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Am J Physiol Lung Cell Mol Physiol 291: L683-L693, 2006. First published May 12, 2006; doi:10.1152/ajplung.00497.2005
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Ectopic expression of C/EBP{alpha} in the lung epithelium disrupts late lung development

Tove Berg, Lukas Didon, and Magnus Nord

Division for Respiratory Medicine, Department of Medicine, Karolinska Institute, Lung Research Laboratory, Karolinska University Hospital-Solna, Stockholm, Sweden

Submitted 28 November 2005 ; accepted in final form 7 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The lung develops from the endoderm through a process of branching morphogenesis. This process is highly active during the pseudoglandular stage of lung development and continues into the canalicular stage, resulting in the formation of terminal sacs. CCAAT/enhancer binding proteins (C/EBPs) are transcription factors regulating central aspects of differentiation and proliferation. We report here the developmental expression of C/EBP{alpha}, -beta, and -{delta} in the lung. C/EBP{alpha} exhibits a dynamic expression pattern and is first detected during the late pseudoglandular stage. At this stage, expression is observed in a subset of epithelial cells in the distal parts of the branching tubules. The expression of C/EBP{alpha} is confined to nonproliferating cells. To examine the role of C/EBP{alpha} in lung development, we generated transgenic mice ectopically expressing C/EBP{alpha} in the lung epithelium using the human surfactant protein C promoter. Lungs from these mice were of normal size but exhibited a phenotype characterized by fewer and larger developing epithelial tubules, indicating that the branching process was affected. No effects on overall proliferation or cellular differentiation were observed. When this phenotype was compared with that of mice carrying a targeted mutation of the Cebpa gene, the Cebpa–/– mice exhibited a similar developmental phenotype. In conclusion, our results show a role for C/EBP{alpha} in lung development and suggest a function in the later stages of lung branching morphogenesis.

CCAAT/enhancer binding proteins; transcription factors; expression pattern; branching morphogenesis; cellular differentiation

MOUSE LUNG DEVELOPMENT starts at embryonic day 9.5 (E9.5), when two primordial buds appear in the ventral foregut endoderm just anterior to the developing stomach. This is followed by growth and branching of the primitive lung endoderm in a process of branching morphogenesis dependent on reciprocal interactions between the endoderm and surrounding mesenchyme. It eventually leads to the development of the highly branched organ that constitutes the mature lung, where the conducting airways lead the inhaled air into the respiratory alveolar region. For reviews on lung development, see, for example, Refs. 9, 14, , 52, and 60.

Lung development can be histologically divided into four stages. The first stage, the pseudoglandular period, reaches from E9.5 to E16.5 in the mouse. During this period of active branching morphogenesis, the respiratory tree is formed from the primordial buds and lined with epithelial precursor cells. Coincident with growth and branching, the lung undergoes proximal-distal patterning. At the end of the pseudoglandular period, extensive cellular differentiation occurs. This results in a change of appearance, and structurally the developing lung is described as having entered the next histological stage, the canalicular period (E16.5–E17.5). In parallel with this burst of cellular differentiation in the late pseudoglandular and canalicular stages, the distal epithelium continues to branch through a process morphologically different from the preceding branching morphogenesis (45). This results in the formation of terminal sacs, and the following stage is referred to as the saccular, or terminal sac, period and spans E17.5 to postnatal day 5 (P5) in the mouse. During these later stages of lung development, differentiation of the epithelial precursor cells leads to the formation of the various cell types lining the proximal and distal airways. This differentiation is reflected by the onset of expression of differentiation-dependent genes necessary for respiration, most notably components of the surfactant system (35, 63). In parallel, future air spaces widen, interstitial tissue thins out, and the vasculature and capillary bed mature. Together, this serves to prepare the lung for respiration after birth. However, complete alveolarization, with septation of the air spaces, finishes after birth during the fourth stage of lung development, the alveolar period (P5–P30 in the mouse).

