SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation

J. S. Torday, H. Sun, L. Wang, and E. Torres

Department of Pediatrics and Obstetrics and Gynecology, Harbor-University of California Los Angeles Research and Education Institute, Torrance, California 90502


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Developing rat lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed mutually exclusively by fetal lung fibroblasts and type II cells, suggesting a paracrine signaling "loop." This hypothesized mechanism is supported by the following experimental data: 1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400% · 100 ng-1 · ml-1 · 24 h-1) and adult human airway epithelial cells (85% · 100 ng-1 · 24 h-1); 2) leptin is secreted by lipofibroblasts in amounts that stimulate type II cell surfactant phospholipid synthesis in vitro; 3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP), PGE2, and dexamethasone stimulate leptin expression by fetal rat lung fibroblasts; 4) PTHrP or leptin stimulate the de novo synthesis of surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants, an effect that is blocked by a leptin antibody; and 5) a PTHrP receptor antagonist inhibits the expression of leptin mRNA by explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop that functionally cooperates to induce alveolar acinar lung development.

lung development; surfactant; type II cell; lipofibroblast


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LIPID-LADEN LIPOFIBROBLASTS were first identified as a morphologically distinct cell type in fetal and neonatal rat lung in 1970 (8). Both lipofibroblasts and adipocytes originate from mesoderm and are physically distinguished by the presence of large pools of triglyceride stores in their cytoplasm (11). The metabolism of lipid by these two cell types is also strikingly similar with respect to their lipogenic pathways, which are regulated by the same signal transduction pathway (11). Mature adipocytes express leptin, a 16-kDa cytokine product of the obesity (ob) gene (30). Fetal lung lipofibroblasts produce fibroblast pneumonocyte factor (FPF), a 10,000 to 20,000 molecular weight peptide that stimulates surfactant phospholipid synthesis by fetal type II cells (17). Differentiation of both cell types is stimulated by glucocorticoids (17, 33) and downregulated by androgens (10, 12). Furthermore, previous studies from our laboratory had suggested that lung epithelial cell secretions such as PGE2 (28) and parathyroid hormone-related protein (PTHrP; see Ref. 29) stimulate type II cell-fibroblast interactions via a soluble fibroblast growth factor of unknown identity. These observations, combined with the similarities between adipocytes and lipofibroblasts, and the recent reports that leptin is expressed in developing lung (9, 30) prompted us to investigate the possible role of leptin as a lipofibroblast growth factor that might stimulate fetal lung development.

It has long been recognized that lung development is mediated by soluble paracrine growth factors that mediate epithelial-mesenchymal interactions (15). These signals are bidirectional (1, 23), and the earliest known signals originate from the epithelium (22). We had previously identified PTHrP as a fetal rat alveolar type II cell product that is expressed beginning in the glandular phase of fetal lung development (16) and stimulates type II cell differentiation indirectly by stimulating fetal lung fibroblast factors (14). PTHrP stimulates fibroblast maturation through a receptor-mediated signal transduction pathway involving the production of cAMP and inositol phosphate (14), both of which induce fibroblast differentiation into adipocytes (34). Based on these observations, we hypothesized that leptin mediates the paracrine effect of PTHrP on alveolar type II cell surfactant synthesis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Leptin (rat, human) and leptin antibody (rat, polyclonal) were acquired from Linco (St. Charles, MO). The leptin RIA kit was obtained from DSL (Webster, TX). PTHrP-(1---34) and PTHrP-(7---34) amide were obtained from Bachem (Torrance, CA). Dexamethasone and PGE2 were purchased from Sigma Biochemicals (St. Louis, MO).

Animals. Time-mated Sprague-Dawley rats [embryonic day (E) 0, the day of mating] were obtained from Charles River Breeders (Hollister, CA). These experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Cell culture. WI-38 and H441 cells were obtained from the American Type Culture Collection (Rockville, MD).

Isolation of lipofibroblasts and type II cells from fetal lung. The following methods have been used extensively in our laboratory (6). Three to five dams were used per preparation. The dams were killed with an overdose of pentobarbital sodium (100 mg/ml ip), and the pups were removed from the uterus by laparotomy and kept on ice. The lungs were removed en bloc in a laminar flow hood using sterile technique and put into ice-cold sterile Hanks' balanced salt solution without calcium or magnesium. The solution was decanted, and 5 vol of 0.05% trypsin (Worthington) were added to the lung preparation.

