubin and Torday, 2000). PTH/PTHrP receptors are localized to the adepithelial mesenchyme (Lee et al., 1995) adjacent to sites of PTHrP synthesis; PTH/ PTHrP receptor mRNA signal density diminishes with increasing distance from the epithelial boundary (Karperien et al., 1994; Lee et al., 1995). This “hand-in-glove” ligand and receptor expression pattern is a distinguishing feature of paracrine regulators of lung maturation and suggests that PTHrP may be a patterning or differentiation factor during pulmonary development. PTHrP stimulates surfactant phospholipid production in type II cells in vitro

by mesenchymal– epithelial interactions (Rubin et al., 1994; Torday et al., 2002; Torday and Rehan, 2002) and, in adult cells, by autocrine mechanisms (Hastings et al., 1994). Lung epithelial PTHrP expression peaks shortly before birth (Rubin and Torday, 2000), cotemporal with the phase of accelerated surfactant production. In addition, PTHrP levels in tracheal fluid of human newborns are positively correlated with pulmonary maturation and are low in infants with surfactant deficiency, or hyaline membrane disease (Speziale et al., 1998; Sasaki et al., 2000). Similarly, lung PTHrP is depleted in the preterm baboon model of bronchopulmonary dysplasia (Torday et al., 2003). To study the physiological role of PTHrP, Karaplis et al. (1994) disrupted the PTHrP gene and generated a mouse strain deficient in PTHrP. Mice heterozygous (haplodeficient, ⫹/⫺) for the disrupted PTHrP allele appear phenotypically indistinguishable from wild-type (WT, ⫹/⫹) littermates at birth, but PTHrP homozygous mutants (⫺/⫺) have a distinctive chondrodystrophy. They usually die of uncertain causes within 30 min after birth, but occasionally survive for 12 to 36 hr. Depending on genetic background, PTH/PTHrP receptor(⫺/⫺) mice also die between E12.5 and E14.5 of unknown causes or within a few minutes after birth (Lanske et al., 1996). The objective of the present study was to characterize the pulmonary pathophysiology of the PTHrP(⫺/⫺) mouse and to use embryonic lung organ cultures from PTHrP(⫺/⫺), (⫹/ ⫺), and (⫹/⫹) mice to determine specific functions of PTHrP during alveolar development. Branching morphogenesis is not significantly altered in PTHrP(⫺/⫺) (Rubin and Torday, 2000) or PTH/PTHrP receptor(⫺/⫺) mouse embryos (Ramirez et al., 2000). However, because PTHrP appears to be an important regulator of cell– cell communication during terminal airway cytodifferentiation, the present studies focused on developmental events taking place in the alveolar ducts and centriacinar regions, where the transition from conducting airways into the respiratory areas occurs. Our findings indicate that ablation of

PTHrP expression causes specific defects or delays in alveolar epithelial and mesenchymal development.

RESULTS Alterations in Alveolar Differentiation in PTHrP(ⴚ/ⴚ) Mice There were no differences among PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) fetuses in mean body weight. Lungs removed from spontaneously delivered PTHrP null (⫺/⫺) animals did not float when placed in saline solution, unlike the lungs from phenotypically normal (⫹/⫺, ⫹/⫹) littermates. The histologic appearances of the PTHrP(⫺/⫺) and normal (⫹/⫺, ⫹/⫹) lungs showed conspicuous differences on embryonic day (E) 18.5 and at term (E19.0 –E19.5). PTHrP(⫺/⫺) newborn lungs (Fig. 1B) were smaller, denser, and less-well aerated than were lungs of normal littermates (Fig. 1A), even though the lung weights were similar (Table 1). Higher magnification of the hematoxylin and eosin (H&E)-stained sections showed that the normal lung tissue (Fig. 2A) had well-distended saccules, thin saccular septae, morphologic differentiation into types I and II cells, and protrusion of capillaries into the air spaces. PTHrP(⫺/⫺) lung (Fig. 2B) showed more immature features that are consistent with arrest at the canalicular stage of lung development. Distinct saccules and thin, type I-like epithelial cells were absent. The cuboidal epithelium in PTHrP(⫺/⫺) lungs had significantly greater nuclear:cell area ratios than PTHrP(⫹/⫹) or PTHrP(⫹/⫺) lungs (P ⬍ 0.005) and significantly fewer lamellar bodies (P ⬍ 0.005) per cell (Table 1). The septal mesenchyme in the PTHrP(⫺/⫺) lungs showed the thickness and cellularity characteristic of canalicular lung, as is the case for spontaneous mutant chondrodysplastic mice (Hepworth et al., 1989). In PTHrP(⫺/⫺) mice, the percentage of lung tissue occupied by potential air spaces and the mean area per saccule were significantly decreased by approximately half (P ⬍ 0.005; Table 1). The sequence of distinctive ultrastructural features in embryonic mouse lungs that appear from the

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Fig. 1. A,B: Cross-sectional thoracic anatomy of embryonic day 18.5 normal [PTHrP(⫹/⫹)] (A) and PTHrP(⫺/⫺) (B) littermates. The PTHrP(⫺/⫺) lungs are smaller and more compact. Hematoxylin and eosin stain; original magnification, ⫻200. PTHrP, parathyroid hormone-related protein.

