1 Department of Medicine, Molecular Cardiology Research Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
2 Department of Pulmonary Biology, Childrens Hospital Medical Center, Cincinnati, Ohio 45229, USA
*Author for correspondence (e-mail: emorrise{at}mail.med.upenn.edu)
Accepted 4 February 2002
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SUMMARY |
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Key words: GATA6, Lung development, Distal epithelium, Surfactant proteins, Fox genes, Mouse
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INTRODUCTION |
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Several transcription factors have been implicated in regulating lung epithelial development including thyroid transcription factor-1 (TTF-1) (also known as Nkx2.1 and Titf1), several Fox gene products including Foxa2 and Foxj1, and the zinc finger transcription factor GATA6. TTF-1 is a member of the Nkx homeodomain gene family and has been shown to bind and trans-activate sequences present in many lung-specific promoters including the surfactant proteins A (SP-A/Sftpa), B (SP-B/Sftpb), and C (SP-C/Sftpc) and CC10 (Utg) (Bruno et al., 1995; Margana and Boggaram, 1997
; Toonen et al., 1996
). Mice harboring a null mutation in the TTF-1 gene exhibit severely attenuated lung epithelial development with a dramatic decrease in airway branching (Kimura et al., 1996
). In addition, lung epithelial cells present in these mice lack expression of certain putative target genes such as SP-C suggesting that TTF-1 is an important and essential transcription factor for lung epithelial gene expression (Minoo et al., 1999
). Members of the winged-helix/forkhead or Fox gene family are expressed in a wide range of cells in the lung including the distal epithelium (Foxp2 and Foxa2), proximal epithelium (Foxj1 and Foxa2), and mesenchyme (Foxf1 and Foxf2) and Fox DNA binding sites are located in the promoters and enhancers of several lung specific genes including CC10, TTF-1 and SP-B (Braun and Suske, 1998
; Clevidence et al., 1994
; Ikeda et al., 1996
; Kaestner et al., 1994
; Mahlapuu et al., 1998
; Margana and Boggaram, 1997
; Miura et al., 1998
; Shu et al., 2001
; Tichelaar et al., 1999b
; Zhou et al., 1996b
). Furthermore, gain-of-function and loss-of-function experiments have demonstrated the necessity of wild-type expression levels of some of these Fox genes during lung differentiation and development including Foxf1, Foxa2 and Foxj1 (Kalinichenko et al., 2001
; Mahlapuu et al., 2001
; Tichelaar et al., 1999a
; Zhou et al., 1997
).
The zinc finger transcription factor GATA6 has recently been implicated in regulating lung gene expression and development. GATA6 belongs to the GATA family of zinc finger transcriptional regulators, several of which have been shown to play important roles in tissue specification and gene expression (Simon, 1995). GATA6 is the only known GATA factor expressed in the distal epithelium of the developing lung where its expression is observed as early as E10.5 (Bruno et al., 2000
; Keijzer et al., 2001
; Morrisey et al., 1996
; Morrisey et al., 1997
). Binding sites for GATA6 are present in several lung-specific promoters and enhancers including SP-A, SP-C, and TTF-1 (Bruno et al., 2000
; Shaw-White et al., 1999
). In addition, GATA6 is able to trans-activate these promoters in non-lung cells suggesting a role in the transcriptional regulation of these genes (Bruno et al., 2000
; Shaw-White et al., 1999
). Previous experiments implicated an essential role for GATA6 in early lung epithelial development because Gata6/ embryonic stem (ES) cells were unable to contribute to the airway epithelium of Gata6//C57BL6 chimeric mice at E13.5 (Morrisey et al., 1998
). However, a recent report suggests that under certain in vivo experimental conditions, Gata6/ ES cells can differentiate down the lung epithelial cell lineage pathway (Keijzer et al., 2001
). These Gata6//C57BL6 chimeric lungs displayed defects in branching morphogenesis and down-regulation of putative target genes such as SP-C (Keijzer et al., 2001
). However, the specific defects in epithelial cell differentiation were not reported. Therefore, much is still not known about the cell intrinsic function(s) of GATA6 transcriptional regulation in lung epithelium, which is important because of the expression of GATA6 in the adjacent mesenchyme of the lung (Bruno et al., 2000
; Keijzer et al., 2001
; Morrisey et al., 1996
).
