1 Division of Pediatric Surgery and Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, Keck School of Medicine, University of Southern California, Los Angeles 90027; 2 The Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles, California 90033; 3 Equipe Morphogénèse Cellulaire et Progression Tumorale, Institut Curie Unité Mixte de Recherche 144 Centre National de la Recherche Scientifique, 75005 Paris; 4 Service de Pédiatrie et Réanimation Néonatales-UPRES EA2704, Hôpital Antoine Béclère, 92141 Clamart; 5 Service de Physiologie Respiratoire, Hôpital Cochin, Université René Descartes, 75005 Paris; 6 Vascular Biology Research Group, University of Alberta, Edmonton, Alberta T6G 2S2, Canada; and 7 Institut National de la Santé et de la Recherche Médicale Unité 319, Universite Jussieu, 75005 Paris, France
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ABSTRACT |
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We evaluated the role of the key pulmonary morphogenetic gene fibroblast growth factor-10 (Fgf10) in murine nitrofen-induced primary lung hypoplasia, which is evident before the time of diaphragm closure. In situ hybridization and competitive RT-PCR revealed a profound disturbance in the temporospatial pattern as well as a 10-fold decrease in mRNA expression level of Fgf10 but not of the inducible inhibitor murine Sprouty2 (mSpry2) after nitrofen treatment. Exogenous FGF-10 increased branching not only of control lungs [13% (right) and 27% (left); P < 0.01] but also of nitrofen-exposed lungs [23% (right) and 77% (left); P < 0.01]. Expression of mSpry2 increased 10-fold with FGF-10 in both nitrofen-treated and control lungs, indicating intact downstream FGF signaling mechanisms after nitrofen treatment. We conclude that nitrofen inhibits Fgf10 expression, which is essential for lung growth and branching. Exogenous FGF-10 not only stimulates FGF signaling, marked by increased mSpry2 expression, in both nitrofen-treated and control lungs but also substantially rescues nitrofen-induced lung hypoplasia in culture.
congenital diaphragmatic hernia; lung hypoplasia; fibroblast growth factor-10; murine Sprouty2
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INTRODUCTION |
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CONGENITAL DIAPHRAGMATIC HERNIA (CDH) occurs in ~1 in 3,000 human live births. Although it is associated with several genetic defects, its exact etiology is not known. Newborns with CDH have a 40-50% mortality, which is primarily caused by the associated pulmonary hypoplasia. The hypoplastic lungs are not capable of providing adequate gas exchange for oxygenation, and persistent pulmonary hypertension leads to refractory hypoxia (right to left shunting). Unlike other causes of neonatal respiratory failure, infants with CDH are often unresponsive to the modern therapeutic armamentarium because it does not solve the basic problem of lung hypoplasia (28).
The hypoplastic lung in CDH is developmentally delayed. There is a marked reduction from 21 generations of airways in the normal human lung to 12-14 generations in the ipsilateral and 16-18 generations in the contralateral lung in CDH (1). There is also a delay in the differentiation of alveolar epithelial cells, with a resultant surfactant deficiency (31), and fewer and more arterialized vascular branches (20). In addition to the effect of mechanical compression by the herniated abdominal viscera, lung hypoplasia in CDH may also result from a primary abnormality in airway branching (11, 15). However, the molecular mechanisms underlying lung hypoplasia in human CDH have not been fully investigated.
Since the first description of nitrofen-induced diaphragmatic hernias in rodents by Iritani (10) in 1984, the murine nitrofen-induced model of CDH has been extensively studied and by now is widely accepted as a well-established model that has many phenotypic similarities to the human condition (7, 16). Using this model in mice, we showed that nitrofen causes primary pulmonary hypoplasia, which is worsened by the presence of a hernia (4). In rats, nitrofen has also recently been shown to reduce branching morphogenesis before diaphragmatic closure both in vitro and in vivo (15). Because nitrofen-exposed embryonic lungs are clearly hypoplastic before the appearance of an actual diaphragmatic defect, we began to evaluate candidate factors known to be required for early lung development (30).