TABLE 1. Lung Weights and Morphometric Characteristics PTHrP(ⴙ/ⴙ), (ⴙ/ⴚ), and (ⴚ/ⴚ) Fetuses on Day E18.5a Parameter

⫹/⫹

⫹/⫺

⫺/⫺

Significance

Wet lung wt (mg) Dry lung wt (mg) Thoracic volume (ml) Saccular area occupied by tissue (%) Number of saccules/HPF Mean saccular area (mm2) Nuclear: cell area ratio epithelial LB/epithelial cell

35.1 ⫾ 4.8 5.4 ⫾ 0.6 52.9 ⫾ 6.8 36 ⫾ 12 17 ⫾ 3 6.4 ⫾ 1.4 0.27 ⫾ 0.08 9⫾3

29.7 ⫾ 8.8 4.7 ⫾ 1.2 48.5 ⫾ 7.9 33 ⫾ 14 16 ⫾ 4 6.7 ⫾ 1.0 0.29 ⫾ 0.07 9⫾3

33.9 ⫾ 4.4 5.1 ⫾ 0.4 23.9 ⫾ 30 13 ⫾ 7 37 ⫾ 8 3.8 ⫾ 0.3 0.39 ⫾ 0.05 1⫾1

NS NS P⬍ P⬍ P⬍ P⬍ P⬍ P⬍

0.005 0.005 0.005 0.005 0.005 0.0005

All values are recorded as means ⫾ SD; n ⫽ 3–7 HPF (high power field) or n ⫽ 10 epithelial cells (LB, lamellar body). Statistical comparisons between groups were performed using one-factor analysis of variance and indicate differences of PTHrP(⫺/⫺) vs. (⫹/⫺) or (⫹/⫹). PTHrP, parathyroid hormone-related protein; NS, not significant.

a

Fig. 2. A: Section from PTHrP(⫹/⫹) newborn lung. The arrow indicates a type II cell, and the arrowhead indicates a type I-like cell. B: Lung tissue from a PTHrP(⫺/⫺) littermate that died 3 hours after birth. Arrowheads indicate amorphous intraluminal material. Hematoxylin and eosin stain; original magnification, ⫻600. PTHrP, parathyroid hormone-related protein.

onset of the pseudoglandular stage (⬃E14.5) to birth (Ten Have-Opbroek et al., 1988, 1990; Hepworth et al.,

1989) facilitated developmental stage comparisons among the genotypes. Electron photomicrographs

of the E18.5 normal lungs showed numerous mature-appearing type II cells (Fig. 3A), i.e., cuboidal cells

PTHRP AND PULMONARY ALVEOLAR DEVELOPMENT 281

Assessment of Functional Alveolar Maturation in PTHrP(ⴚ/ⴚ) Lungs: Pulmonary Surfactant Production

Fig. 3. A,B: Electron photomicrographs (original magnification, ⫻16,200) of PTHrP(⫹/ ⫹)(A) and PTHrP(⫺/⫺) (B) lung tissue on embryonic day 18.5. The wild-type lung has normal type II cell maturation. G indicates glycogen. Arrows indicate forming, intracellular and intraluminal lamellar bodies. B: The PTHrP depleted lung shows immature type II cell precursors with large nuclei, large glycogen lakes, and few lamellar bodies or apical microvilli. C,D: Electron photomicrographs (original magnifications, ⫻2,856) of PTHrP(⫹/ ⫹)(C) and PTHrP(⫺/⫺) (D) saccular septae. C: The thick arrow points to a type II cell, and curved arrows indicate intracellular and intraluminal lamellar bodies. D: The stars indicate lipid vesicles in an interstitial fibroblast; the arrows indicate areas of unremodeled basal lamina. PTHrP, parathyroid hormone-related protein.

having a large and roundish nucleus, apical microvilli, glycogen fields, and numerous lamellar bodies (LBs, arrows in Fig. 3A). Most lumens of these saccular stage lungs also contained extruded LBs (Fig. 3A). In contrast, distal airways in the PTHrP(⫺/⫺) newborns (Fig. 3B) showed canalicular morphology with immature type II cell precursors, i.e., rounded to columnar cells having poorly differentiated cytoplasm, rare apical microvilli, large glycogen lakes, and few precursor multilamellar inclusion bodies or mature LBs. The interstitia of normal mice on E18.5 contained mature saccular septa (Fig. 3C), while the PTHrP(⫺/⫺) lung interstitia contained immature

fibroblastic cells that lacked a conspicuous endoplasmic reticulum and Golgi complex. The acinar mesenchymal– epithelial boundaries in PTHrP(⫺/⫺) specimens were composed of thick basement membranes containing collagen fibrils. Laminar remodeling, which is important for type II cell cytodifferentiation and pulmonary surfactant production (Adamson, 1992), was less apparent in PTHrP(⫺/⫺) lungs. Finally, many interstitial fibroblasts in PTHrP(⫺/⫺) lungs, but not in PTHrP(⫹/⫺) or (⫹/⫹) lungs, were packed with abundant lipid droplets (Fig. 3D, stars), which stained with oil red-O by light microscopy (LM; data not shown).