CCAAT/enhancer binding proteins (C/EBPs) are a family of related basic region leucine zipper transcription factors regulating central aspects of differentiation such as gene expression and proliferation; for recent reviews, see, for example, Refs. 12 and 47. Typically, C/EBPs are involved in stimulating the transcription of genes characteristic of the mature differentiated organ, and their critical role in these processes has been well established in the liver and fat (7, 15, 27, 48). Studies in lung epithelial cell lines have shown that C/EBPs regulate the expression of several lung-enriched genes including surfactant proteins A and D (22, 49), the airway secretory protein secretoglobin 1a1/Clara cell secretory protein (CCSP) (38), and the cytochrome P-450 enzyme Cyp2b1 (11). All these genes exhibit a developmental expression pattern paralleling the extensive cellular differentiation occurring during the canalicular and saccular stages of lung development leading to the formation of the different specialized cell types of the proximal and distal lung.

Because of their highly related structure, considerable overlap in target gene specificity of the different C/EBPs exist, and different C/EBPs can, to a large extent, functionally replace each other with regard to their transactivating functions (47). In contrast, with regard to the effects of different C/EBP members on proliferation, a clear distinction is evident. Here, C/EBP{alpha} is a strong inhibitor of proliferation and is downregulated in proliferating cells, whereas C/EBPbeta and C/EBP{delta} support proliferation (7, 34). These opposing roles are apparent during adipose differentiation, where C/EBPbeta and C/EBP{delta} are expressed during mitotic expansion and C/EBP{alpha} is turned on later, inducing terminal differentiation (8, 57). Similar mechanisms are seen during liver regeneration, where C/EBPbeta and C/EBP{delta} increase and C/EBP{alpha} decreases during the proliferative phase (17, 36).

In this study, we investigated the expression pattern of C/EBP{alpha}, C/EBPbeta, and C/EBP{delta} during lung development. C/EBP{alpha} displayed a dynamic expression pattern with expression initially observed in a subset of cells in the distal parts of the branching tubules. By generating and studying lung development in a transgenic C/EBP{alpha} gain of function mouse model, in combination with a C/EBP{alpha} loss of function mouse model, we found results indicating a role for C/EBP{alpha} in lung development with a function in the later stages of branching morphogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. To generate the construct for the lung-specific C/EBP{alpha} gain of function mouse model [surfactant protein C (Sftpc)-Cebpa], we cloned an EcoRI fragment encoding murine C/EBP{alpha} from pCMVC/EBP{alpha} (10) into the EcoRI site of the 3.7SPC/SV40 vector (a kind gift from Drs. Jeffrey A. Whitsett and Stephan W. Glasser, Cincinnati Children's Hospital Medical Center). In the resulting construct, the 3.7-kb human Sftpc promoter contained in the vector drives the expression of the Cebpa transgene only in embryonic lung epithelial cells. After linearization of the DNA construct with NdeI and NotI, transgenic animals were generated by a standard pronuclear injection into one-cell CBB6F2 embryos. Embryos were obtained at E15.5. For genotyping, DNA was extracted from the placental tissue by a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and PCR was carried out using the following primers: 5'-CGC CAG GAA GAC ACC CAT-3' and 5'-AAA CCT GGG TGA GTT GAG CAA-3'. The C/EBP{alpha} loss of function mice (Cebpa–/– mice) (18) were kindly provided by Drs. Per Flodby and Kleanthis G. Xanthopoulos. Cebpa+/– embryos were obtained by standard procedures from cryopreserved stocks at the Karolinska Institute Mouse Embryo Bank. The strain was kept as heterozygous and intercrossed to obtain Cebpa–/– embryos. Because Cebpa–/– mice are not viable after birth, embryos were analyzed during the embryonic period. For genotyping, DNA was obtained from tail biopsies or, in the case of embryos, from parts of the liver. All other mice were C57BL6. The animal studies were approved by the Southern Stockholm Animal Welfare Ethics Committee and performed in compliance with international guidelines and Swedish law.