During mouse lung morphogenesis, the distal mesenchyme has long been known to regulate the growth and branching of the adjacent endoderm through the secretion of soluble factors [recently reviewed by Warburton et al. (30)]. Bellusci et al. (2) reported that fibroblast growth factor (FGF)-10 is a mesenchyme-derived factor that plays a critical role in patterning the early branching events in lung development. Fgf10-null mutant mice and transgenic mice expressing dominant negative forms of the FGF-10 receptor Fgfr2-IIIb have a dramatic inhibition of bronchial branching (19, 22). Fgf10 is expressed in a temporospatially specific pattern in the peripheral embryonic lung mesenchyme near the positions where primary, secondary, and tertiary bronchi bud (2). The buds grow toward these areas of Fgf10 expression. Thus Fgf10 appears to stimulate and direct early bronchial branching. FGF pathway signaling is modified at each stage of branching by genetic feedback controls. Sonic hedgehog (Shh), which is strongly expressed in the distal epithelium, may function as a negative signal for Fgf10 (3, 8). Shh inhibits Fgf10 expression in the mesenchyme near the growing tips where the initial Fgf10 expression domain splits laterally into two domains. Two new buds then sprout, each targeting one of the lateral subdomains of Fgf10 expression. Mice in which Shh has been inactivated also have profound impairments of lung branching (21). Other key antagonists of the FGF pathway include members of the Sprouty gene family. Murine Sprouty2 (mSpry2) is an inducible negative regulator of FGF receptor tyrosine kinase signaling that is expressed in the distal epithelium of the embryonic mouse lung adjacent to the mesenchymal loci of Fgf10 expression at embryonic stages when the lung epithelial buds are highly responsive to FGF-10. Abrogation of mSpry2 expression in lung organ cultures with antisense oligonucleotides increases branching morphogenesis and surfactant gene expression (27).
Here we determined that the temporospatial pattern of Fgf10 expression in mouse embryonic lungs is severely disrupted in the presence of nitrofen-induced lung hypoplasia. Nevertheless, taking advantage of highly inducible mSpry2 expression by FGF-10, we were able to determine that downstream FGF signaling was intact. We also determined that exogenous FGF-10 could increase branching morphogenesis in both wild-type and nitrofen-exposed embryonic lungs in culture. Thus nitrofen-induced lung hypoplasia may be at least partially explained by downregulation of Fgf10, whereas exogenous FGF-10 can substantially rescue nitrofen-induced mouse embryonic lung hypoplasia in culture. These findings may have potential value in devising novel approaches to correcting lung hypoplasia in human CDH because only a small increase in diffusion capacity could be lifesaving.
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MATERIALS AND METHODS |
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Nitrofen-exposed lungs. Timed-pregnant Swiss-Webster mice (Simonsen Laboratories, Gilroy, CA) were gavage fed 25 mg of nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether; Radian International, Austin, TX) on day 8 of gestation (presence of a vaginal plug = day 0). The control animals received olive oil. With the use of aseptic technique, the mouse embryos were harvested by cesarean section on embryonic day (ED) 12. On retrieval, the embryos were transferred to an isotonic Hanks' balanced salt solution and cooled on ice. They were then microdissected from their extraembryonic membranes, and with the use of a stereomicroscope and microsurgical instruments, the lungs were excised and the right and left lobes were separated and placed in Hanks' balanced salt solution. The University of Southern California (Los Angeles, CA) Institutional Animal Care and Use Committee approved the use of animals in this study.
Whole mount in situ hybridization. The whole mount in situ hybridization technique was based on that previously described by Sasaki and Hogan (24). The following murine cDNAs were used as templates for synthesizing digoxin-labeled riboprobes: 584-bp FGF-10 and 948-bp full-length Spry2. To provide a qualitative comparison of levels of gene expression between control and nitrofen-exposed lungs between ED12 and ED15, the lungs were fixed and processed under the same conditions with respect to probe concentration and specific activity and washed at the same temperature and stringency. Photomicrographs were taken with the same exposure time.
Organ culture. ED12 lungs were cultured at the air-fluid interface by placing them on 0.8-µm MF-Millipore filters (Millipore, Bedford, MA) supported by stainless steel grids in culture dishes containing BGJb medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 1 mg/ml of ascorbic acid and 50 U/ml of penicillin-streptomycin. FGF-10 was added to the culture medium at a concentration of 500 ng/ml (R&D Systems, Minneapolis, MN). FGF-10 is not very bioactive, and dose-response curves have demonstrated that at 500 ng/ml, there is the maximum amount of branching morphogenesis. Organ cultures were maintained at 37°C in 100% humidity and 95% air-5% carbon dioxide for 4 days, with the medium changed after 2 days.
