1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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GATA-6, a member of the GATA family of
zinc finger proteins, is the only family member known to be expressed
in the epithelial cells of the developing airway epithelium. To
determine the role of GATA-6 in lung morphogenesis, a chimeric fusion
protein containing GATA-6 and the strong transcriptional inhibitor,
engrailed, was conditionally expressed in mice under control of a
doxycycline-inducible transgene. Expression of GATA-6-engrailed was
initiated at embryonic day (E)
6-7 by treatment of the dam with doxycycline. Although branching morphogenesis of the proximal airways proceeded normally to
E16.5, maturation of terminal airways and alveoli that
normally occurs before birth was inhibited. At
E17.5-18.5, aquaporin-5 mRNA and type I cell
marker- staining, both markers of type I cells, were
decreased. Homogenous distribution of the thyroid transcription
factor-1, decreased expression of surfactant proteins, delayed thinning
of the walls of the peripheral airways, and lack of squamous
differentiation of epithelial cells were observed in the lung periphery
after expression of GATA-6-engrailed. Activity of GATA-6 is required
for maturation of the gas exchange area before birth.
lung development; transcription factors
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INTRODUCTION |
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GATA TRANSCRIPTION
FACTORS include a family of zinc finger domain-containing
polypeptides, GATA-1 through -6, that are involved in gene regulation,
organogenesis, and differentiation in various developing organ systems
(reviewed in Ref. 19). GATA-1, -2, and -3 are expressed
primarily in hematopoietic tissues; GATA-4, -5, and -6 are expressed in
many organs, including heart, lung, and gastrointestinal tract
(2, 7, 10, 16, 17, 23). In the lung, GATA-6 is selectively
expressed in endodermally derived cells early in lung morphogenesis,
being detected in subsets of both conducting and peripheral airway
epithelial cells (9, 12, 18). The levels and extent of
expression of GATA-6 in the developing respiratory epithelium are
similar to that of thyroid transcription factor-1 (TTF-1) in fetal
lung, and levels of both factors decrease perinatally and postnatally
(28 and unpublished observation). GATA-6 is coexpressed with a
number of genes that mark epithelial cell differentiation, including
Clara cell secretory protein (CCSP) and surfactant protein (SP)-B,
SP-C, and SP-A (28), the latter being components of the
surfactant system that are required for postnatal lung function and
host defense. In vitro studies demonstrated that GATA-6 enhances
transcription of SP-A, SP-C, and TTF-1, supporting its potential role
in the regulation of gene expression and/or differentiation in type II
cells of the developing lung (1, 14, 22). Although GATA-6
gene-targeted mice do not survive to a stage in which lung
morphogenesis is initiated, studies with GATA-6(/
) chimeric
embryonic stem cells (ES) cells support the concept that GATA-6
expression is required for the contribution of the ES cells to the
conducting airways during lung morphogenesis, although this has been
questioned recently (9, 18). Because GATA-6 gene-targeted
mice die early in gestation, before formation of many organs, its role
in lung morphogenesis or function remains unclear. Although recent
studies support the concept that GATA-6 is essential for formation of
the airways during early lung morphogenesis (9), it
remains unclear whether GATA-6 may play a role later in development.
The present study was designed to discern the role of GATA-6 in lung morphogenesis using a conditional system to express a GATA-6-engrailed fusion protein using the reverse tetracycline transactivator (rtTA) controlled by the human SP-C promoter. Previous studies demonstrated that GATA-6-engrailed selectively inhibited GATA-6-dependent activation of target genes, including SP-A (1). The SP-C promoter element is expressed in a lung-specific manner and is active as early as embryonic day (E) 10, being expressed thereafter in epithelial cells of developing respiratory tubules at sites consistent with the expression of endogenous GATA-6 (26). In the present study, the GATA-6-engrailed fusion protein did not alter branching morphogenesis but inhibited maturation of the gas-exchange region of the lung in the saccular period of development.
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MATERIALS AND METHODS |
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Plasmid construction and transgenic mice.