The lungs were dissociated in a 37°C water bath using a Teflon stirring bar to physically disrupt the tissue. When the tissue had been completely dispersed in a unicellular suspension (~20 min), the cells were spun down at 500 g for 10 min at room temperature in a 50-ml polystyrene centrifuge tube. The supernatant was decanted, and the cell pellet was resuspended in MEM (GIBCO) containing 10% FBS to yield a mixed cell suspension of ~3 × 108 cells as determined by a Coulter particle counter (Hialeah, FL). The cell suspension was added to 75-cm2 culture flasks (Corning Glass Works, Corning, NY) for 30-60 min at 37°C in a CO2 incubator to allow for differential adherence of the fibroblasts (20). Fibroblasts were maintained in MEM until further processing.

Preparation of type II cell cultures. The unattached cells from the above-described cell preparation were transferred to another 75-cm2 culture flask for an additional 60-min period. After this second culture period, the medium and nonadherent cells were removed from the flask and diluted with 1 vol of culture medium. This diluted suspension was cultured overnight in a 75-cm2 flask at 37°C in a CO2 incubator to allow the type II cells to adhere (2). Type II cells were identified by their appearance in culture under phase-contrast microscopy, lamellar body content and microvillar processes, or by cytokeratin-positive staining.

Explant culture. Fetal rat lung tissue was cut into 1-mm cubes with a McIlwain tissue chopper (Brinkmann Instruments, Westbury, NY) and incubated in 0.5 ml Waymouth's MB-252/l medium containing penicillin (100 U/ml), streptomycin (100 U/ml), and fungizone (2.5 µg/ml) while rocking on an oscillating platform (3 cycles/min) in an atmosphere of 5% CO2-95% air at 37°C (25).

mRNA extraction. Total cellular RNA was isolated using previously described methods (5).

Semiquantitative RT-PCR. The appropriate cDNA fragments were amplified using 400 ng of total RNA from lung tissues or cells, avian myeloblastosis virus RT, and random hexamers and deoxyribonucleotides. The reactions were run at 42°C for 75 min and terminated by heating at 95°C for 5 min. Coamplification with glyceraldehyde-3-phosphate dehydrogenase cDNA was used as an internal standard. PCR was initiated by Taq DNA polymerase and allowed to proceed for 30 cycles with an annealing temperature of 50°C. The following primers were used in the RT-PCR assay: rat surfactant protein (SP) B (sense, 5'-TACACAGTACTTCTACTAGATG; antisense, 3'-ATAGGCTGTTCACTGGTGTTCC); human leptin (sense, 5'-CCTATCTTTTCTATGTCCAAGC; antisense, 3'-GTGAGGATCTGTTGGTAGACTG); human leptin receptor designed to detect all of the known isoforms (sense, 5'-TACTTTGGAAGCCCCTGATG; antisense, 3'-AAGCACTGAGTGACTGCACG); rat leptin (sense, 5'-TTATGTTCAAGCAGTGCCTATC; antisense, 3'-CATCCAACTGTTGAAGAATGTC); and rat leptin receptor(sense, 5'-ACCTTCAGTTCCAGATTCGA; antisense, 3'-TGAGATTGGTCTGATTTCCC). The identities of all RT-PCR products were confirmed by Southern blotting. mRNA expression was quantitated by densitometry (Eagle Eye; Stratagene).

Phospholipid assay. The rate of saturated phosphatidylcholine synthesis was determined as previously described (6) with modifications. Briefly, confluent monolayer cultures of fetal rat type II cells, H441 cells, or fetal rat lung explants were treated with leptin, PTHrP, leptin antibody, or PTHrP receptor antagonist and subsequently incubated with [3H]choline chloride (1 µCi/ml) for 4 h at 37°C in 5% CO2-95% air. Lamellar body fractions were prepared from the cells and tissues as described by Snyder et al. (21). To correct for procedural losses, fetal rat lung tissue was incubated with [U-14C]glycerol (10 µCi/ml, 10 mCi/mmol; NEN, Boston, MA) for 24 h, and lamellar bodies were prepared as previously described (21). This lamellar body preparation (10,000 dpm) was added to each sample before processing for [3H]phosphatidylcholine. Phospholipids were extracted from the lamellar body fractions by the method of Bligh and Dyer (3) and were dried under a stream of nitrogen at 50°C. The phospholipid extracts were mixed with 0.5 ml of an osmium tetroxide solution (70 mg/100 ml carbon tetrachloride) and reacted for 15 min at room temperature (26). The reaction mixture was dried under a stream of nitrogen at 60°C, and the dried extract was chromatographically separated by TLC in chloroform-methanol-water (65:25:4 vol/vol/vol; see Ref. 26). The phospholipids were subsequently scraped from the chromatography plates and analyzed for their radioactive content by liquid scintillation spectrometry.