Histology and immunohistochemistry. Embryos were isolated from the embryonic stages described. Fixation was done with either PBS with 4% paraformaldehyde (≤E16.5) or PBS with 4% paraformaldehyde plus 0.5% Triton (>E16.5) for 24 h at 4°C. After dehydration and paraffin embedding, 4-µm sections were cut and deparaffinated in xylene followed by rehydration through ethanol to water. Antigen retrieval was performed by microwaving in 10 mM citrate buffer (pH 6.0) for 30 min at 200 W. Endogenous peroxidase activity was blocked by 0.3% H2O2 in PBS for 30–45 min at room temperature. After being washed, sections were blocked in 5% serum in PBS with 0.3% Tween (PBS-T) for 1 h at room temperature. Sections were incubated with primary antibody overnight at 4°C, washed in PBS-T, and incubated with secondary antibody for 1.5 h at room temperature. After being washed in PBS-T, the immune complexes were detected with a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine (DAB) tablets (Sigma, St. Louis, MO); both were used according to instructions from the manufacturer. Sections were briefly counterstained with Mayer's hematoxylin (Histolab, Stockholm, Sweden) and washed in tap water followed by dehydration and mounting. Controls excluding the primary antibody were in all cases negative. For histology, tissues were treated identically as for immunohistochemistry but stained with hematoxylin-eosin (Histolab).

Antibodies. The primary antibodies used were as follows: anti-C/EBP{alpha} (14AAx), anti-C/EBPbeta (C19x), anti-C/EBP{delta} (C-22x), anti-SP-A (N-19), anti-SP-C (M-20), and anti-cyclin A (H-432) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-proliferating cell nuclear antigen (PCNA; Ab-1) from Oncogene Research Products (San Diego, CA); anti-thyroid transcription factor-1 (TTF-1; clone 8G7G3/1) from NeoMarkers (Fremont, CA); anti-{alpha}-smooth muscle actin (clone 1A4) and anti-fibronectin (A0245) from DAKO (Glostrup, Denmark); and anti-phospho-histone H3 (Ser10) from Upstate (Charlottesville, VA). The anti-secretoglobin 1a1/CCSP antibody has been previously described (38), as has the anti-cytokeratin 8 antibody (6). For the detection of apoptotic cells, the APO Direct Kit from BD Pharmigen (San Diego, CA) was used together with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-peroxidase antibody (Roche, Penzberg, Germany). Biotinylated secondary antibodies were all from Vector laboratories.

Immunofluorescence. Sections were treated as above with the following exceptions: anti-C/EBP{alpha} was detected using a biotinylated secondary antibody (Vector) and FITC-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA). Anti-PCNA was detected by an incubation with a secondary Cy3-conjugated antibody (Jackson ImmunoResearch). All washes and antibody dilutions were made in PBS. Slides were mounted with Vectashield Hard Set (Vector Laboratories).