The GATA-6-engrailed-bovine growth hormone poly A construct contains
the GATA-6 zinc finger DNA-binding domain (amino acids 228-351),
Drosophila engrailed repressor domain (amino acids
1-298), and the bovine growth hormone polyadenylation
signal. The 1.8-kb transgene was placed under the control of
(tetracycline operator)7-cytomegalovirus [(tetO)7-CMV]
promoter. Plasmid constructs were verified by sequencing and
then were microinjected in mouse oocytes using standard transgenic procedures. These mice were mated to SP-C-rtTA transgenic mice to place
the GATA-6-engrailed gene under conditional control of exogenous
doxycycline (Ref. 24 and Fig.
1A). In this system, expression of target genes is induced by doxycycline in the developing lung as early as E10 (21). Heterozygous
SP-C-rtTA were mated to heterozygous
(tetO)7-CMV-GATA-engrailed mice to generate double and
single transgenic mice and wild-type littermates.
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PCR genotyping. Primers used for identification of SP-C-rtTA transgenic mice were as follows: 5'-primer in SP-C promoter, 5'-GAC ACA TAT AAG ACC CTG GTC A-3'; 3'-primer in rtTA coding sequence, 5'-AAA ATC TTG CCA GCT TTC CCC-3'. Primers used for identification of (tetO)7-CMV-GATA-6-engrailed transgene were as follows: 5'-primer in GATA-6 zinc finger, 5'-TGG CGT AGA AAT GCT GAG GG-3'; 3'-primer in engrailed repressor, 5'-TTG GTG GTG TGC GTC TGA TTG-3'. Amplification of PCR product for SP-C-rtTA was performed by denaturation at 94°C for 10 min and then 30 cycles of amplification at 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by a 7-min extension at 72°C. Detection of (tetO)7-CMV-GATA-6-engrailed was identical except that the annealing temperature was 59°C and required 25 cycles.
Animal use and administration of doxycycline. Animals were maintained in a pathogen-free vivarium in filtered cages in an Association for the Assessment and Accreditation of Laboratory Animal Care-approved facility. Oral doxycycline was administrated in drinking water at a final concentration of 0.5 mg/ml. Because of the light sensitivity of doxycycline, doxycycline-containing water was replaced three times per week. Sentinal mice were free of common viral and bacterial pathogens.
RNA isolation and RT-PCR.
Lung tissue was homogenized in Trizol reagent (Life Technologies, San
Francisco, CA), and RNA was isolated following the manufacturer's specifications. RNA (10 µg) was treated with 2 units of DNase at
37°C for 30 min before cDNA synthesis. DNase-treated total lung DNA
(5 µg) was reverse transcribed and analyzed by PCR for GATA-6-engrailed transgene and -actin mRNA. PCR primers for
-actin were as follows:
-actin primer 1, 5'-GTG GGC CGC TCT AGG
CAC CAA-3';
-actin primer 2, 5'-CTC TTT GAT GTC ACG CAG GAT TTC-3'; PCR conditions were 94°C denaturation for 10 min, followed by 25 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for
30 s, and then a 7-min extension at 72°C. Primers and PCR
conditions for GATA-6-engrailed were identical to that used for
genotyping except that 30 cycles were used. All RT-PCR reactions were
performed with controls lacking reverse transcriptase. Amplification
was not seen in reactions lacking reverse transcriptase (data not shown). Quantitation of aquaporin-5 (Aqp5), SP-C, and CCSP mRNAs was
performed by real-time RT-PCR.
Lung histology and immunohistochemistry.
To obtain lung tissue, the fetus was isolated from a pregnant female
after injection of ketamine-xylazine-acepromazine to the dam. The chest
was opened, and the lung was fixed with 4% paraformaldehyde at 4°C.
Lungs from postnatal animals were inflation fixed at 25 cmH2O pressure via a tracheal cannula with 4%
paraformaldehyde. Tissue was processed according to a standard method
(3) and embedded in paraffin. Antibodies and procedures
for immunostaining of TTF-1, pro-SP-C, and SP-B have been described
previously (28). Rabbit polyclonal antibody against amino
acids 110-122 of rat TTF-1 was kindly provided by Dr. Roberto
DiLauro and was used at a dilution of 1:1,000. Rabbit polyclonal
antisera against human pro-SP-C (R68514) and bovine mature SP-B
(R28031) were generated in this laboratory and used at dilutions of
1:1,000. A hamster monoclonal antimouse type I cell marker-
(T1
) antibody (Developmental Studies Hybridoma Bank, University of
Iowa, Hybridoma no. 8.1.1, www.uiowa.edu/~dshbwww, courtesy of Dr.