Protein determination. Protein determination was made using the Bradford (4) dye-binding method.

Statistical analyses. ANOVA was used to compare data within experimental groups. The null hypothesis was rejected when P < 0.05 was not obtained.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initially, we examined the expression and ontogeny of leptin by fetal rat lung in the last third of gestation, during the phase of lung development when the fibroblast and type II cell mature. As can be seen in Fig. 1, the expression of leptin increases progressively between E17 and E21, which corresponds to the transition from the glandular (<= E17) to the canalicular (E18 and E19) and saccular (<= E20) stage of fetal rat lung development. The apparent decrease in leptin expression on E22 was observed consistently (n = 5). Leptin expression by E19 fetal rat lung fibroblasts (Fig. 2, top) was significantly increased by dexamethasone (8-fold), PTHrP (6-fold), and PGE2 (6-fold), which are endocrine and paracrine factors known to stimulate lipofibroblast development. Similar effects on leptin expression were obtained (Fig. 2, bottom) by incubating human embryonic lung fibroblasts (WI-38) with these same agonists. H441 human small cell carcinoma cells (Fig. 3) and E18 fetal rat type cells expressed the leptin receptor, whereas expression of the leptin receptor was faint in fetal rat lung fibroblasts and WI-38 cells, consistent with the hypothesized paracrine nature of the leptin mechanism within the alveolar acinus. Fetal rat lung fibroblasts cumulatively secrete leptin in the culture medium over a 24-h period (Fig. 4), increasing from 23 ± 8 ng · ml-1 · 4 h-1 · 106 cells to 57 ± 12 ng · ml-1 · 8 h-1 · 106 cells and 97 ± 58 ng · ml-1 · 24 h-1 · 106 cells, equivalent to ~5 ng · ml-1 · h-1 · 106 cells; incubation of these cells with PTHrP increased secretion of leptin by greater than twofold more than the 6-h rate. Incubation of fetal rat type II cells with leptin concentrations comparable to the amounts we had observed being produced by the fetal rat lung fibroblasts over a 24-h period stimulated the incorporation of [3H]choline into lamellar body-saturated phosphatidylcholine at 10, 50, and 100 ng/ml by 150-400% (Fig. 5). Leptin also stimulated choline incorporation into lamellar body-saturated phosphatidylcholine by H441 cells, although the effect was lower than that of leptin on the fetal type II cells, i.e., there was no effect at 10 ng/ml, 40% at 50 ng/ml, and 85% at 100 ng/ml (Fig. 6). The stimulation of surfactant synthesis by these cells of human adult lung origin is evidence for the presence of functional leptin receptors on adult type II cells, which is consistent with the observed expression of leptin receptor mRNA by these cells (see Fig. 3).


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Fig. 1.   Leptin ontogeny in fetal rat lung. Semiquantitative RT-PCR was performed using RNA from fetal rat lung at the indicated gestational ages [embryonic day (E) 17-E22]. Coamplification with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as an internal standard for normalization of aliquot volumes for leptin mRNA expression.



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Fig. 2.   Semiquantitative RT-PCR of RNA extracts from fetal rat lung fibroblasts and WI-38 human embryonic lung fibroblasts. Primary fetal rat lung fibroblasts (E18) and WI-38 human embryonic lung fibroblasts were treated with either dexamethasone (Dex, 1 × 10-8 M), parathyroid hormone-related protein (PTHrP, 5 × 10-7 M), or PGE2 (5 × 10-7 M) for 24 h and subsequently analyzed for leptin mRNA expression using semiquantitative RT-PCR. Coamplification with GAPDH cDNA was used as an internal standard for normalization of the aliquot volumes for leptin mRNA expression. Center: representative blots for RT-PCR of leptin mRNA. Top and bottom, densitometric quantitation of the corresponding blots. Each bar represents the mean ± SD; n = 5 experiments. C, control; OD, optical density. ***P < 0.0001 by ANOVA.



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Fig. 3.   Semiquantitative RT-PCR for leptin receptor mRNA. Primary fetal rat lung fibroblasts (FrLf) and type II cells (TII), WI-38 human embryonic lung fibroblasts, and H441 adult lung epithelial cells were assayed for leptin receptor mRNA expression. Coamplification with GAPDH cDNA was used as an internal standard for the normalization of the aliquot volumes for leptin receptor mRNA expression.



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Fig. 4.   Fetal rat lung fibroblasts secrete leptin under the influence of PTHrP. E21 fetal rat lung fibroblasts were cultured in 2-cm2 wells in serum-free DMEM containing 0.1% fatty acid-free BSA for up to 6 h with or without PTHrP (5 × 10-7 M). Leptin concentrations were determined by an ELISA for rat leptin. Each bar represents the mean ± SD; n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.