Image analysis. Coronal sections of comparable depth from lungs of six Sftpc-Cebpa transgenic mice and six wild-type littermates were immunohistochemically and hematoxylin stained for morphometric analysis. For Cebpa–/– mice, coronal sections of comparable depth from three Cebpa–/– mice and five wild-type littermates were analyzed. All analyses were carried out as described below using a Nikon Eclipse E1000 microscope with a Nikon Digital camera Dxm 1200 (Nikon, Tokyo, Japan). Pictures were captured and saved by ACT-1 software (Nikon). Total lung area and numbers and areas of the epithelial tubules were measured using Easy Image Analysis 2000 software (Tekno Optik, Stockholm, Sweden). For each animal, eight fields were analyzed. The numbers of tubules were normalized to total lung area, and mean tubule areas were calculated from the total tubule area divided by the total number of tubules. Quantification of the total lung mesenchymal area was carried out by subtracting the airway tubule area from the total lung area. All analyses were blinded and carried out in duplicate. Cells staining positive for cyclin A in the epithelium were counted by hand and normalized to the total number of nuclei to determine the fraction of cyclin A-positive cells. Student's t-test was used to assess statistical significant differences between groups. Significant difference was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
C/EBP{alpha}, C/EBPbeta, and C/EBP{delta} are expressed during lung development. No detailed information exists on the developmental expression pattern of C/EBP transcription factors in lung development. In the lung, C/EBP{alpha}, C/EBPbeta, and C/EBP{delta} are expressed (12), and we therefore investigated their expression pattern during lung development with immunohistochemistry. Lung sections of C57BL6 embryos from pseudoglandular, canalicular, and saccular stages of lung development were stained with antibodies for C/EBP{alpha}, C/EBPbeta, and C/EBP{delta}. We found the expression of both C/EBP{alpha} and C/EBPbeta at the late pseudoglandular stage at E15.5, albeit with different patterns. At this stage, C/EBP{alpha} expression (see Fig. 1, A–C) was mainly detected in the epithelium, and here it was restricted to a subset of cells in the distal parts of the developing tubules. C/EBPbeta was expressed in mesenchymal cells, and expression was also evident in most cells of the epithelium (Fig. 1I). At E17.5, after the pseudoglandular-canalicular transition (Fig. 1E), the expression of C/EBP{alpha} was more widespread in the future alveolar region and also seen in a few cells in the epithelium lining the future distal conducting airways. At this stage, C/EBPbeta continued to be expressed in mesenchymal cells as well as in most cells of the developing epithelium (Fig. 1J). During the saccular stage (Fig. 1F), the expression of C/EBP{alpha} was increased and started to resemble the adult expression pattern. At that time, C/EBP{alpha} exhibited higher levels of expression in the alveolar region as well as expression in the conducting airway epithelium (Fig. 1F). C/EBPbeta and also C/EBP{delta} were expressed in the saccular stage lung, with expression detected in epithelial cells lining the developing conducting airways as well as in cells in the future alveolar region (Figs. 1, K and O). In the adult lung, low-level C/EBP{alpha} expression was detected in most epithelial cells lining the distal conducting airways. In the alveolar region, higher levels of expression were confined to cells suggestive of alveolar type II cells (Fig. 1G). Also, C/EBPbeta (Fig. 1L) and C/EBP{delta} (Fig. 1P) were expressed in the alveolar region as well as in the conducting airways in the adult lung. Compared with C/EBPbeta and C/EBP{delta}, C/EBP{alpha} exhibited a more dynamic expression pattern in the epithelium of the embryonal lung, and we thus included additional time points during development. Compared with the low-level expression detected at E15.5 (Fig. 1, A–C), the specific pattern of C/EBP{alpha} close to the distal tips was more pronounced 1 day later, at E16.5 (Fig. 1D). No C/EBP{alpha} expression could be detected at E13.5 (data not shown). In summary, C/EBP{alpha} exhibits a dynamic pattern of expression during lung development. Initially, during the later pseudoglandular stages, which are characterized by growth and branching of the epithelium, expression of C/EBP{alpha} is observed in a subset of cells in the distal tubules. Later, after the pseudoglandular-canalicular transition, expression increases and exhibits a more widespread pattern in the developing lung epithelium, correlating with the extensive cellular differentiation occurring in this period (12).


Figure 1
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  		Fig. 1. Developmental expression pattern of CCAAT/enhancer binding protein (C/EBP){alpha}, C/EBPbeta, and C/EBP{delta} in the lung. Immunohistochemical staining (DAB; brown color) for C/EBPs during the pseudoglandular, canalicular, and saccular stages of lung development as well as in the adult lung is shown. Sections were slightly counterstained with hematoxylin. H: control excluding primary antibody. Numbers refer to embryonic days (E; plug day: E0.5). In A-F, I-K, and M-O, arrows point out the expression of respective C/EBPs in the distal, future respiratory epithelium; arrowheads indicate the expression in the proximal, future conducting airway epithelium; and open arrowheads indicate C/EBP expression in mesenchymal cells. In G, L, and P, arrows indicate the expression in the respiratory (alveolar) epithelium, and arrowheads indicate the expression in the conducting airway (bronchiolar) epithelium. Bars indicate magnification.