Branching morphogenesis. Branching morphogenesis was quantified by counting the number of terminal branches visible around the periphery of each lung. This was performed before and after 4 days in culture with transillumination to visualize the structures and photomicrography to record permanent images (30a).
RNA extraction and reverse transcription.
Individual cultured explants were homogenized by repeated pipetting in
4 M guanidinium isothiocyanate. Total RNA was then extracted with the
Rapid Total RNA Isolation Kit (5 Prime 3 Prime, Boulder, CO).
Reverse transcription (RT) was performed by incubating samples of
individual lung RNA at 37°C in 10 mM Tris (pH 8.4), 50 mM KCl, 2 mM
MgCl2, 1 mM dithiothreitol, 5 U of ribonuclease inhibitor,
0.5 mM deoxynucleotide triphosphates, 100 pmol of
oligo(dT)12-18, and 200 U of Moloney murine leukemia
virus reverse transcriptase (USB, Cleveland, OH). The reaction was
terminated by heating for 5 min at 100°C. Reverse-transcribed products were then used for competitive PCR.
Competitive PCR.
PCR amplification was performed with a DNA Robocycler (Stratagene, La
Jolla, CA), with an initial denaturation at 94°C for 3 min followed
by 35 cycles of denaturation at 93°C for 2 min, annealing at 62°C
for 2 min, and extension at 72°C for 2 min. The final cycle concluded
with a 5-min extension step. The reaction mixture contained 10 mM Tris
(pH 8.4), 50 mM KCl, 2 mM MgCl2 (optimized), 0.01% Triton
X-100, 20 pmol of primer sets, 100 µM deoxynucleotide triphosphate,
and 0.5 U of Taq thermostable DNA polymerase (Promega, Madison, WI). A reaction mixture containing 1 pg/µl of the
appropriate competitor DNA was added to the reverse-transcribed samples
derived from 50 ng of total RNA. The concentration of cDNA standard
solutions was determined spectrophotometrically by absorbance at 260 nm. The equations drawn from the linear regressions for each of the standard curves were used to interpolate the mRNA amounts from their
respective cDNA equivalents in each lung sample. To control for
potential variations due to the efficiency of RNA extraction and RT,
-actin mRNA was also quantified in the same samples.
Competitive RT-PCR quantification. The same primers for mouse mSpry2 were used to amplify both the cDNA and competitor for each gene of interest. The upstream primer of cDNA synthesis was 5'-TGTGAGGACTGTGGCAAGTGC-3' and the downstream primer was 5'-TTTAAGGCAACCCTTGCTGG-3', resulting in a 300-bp PCR product. Two composite primers were synthesized to construct the mSpry2 competitor. Each set of composite primers contained the mSpry sequence as well as a short sequence designed to hybridize to the cDNA of interest. This allowed the incorporation of the mSpry2 sequence into the DNA during the PCR. The competitor was v-erbB DNA. The competitor was then sequenced to verify the incorporation of the gene-specific primers and was 400 bp long. The same primers were used to amplify 1 fg of competitor and scaled concentrations of cDNA. The log of cDNA/competitor was plotted against the target concentrations, producing a coefficient (r2) > 0.98 (data not shown). The same assay was developed for Fgf10. Competitive PCR quantification allows accurate assessment of mRNA levels and is reliable without contaminating DNA species.
Electrophoresis and densitometric analysis.
Target and competitor PCR products were separated by size with
electrophoresis in 3% agarose gels (NuSieve, FMC BioProducts, Rockland, ME). Gels were stained with 5 µg/ml of ethidium bromide and
photographed with a digital camera (Cohu, San Diego, CA). Band
intensities were determined by densitometric analysis with ImageQuant
band-analyzing software (Molecular Dynamics, Sunnyvale, CA). -Actin
mRNA levels were measured in an identical fashion in both groups as an
internal control for RNA extraction and cDNA production. All mRNA
values were normalized to
-actin mRNA levels, which were the same
for nitrofen-exposed and control lungs.
Statistical analysis. Morphometric data are reported as means ± SD. Densitometric data are reported as the mean ratios of control values. Data from nitrofen-exposed lungs were compared with those of control lungs with two-tailed Student's t-test. A P value of <0.05 was considered significant.
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RESULTS |
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Decreased temporospatial expression of Fgf10 in hypoplastic lungs.