Andrew Farr; see Refs. 6 and 11) was used at dilution of
1:2,000 after blocking the sections with 5% goat serum in PBS.
Platelet endothelial cell adhesion molecule (PECAM-1; CD31) antibody
(Pharmingen, San Diego, CA) was used as previously described at 1 µg/ml (27). Proliferating cell nuclear antigen (PCNA)
was detected using a staining kit from Zymed Laboratories (Grand
Island, NY). For bromodeoxyuridine (BrDU) labeling, pregnant females
were injected 10 mg/kg body wt BrDU (Zymed Laboratories) 2 h
before death. BrDU incorporation in fetal lung was detected by
immunostaining using a BrDU staining kit from Zymed Laboratories.
Real-time RT-PCR.
Lung mRNA was isolated and reverse transcribed. The Smart Cycler System
(Cepheid, Sunnyvale, CA) was used to determine the cDNA concentration
of Aqp5, SP-C, and CCSP in mouse lung. The concentration of SP-C, CCSP,
and Aqp5 cDNA was read from standard curves generated using a series of
dilutions of each cDNA and normalized to the concentration of -actin
in each sample. The primers used for Aqp5 were as follows: 5'-primer,
5'-CAG TTC AGG ACC ATC CCA GAA AG-3' and 3'-primer, 5'-AAA CGC CCA ACC
CGA ATA CC-3'; for SP-C, 5'-primer, 5'-CAT CGT TGT GTA TGA CTA CCA
GCG-3' and 3'-primer, 5'-GAA TCG GAC TCG GAA CCA GTA TC-3'; and for
CCSP, 5'-primer, 5'-ATC ACT GTG CTC ATG CTG TCC-3' and 3'-primer,
5'-GCG TCG AAT ATC TCT GAA ATC-3'.
In situ hybridization. In situ hybridization analyses for GATA-6 mRNA were performed on lung from fetal mice on E17.5 and E18.5 using 35S-labeled cDNA probes described previously (1). A 3.1-kb pair mouse GATA-6 cDNA template was used to generate 35S-labeled probe, which was reduced to an average size of 200 bp by alkaline hydrolysis.
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RESULTS |
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Conditional expression of GATA-6-engrailed mRNA. Transgenic mice bearing the (tetO)7-GATA-6-engrailed transgene were produced by oocyte injection and bred to transgenic mice bearing the human SP-C-rtTA transgene, as previously described (3, 24 and Fig. 1, A and B). In SP-C-rtTA transgenic mice, rtTA is expressed selectively in epithelial cells in the lung by the 3.7-kb human SP-C promoter. Double-transgenic mice were produced that were heterozygous for SP-C-rtTA and (tetO)7-GATA-6-engrailed. In double-transgenic mice, expression of the targeted gene in the minimal (tetO)7-CMV promoter is induced by doxycycline (24). In the absence of doxycycline, single- and double-transgenic mice survived and were phenotypically normal. GATA-6-engrailed mRNA in the lung was barely detectable in the absence of doxycycline and was markedly enhanced by exposure of the animals to doxycycline (Fig. 1C). When the four dams were placed on doxycycline from E6 to birth, survival of double-transgenic mice was only 60% of expected.
Effects of GATA-6-engrailed on fetal lung morphogenesis.