We have examined the role of GATA6 in distal lung epithelial development by expressing a GATA6/Engrailed dominant-negative fusion protein (GATA6/En) in transgenic mice using the well-characterized human 3.7 kb SP-C promoter which drives expression exclusively in distal lung epithelium (Koutsourakis et al., 2001; Lu et al., 2001
; Weaver et al., 1999
; Wert et al., 1993
; Zhou et al., 1997
). Several reports, from our laboratory and others, have shown that the GATA6/En fusion protein can repress GATA6 transcriptional activation (Bruno et al., 2000
; Liang et al., 2001
). In addition, similar Engrailed repressor domain/transcription factor fusion protein strategies have been used to perturb the function of c-myb and Nkx2.5 (Badiani et al., 1994
; Fu et al., 1998
). Although the GATA6/En dominant-negative may repress the function of most if not all GATA factors, GATA6 is the only known GATA factor expressed in distal lung epithelium (Morrisey et al., 1996
; Morrisey et al., 1997
). The morphological characteristics of the surfactant protein C/GATA6-Engrailed transgenic (SP-C/G6en) embryos are distinct from other lung transgenic models previously reported. SP-C/G6en transgenic mice exhibited a lack of squamous epithelium in the distal regions of the lung during late alveolar development indicating a defect in AEC differentiation. In particular, AEC-1 cells were absent in the distal airways of SP-C/G6en embryos as demonstrated by electron microscopy and attenuated aquaporin 5 gene expression. AEC-2 cells had been properly specified as shown by normal TTF-1 gene expression in these embryos. Furthermore, the presence of lamellar bodies, secreted surfactant and normal expression of SP-A suggests that AEC-2 differentiation had progressed beyond the pseudoglandular stage (E12.5-E16.0) of lung development in SP-C/G6en embryos. However, decreased endogenous SP-C expression as well as increased Foxp2 gene expression after E17.5 indicates that certain aspects of AEC-2 cell gene expression were perturbed. Proximal airway development was also disrupted as shown by a decrease in CC10- and Foxj1-positive proximal airway tubules. Finally, we show that binding sites for GATA factors are conserved in the aquaporin 5 promoters from mouse and rat and that GATA6 can trans-activate the mouse aquaporin 5 promoter, supporting a role for GATA function in AEC-1 cell gene expression. Taken together, these data implicate a role for GATA6 in the regulation of distal airway epithelial differentiation, leading to a disruption in AEC-1 differentiation and proximal airway development.
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MATERIALS AND METHODS |
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Histology
E17.5 embryos were dissected from the uterus and fixed in 4% paraformaldehyde for 48 hours. The lungs from E19.5 embryos and P0 neonates were dissected and removed from the chest cavity and fixed for 24 hours in 4% paraformaldehyde. All tissue samples were dehydrated in a series of ethanol washes and embedded in paraffin blocks. Hematoxylin and Eosin (H+E) staining was performed as previously described (Soudais et al., 1995). The in situ probes for Gata6, SV40 poly(A)+ intron, engrailed, SP-A, SP-C, Foxp2, CC10 and Foxj1 have been described (Lu et al., 2001
; Morrisey et al., 1996
; Shu et al., 2001
). The in situ probe for mouse aquaporin 5 was generated by RT-PCR from adult mouse lung mRNA using the following primers: sense 5'-GCTGTGGTCAAAGGCACATATGAG-3', antisense w/T7 site 5'-TGTAATACGACTCACTATAGGGCGACTGAACTGCTGTGAGCTTGCAC-3'. Radioactive in situ hybridization was performed as previously described (Soudais et al., 1995
). To quantitate the numbers of CC10-positive airways in wild-type and SP-C/G6en mice, five sections corresponding to the same region of the left lung from three wild-type and three SP-C/G6en transgenic littermates at E17.5 and E19.5 were probed with the CC10 riboprobe and the resulting positive airways were counted and analyzed using the NIH Image 1.62 software.
Non-radioactive in situ hybridization was performed by probing deparaffinized tissue sections with a digoxigenin-labeled riboprobe and developing and immunologically detecting the positive signal with an anti-digoxigenin monoclonal antibody (Roche Biochemicals, Inc.). Non-radioactive in situ hybridization was used to detect SP-C gene expression because the signal from radioactive in situ hybridization was too intense for high resolution analysis of expression. Periodic acid-Schiff staining (PAS) was performed by soaking deparaffinized embryo sections in 0.5% Periodic acid for 5 minutes, rinsing with dH2O and then staining in Schiffs reagent for 60 seconds. PAS-stained slides were then counterstained with Hematoxylin. For amylase treatment, slides were predigested in diastase (with -amylase) solution for 15 minutes prior to PAS staining. Further details of all histological procedures can be obtained at the University of Pennsylvania Molecular Cardiology Research Center Web page-Histology Core Facility Protocols (http://www.med.upenn.edu/mcrc/histology/histologyhome.html).
Ultrastructural analysis of mouse lung tissue
Electron microscopy procedures were performed as previously described (Soudais et al., 1995). Briefly, lung tissue was dissected from E19.5 SP-C/G6en, SP-C/en and wild-type littermates and fixed in 2% glutaraldehyde with 0.1 M sodium cacodylate pH 7.4 for 48 hours at 4°C. Samples were then incubated with 2% osmium tetroxide and 0.1 M sodium cacodylate pH 7.4 for 1 hour at 4°C. Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a JEM 1010 microscope. Digital images were captured on a Hamamatsu HamC4742-95-12 CCD camera using AMT Advantage software.
