GATA-6 is required for maturation of the lung in late gestation

Cong Liu1, Edward E. Morrisey2, and Jeffrey A. Whitsett1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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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Fig. 1.   Design of the transgene used to generate conditional expression of GATA-6-engrailed in the lung. A: 3.7-kb human (h) surfactant protein (SP)-C 5'-flanking region was used to express the reverse tetracycline transactivator (rtTA) in respiratory epithelial cells. GATA-6-engrailed was inserted 3' to the tet operator concatamer and a minimal promoter from CMV as described by Gossen and Bujard (8). Transgenic mice bearing the SP-C-rtTA transgene were bred to single transgenic mice bearing (tetracycline operator)7-cytomegalovirus [(tetO)7-CMV] GATA-6-engrailed to generate double-transgenic mice expressing GATA-6-engrailed mRNA in the lung in a doxycycline-inducible manner. SV40, Simian virus 40; bGH, bovine growth hormone. B: transgenic mice were identified using PCR primers specific for each transgene. Single-transgenic or wild-type littermates were used as the controls for the double-transgenic mice. C: RT-PCR was used to detected transgene expression in both embryonic and adult lung. On embryonic day (E) 17.5, double-transgenic mice, but not single-transgenic or wild-type littermates, expressed GATA-6-engrailed transgene in the lung after 11 days of doxycycline treatment (lanes 1-4). In adult double-transgenic mice, the expression of GATA-6-engrailed transgene in the lung was detected at low levels in the absence of doxycycline (0) and was increased after 2 days of doxycycline treatment (2; lanes 5 and 6). Arrows showed the position of the primers used in genotyping and RT-PCR.

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 beta -actin mRNA. PCR primers for beta -actin were as follows: beta -actin primer 1, 5'-GTG GGC CGC TCT AGG CAC CAA-3'; beta -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-alpha (T1alpha ) 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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Lung histology in GATA-6-engrailed transgenic mice. Lung tissue from E16.5 (A and B) or E17.5 (C and D) mice were stained with hematoxylin and eosin. At E16.5, normal lung histology was observed in wild-type pups (A) and an SP-C-rtTA × (tetO)7-CMV-GATA-6-engrailed bitransgenic littermate (B) whose dam was treated with doxycycline since E6.5. On E17.5, peripheral saccules were fewer, and the peripheral airway wall was thicker in lungs from bitransgenic (D) compared with wild-type littermates (C) after exposure to doxycycline for 11 days. Data are representative of findings for at least 4 animals/genotype. Original magnification ×10 in A and B and ×4 in C and D. Bars = 100 µm.



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Fig. 3.   Immunohistochemical staining for thyroid transcription factor-1 (TTF-1) and type I cell marker-alpha (T1alpha ). SP-C-rtTA × (tetO)7-CMV-GATA-6-engrailed bitransgenic mice (B and D) and wild-type littermates (A and C) were treated with doxycycline from E6.5 to E16.5 (A and B) or E6.5 to E17.5 (C-F). On E16.5, TTF-1 was expressed in nearly all peripheral respiratory epithelial cells in both wild-type and double-transgenic mice (A and B). In wild-type mice on E17.5, TTF-1 was detected in nuclei of a subset of cuboidal epithelial cells but was not detected or decreased in squamous cells in peripheral tubules (C). In double-transgenic mice, TTF-1 staining was detected in the nucleus of all cells lining terminal airways (D), the lungs lacking squamous cells. T1alpha staining was assessed in nontransgenic control and GATA- and 6-engrailed-expressing littermates at 17.5 days gestation. E: on E17.5, T1alpha staining in wild-type pups demonstrated an intense staining in lung epithelial cells. F: in contrast, in double-transgenic mice, T1alpha staining was dramatically decreased. Data are representative of at least 4 animals/genotype. Original magnification ×20. Bar = 100 µm.