We assessed pulmonary surfactant production, because surfactant is a distinctive secretory product of type II pneumocytes and is a functional maturation marker in the developing alveolus. Minced lung tissues obtained from E17.5 and E18.5 fetuses were incubated with [3H]choline chloride for 4 hr to estimate the rates of surfactant phospholipid synthesis. As shown in Figure 4, 3H-saturated phosphatidylcholine (PC) synthesis was significantly decreased in PTHrP(⫺/⫺) lungs to approximately one third the values of (⫹/⫺) or (⫹/⫹) littermate lung tissues. Because glycogen is a substrate for type II cell surfactant phospholipid production, the greater glycogen content of the PTHrP(⫺/⫺) specimens (Fig. 3B) is also consistent with inhibited surfactant biosynthesis. However, in contrast to the decreased [3H]choline incorporation into surfactant phospholipid, mRNA levels for surfactant protein (SP)-A, SP-B, and SP-C were quite variable and did not significantly differ among PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) littermates near and at term (Fig. 5). This finding is similar to results obtained in PTH/PTHrP receptor(⫺/⫺) lungs (Ramirez et al., 2000).

PTHrP Depletion and Impairment of Lung Differentiation In Vitro To study more directly the roles of PTHrP in lung morphogenesis excluding potential confounding effects from thoracic chondrodystrophy and the systemic absence of PTHrP, we cultured lung buds from PTHrP(⫹/ ⫹), (⫹/⫺), and (⫺/⫺) E13.5 and E14.5 embryos and maintained the organs in serumless medium for 5 to 6 days. Importantly, these gestational ages antedate the appearance of the thoracic skeletal abnormalities in PTHrP(⫺/⫺) fetuses (Karaplis et al., 1994) and effectively eliminate genotype-specific pulmonary consequences due to progressive thoracic volume constriction. At the end of incubation, the right lungs

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Fig. 4. Saturated phosphatidylcholine (PC) synthesis in PTHrP(⫺/⫺; open bars), (⫹/⫺; gray bars), and (⫹/⫹; black bars) fetal lungs. Embryonic day (E) 7.5 and E18.5 lungs were isolated, separately genotyped, and incubated with [3H]choline chloride (10 Ci/ml) for 4 hr. [3H]saturated PC content was assayed by thin layer chromatography. Each bar represents the mean ⫾SD of five to eight fetuses per group. Asterisks indicate P ⬍ 0.005 vs. normals (⫹/⫺ or ⫹/⫹) by analysis of variance PTHrP, parathyroid hormone-related protein.

Fig. 5. Northern blot analysis of steady-state mRNA levels for surfactant protein (SP)–A (open bars), SP-B (gray bars), and SP-C (black bars) in representative litters (one of three) for embryonic day 19 PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) lung tissue (see Experimental Procedures section). Signal intensities of surfactant protein mRNAs were normalized to the intensity of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase signal. PTHrP, parathyroid hormone-related protein.

were frozen for RNA analysis and the left (unilobar) lungs were fixed and examined by LM and electron microscopy (EM). Consistent with previous findings (e.g., Jaskoll et al., 1988), embryonic lungs cultured at an air-fluid interface flattened during culture and maintained a glandular appearance (not shown). Because PTHrP is transcriptionally regulated by me-

chanical stretch in numerous tissues, including pulmonary epithelium (Torday et al., 1998; Torday and Rehan, 2002), these culture conditions may inhibit PTHrP expression. Therefore, in a second series of experiments, lung buds were cultured in a buoyant environment having minimal surface tension (McAteer et al., 1983). These fetal lungs cultured fully submerged in serumless Waymouth’s medium

developed as hollow, septated organs with outpouchings from each central lumen lined by a peripheral margin of epithelial cells and subjacent fibroblastic cells. Figure 6 illustrates representative EM sections of PTHrP(⫹/⫹) (Fig. 6A) and PTHrP(⫺/⫺) (Fig. 6B) embryonic (E14.5) lung cultured for 5 days in serumless medium. The central acini of PTHrP(⫺/⫺) lungs typically showed less-mature epithelium with fewer lamellar bodies and greater glycogen content. The differences among E13.5 and E14.5 cultured lungs from PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) littermates were quantified by using stereologic morphometry. Table 2 indicates that PTHrP(⫺/⫺) tissue in vitro exhibited several features characteristic of less-mature glandular epithelium, specifically, an increased nuclear:cell area ratio (i.e., larger nuclei) and a decreased number (or absence) of lamellar bodies. These results suggest that delayed lung development observed in the PTHrP(⫺/⫺) mouse in vivo at least partly results from absence of PTHrP per se. Automaturation occurring during ex vivo lung culture for 5 days also permitted us to determine the direct effects of pulmonary PTHrP expression on surfactant phospholipid production and lung-specific gene expression. E14 PTHrP(⫺/⫺) cultured lungs showed decreased [3H]choline incorporation into saturated PC of 44% vs. PTHrP(⫹/⫺) and 36% vs. PTHrP(⫹/⫹) (P ⬍ 0.05) littermate tissue (Fig. 7). In contrast, when embryonic lungs were maintained in culture, there were no significant differences in steady-state mRNA levels for SP-A, SP-B, or SP-C among genotypes within litters (n ⫽ 3; Fig. 8). These data indicate the discordance observed in vivo at term in PTHrP(⫺/⫺) lung between the significantly impaired saturated phosphatidylcholine synthesis and the normal mRNA levels of surfactant-associated proteins also is present in PTHrP(⫺/⫺) lung tissue cultured ex vivo.