Northern blot analysis
SP-C/G6en transgenic mice were harvested at E19.5 and the whole lung was dissected from the fetus and frozen at 80°C. Two genotype positive and wild-type lung samples from the same litter were homogenized in Trizol and total RNA was extracted according to the manufacturers protocol (Invitrogen, Inc.). 10 µg of total RNA was resolved on a 1.5% formaldehyde agarose gel and blotted to a Hybond-N membrane (Amersham, Inc.). Generation of radiolabeled probes and hybridization was performed as described previously (Morrisey et al., 1996). The DNA fragments used to generate probes for aquaporin 5 (Aqp5), Foxp2 and engrailed were the same as described for the in situ hybridization analysis.
Co-transfection experiments
The 1437 bp mouse Aqp5 promoter was amplified from mouse genomic DNA and subcloned into the MluI/XhoI sites of pGL3basic (Promega) to generate pGL3/Aqp5-1.4 using the following primers: sense 5'-CACACGCGTAAACCTAGAAGGTCCTCCCTCC-3', antisense 5'-CACCTCGAGTTCTGCGAGACGTGCGGTGCCC-3'. A truncated version lacking the four potential GATA6 binding sites (pGL3/Aqp5-0.5) was generated by PCR from pGL3/Aqp5-1.4kb using the same antisense primer described above and the following sense primer: 5'-CACACGCGTGGCGCTGTCCTCAGAAACTCATC-3'. The fidelity of both constructs was verified by DNA sequence analysis. These reporter constructs (0.5 µg) were co-transfected into NIH-3T3 cells along with the pcDNA3G6 construct (Morrisey et al., 1996) (2.5 µg) containing the entire open reading frame of the mouse Gata6 cDNA using Fugene 6 (Roche Biochemicals, Inc.). To test whether the GATA6 activation of the Aqp5 promoter could be repressed with the GATA6/Engrailed fusion protein, pcDNA3 plasmids containing either the GATA6/Engrailed fusion protein (pcDNA3G6/En), the Engrailed repressor domain (pcDNA3En) or a mutant GATA6/Engrailed fusion protein (pcDNA3G6mut/En), which contains mutations changing aa293-294 of GATA6 cysteine-alanine to serine-arginine (eliminating DNA binding), were transfected along with the pGL3/Aqp5-1.4kb reporter plasmid and the pcDNA3G6 expression plasmid (1 µg). All transfections contained 0.5 µg of the pMSVßgal vector to control for transfection efficiency. Forty-eight hours after transfection, cells were harvested and luciferase and ß-galactosidase assays were performed using commercially available kits (Promega).
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RESULTS |
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Expression of TTF-1 and SP-C in SP-C/G6en embryos
To further investigate pulmonary epithelial cell differentiation in SP-C/G6en embryos, in situ hybridization utilizing gene- specific riboprobes for SP-C, a marker for AEC-2 cells, and TTF-1 an important transcription factor expressed in both AEC-2 cells and in proximal Clara epithelial cells, was performed (Glasser et al., 1991; Wert et al., 1993
; Zhou et al., 1996b
). Both of these genes have been used previously as specific markers of lung epithelial cell specification (Zhou et al., 1996b
). TTF-1 null embryos die during gestation and exhibit severe defects in lung epithelial development and lack SP-C expression (Minoo et al., 1999
). In addition, the mouse TTF-1 promoter contains an important GATA factor DNA binding site suggesting that GATA factors may regulate its expression (Shaw-White et al., 1999
). TTF-1 expression was observed in both wild-type and SP-C/G6en lung epithelium at E17.5 and E19.5 (Fig. 6A-D). No differences were observed in the level or pattern of TTF-1 expression between wild-type and transgenic littermates (Fig. 6A-D). At E17.5, expression of endogenous SP-C was observed throughout the distal airway epithelium of wild-type embryos (Fig. 6E,G). Interestingly, endogenous SP-C expression was not observed in the thick cuboidal epithelium where the SP-C/G6en transgene was expressed (Fig. 6H, bracket). This observation was consistent in all five SP-C/G6en embryos examined. However, endogenous SP-C was expressed in regions of the distal lung epithelium that lacked expression of the SP-C/G6en transgene (Fig. 6F,H arrowheads). These data suggest that pulmonary epithelium has been specified but expression of endogenous SP-C is attenuated in SP-C/G6en embryos. However, attenuated endogenous SP-C expression in SP-C/G6en embryos is not due to the loss of TTF-1, which is expressed at wild-type levels.