 
Expression of C/EBP{alpha} in nonproliferating cells of the developing lung. C/EBP{alpha} is an inhibitor of proliferation and is absent from proliferating cells in other organs such as the liver and fat (34). To investigate the relationship between cellular proliferation and C/EBP{alpha} expression during lung development, we stained for C/EBP{alpha} together with PCNA as a marker for cell proliferation by immunofluorescence. As shown in Fig. 2, C/EBP{alpha} only rarely colocalized with PCNA in the growing epithelial tubules at E17.5. This suggests that C/EBP{alpha} is expressed only in growth-arrested cells within the epithelium, and this is in agreement with a role for C/EBP{alpha} as an inhibitor of proliferation in lung development as well (18). It is thus possible that the restricted expression of C/EBP{alpha} at these stages of lung development defines cells within the growing epithelial tubules that are not allowed to proliferate. This, together with the dynamic expression pattern of C/EBP{alpha} and the previously demonstrated role for C/EBP{alpha} in regulating lung-specific gene expression (12), suggested to us that C/EBP{alpha} has an important role in lung development.


Figure 2
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  		Fig. 2. Expression of C/EBP{alpha} and the proliferation marker proliferating cell nuclear antigen (PCNA) in the developing mouse lung. A: expression of C/EBP{alpha} at E17.5 detected by immunofluorescence with FITC (green fluorescence). B: expression of PCNA (detected by immunofluorescence with Cy3, red). C: merged image. The yellow color indicates coexpression of C/EBP{alpha} and PCNA.

 
Ectopic expression of C/EBP{alpha} affects lung development. To continue our investigations of the role of C/EBP{alpha} in lung development, we generated transgenic animals ectopically expressing C/EBP{alpha} in the lung epithelium. To drive the expression of the murine Cebpa gene, we used the human 3.7-kb Sftpc promoter (20, 62). This well-characterized promoter is specifically activated in the lung epithelium and gives expression from at least E10. Thus the use of this promoter to drive Cebpa provides an expression of C/EBP{alpha} starting at least 4 days earlier than normal but still epithelium specific. It also gives a more widespread expression than the discrete expression pattern normally seen in the distal epithelium during the later pseudoglandular stages. Sftpc-Cebpa transgenic mice were analyzed during the late pseudoglandular period. Of the 20 transgenic embryos analyzed at E15.5, 6 embryos were found to express the Cebpa transgene. In all cases, expression was specific for the developing lung epithelium, with the highest levels in the distal epithelium (see GoFig. 4A). Lungs from Sftpc-Cebpa transgenic embryos showed an abnormal phenotype that was histologically characterized by a decreased number of growing epithelial tubules. The epithelial tubules were also larger in size compared with wild-type littermates (compare Fig. 3, A and B with C and D). However, the overall size of the lungs was not affected. Quantitative morphology corroborated that ectopic expression of C/EBP{alpha} reduced the number of growing airways and increased their mean area (Fig. 3E).


Figure 3
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  		Fig. 3. Histology of surfactant protein C (SP-C; Sftpc)-Cebpa transgenic and wild-type (WT) littermate lungs. A–D: histological appearance of E15.5 lungs from Sftpc-Cebpa mice (A and B) and WT littermates (C and D). Sections were stained with hematoxylin-eosin. Bars indicate magnification. E: quantitative morphometry of Sftpc-Cebpa transgenic and WT littermate lungs. Numbers and mean areas of the developing epithelial tubules in E15.5 Sftpc-Cebpa and WT littermate lungs were determined in hematoxylin-eosin-stained sections as described in MATERIALS AND METHODS. Values are means ± SD; n = 6. **P < 0.001.