No abnormalities were observed in the doxycycline-treated
double-transgenic mice obtained at E17.5-18, except in
the lung. After exposure to doxycycline, lung morphology of
single-transgenic and double-transgenic mice was normal at
E16.5, consistent with no observable effect of the transgene
on branching morphogenesis. In contrast, consistent abnormalities were
seen in doxycycline-exposed double-transgenic mice on E17.5
(Fig. 2). Although lung size was similar
among wild-type, single-, and double-transgenic mice, peripheral air
spaces in double-transgenic mice contained fewer but larger saccules
with a relatively thick mesenchyme, morphological features similar to
those seen ~1-2 days earlier in wild-type or single-transgenic
mice. Staining for TTF-1, a homeodomain-containing transcription factor
critical for formation of the lung periphery, demonstrated homogenous
staining of nuclei in virtually all epithelial cells lining the
terminal air spaces of the double-transgenic mice (Fig.
3). Epithelial cells lining peripheral
airways were primarily cuboidal and lacked the squamous features
typical of the peripheral lung saccules in late gestation. In contrast,
terminal airways in control mice were lined by both cuboidal and
squamous cells, the latter lacking TTF-1 staining, consistent with the normal decrease in TTF staining that accompanies the differentiation of
cuboidal type II cells into squamous type I epithelial cells that
occurs in late gestation and postnatally (28).
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GATA-6-engrailed decreased SP expression.
To assess whether GATA-6-engrailed altered the expression of
differentiation-dependent markers in respiratory epithelial cells, immunohistochemistry for pro-SP-C, the active SP-B peptide, and CCSP,
markers for type II cells and nonciliated bronchial and bronchiolar
respiratory epithelial cells, respectively, was performed (Fig.
5). On E17.5, the intensity of
staining for pro-SP-C and SP-B was decreased in the peripheral airway
epithelial cells in the GATA-6- and engrailed-expressing mice. However,
most epithelial cells were cuboidal and stained lightly, consistent
with the inhibition of type II and type I cell differentiation.
Likewise, both SP-C and CCSP mRNA concentrations were significantly
decreased by GATA-6-engrailed at E18.5 (Fig. 4). In
contrast, in wild-type mice, pro-SP-C and SP-B staining was intense in
the cuboidal subsets of peripheral respiratory epithelial cells (type
II cells) and absent in the squamous type I cells, a finding consistent
with the normal maturation and differentiation of type II cells to type
I cells in late gestation. Likewise, staining for CCSP, normally
intense in the conducting airways at E17.5-18, was
markedly decreased in GATA-6- and engrailed-expressing mice (Fig. 5).
Staining for pro-SP-C and pro-SP-B was relatively weak on
E16 and was not influenced by the GATA-6-engrailed transgene (data not shown). The abundance and sites of PCNA, a marker of cell
proliferation, were similar in the GATA-6-engrailed and control mice at
E17.5. BrDU labeling also failed to show differences in cell
proliferation between GATA-6-engrailed and control mice at E17.5 (data not shown).
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Abnormalities in lung mesenchyme.
The relative abundance of mesenchyme was increased in the GATA-6- and
engrailed-expressing mice at E17.5 and E18,
consistent with the general arrest in differentiation and morphogenesis
of the lung. Vascular development proceeds rapidly in the
saccular-alveolar stage of late gestation and is associated with marked
thinning of the pulmonary mesenchyme. Increasingly, close apposition of the pulmonary vasculature to the squamous cells occurs in the lung
periphery during the saccular-alveolar period. PECAM staining demonstrated that pulmonary vascular tissues were embedded in the
relatively thick mesenchyme in the GATA-6- and engrailed-expressing mice, and the endothelial cells were not in close relationship with
epithelial cells in the lung periphery, morphological findings consistent with immaturity (Fig. 6).
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Lack of effect of GATA-6-engrailed in the postnatal period.
When the double-transgenic animals were placed on doxycycline for 2 days at 6 wk of age, GATA-6-engrailed RNA was readily detected.
Histology of the lungs from adult double-transgenic mice was normal
when assessed after 2 wk of doxycycline. Endogenous GATA-6 mRNA was
readily detected in epithelial cells of the fetal lung by in situ
hybridization on E17.5, decreased at E18.5 (Fig. 7) and was absent on postnatal days
5 and 21 (data not shown). These findings are
consistent with the lack of GATA-6 mRNA in the postnatal lung.