Effects of Exogenous PTHrP on PTHrP(ⴚ/ⴚ) Lung Tissue In Vitro We next tested the hypothesis that exogenous PTHrP would reverse the structural and functional abnormali-

PTHRP AND PULMONARY ALVEOLAR DEVELOPMENT 283

Fig. 6. A,B: Electron photomicrographs of glandular structures in embryonic day 14.5 PTHrP(⫹/⫹) (A) and PTHrP(⫺/⫺) (B) lungs after submersion culture in serumless medium for 5 days. A depicts maturing epithelial cells lining a glandular structure. Arrows indicate lamellar bodies. B shows greater glycogen content and less-differentiated morphology of PTHrP(⫺/⫺) epithelium. Some lamellar bodies are present (arrow). PTHrP, parathyroid hormone-related protein.

TABLE 2. Morphometric Analysis of Cultured PTHrP(ⴙ/ⴙ), (ⴙ/ⴚ), (ⴚ/ⴚ) Plus 100 nM PTHrP Embryonic Lungsa

a

Gestation/genotype

Parameter

E13 ⫹/⫹ E13 ⫹/⫺ E13 ⫺/⫺

Nuclear: cell area ratio (mean ⫾ SD) 0.16 ⫾ 0.02 0.23 ⫾ 0.02 0.38 ⫾ 0.10

E13 ⫹/⫹ E13 ⫹/⫺ E13 ⫺/⫺

LB/cell (mean ⫾ SD) 6⫾2 3⫾1 1⫾1

E14 ⫹/⫹ E14 ⫹/⫺ E14 ⫺/⫺

Nuclear: cell area ratio (mean ⫾ SD) 0.12 ⫾ 0.02 0.26 ⫾ 0.07 0.34 ⫾ 0.02

E14 ⫺/⫺ plus PTHrP

0.16 ⫾ 0.03

E14 ⫹/⫹ E14 ⫹/⫺ E14⫺/⫺

LB/cell (mean ⫾ SD) 5⫾1 4⫾1 1⫾1

E14⫺/⫺ plus PTHrP

3⫾1

Significance — P ⬍ 0.05 vs. ⫹/⫹ P ⬍ 0.0005 vs. ⫹/⫹; P ⬍ 0.01 ⫹/⫺ — P ⬍ 0.005 vs. ⫹/⫹ P ⬍ 0.0005 vs. ⫹/⫹; P ⬍ 0.01 vs. ⫹/⫺ — P ⫽ NS vs. ⫹/⫹ P ⬍ 0.005 vs. ⫹/⫹; P ⬍ 0.05 vs. ⫹/⫺ P ⬍ 0.05 vs. ⫺/⫺; P ⫽ NS vs. ⫹/⫹ — P ⫽ NS vs. ⫹/⫹ P ⬍ 0.001 vs. ⫹/⫹; P ⬍ 0.01 vs. ⫹/⫺ P ⬍ 0.005 vs. ⫺/⫺; P ⫽ NS vs. ⫹/⫹

E13 and E14 lung buds were dissected and maintained in serumless medium for 5 days. Statistical comparisons between groups were performed using one-factor analysis of variance (n ⫽ 3–5 litters each for E13 and E14). PTHrP, parathyroid hormone-related protein; E, embryonic day; NS, not significant.

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Fig. 7. Saturated phosphatidylcholine (PC) synthesis in PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) embryonic (E14.5) lungs maintained in serumless organ culture for 4 days (see Experimental Procedures section). At the end of the culture period, lungs were minced and incubated with [3H]choline chloride (10 ␮Ci/ml) for 4 hr. 3H-saturated PC content was assayed by thin layer chromatography. Each bar represents the mean ⫾ SD of three to five fetuses per group. P ⬍ 0.01 vs. normals (⫹/⫺ or ⫹/⫹) by analysis of variance. PTHrP, parathyroid hormone-related protein.

ties observed in the PTHrP(⫺/⫺) embryonic lungs maintained in vitro. PTHrP(⫺/⫺) embryonic lung cultures were treated ⫾ 100 nM PTHrP in culture medium; medium was changed daily for 4 days. In fetal rat lung cells, this concentration of PTHrP produces maximal effects on surfactant phospholipid production and maximally stimulates fibroblast PTH/PTHrP receptor-dependent second messenger pathways (Rubin et al., 1994). We determined that addition of PTHrP to PTHrP(⫺/⫺) lung increased [3H]choline incorporation into saturated PC to the levels observed in PTHrP(⫹/⫹) lung tissue (Fig. 9). In addition, addition of exogenous PTHrP resulted in an increase in epithelial nuclear:cell area ratio and in number of lamellar bodies per cell (Table 2). These data demonstrate that correction of pulmonary tissue PTHrP deficiency normalized the specific defects in type II cell surfactant phospholipid synthesis. There were no significant changes in steadystate mRNA levels for the surfactant apoproteins (data not shown).