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At E17.5, Aqp5 gene expression was observed at low levels in the distal airways of wild-type lungs as has been previously demonstrated (Fig. 9A) (Kreda et al., 2001). However, Aqp5 expression was not observed in the distal airways in any of the E17.5 SP-C/G6en embryos (Fig. 9B). By E19.5, expression was very robust in the distal airways of wild-type embryos but severely attenuated in all E19.5 SP-C/G6en embryos (Fig. 9C,D). SP-C/en embryos showed no difference in Aqp5 gene expression from their wild-type littermates (Fig. 9E,F). These data suggest that aquaporin-5 gene expression is significantly down-regulated in SP-C/G6en embryos.
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To confirm the in situ hybridization results showing a decreased level of Aqp5 expression and an increased level of Foxp2 expression in the airways of SP-C/G6en embryos, equal amounts of total RNA from the lungs of two E19.5 SP-C/G6en transgenic embryos and two wild-type littermates was analyzed by northern blotting to quantitate the changes in Foxp2 and Aqp5 expression. Both of the independent F0 SP-C/G6en founder embryos expressed high levels of the SP-C/G6en transgene (Fig. 10). Phosphoimager analysis shows that Aqp5 gene expression is decreased an average of 7.8-fold while Foxp2 gene expression is increased by 3.5-fold in SP-C/G6en transgenic embryos at E19.5 (Fig. 10). These data support the in situ hybridization analysis showing a significant decrease in Aqp5 and increase in Foxp2 expression in the lungs of SP-C/G6en embryos.
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DISCUSSION |
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Other loss-of-function experiments have revealed phenotypes that are related but distinct to that observed in the lungs of SP-C/G6en embryos. Parathyroid hormone-related peptide (PTHrP) mice also lack Aqp5 expression and die at birth from respiratory failure (Karaplis et al., 1994; Ramirez et al., 2000
). However, these mice appear to contain squamous epithelium in their lungs (Karaplis et al., 1994
; Ramirez et al., 2000
). In addition, since mice homozygous for an Aqp5 null allele do not exhibit a postnatal lung phenotype, the lack of Aqp5 in the PTHrP mice would not account for perinatal lethality (Ma et al., 1999
). Mouse lungs containing a null mutation in the tumor necrosis factor-
converting enzyme (TACE) also exhibit attenuated SP-C and Aqp5 gene expression and display dilated airways similar to those observed in SP-C/G6en embryos (Zhao et al., 2001
). As with SP-C/G6en embryos, TACE mice die soon after birth as a result of respiratory failure (Zhao et al., 2001
). However, Tace/ mice have defects in lung vascular development which were not observed in SP-C/G6en embryos (Zhao et al., 2001
). Therefore, the SP-C/G6en transgenic embryos have a unique phenotype that correlates well with a specific loss in AEC-1 cell differentiation during late lung development. This may contribute to the lethality observed in the transgenic mice because of the increased diffusion distance for gas exchange caused by the thicker AEC-2 cells lining the airways. Although AEC-2 differentiation was disrupted as shown by reduced SP-C expression, high levels of transgene expression are not observed with the human SP-C promoter until after E15.5 (Wert et al., 1993
). Thus, the SP-C/G6en transgene is not likely to have affected GATA6 function until late lung development, further supporting a defect in the AEC-2 to AEC-1 cell differentiation process.
GATA6 is the only GATA factor expressed in distal lung epithelium and its ability to trans-activate the mouse aquaporin-5 promoter through GATA factor DNA binding sites conserved in the mouse and rat promoters, further reinforces a model wherein GATA6 regulates AEC-1 differentiation and gene expression at the transcriptional level. These data suggest that GATA6 can regulate AEC-1 cell-specific gene expression in addition to the differentiation process leading to AEC-1 cell formation. Indeed, one of the hallmark functions of GATA factors is their ability to regulate cell-specific gene expression programs crucial for tissue-specific differentiation processes. For instance, GATA1 is absolutely required for terminal erythrocyte differentiation while GATA2 and GATA3 are essential for hematopoeitic stem cell and T-cell differentiation, respectively (Pevny et al., 1991; Ting et al., 1996
; Tsai et al., 1994
). Our data suggests that the AEC-2 to AEC-1 cell differentiation process and AEC-1 cell-specific gene expression may be added to the repertoire of the roles performed by GATA6 during development.