To further assess type I epithelial cell differentiation, immunohistochemical staining of T1alpha , a type I cell-specific marker, was performed on sections from double-transgenic pups and wild-type littermates at E17.5 and E18 (6, 11). On E17.5, T1alpha was seen at the apical regions of virtually all peripheral epithelial cells in normal littermates, whereas T1alpha staining in double-transgenic mice was decreased throughout the respiratory epithelium, suggesting that the differentiation of type I epithelial cells was inhibited in GATA-6- and engrailed-expressing mice (Fig. 3). However, on E18, no difference in the intensity of T1alpha staining was observed in double-transgenic compared with control mice, suggesting that the differentiation of type I epithelial cells was delayed but not completely inhibited by expressing GATA-6-engrailed transgene (data not shown). Aqp5 mRNA, also a type I cell marker in the lung (25), was determined by real-time RT-PCR. After 2 days treatment of doxycycline, the lung mRNA concentration of Aqp5 in double-transgenic mice was decreased ~50% compared with wild-type littermates at E18.5, suggesting that type I cell differentiation was inhibited by expression of GATA-6-engrailed in lung epithelium (Fig. 4).


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Fig. 4.   GATA-6-engrailed inhibits SP-C, Clara cell secretory protein (CCSP), and aquaporin-5 (Aqp5) mRNA. Real-time PCR was used to quantitate concentrations of SP-C, CCSP, and Aqp5 mRNA in whole lung isolated from GATA-6- and engrailed-expressing and wild-type (wt) littermates on E18.5. Levels were normalized to beta -actin mRNA and are expressed in relation to control levels (100%). Differences in each mRNA were assessed by ANOVA, n = 3 animals/group. TG, transgenic.

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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Fig. 5.   Immunohistochemical staining for pro-SP-C, SP-B, and CCSP. SP-C-rtTA × (tetO)7-CMV-GATA-6-engrailed double-transgenic mice (B, D, and F) and wild-type littermates (A, C, and E) were treated with doxycycline from E6.5 to E17.5. Staining for pro-SP-C and SP-B, markers for type II alveolar epithelial cells, was decreased in bitransgenic mice (B and D) compared with wild-type littermates (A and C). Intense staining for both proteins was observed in the cuboidal subsets of epithelial cells in wild-type pups. In contrast, in double-transgenic mice, most epithelial cells in peripheral tubules were lightly stained, and squamous (type I) cells lacking staining were not observed. CCSP, a marker for nonciliated bronchial and bronchiolar respiratory epithelial cells, was decreased in conducting airways of double-transgenic mice (E and F). Data are representative of at least 4 animals/genotype. Original magnification ×20. Bar = 100 µm.

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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Fig. 6.   Immunohistochemical staining for platelet endothelial cell adhesion molecule (PECAM). SP-C-rtTA × (tetO)7-CMV-GATA-6-engrailed double-transgenic mice (B) and wild-type littermates (A) were treated with doxycycline from E6.5 to E17.5. On E17.5, PECAM staining in wild-type pups demonstrated an extensive vascular network in the lung saccules, with staining observed in close proximity to epithelial cells (A). PECAM staining in double-transgenic mice revealed a relatively undeveloped pulmonary capillary bed in which stained cells were embedded in a relatively thick mesenchyme and did not come into close apposition to the respiratory tubules (B). Data are representative of at least 4 animals from each genotype. Original magnification ×20. Bar = 100 µm.

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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Fig. 7.   In situ hybridization for mouse GATA-6 mRNA in developing mouse lung. Antisense and sense probe covering a 3.1-kb GATA-6 mRNA sequence were used to detect the expression pattern of GATA-6 in the embryonic and adult lung. On E17.5, GATA-6 was expressed at high levels in the distal lung epithelium and blood vessels (A and B). On E18.5, the expression levels of GATA-6 decreased in the epithelium but were still high in the blood vessels (C and D). Original magnification ×4. Bar = 100 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 T1alpha , 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 T1alpha 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 T1alpha 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.


    ACKNOWLEDGEMENTS

We thank Dr. Susan Wert for histology analysis and in situ hybridization, and Ann Maher for manuscript preparation.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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