DISCUSSION Based on in vitro studies using exogenous PTHrP and PTHrP antagonists (Rubin et al., 1994; Hastings et al., 1994, 1997; Torday et al., 2002), we hypothesized that lungs of PTHrP(⫺/⫺) mice would show a delayed temporal sequence of mesenchymal– epithelial interactions, arrested type II cell differentiation, deficient alveolar maturation, and reduced surfactant production. These predictions were verified by the findings in PTHrP geneablated embryonic mouse lung described in this report. Structural and functional analysis of the PTHrP(⫺/⫺) mouse lung in vivo and in organ culture demonstrates the importance of PTHrP as a regulator of mammalian alveolar duct and centriacinar development. Disruption of the PTHrP gene in mice is associated with persistence until birth of a primitive-appearing, compact lung containing acini lined with glycogen-rich, cuboidal epithelial cells, few or no lamellar bodies, and sparse endoplasmic reticulum.

The acinar mesenchyme in the PTHrP(⫺/⫺) newborn mice is loosely packed and poorly differentiated, consistent with developmental arrest in the canalicular stage of lung development. The PTHrP(⫺/⫺) lungs also lack the basement membrane discontinuities and epithelial foot processes extending from cuboidal epithelial cells to underlying interstitial cells that are maximal in the perinatal period, coincident with the appearance of lamellar bodies and surfactant synthesis (Grant et al., 1983; Adamson, 1992). These gaps may allow direct mesenchymal– epithelial interaction and may be important for PTHrP juxtacrine signaling and surfactant synthesis. The PTHrP(⫺/⫺) lung exhibits several singular features. One is the abundance of lipid-laden interstitial cells. Based on lipid content, pulmonary interstitial cells may be divided into lipid interstitial cells (or lipofibroblasts) and a nonlipid interstitial cell population, which lacks the characteristic lipid droplets and is located more peripherally in the alveolar septum (Kaplan et al., 1985; Penney et al., 1992; McGowan and Torday, 1997). The perialveolar lipofibroblasts maximally accumulate triglyceride (TG) just before the appearance of surfactant-containing lamellar bodies in neighboring type II cells (Maksvytis et al., 1981). In mixed cell culture systems, pulmonary fibroblast TG is transported to type II cells and incorporated into surfactant phospholipids (Nunez and Torday, 1995). Consequently, the profusion of lipid interstitial cells in the PTHrP(⫺/⫺) mouse fetal lung could indicate a defect in lipid transfer from lipofibroblasts to type II pneumocytes. We speculate that these lipid-laden fibroblasts may be the morphologic correlate of our previous findings (Torday et al., 1998, 2002) that glucocorticoids and PTHrP both stimulate pulmonary fibroblast TG uptake in vitro, but PTHrP specifically enhances TG transfer to type II cells. In fact, lipid TG uptake by fibroblasts appears to be unimpeded in the PTHrP-deficient, but glucocorticoidsufficient mouse fetuses, but the absence of epithelial PTHrP production interferes with fibroblast TG transfer to type II cells.

PTHRP AND PULMONARY ALVEOLAR DEVELOPMENT 285

Fig. 8. Northern blot analysis of steady-state mRNA levels for surfactant protein (SP) -A, SP-B, SP-C, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for PTHrP(⫺/⫺), (⫹/⫺), and (⫹/⫹) embryonic day 14.5 embryonic lungs maintained in serumless organ culture for 5 days. The figure depicts data from a representative litter (n ⫽ 3 litters). There are no significant genotype-specific differences in steady-state mRNA levels for these surfactant-associated proteins. PTHrP, parathyroid hormone-related protein.

The apparently normal surfactant protein gene expression at term in the PTHrP(⫺/⫺) mice might be attributable to intrauterine glucocorticoid action or to compensatory activity of PTH as a PTH/PTHrP receptor ligand, or both. Blood levels of PTH are elevated in PTHrP(⫺/⫺) mice (Kovacs et al., 2001). However, when we cultured PTHrP(⫺/⫺) lung buds for several days in serumless medium (i.e., without exposure to circulating corticosterone or PTH), SP-A, SP-B, and SP-C mRNA were still similar to heterozygote and wildtype littermate lungs. This discordance between delayed surfactant phospholipid synthesis and the normal surfactant protein expression is unusual but not unprecedented. Regulation of surfactant-associated protein gene expression appears to be under separate control from increased surfactant phospholipid synthesis and lamellar body formation (Slavkin et al., 1989; Wright and Dobbs, 1991). A confounding factor in interpret-