Disruption of distal lung epithelial differentiation leads to attenuated proximal airway development
Histological and gene expression data using the proximal airway epithelial marker genes Foxj1 and CC10 suggests a defect in late stage proximal airway development in SP-C/G6en mice as exhibited by reduced numbers of proximal airway tubules. A previously reported transgenic model using the SP-C promoter to over-express a constitutively active form of the signaling protein TGFß1 resulted in decreased expression of the proximal epithelial gene CC10 (Zhou et al., 1996a). This result led the authors to conclude that the lung epithelium of SP-C/Tgfß1 mice was arrested in the pseudoglandular stage of development. In addition, over-expression of Foxa2 (HNF3ß) in the distal airway epithelium led to decreased levels of CC10 expression in lung epithelium (Zhou et al., 1997
). Because the SP-C promoter drives expression in the distal and not proximal airway epithelium, these reports suggest that disruption of distal airway development and differentiation results in aberrant proximal airway development. In SP-C/G6en mice, normal expression levels of CC10 and Foxj1 in the upper airways indicates that lung development has not been arrested in the pseudoglandular stage of development. In contrast, the number of proximal airways is decreased in these embryos compared to wild-type littermates. In light of these results, it is interesting to note that a previous model of lung development proposed that proximal airway epithelium develops from more distal epithelial cell types through combined proliferation and differentiation events (Weaver et al., 1999
). Because we have not observed any differences in epithelial cell proliferation or programmed cell death in SP-C/G6en mice as compared to wild-type or SP-C/en mice (data not shown), the decreased numbers of CC10-positive airways probably result from a disruption of distal epithelial cell differentiation in SP-C/G6en mice. Low levels of transgene expression in some part of the proximal airways may also confer this phenotype. However, endogenous GATA6 is not expressed in proximal airway epithelium and the SP-C promoter does not drive expression in these cells (Morrisey et al., 1997
; Weaver et al., 1999
; Wert et al., 1993
). Therefore, our results correlate with those observed in a previous study showing that CC10 expression was attenuated in chimeric lung tissue made from Gata6/ ES cells (Keijzer et al., 2001
). However, in contrast to that report, our data suggests that reduced CC10 expression is due to a reduction in proximal airway development and not to direct control of CC10 expression by GATA6, indicating a role for GATA6 in distal-proximal airway epithelial differentiation.
Regulation of AEC-2 cell-specific gene expression and branching morphogenesis by GATA6
The relatively normal expression of SP-A suggests that SP-C/G6en embryos are not arrested at the pseudoglandular stage of development as has been observed in other transgenic models of disrupted lung epithelial differentiation (Zhou et al., 1996a; Zhou et al., 1997
). In contrast, Foxp2 expression was elevated during late gestation and SP-C expression was attenuated, indicating aberrant AEC-2 development. These data suggest a model in which GATA6 plays an important role in certain aspects of AEC-2 development, in particular the regulation of SP-C at the transcriptional level (Fig. 12). These results also support a hypothesis wherein inhibition of GATA6 function leads to a broader and earlier inhibition of lung epithelial cell differentiation that results in the later observed defects in proximal airway development and AEC-1 cell differentiation. Although the human SP-C promoter does not produce high levels of transgene expression until E15.5 and later (Wert et al., 1993
), tissue-restricted inactivation of the mouse Gata6 gene will be required to discern the difference between an early and broad role and/or a later and more specific role for GATA6 in lung epithelial differentiation as is suggested by our results.
SP-C expression is initiated as early as E11.0 in the mouse where it is restricted to the distal tips of developing airway epithelium (Wert et al., 1993; Zhou et al., 1996b
). Later in development SP-C expression is restricted to AEC-2 cells in the lung (Zhou et al., 1996b
). The transcriptional regulation of SP-C has been previously examined and several lung restricted transcription factors have been implicated in its regulation including TTF-1 and Foxa2 (Glasser et al., 2000
; Glasser et al., 1991
; Wert et al., 1993
). A recent study has shown that SP-C expression was down-regulated in chimeric lung tissue derived from Gata6/ ES cells (Keijzer et al., 2001
) and our results corroborate this finding. These observations are supported by the presence of conserved GATA factor DNA binding sites in the proximal promoter region of both mouse and human SP-C genes, which can bind GATA6 and mediate GATA6-dependent trans-activation of these promoters (data not shown). Another study showed that over-expression of GATA6 in distal lung epithelium resulted in decreased levels of SP-C expression (Koutsourakis et al., 2001
). Together, these data indicate that wild-type GATA6 activity is essential for SP-C expression.
Another transcription factor that has been shown to be essential for SP-C expression in a loss-of-function experiment is TTF-1 (Minoo et al., 1999). TTF-1 null mice exhibit arrested lung development and lack expression of SP-C (Kimura et al., 1996
; Minoo et al., 1999
). Interestingly, TTF-1 (Nkx2.1) belongs to the same family of homeodomain proteins as Nkx2.5, an important regulator of cardiac gene expression, which has been shown to interact and synergize with GATA4 to activate cardiac-specific gene expression (Durocher et al., 1997
; Sepulveda et al., 1998
). It will be interesting to determine whether GATA6 and TTF-1 are also capable of physically interacting and synergistically regulating lung epithelial gene expression.