Fgf10 transcripts were studied during lung development by
whole mount in situ hybridization. From ED12.5 through ED15.5, high levels of Fgf10 expression were present in the mesenchyme
adjacent to the epithelial buds of wild-type lungs (Fig.
1, A and C),
correlating with the sites of future dichotomous branching. In
contrast, in nitrofen-treated lungs, the temporospatial pattern
expression of Fgf10 was markedly impaired (Fig.
1D). Interestingly, loss of Fgf10 expression
appeared to correlate well with the severity of lung hypoplasia (Fig.
1B); Fgf10 transcripts could be detected only in the caudal part of the left lobe in moderately severe hypoplasia (Fig. 1F), whereas expression was nearly totally
abolished in examples with more severe hypoplasia (Fig. 1E).
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Nitrofen exposure, FGF-10, and relative mRNA expression of Fgf10
and mSpry2.
Fgf10 and mSpry2 expression levels were measured
by competitive RT-PCR and compared between wild-type and
nitrofen-exposed and between right and left ED12 lungs after 4 days in
culture. The results of this analysis are shown in Figs.
3 and 4
and were all corrected as ratios to -actin
mRNA. The mean ratios of Fgf10 to
-actin
expression were 2.65 in the right and 1.26 in the left wild-type lungs.
These relative levels of expression decreased to 0.4 (right) and 0.3 (left) after exposure to exogenous FGF-10 (all P < 0.05). It is also interesting to note that Fgf10 mRNA levels
were reduced significantly after in utero exposure to nitrofen in both
the right and left lungs. On the other hand, the mean level of
expression of mSpry2 mRNA did not differ significantly between the right and left lungs in wild-type or nitrofen-exposed embryos. However, levels of mSpry2 expression did increase
4- to 10-fold in the presence of exogenous Fgf10 under all
conditions examined.
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Nitrofen exposure, lung branching morphogenesis, and FGF-10 rescue.
Nitrofen exposure produced a profound decrease in branching
morphogenesis, which was already evident at ED12 and persisted when ED12 lungs were cultured for 4 days, as illustrated in
Fig. 5 and quantified in Fig.
6. Nitrofen exposure resulted in an
almost complete arrest of lung budding in the left lung (Fig.
5B). The effects on branching in the right lung were also
very striking; budding over 4 days in culture was very significantly
decreased in the right lungs after nitrofen exposure (Fig.
5A).
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DISCUSSION |
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Nitrofen-induced early lung hypoplasia that occurs before closure of the diaphragm and that persists for up to 72 h in culture was recently reported in the rat model (11). Here we showed that this is also the case in the mouse. However, in contrast to the rat, where the incidence of hypoplasia and CDH is reported to be the same at ~36%, Coleman et al. (4) and Iritani (10) found that hypoplasia occurs in >90% of mice, whereas the incidence of CDH is about half that in the rat. Thus, as Coleman et al. (4) have reported before, nitrofen-induced lung hypoplasia does not predict which mice will get CDH, but the degree of hypoplasia is greatly exacerbated by the presence of an ipsilateral CDH. This has recently been referred to by Keijzer et al. (15) as the two-hit hypothesis. In the present study, we killed our mice on ED12 so we can only comment here on the primary lung hypoplasia induced by nitrofen, which has recently been reported to be associated with decreased cell proliferation (12).
The temporospatial pattern of Fgf10 expression in the wild-type murine embryonic lung supports the concept that FGF-10 plays a key role in the directional outgrowth and possibly the induction of epithelial buds. By using whole mount in situ hybridization and competitive RT-PCR, we show that Fgf10 mRNA expression is severely temporospatially disrupted and significantly decreased in nitrofen-exposed lungs compared with that in control lungs. We further show that exogenous FGF-10 induces significant lung growth by stimulating lung branching morphogenesis in both control and nitrofen-exposed lungs in culture. These observations suggest that abnormal FGF-10 signaling may account substantially for the decreased branching in nitrofen-induced lung hypoplasia before the onset of CDH.
Jesudason et al. (13) recently worked with FGF-1 and FGF-2
and also concluded that there is an intrinsic abnormality of the FGF
signaling system in hypoplastic lungs after nitrofen treatment. However, an important caveat is that decreased expression and/or function of several other key morphogenetic genes has been reported previously in this model. These include thyroid transcription factor-1
(17), endothelin-converting enzyme neutral endopeptidase (14), basic FGF, platelet-derived growth factor
(9), and tumor necrosis factor- (25, 26).