 

Figure 4
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  		Fig. 4. Immunohistochemistry of Sftpc-Cebpa transgenic and WT littermate lungs. A and B: expression of C/EBP{alpha} in E15.5 Sftpc-Cebpa transgenic (A) and WT littermate (B) lungs. C and D: immunohistochemical staining for the lung epithelial cell marker thyroid transcription factor-1 (TTF-1)/Nkx2.1. E–H: stainings for pro-SP-C and cytokeratin 8 (CK-8) as markers for distal and proximal differentiation, respectively. I and J: immunohistochemical staining for the mesenchymal marker {alpha}-smooth muscle actin ({alpha}SMA). K and L: staining for the extracellular matrix protein fibronectin. M–P: immunohistochemical stainings for the proliferation markers phospho (P)-histone H3 (a marker of mitosis) and cyclin A (which is expressed in the S and G2 phases of the cell cycle). For the immunohistochemical stainings, DAB (brown) was used for visualization. All sections except C and D were slightly counterstained with hematoxylin. Bars indicate magnification. Q: quantitative morphometry of cyclin A-positive cells in E15.5 Sftpc-Cebpa transgenic and WT littermate lung. Shown is the fraction of cyclin A-reactive cell nuclei per total number of nuclei in the lung epithelium. Values are means ± SD; n = 6. R: quantitative morphometry of the mesenchymal area in Sftpc-Cebpa transgenic and WT littermate lungs. Total areas of the developing mesenchyme in E15.5 Sftpc-Cebpa transgenic and WT littermate lungs were determined in hematoxylin-eosin-stained sections as described in MATERIALS AND METHODS. Values are means ± SD; n = 6.

 
Effects of ectopic expression of C/EBP{alpha} on cellular differentiation. The first morphological sign of cellular differentiation in the developing lung epithelium is a proximal-distal patterning that results in a subdivision between the future conducting airway epithelium and alveolar epithelium, which occurs in mice at E14.5 (54). Still, an in vivo cell lineage study (42) has indicated that the epithelial precursors for the trachea and large bronchi versus more distal airways are segregated at an even earlier stage (before E11.5). Because C/EBP{alpha} is involved in controlling differentiation and differentiation-dependent gene expression in several organs, we investigated epithelial differentiation in Sftpc-Cebpa transgenic mice. No histological signs of affected differentiation were observed in the lungs of Sftpc-Cebpa mice (Fig. 3), and staining for the lung epithelial marker TTF-1 (Nkx2.1) was evident in all nuclei of the developing epithelial tubules (Fig. 4, C and D). The morphological proximodistal differentiation occurring at E14.5 is accompanied by changes in gene expression (54). To investigate this differentiation step in the transgenic mice, their lungs were stained with antibodies against markers for proximodistal differentiation. As seen in Fig. 4, E and F, staining for the distal marker pro-surfactant protein C was evident in the future alveolar region only, in both Sftpc-Cebpa mice and wild-type littermates, and expression of the proximal marker cytokeratin 8 was confined to the future conducting airway epithelium in both wild-type and transgenic lungs (Fig. 4, G and H). Thus this indicates that proximal-distal differentiation of the lung epithelium was intact. To investigate whether ectopic expression of C/EBP{alpha} resulted in premature differentiation, we analyzed the expression pattern of surfactant protein A as a marker for differentiation of the alveolar epithelium and secretoglobin 1a1/CCSP as a marker for the conducting airway epithelium using immunohistochemistry. Both of these are known target genes for C/EBP{alpha} in the lung epithelium, and neither is normally expressed at E15.5. No premature expression of these genes could be seen in the lungs of Sftpc-Cebpa mice (data not shown). We also investigated the effects of ectopic C/EBP{alpha} expression on the development of the mesenchyme. As seen in Fig. 4, I and J, staining for the mesenchymal marker {alpha}-smooth muscle actin showed a pattern reflecting the epithelial phenotype with fewer and larger developing tubules; however, within the mesenchymal compartment, the expression pattern was not affected. Moreover, morphometric quantification of the lung mesenchymal area showed that it was not altered in Sftpc-Cebpa transgenic mice compared with their wild-type littermates (Fig. 4R). Thus, together, this indicates that mesenchymal development was not primarily affected by transgenic overexpression of C/EBP{alpha} in the lung epithelium.