SP-C/G6en embryos also exhibited a reduction in airway branching morphogenesis as shown by the dilated distal airways containing fewer interalveolar septa at E17.5 and E19.5. Branching morphogenesis is a complex process that is regulated by both autocrine/paracrine signaling pathways and regulation of gene expression at the transcriptional level (for reviews, see Hogan et al., 1997; Metzger and Krasnow, 1999
). Thus, this process is extremely sensitive to both cell intrinsic and extrinsic perturbations. Many in vitro and in vivo loss-of-function models of lung development involve some level of disrupted branching morphogenesis (Arman et al., 1999
; Bellusci et al., 1997
; Miettinen et al., 1997
; Pepicelli et al., 1998
; Serra et al., 1994
; Volpe et al., 2000
). These findings are not surprising since airway branching and epithelial cell differentiation and development are closely linked (Warburton et al., 2000
). Therefore, GATA6 may regulate lung branching morphogenesis directly through pertinent signaling pathways, but it is equally possible that disrupted branching observed in SP-C/G6en embryos is a secondary effect of disrupted distal and proximal airway differentiation.
In summary, SP-C/G6en transgenic embryos exhibit a unique phenotype during late lung epithelial development, leading to a failure of AEC-1 cell differentiation and disruption in proximal airway development. The SP-C/G6en transgenic model provides key information on the role of GATA6 during lung development, both during late processes (AEC-1 differentiation) and earlier epithelial development (SP-C gene regulation). Disrupted proximal airway development in these embryos also suggests that GATA6 plays an important role in the distal-proximal differentiation of lung airway epithelium.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Arman, E., Haffner-Krausz, R., Gorivodsky, M. and Lonai, P. (1999). Fgfr2 is required for limb outgrowth and lung-branching morphogenesis. Proc. Natl. Acad. Sci. USA 96, 11895-11899.
Badiani, P., Corbella, P., Kioussis, D., Marvel, J. and Weston, K. (1994). Dominant interfering alleles define a role for c-Myb in T-cell development. Genes Dev. 8, 770-782.[Abstract]
Bellusci, S., Grindley, J., Emoto, H., Itoh, N. and Hogan, B. L. (1997). Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124, 4867-4878.
Bohinski, R. J., Di Lauro, R. and Whitsett, J. A. (1994). The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol. Cell. Biol. 14, 5671-5681.[Abstract]
Braun, H. and Suske, G. (1998). Combinatorial action of HNF3 and Sp family transcription factors in the activation of the rabbit uteroglobin/CC10 promoter. J. Biol. Chem. 273, 9821-9828.
Bruno, M. D., Bohinski, R. J., Huelsman, K. M., Whitsett, J. A. and Korfhagen, T. R. (1995). Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1. J. Biol. Chem. 270, 6531-6536.
Bruno, M. D., Korfhagen, T. R., Liu, C., Morrisey, E. E. and Whitsett, J. A. (2000). GATA-6 activates transcription of surfactant protein A. J. Biol. Chem. 275, 1043-1049.
Clevidence, D. E., Overdier, D. G., Peterson, R. S., Porcella, A., Ye, H., Paulson, K. E. and Costa, R. H. (1994). Members of the HNF-3/forkhead family of transcription factors exhibit distinct cellular expression patterns in lung and regulate the surfactant protein B promoter. Dev. Biol. 166, 195-209.[Medline]
Costa, R. H., Kalinichenko, V. V. and Lim, L. (2001). Transcription factors in mouse lung development and function. Am. J. Physiol. Lung Cell. Mol. Physiol. 280, L823-838.
Durocher, D., Charron, F., Warren, R., Schwartz, R. J. and Nemer, M. (1997). The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 16, 5687-5696.
Evans, M. J., Cabral, L. J., Stephens, R. J. and Freeman, G. (1975). Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp. Mol. Pathol. 22, 142-150.[Medline]
Fu, Y., Yan, W., Mohun, T. J. and Evans, S. M. (1998). Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in a functionally redundant manner. Development 125, 4439-4449.
Glasser, S. W., Burhans, M. S., Eszterhas, S. K., Bruno, M. D. and Korfhagen, T. R. (2000). Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 278, L933-945.
Glasser, S. W., Korfhagen, T. R., Wert, S. E., Bruno, M. D., McWilliams, K. M., Vorbroker, D. K. and Whitsett, J. A. (1991). Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice. Am. J. Physiol. 261, L349-356.
Hogan, B. L., Grindley, J., Bellusci, S., Dunn, N. R., Emoto, H. and Itoh, N. (1997). Branching morphogenesis of the lung: new models for a classical problem. Cold Spring Harb. Symp. Quant. Biol. 62, 249-256.[Medline]
Ikeda, K., Shaw-White, J. R., Wert, S. E. and Whitsett, J. A. (1996). Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Mol. Cell. Biol. 16, 3626-3636.[Abstract]
Kaestner, K. H., Hiemisch, H., Luckow, B. and Schutz, G. (1994). The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution. Genomics 20, 377-385.[Medline]
Kalinichenko, V. V., Lim, L., Stolz, D. B., Shin, B., Rausa, F. M., Clark, J., Whitsett, J. A., Watkins, S. C. and Costa, R. H. (2001). Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the forkhead box f1 transcription factor. Dev. Biol. 235, 489-506.[Medline]
Karaplis, A. C., Luz, A., Glowacki, J., Bronson, R. T., Tybulewicz, V. L., Kronenberg, H. M. and Mulligan, R. C. (1994). Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev. 8, 277-289.[Abstract]
Keijzer, R., van Tuyl, M., Meijers, C., Post, M., Tibboel, D., Grosveld, F. and Koutsourakis, M. (2001). The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development 128, 503-511.