ing the pulmonary findings is that PTHrP(⫺/⫺) fetuses have abnormal endochondral bone formation (Karaplis et al., 1994). However, despite the similar skeletal and thoracic phenotype to nonallelic spontaneous chondrodystrophic mouse strains (Rittenhouse et al., 1978; Brown et al., 1981; Foster et al., 1994; Li et al., 1995), arrested maturation to the degree observed in the PTHrP knockout, to our knowledge, has not been a conspicuous feature of restrictive lung hypoplasias resulting from osteochondrodystrophies in rodents (Houghton et al., 1989; Hepworth and Seegmiller, 1989; Foster et al., 1994) or humans (Finegold et al., 1971; Wigglesworth and Desai, 1982). Nonetheless, to determine the direct effects of PTHrP on development of the lung, we studied the effect of PTHrP gene ablation in embryonic lung organ culture. We principally used a submersion culture technique in which the intact fetal lung is exposed to low surface

tension and the pulmonary acini develop around large, fluid-filled lumens. These experiments permitted investigation of PTHrP depletion in the absence of thoracic volume restriction, systemic sequelae of PTHrP deficiency such as hypocalcemia (Kovacs et al., 1996), intrauterine alterations of the pituitary–adrenal or thyroid axes, or compensating effects due to circulating PTH (Kovacs et al., 2001). Although the parathyroid gland PTH synthesis and release are relatively suppressed in late gestation (Rubin et al., 1991; Kovacs and Kronenberg, 1997), hypocalcemia in the PTHrP(⫺/⫺) and PTHrP receptor(⫺/⫺) fetal environments induce overexpression of PTH (Kovacs et al, 1997, 2001); this hyperparathyroidism, in turn, may stimulate lung development in the PTHrP(⫺/⫺) mice. Our analysis of the cultured PTHrP(⫺/⫺) fetal lungs has shown that PTHrP depletion is associated with specific abnormalities in structural and functional alveolar devel-

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Fig. 9. Saturated phosphatidylcholine (PC) synthesis in PTHrP(⫺/⫺), (⫹/⫹), and (⫺/⫺) plus 100 nM PTHrP embryonic day 14.5 lungs maintained in serumless organ culture for 4 days (see Experimental Procedures section). At the end of the culture period, lungs were minced and incubated with [3H]choline chloride (10 ␮Ci/ml) for 4 hr. 3H-saturated PC content was assayed by thin layer chromatography. Each bar represents the mean ⫾ SD of three to five fetuses per group. P ⫽ not significant vs. normals (⫹/⫺ or ⫹/⫹) by analysis of variance. PTHrP, parathyroid hormone-related protein.

opment. Treating lung cultures with exogenous PTHrP reversed these cytologic abnormalities and corrected the deficiency in surfactant phospholipid synthesis. The absence of significant effects of PTHrP ablation on surfactant protein expression also points to the complexity of alveolar regulatory pathways. Ramirez et al. (2000) determined that expression of a marker of type I cell formation, aquaporin-5, was decreased in PTH/ PTHrP receptor(⫺/⫺) newborn lungs, but, as is the case for the PTHrP(⫺/⫺) mouse, expression of surfactant protein genes was not affected. Late embryonic PTHrP(⫺/⫺) and PTH/ PTHrP-receptor(⫺/⫺) lungs both exhibit a similar compact canalicularto-saccular histology. Distal lung ultrastructure and surfactant phospholipid production were not examined (Ramirez et al., 2000). We have determined that ablation of the PTHrP gene is associated with delayed maturation of epithelial and mesenchymal cells in the developing alveolus. In terms of surfactant production, our findings indi-

cate that PTHrP specifically regulates the metabolic pathways leading to synthesis of saturated phosphatidylcholine. Glycogen, a principal substrate for type II cell fatty acid synthesis, accumulates in PTHrP(⫺/⫺) alveolar epithelial cells in vivo and in vitro. Similarly, incorporation of choline into surfactant phospholipid is reduced under both of these conditions. It is of interest that chondrocytes of PTHrP(⫺/⫺) and PTH/PTHrP receptor(⫺/⫺) mice also have increased glycogen deposition (Amizuka et al., 1994; Lanske et al., 1998), perhaps suggesting a wider role for PTHrP action in glycogen metabolism. We currently are investigating the roles of PTHrP in regulation of fatty acid synthase, choline kinase, and choline-phosphate cytidyltransferase and their activities in fetal lung. Expression of a constitutively active PTH/PTHrP receptor targeted to the growth plate by the rat 1 (II) collagen promoter corrects the major skeletal abnormalities of PTHrP(⫺/⫺) mice and allows their survival at

birth, although the “bone rescued” animals die before 2 months of age (Schipani et al., 1997). The lungs of these animals show pulmonary interstitial abnormalities (unpublished observations). In light of the association between PTHrP “depletion” and lung disease in respiratory distress syndrome (Speziale et al., 1998), bronchopulmonary dysplasia (Torday et al., 2003), and adult lung injury (Hastings et al., 2001; Stern et al., 2002), characterization of the pulmonary phenotype in these mutant animals may be important. In summary, these findings suggest that PTHrP plays an important role in epithelial cytodifferentiation and epithelial–mesenchymal communications in the developing pulmonary alveolus. We speculate that endogenous expression of PTHrP in the developing mammalian lung may influence commitment to a mature type II cell phenotype.