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DISCUSSION |
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Conditional expression of GATA-6-engrailed fusion protein delayed
lung morphogenesis in the saccular period but did not alter branching
of the embryonic lung or influence postnatal alveolarization. Delayed
sacculation and differentiation of type II and type I cells were
accompanied by decreased expression of T1, Aqp5, SPs, and CCSP.
Thinning of the pulmonary mesenchyme and formation of peripheral airway
capillaries was inhibited. Taken together, these findings support a
critical role for GATA-6 in maturation of the gas exchange region of
the lung in late gestation.
The GATA-6-engrailed fusion protein used in the present study inhibited GATA-6 activity in vitro, decreasing GATA-6-dependent activation of SP-A and SP-C promoters in HeLa and mouse lung epithelial cells (1, 14), demonstrating the selective inhibitory activity of the chimeric gene on GATA-induced gene transcription. The actions of GATA-6 on these target genes are modulated by binding to cis-acting elements located in the 5'-flanking region of the target genes. However, GATA-6 also acts synergistically with TTF-1 on the transcriptional activities mediated by various cis-acting elements. Although GATA-6 may activate respiratory epithelial gene transcription by mechanisms independent of TTF-1, recent studies demonstrated that GATA-6 and TTF-1 directly interact, binding each other via the carboxy zinc-finger domain of GATA-6 and the homeodomain region of TTF-1 (14). Interactions between TTF-1 and GATA-6 are similar to those by which Nkx2.5 (tinman) and GATA-4 interact in the heart to regulate gene expression (5). In the present study, no effects of GATA-6-engrailed protein were observed at E16, a time at which branching morphogenesis is nearly completed. It is unclear, however, whether this observation is related to the site and levels of expression of the transgene rather than the processes that are influenced by GATA-6. Because the timing and levels of expression of SP-C-rtTA in these transgenic mice increase developmentally and change spatially, GATA-6-engrailed may not be as active early in gestation. SP-C transgenes, including the rtTA, are expressed as early as E10 (21, 24, 26) and are maintained at high levels in type II epithelial cells and in bronchiolar and type II cells postnatally (28). In the present study, the GATA-6-engrailed fusion protein was expressed with the SP-C promoter that is itself regulated by GATA-6. Thus GATA-6-engrailed may influence the levels or timing of expression of the transgene. In spite of such potential autoregulation, GATA-6-engrailed mRNA was readily detected in the transgenic mice. In studies in which the SP-C-rtTA mice were used for expression of luciferase, expression of the (tetO)7 target gene was induced 12 h after exposure of the dam to doxycycline. Luciferase gene expression was terminated 24-48 h after removal from doxycycline in postnatal mice (3, 21, 24), demonstrating the reversibility of doxycycline-regulated expression in this system. The lack of postnatal effects of GATA-6-engrailed is consistent with the paucity or lack of endogenous GATA-6 expression in the postnatal lung but may have been influenced by the extent or levels of expression of the transgene in the adult lung.
The GATA-6-engrailed gene inhibited the expression of SP-C and SP-B in
lung saccules and CCSP in bronchioles in vivo. Because the level of
expression of each of these genes increases normally in late gestation,
it is unclear whether this represents a direct inhibitory effect of the
chimeric gene on gene transcription or represents a more generalized
delay in lung maturation. GATA-6 stimulated SP-A and SP-C gene
transcription in vitro, acting synergistically with TTF-1 to enhance
SP-C gene transcription (1, 14). Thus the GATA-engrailed
transgene may directly inhibit transcription of the SP genes in this
transgenic model. Alternatively, decreased SP expression may be
mediated by generalized effects of GATA-engrailed on lung maturation
and epithelial differentiation. Thinning of the pulmonary mesenchyme,
transition of cuboidal type II to squamous type I epithelial cells, and
alveolar-capillary development were all inhibited by the
GATA-6-engrailed transgene. Decreased T1 expression and decreased
Aqp5 mRNA at E17.5 and E18.5, respectively, seen
in the GATA-6-engrailed mice are also consistent with a delay in
differentiation of type I cells. The distinct temporal effects of
GATA-6-engrailed on T1
and Aqp5 may reflect intrinsic differences in
their regulation or the levels of GATA-6-engrailed required to inhibit
their expression. Because the GATA-6-engrailed protein is expressed and
active only within respiratory epithelial cells, the abnormalities in
maturation of the pulmonary vasculature and pulmonary mesenchyme
support the concept that GATA-6-engrailed has influenced morphogenesis,
at least in part, via the paracrine communication between epithelial
and mesenchymal cells in the lung.