Kimura, S., Hara, Y., Pineau, T., Fernandez-Salguero, P., Fox, C. H., Ward, J. M. and Gonzalez, F. J. (1996). The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 10, 60-69.[Abstract]
Koutsourakis, M., Keijzer, R., Visser, P., Post, M., Tibboel, D. and Grosveld, F. (2001). Branching and differentiation defects in pulmonary epithelium with elevated Gata6 expression. Mech. Dev. 105, 105-114.[Medline]
Krane, C. M., Towne, J. E. and Menon, A. G. (1999). Cloning and characterization of murine Aqp5: evidence for a conserved aquaporin gene cluster. Mamm. Genome 10, 498-505.[Medline]
Kreda, S. M., Gynn, M. C., Fenstermacher, D. A., Boucher, R. C. and Gabriel, S. E. (2001). Expression and localization of epithelial aquaporins in the adult human lung. Am. J. Respir. Cell. Mol. Biol. 24, 224-234.
Lee, M. D., King, L. S., Nielsen, S. and Agre, P. (1997). Genomic organization and developmental expression of aquaporin-5 in lung. Chest 111, 111S-113S.
Liang, Q., De Windt, L. J., Witt, S. A., Kimball, T. R., Markham, B. E. and Molkentin, J. D. (2001). The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J. Biol. Chem. 276, 30245-30253.
Lu, M. M., Yang, H., Zhang, L., Shu, W., Blair, D. G. and Morrisey, E. E. (2001). The BMP antagonist gremlin regulates proximal-distal patterning of the lung. Dev. Dyn. 222, 667-680.[Medline]
Ma, T., Song, Y., Gillespie, A., Carlson, E. J., Epstein, C. J. and Verkman, A. S. (1999). Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J. Biol. Chem. 274, 20071-20074.
Mahlapuu, M., Enerback, S. and Carlsson, P. (2001). Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128, 2397-2406.
Mahlapuu, M., Pelto-Huikko, M., Aitola, M., Enerback, S. and Carlsson, P. (1998). FREAC-1 contains a cell-type-specific transcriptional activation domain and is expressed in epithelial-mesenchymal interfaces. Dev. Biol. 202, 183-195.[Medline]
Margana, R. K. and Boggaram, V. (1997). Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell- specific activation of SP-B gene transcription. J. Biol. Chem. 272, 3083-3090.
Mendelson, C. R. (2000). Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu. Rev. Physiol. 62, 875-915.[Medline]
Merika, M. and Orkin, S. H. (1995). Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and EKLF. Mol. Cell Biol. 15, 2437-2447.[Abstract]
Metzger, R. J. and Krasnow, M. A. (1999). Genetic control of branching morphogenesis. Science 284, 1635-1639.
Miettinen, P. J., Warburton, D., Bu, D., Zhao, J. S., Berger, J. E., Minoo, P., Koivisto, T., Allen, L., Dobbs, L., Werb, Z. et al. (1997). Impaired lung branching morphogenesis in the absence of functional EGF receptor. Dev. Biol. 186, 224-236.[Medline]
Minoo, P., Su, G., Drum, H., Bringas, P. and Kimura, S. (1999). Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(/) mouse embryos. Dev. Biol. 209, 60-71.[Medline]
Miura, N., Kakinuma, H., Sato, M., Aiba, N., Terada, K. and Sugiyama, T. (1998). Mouse forkhead (winged helix) gene LUN encodes a transactivator that acts in the lung. Genomics 50, 346-356.[Medline]
Morrisey, E. E., Ip, H. S., Lu, M. M. and Parmacek, M. S. (1996). GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev. Biol. 177, 309-322.[Medline]
Morrisey, E. E., Ip, H. S., Tang, Z., Lu, M. M. and Parmacek, M. S. (1997). GATA-5: a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development. Dev. Biol. 183, 21-36.[Medline]
Morrisey, E. E., Tang, Z., Sigrist, K., Lu, M. M., Jiang, F., Ip, H. S. and Parmacek, M. S. (1998). GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev. 12, 3579-3590.