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Fig. 5.   Leptin stimulates surfactant phospholipid synthesis by fetal lung type II cells. E19 fetal rat lung type II cells were incubated with leptin for 24 h and exposed to [3H]choline chloride for the last 4 h. The type II cell lamellar body fractions were subsequently assayed for [3H]phosphatidylcholine content. Each bar represents the mean ± SD; n = 5. *P < 0.05 and **P < 0.01, control (C) vs. leptin treated by ANOVA.



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Fig. 6.   Leptin stimulates surfactant phospholipid synthesis by adult lung epithelial cells. H441 adult lung epithelial cells were incubated with leptin for 24 h and exposed to [3H]choline chloride for the last 4 h. The H441 cell lamellar body fractions were subsequently assayed for [3H]phosphatidylcholine content. Each bar represents the mean ± SD; n = 5. *P < 0.05 and **P < 0.01, control vs. leptin treated by ANOVA.

In subsequent experiments, we examined the interaction of PTHrP and leptin in fetal rat lung explant culture. Incubation of E18 fetal rat lung explants with leptin (200 ng · ml-1 · 24 h-1) or PTHrP (5 × 10-7 M/24 h) significantly increased the incorporation of [3H]choline into [3H]phosphatidylcholine (>2-fold; Fig. 7). The concentration of leptin was chosen based on its maximally effective dose in monolayer culture [see effect of leptin on type II cells in monolayer culture (Fig. 4)] and was doubled to compensate for tissue penetration. The PTHrP dose was based on previous studies of its effect on surfactant synthesis in explant culture (14). The stimulatory effect of PTHrP was blocked by coincubation with leptin antibody (1:100 dilution). However, the stimulatory effect of leptin was not blocked by coincubation with the PTHrP receptor antagonist. Maintaining E18 fetal rat lung tissue in explant culture for 3 days resulted in a 10-fold increase in leptin mRNA expression (Fig. 8), consistent with its developmental upregulation during this phase of lung maturation. This increase in leptin mRNA expression was blocked by coincubation with the PTHrP receptor antagonist [PTHrP-(7---34) amide, 5 × 10-6 M]. Incubation of E18 fetal rat lung explants with leptin (200 ng · ml-1 · 24 h-1) or PTHrP (5 × 10-7 M/24 h) also stimulated the expression of SP-B (Fig. 9); coincubation of PTHrP-treated E18 explants with leptin antibody (1:100 dilution) blocked the PTHrP stimulation of SP-B expression. The same effect was observed when leptin-treated explants were coincubated with leptin antibody (data not shown), as expected. In contrast to this, coincubation of leptin-treated explants with the PTHrP receptor antagonist (5 × 10-7 M/24 h) had no effect on leptin stimulation of SP-B expression.


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Fig. 7.   Leptin antibody inhibits PTHrP stimulation of surfactant phospholipid. E18 fetal rat lung explants were maintained in culture for 24 h. Explants were incubated with leptin during the 24-h culture period and were exposed to [3H]choline chloride for the last 4 h. Leptin and PTHrP significantly increased [3H]phosphatidylcholine synthesis (n = 5). *P < 0.01, controls vs. treated, by ANOVA. Coincubation of PTHrP-treated explants with leptin antibody (LeptinAb, 1:100 dilution) blocked the stimulatory effect of PTHrP on saturated phosphatidylcholine synthesis; coincubation of leptin-treated explants with PTHrP receptor antagonist (PTHrP Ant, 5 × 10-6 M) had no effect on saturated phosphatidylcholine synthesis.



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Fig. 8.   Leptin mRNA expression is PTHrP dependent. E18 fetal rat lung explants were maintained in culture for 3 days with or without PTHrP receptor antagonist (1 × 10-6 M). Explants were subsequently analyzed for leptin mRNA expression by semiquantitative RT-PCR. Coamplification with GAPDH cDNA was used as an internal standard for normalization of the aliquot volumes for leptin mRNA expression. Bar graph represents densitometric quantitation of the leptin mRNA bands; each bar is the mean ± SD; n = 5. ***P < 0.0001 by ANOVA.