Formation of the mouse lung, per se, begins on E9 as an outpouching of the foregut endoderm along the laryngeal-esophageal sulcus. The trachea elongates, and bronchi and larger bronchioles form by E15, during the pseudoglandular and cannalicular stages of development. During lung sacculation, at approximately E17-18, the pulmonary mesenchyme thins, and the pulmonary capillary network expands in the lung periphery. Septation of the alveoli begins in late gestation and continues during neonatal and postnatal lung morphogenesis. It is increasingly clear that complex transcriptional and signaling events mediate proliferation, migration, and differentiation of cells in both endodermal and mesenchymal compartments of the developing lung. TTF-1, GATA-6, and forkhead family members (Fox genes) have been implicated in lung formation and transcriptional control of lung-specific gene expression (see Ref. 20 for review). TTF-1 is required for formation and differentiation of the peripheral lung parenchyma and for the expression of SPs and CCSP. Likewise, GATA-6 regulates TTF-1 and SP gene expression in vitro. GATA-6 is selectively expressed in the respiratory epithelial cells of the developing lung. GATA-6 mRNA decreases with advancing gestation (22). The lack of effect of GATA-6-engrailed in the adult is not likely related to lack of expression of the transgene, since the SP-C promoter used to express rtTA remains highly active in the bronchiolar and alveolar regions of the postnatal lung (21, 24). Recently, GATA-6 was overexpressed in the lung of transgenic mice with the SP-C promoter, resulting in abnormalities in branching and loss of peripheral lung saccules, consistent with an important role of GATA-6 in the regulation of early lung morphogenesis.
Postnatal survival of GATA-6- and engrailed-expressing transgenic mice was decreased by treatment of the dam with doxycycline. Although the mechanisms causing death after birth were not determined with certainty, changes in SPs or the generalized delay in lung morphogenesis likely contributed to perinatal death in the GATA-6-engrailed pups. SP-B-deficient mice die of respiratory failure at birth (4). The observed decrease in SP-B seen in the GATA-6-engrailed mice likely contributed to the perinatal lethality observed when the mice were placed on doxycycline. It is of considerable clinical interest that preterm infants are frequently born at times in which the lung has not matured to the saccular-alveolar stage. These infants suffer respiratory distress based on surfactant deficiency and morphological immaturity with features similar to those caused by the GATA-6-engrailed transgene.
The finding that survival of double-transgenic mice was not altered in the absence of doxycycline supports the concept that lower levels of expression of the transgene in the absence of doxycycline are insufficient to inhibit endogenous GATA-6 activity. Because the levels of GATA-6 mRNA are generally higher earlier in lung morphogenesis, the lack of effects on earlier processes, i.e., branching morphogenesis, may represent inadequate levels of GATA-6-engrailed fusion protein that were not sufficient to compete with the high levels of endogenous GATA-6.
In summary, conditional regulation of GATA-6-engrailed inhibitory protein demonstrates a requirement for GATA-6 activity for maturation of the gas exchange area in the saccular-alveolar transition before birth. This conditional system of gene regulation allows generation of transgenic mice that survive perinatally and postnatally, which can be used to assess the importance of gene function in developmental processes when targeted ablation is otherwise lethal earlier in development.
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ACKNOWLEDGEMENTS |
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We thank Dr. Susan Wert for histology analysis and in situ hybridization, and Ann Maher for manuscript preparation.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-56387 and HL-38859 (to J. A. Whitsett).
Address for reprint requests and other correspondence: J. A. Whitsett, Children's Hospital Medical Center, Div. of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jeff.whitsett{at}chmcc.org).
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.
March 22, 2002;10.1152/ajplung.00044.2002
Received 29 January 2002; accepted in final form 15 March 2002.
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