Nielsen, S., King, L. S., Christensen, B. M. and Agre, P. (1997). Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am. J. Physiol. 273, C1549-1561.[Medline]
Pepicelli, C. V., Lewis, P. M. and McMahon, A. P. (1998). Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr. Biol. 8, 1083-1086.[Medline]
Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., DAgati, V., Orkin, S. H. and Costantini, F. (1991). Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349, 257-260.[Medline]
Ramirez, M. I., Cao, Y. X. and Williams, M. C. (1999). 1.3 kilobases of the lung type I cell T1alpha gene promoter mimics endogenous gene expression patterns during development but lacks sequences to enhance expression in perinatal and adult lung. Dev. Dyn. 215, 319-331.[Medline]
Ramirez, M. I., Chung, U. I. and Williams, M. C. (2000). Aquaporin-5 expression, but not other peripheral lung marker genes, is reduced in PTH/PTHrP receptor null mutant fetal mice. Am. J. Respir. Cell. Mol. Biol. 22, 367-372.
Ramirez, M. I., Rishi, A. K., Cao, Y. X. and Williams, M. C. (1997). TGT3, thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of T1alpha, a lung alveolar type I cell gene. J. Biol. Chem. 272, 26285-26294.
Sepulveda, J. L., Belaguli, N., Nigam, V., Chen, C. Y., Nemer, M. and Schwartz, R. J. (1998). GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression. Mol. Cell. Biol. 18, 3405-3415.
Serra, R., Pelton, R. W. and Moses, H. L. (1994). TGF beta 1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures. Development 120, 2153-2161.
Shaw-White, J. R., Bruno, M. D. and Whitsett, J. A. (1999). GATA-6 activates transcription of thyroid transcription factor-1. J. Biol. Chem. 274, 2658-2664.
Shu, W., Yang, H., Zhang, L., Lu, M. M. and Morrisey, E. E. (2001). Characterization of a new subfamily of winged-helix/forkhead (Fox) genes which are expressed in the lung and act as transcriptional repressors. J. Biol. Chem. 276, 27488-27497.
Simon, M. C. (1995). Gotta have GATA. Nat. Genet. 11, 9-11.[Medline]
Soudais, C., Bielinska, M., Heikinheimo, M., MacArthur, C. A., Narita, N., Saffitz, J. E., Simon, M. C., Leiden, J. M. and Wilson, D. B. (1995). Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development 121, 3877-3888.
Ten Have-Opbroek, A. A. (1991). Lung development in the mouse embryo. Exp. Lung Res. 17, 111-130.[Medline]
Tichelaar, J. W., Lim, L., Costa, R. H. and Whitsett, J. A. (1999a). HNF-3/forkhead homologue-4 influences lung morphogenesis and respiratory epithelial cell differentiation in vivo. Dev. Biol. 213, 405-417.[Medline]
Tichelaar, J. W., Lu, W. and Whitsett, J. A. (2000). Conditional expression of fibroblast growth factor-7 in the developing and mature lung. J. Biol. Chem. 275, 11858-11864.
Tichelaar, J. W., Wert, S. E., Costa, R. H., Kimura, S. and Whitsett, J. A. (1999b). HNF-3/forkhead homologue-4 (HFH-4) is expressed in ciliated epithelial cells in the developing mouse lung. J. Histochem. Cytochem. 47, 823-832.
Ting, C. N., Olson, M. C., Barton, K. P. and Leiden, J. M. (1996). Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 384, 474-478.[Medline]
Toonen, R. F., Gowan, S. and Bingle, C. D. (1996). The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene. Biochem. J. 316, 467-473.[Medline]
Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W. and Orkin, S. H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371, 221-226.[Medline]
Volpe, M. V., Vosatka, R. J. and Nielsen, H. C. (2000). Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung. Biochim. Biophys. Acta 1475, 337-345.[Medline]
Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K. D. and Cardoso, W. V. (2000). The molecular basis of lung morphogenesis. Mech. Dev. 92, 55-81.[Medline]
Weaver, M., Yingling, J. M., Dunn, N. R., Bellusci, S. and Hogan, B. L. (1999). Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 126, 4005-4015.
Wert, S. E., Glasser, S. W., Korfhagen, T. R. and Whitsett, J. A. (1993). Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev. Biol. 156, 426-443.[Medline]
Zhao, J., Chen, H., Peschon, J. J., Shi, W., Zhang, Y., Frank, S. J. and Warburton, D. (2001). Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Dev. Biol. 232, 204-218.[Medline]
Zhou, L., Dey, C. R., Wert, S. E. and Whitsett, J. A. (1996a). Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene. Dev. Biol. 175, 227-238.[Medline]
Zhou, L., Dey, C. R., Wert, S. E., Yan, C., Costa, R. H. and Whitsett, J. A. (1997). Hepatocyte nuclear factor-3beta limits cellular diversity in the developing respiratory epithelium and alters lung morphogenesis in vivo. Dev. Dyn. 210, 305-314.[Medline]
Zhou, L., Lim, L., Costa, R. H. and Whitsett, J. A. (1996b). Thyroid transcription factor-1, hepatocyte nuclear factor-3beta, surfactant protein B, C, and Clara cell secretory protein in developing mouse lung. J. Histochem. Cytochem. 44, 1183-1193.