EXPERIMENTAL PROCEDURES PTHrP(ⴚ/ⴚ) Mice Mice carrying a disrupted, nonfunctional PTHrP gene were derived by homologous recombination in embryonic stem cells (Karaplis et al., 1994). The data presented below were derived from the original hybrid C57BL/6129/SvJ genetic background. Mice heterozygous for the PTHrP gene ablation were mated overnight; the presence of a vaginal mucus plug on the morning after mating marked gestational day (E) 0.5. Normal gestation in these mice is 19 days. All mice were fed a standard chow diet and had ad libitum access to water. Fetuses were allowed to deliver spontaneously or were delivered by hysterotomy at 13.5 to 18.5 days postcoitum. For weight determinations, the fetal lungs were blotted, weighed, lyophilized overnight, and weighed again dry. Genotypes of mice were determined from tail DNA by polymerase chain reaction (Karaplis et al., 1994) or, in some experiments, by Southern blotting (Kovacs et al., 1996). All procedures were approved by the Lifespan/Women and Infants Hospital Academic Medical Center and Massachusetts General Hospital institutional animal care and use committees.

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Mouse Embryonic Lung Culture Embryonic mouse lungs were dissected from day E13.5 to E14.5 embryos. A modified Trowell culture method was used (Jaskoll et al., 1988). In other experiments, the lung primordia were cultured in 12-well cluster dishes on a rocker platform (6 cycles/min) for 4 to 5 days in serumless Dulbecco’s MEM. Culture medium was changed daily. In some experiments, embryonic lung cultures were exposed to 100 nM PTHrP(1-34) (Bachem, Torrance, CA) for up to 4 days with daily medium changes. Lung tissues were snap frozen in liquid N2 or were fixed in 10% neutral-buffered formalin or Karnovsky’s buffer for further analysis. Representative peripheral regions showing growth independent of necrosis were photographed at ⫻200 magnification and assessed by a masked observer for Theiler developmental stage and ultrastructural characteristics.

RNA Isolation, Northern Blot Analysis, and Ribonuclease Protection Assay Frozen tissue samples (30 –100 mg) were diluted in lysis solution (2 M guanidine thiocyanate, 12.5 mM sodium citrate [pH 7.0], 0.25% sarkosyl, 50 mM 2-mercaptoethanol, and 50% [v/v] water-saturated phenol) and disrupted on ice by using five to six pulses with a hand-held tissue homogenizer (Omni International, Waterbury, CT). Total cellular RNA isolation, electrophoresis, and blotting onto Gene Screen nylon membranes (Dupont, Wilmington, DE) were performed as previously described (Sanchez-Esteban et al., 1998). Rat SP-A, SP-B, and SP-C cDNAs and a 109-bp human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were linearized at the 5⬘ ends of the cloned inserts and antisense cRNA probes were synthesized by using an in vitro transcription kit (Promega, Madison, WI), the appropriate RNA polymerase, and [␣-32P]UTP (Amersham Biosciences, Piscataway, NJ). Unincorporated nucleotides were separated from the RNA probes by affinity chroma-

tography on Elutip columns (Schleicher & Schuell, Keene, NH). Blots were hybridized to 106–107 cpm of probe for 18 to 22 hr at 68 –70°C, washed in 0.1⫻ standard saline citrate/1% sodium dodecyl sulfate at the same temperatures, and exposed to X-ray film with intensifying screens at ⫺70°C. The intensity of mRNA bands of interest in each lane was normalized to the intensity of the corresponding GAPDH bands to control for differences in sample loading, RNA integrity, and transfer efficiency among lanes. In preliminary experiments, GAPDH mRNA band intensities correlated well with ethidium bromide rRNA fluorescence (data not shown). Scanned autoradiograms were analyzed by using NIH Image (version 1.62) software. The presence or absence of PTHrP mRNA in genotyped PTHrP(⫺/ ⫺), (⫹/⫺), and (⫹/⫹) lung tissue was verified by ribonuclease protection assay as previously described (Torday et al., 1998).

Determination of 3H-Saturated PC 3

The incorporation of [methyl- H]choline chloride (60 –90 Ci/mmol; Dupont, Wilmington, DE) into saturated PC was studied in minced embryonic lung tissue by using previously reported methods (Rubin et al., 1994) with minor modifications. Minced tissue samples (separately genotyped) were cultivated in 12well cluster dishes on a rocker platform (6 cycles/min) in serumless Waymouth’s medium containing 0.1% bovine serum albumin for 24 hr at 37°C. A total of 10 ␮Ci/ml of [methyl-3H]choline chloride was added to each well for an additional 4 hr. Lipids were extracted with chloroform and methanol, dried, and resuspended in 0.5 ml of carbon tetrachloride containing 3.5 mg of osmium tetroxide, redried, and resuspended in 70 ␮l of chloroform/ methanol (9:1, v:v). Saturated PC synthesis was assayed by Silica Gel H (Eastman Kodak, Rochester, NY) thin layer chromatography using a purified D-L-dipalmitoyl phosphatidylcholine (Sigma, St. Louis, MO) authentic standard and a chloroform/ methanol/water (65:25:4) solvent

system. Chromatogram spots corresponding to the migration of saturated PC were scraped and counted by scintillography.