We think that some of the most attractive pieces of data supporting a unifying theory that nitrofen-induced lung hypoplasia is caused by abnormalities of Fgf10 expression are as follows: 1) Fgf10 signaling is clearly necessary for lung morphogenesis distal to the trachea because null mutation of Fgf10 completely abrogates this process (19); 2) many of the associated tracheobronchial and skeletal abnormalities caused by nitrofen exposure, including skull hypoplasia, axial appendicular, and rib anomalies, can also result from abnormalities in the FGF receptor signaling pathway (18, 32); and 3) the findings reported herein, that exogenous FGF-10 both stimulates wild-type lung morphogenesis and substantially rescues nitrofen-induced embryonic lung hypoplasia in culture, further support the concept that nitrofen may interfere with Fgf10 expression. It seems unlikely that nitrofen interferes with downstream FGF-10 signaling because mSpry2 expression was still induced normally in the presence of exogenous FGF-10 in nitrofen-exposed embryonic lungs in culture. However, mSpry2 may also play a role in nitrofen-induced lung hypoplasia because its expression level remains unchanged, allowing relative accumulation of this negative regulator in excess of available FGF-10. On the other hand, the brisk induction of mSpry2 expression by exogenous FGF-10 in both wild-type and nitrofen-exposed lungs indicates that FGF signaling can still activate mSpry2 even in the presence of nitrofen-induced hypoplasia. Thus mSpry2 probably functions normally as a readily inducible negative modulator of FGF signaling even in the presence of nitrofen-induced hypoplasia.
Previous experimental approaches for rescuing lung hypoplasia associated with nitrofen exposure and CDH have included tracheal ligation and glucocorticoid, retinoid, and antioxidant treatment. Tracheal ligation certainly does increase the overall size and wet weight of the murine embryonic lung in culture, as it does when carried out in utero in sheep and also in humans (6). One rationale behind tracheal ligation is that it traps tracheal effluent, which is driven by active chloride secretion during embryonic and fetal life, thereby expanding the epithelial surface by positive hydraulic pressure. However, as recently reported (6), mesenchymal and epithelial maturation in the lung does not proceed normally under these conditions, and therapeutic results in humans have, for the most part, been disappointing. Glucocorticoids, on the other hand, are now considered a standard of care as preventive therapy to accelerate lung maturation in human premature labor (5). They have been reported to have many possible beneficial effects in the nitrofen model, most notably with respect to the current study, by inducing the expression of several peptide growth factors including FGF and platelet-derived growth factor. However, the effects of tracheal ligation and/or glucocorticoids may have negative consequences on postnatal alveolar development. Retinoids, on the other hand, appear to decrease the incidence and severity of nitrofen-induced CDH and may even have beneficial effects on alveolarization (23, 29).
Here we show for the first time that nitrofen-induced embryonic mouse lung hypoplasia is associated with decreased and temporospatially abnormal expression of Fgf10. Finally, nitrofen-induced lung hypoplasia is present on ED12, is worse in the left than in the right lung, persists in embryonic lung culture, and, most importantly, can be substantially rescued in culture by exogenous FGF-10. Taken together, these results lead us to speculate that nitrofen-induced lung hypoplasia may be caused, at least in part, by abnormalities in Fgf10 expression. Furthermore, we speculate that novel therapeutic approaches designed to achieve either direct or indirect correction of FGF-10 signaling may, in the future, have the potential to contribute to the substantial rescue of in utero lung hypoplasia as seen in human neonates with CDH.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-60231-02S2 (to J. M. Acosta), HL-60231, HL-44977, and HL-44060; a Fogarty Senior International Fellowship; a Rothschild-Mayant Fellowship de L'Institut Curie (all to D. Warburton); Association pour la Recherche contre le Cancer Grant ARC 5214; and Human Frontier Science Program Grant RG0051/1999-M (both to S. Bellusci).
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FOOTNOTES |
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* Juan M. Acosta and Bernard Thébaud contributed equally to this work.
Address for reprint requests and other correspondence: D. Warburton, Developmental Biology Program, Childrens Hospital Los Angeles, 4650 Sunset Blvd., MS 35, Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.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 16 October 2000; accepted in final form 22 December 2000.
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