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Fig. 9.   PTHrP paracrine stimulation of surfactant protein-B (SP-B) is leptin dependent. E18 fetal rat lung explants were maintained in culture for 24 h with or without leptin (200 ng/ml), PTHrP (5 × 10-7 M), PTHrP plus leptin antibody (PTHrP/LeptinAb; 1:100 dilution), or leptin plus PTHrP receptor antagonist (Leptin/PTHrP Ant, 5 × 10-6 M). SP-B mRNA expression was determined using semiquantitative RT-PCR as shown in the representative blot. Coamplification with GAPDH cDNA was used as an internal standard for normalization of the aliquot volumes for leptin mRNA expression. Bar graph represents the mean ± SD for control vs. treated groups; n = 5. ***P < 0.0001 by ANOVA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hypothesized role of leptin as a paracrine mediator of the lung type II cell-fibroblast interaction resulting in surfactant synthesis was supported by our experimental results. Leptin and its receptor are expressed by fetal rat lung fibroblasts and type II cells, respectively, before the onset of type II cell maturation, beginning on E19-E20, and leptin expression is under both endocrine and paracrine control. Leptin stimulates both surfactant phospholipid and protein synthesis by type II cells by a PTHrP-dependent signaling pathway, based on the observation that the PTHrP receptor antagonist inhibits the developmental upregulation of leptin expression in explant culture; also, the leptin antibody blocks the PTHrP stimulation of surfactant phospholipid and protein expression, although the PTHrP receptor antagonist has no effect on (downstream) leptin-stimulated SP-B expression.

In the present series of experiments, we have observed that leptin mRNA expression in the developing fetal rat lung increases during the period of alveolar lung differentiation when pulmonary surfactant phospholipid synthesis is induced (E18-E20). Leptin expression appears to peak at E20 and then progressively declines on E21 and E22. This phenomenon has also been observed for FPF (18) and may be due to the thinning of the alveolar wall, which occurs during the process of alveolarization. The process of alveolar differentiation is a well-recognized hormonally regulated paracrine mechanism (17) that depends upon interactions between the type II epithelial cell and mesenchymal fibroblast. Type II cells elaborate PTHrP (29), which promotes the differentiation of the mesenchyme into lipofibroblasts in a manner similar to adipocyte differentiation, including the expression of key adipocytic enzymes, triglyceride uptake, storage, and secretion (11). These PTHrP-induced lipofibroblasts then apparently produce a soluble growth factor that stimulates type II cell surfactant phospholipid synthesis (14, 29) and SP-B expression, as shown in this study; the other surfactant proteins (SP-A, -C, and -D) were not examined, although they also play an essential role in surfactant metabolism. With these principles in mind, we hypothesized that the mature lipofibroblast, like the mature adipocyte, would express leptin, a secreted product of the mature adipocyte. Because leptin belongs to the interleukin-6 family of cytokines (32), and interleukin-6 stimulates the synthesis of pulmonary surfactant by type II cells (13), PTHrP-stimulated expression of leptin by lipofibroblasts provides a closed paracrine loop from the type II cell to the lipofibroblast and back to the type II cell.

It has long been recognized that alveolar type II cell differentiation is dependent on mesenchymal-epithelial interactions (15), which are mediated by low-molecular-weight soluble factors (7). Smith (18) was the first investigator to identify a specific hormonally induced mesenchymal factor that stimulated surfactant phospholipid synthesis over 20 years ago (19), although the identity of this factor has remained undetermined. He termed this differentiation factor FPF. We subsequently discovered that FPF was downregulated by androgens (6, 12), providing further evidence for a biological role of FPF in the timing of lung development, since there is a spontaneous sex difference in the rate of lung development and pulmonary surfactant synthesis (6, 27) that is androgen dependent (24). Leptin has many of the same characteristics as FPF: 1) its molecular weight (16,000) is within the range previously reported for FPF (19); 2) it is expressed by lung mesenchymal cells during fetal development in a pattern like that reported for FPF (i.e., beginning in the canalicular phase, peaking on E21, and then declining on E22; see Ref. 19); and 3) its expression is stimulated by glucocorticoids (19) and inhibited by both androgens (9) and transforming growth factor-beta (25, 27). These similarities between leptin and FPF strongly support their common identity. Their functional similarities are further reinforced by their common cellular origins; leptin is produced by the ob gene of the mature adipocyte (31), which lung lipofibroblasts bear a strong resemblance to, both structurally and functionally (11). FPF is also produced by adepithelial lung fibroblasts (M. Post, personal communication). In summary, leptin may be the long-sought-after soluble factor that mediates the hormonal effects on fetal lung development, which govern surfactant production.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-55268.


    FOOTNOTES

Address for reprint requests and other correspondence: J. S. Torday, Dept. of Pediatrics/OB-GYN, Center for Developmental Biology, RB-1, 1124 W. Carson St., Torrance, CA 90502 (E-mail: jtorday{at}prl.humc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 August 2000; accepted in final form 10 January 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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