Electron Microscopy Whole embryos or lungs were fixed by immersion in Karnovsky’s medium for 24 hr at 4°C and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 2 hr at room temperature. Tissue blocks were dehydrated with graded ethanols, moved to propylene oxide, infiltrated, and embedded in Spurr’s epoxy resin (Polyscience, Warrington, PA) in BEEM capsules (SPI Supplies, West Chester, PA). Semithin sections (1 ␮m) were stained with azure II-methylene blue for light microscopy. Ultrathin sections (⬃75 nm) from selected blocks were mounted on 1- ⫻ 2-mm grids, stained with uranyl acetate and lead citrate, and examined in a Philips EM 301 electron microscope (Philips, Mahwah, NJ) with an accelerating voltage of 60 kV.

Histochemistry and Immunohistochemistry For LM and immunohistochemistry (IHC), whole fetuses or fetal tissues were fixed in 10% buffered neutral formalin solution for 24 hr and embedded in Surgipath polymer (Richmond, IL). Sections were deparaffinized and rehydrated through graded alcohols by using standard procedures. Serial sections (5 ␮m) were stained with H&E, Alcian blue/ 0.1% nuclear Fast Red, or Oil Red-O (for assessment of lipid content). For IHC, the primary antibodies were detected by using an amplified streptavidin-biotin-peroxidase procedure (BioGenex, San Ramon, CA) according to manufacturer’s instructions with 3,3⬘-diaminobenzidine as the chromogen substrate. Slides were rinsed with water and counterstained with hematoxylin. Polyclonal antibodies to SP-A and SP-B (gifts from Profs. D. Phelps, Pennsylvania State University, Hershey, PA, and J.A. Whitsett, University of Cincinnati, Cincinnati, OH) and to PTHrP (Phoenix Pharmaceuticals, Mountain View, CA) were used as previously described (De Paepe et

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al., 1998), except that microwave treatment was used for antigen enhancement. Nonspecific binding sites were blocked by incubation with 5% goat serum. Control slides for specificity were performed with irrelevant antibodies.

Stereology and Morphometry To minimize regional differences in lung morphology, samples for EM were taken from sagittal sections of the murine unilobar left lung. For LM and EM, the observer was masked to sample genotype; at least 10 –15 peripheral lung areas/specimen were viewed. Light photomicrographs (excluding regions containing large blood vessels and bronchi) were taken with a Zeiss microscope CCD video camera attachment (KP-161, Hitachi, Norcross, GA). Images were digitized by using a flat bed scanner and Photoshop (version 4.0) software. Standard stereological volumetric techniques (Bolender et al., 1993; DePaepe et al., 1998) and image processing (NIH Image version 1.62) were used. Saccular septal wall morphometry was analyzed by using a modification of published methods (Mercer et al., 1994) at low magnification (⫻7,600). The arithmetic mean thicknesses of the epithelium, interstitium, and endothelium were calculated by using the volumes and surface densities of the tissue compartments, which were simply obtained by using point and intercept counts (Weibel, 1979). The interstitial thickness is defined as the average thickness of the interstitium across the whole septum. Distal airway epithelial cytodifferentiation (type II cell maturation) was scored by using the criteria developed for fetal mice by Ten Have-Opbroek et al. (1988, 1990), i.e., low-columnar to cuboidal cell shape, glycogen fields, apical microvilli, lamellar bodies, and cytoplasmic staining for surfactant-associated proteins. The number and proportion of type II cells in the airway epithelium were determined from low-magnification EM of randomly selected sections obtained in different levels from ⬎3 genotypeidentical lungs.

Statistics Each experiment was repeated by using at least three different litters to confirm the validity of observations. Student’s t-tests or Wilcoxon signed rank tests were used to determine differences between experimental groups. For morphometric comparisons, a single-factor analysis of variance was used to test the hypothesis that the means of the different groups were equal. If the hypothesis was rejected at the 1% significance level, a multiple comparison test (Student– Newman–Keul’s test or Scheffe’s test) was used to determine which means were different from each other. The 5% level was used to determine significant differences.

ACKNOWLEDGMENTS Surfactant protein cDNAs were provided by Dr. Joanna Floros, Pennsylvania State University School of Medicine, Hershey, PA. Technical assistance was provided by Brian E. Johnson, James Qin, and J. Sang. We also thank Betsey Mottershead for assistance with the preparation of the manuscript. J.S.T., L.P.R., and H.M.K. were funded by the NIH, J.S.T. was funded by the American Heart Association, M.E.D. was funded by the Rhode Island Foundation, and C.S.K. was funded by the Medical Research Council of Canada (now known as the Canadian Institutes of Health Research).

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