Several mechanisms could underlie the structural phenotype caused by the ectopic expression of C/EBP{alpha}, i.e., fewer and larger developing epithelial tubules. Because C/EBP{alpha} inhibits proliferation (34), one possibility could be that the ectopic expression of C/EBP{alpha} induces a general growth inhibition in the epithelium and thus affects development. This seems unlikely, because an overall growth inhibition would be expected to lead to smaller lungs, which was not observed. In addition, a general growth inhibition in the epithelium would be expected to result in an unaltered, or perhaps a decreased, number of tubules smaller in size instead of the observed decreased number of growing epithelial tubules larger in size. Still, to investigate whether an overall growth inhibition had occurred, we stained Sftpc-Cebpa transgenic and wild-type lungs for the proliferation markers phospho-histone H3, a marker for mitosis, and cyclin A, which is expressed in the S and G2 phases of the cell cycle. At E15.5, a large proportion of epithelial cells was proliferating as evidenced by cyclin A reactivity (Fig. 4, O and P). As shown in Fig. 4Q, the overall number of proliferating cells was not different between transgenic and wild-type lungs. As expected, a smaller fraction of cells was positive for the mitosis marker phospho-histone H3. Also with regard to this proliferation marker, no differences were seen between wild-type and transgenic lungs (Fig. 4, M and N). Because the expression of C/EBP{alpha} in lung cell lines has been reported to induce apoptosis (21), we also investigated apoptosis by TUNEL staining. However, no differences in the numbers of apoptotic cells could be detected between Sftpc-Cebpa and wild-type littermate lungs (data not shown). This shows that an overall growth inhibition, or an altered rate of apoptosis, caused by ectopic expression of C/EBP{alpha} is not likely to underlie the observed phenotype with fewer and larger developing epithelial tubules. An alternative explanation could be that the branching process during the later stages of lung branching morphogenesis is affected by the ectopic C/EBP{alpha} expression. This would then cause insufficient formation of branches leading to the observed phenotype with fewer and larger epithelial tubules. We also investigated the deposition of the extracellular matrix protein fibronectin in the embryonic lungs. Fibronectin is secreted by the developing lung epithelium and has been shown to be important for lung branching morphogenesis (51). As seen in Fig. 4, K and L, fibronectin deposition in Sftpc-Cebpa transgenic lungs displayed an abnormal pattern characterized by bundles of increased thickness around the enlarged developing epithelial tubules. Because an abnormal pattern of fibronectin deposition has previously been shown to be associated with disturbed branching (16), this adds support to the hypothesis that ectopic C/EBP{alpha} expression in the epithelium affects the later stages of branching morphogenesis leading to insufficient formation of branches and the observed phenotype with fewer and larger epithelial tubules.

Mice lacking C/EBP{alpha} have defects in late embryonal lung development. To continue investigating the role of C/EBP{alpha} in lung development, we investigated lung development in mice carrying a targeted mutation of the gene for C/EBP{alpha} (Cebpa–/– mice). Cebpa–/– mice have developmental defects in their liver and adipose tissue and die within a few hours after birth due to hypoglycemia (18, 59). As previously described, a lung phenotype is present in Cebpa–/– mice at birth; their lungs exhibit alveolar abnormalities characterized by a hyperproliferation of epithelial cells (18). However, embryonal lung development in Cebpa–/– mice has not been studied in detail. We thus investigated the lungs of Cebpa–/– mice during the developmental period compared with wild-type and Cebpa+/– littermates. At E16.5, the first indications of a difference were visible, and, at E17.5, a phenotype could readily be observed in Cebpa–/–mice. This phenotype was characterized by a decreased number of developing airways (Fig. 5, A, B, D, and E). The airway spaces were also larger in size. The morphometric analyses shown in Fig. 5G further corroborated a structural phenotype characterized by fewer developing airways with an increased mean area. At later developmental stages, the lung phenotype seen at E17.5 was obscured by the hyperproliferation of epithelial cells in the distal respiratory portion of the developing lung. This latter phenotype has already been described in Cebpa–/– mice and most probably accounts for the immediate lethality observed in a fraction of newborn Cebpa–/– mice (18, 59). To investigate whether the absence of C/EBP{alpha} in Cebpa–/– mice affected proximal-distal patterning of the lung epithelium, the expression of pro-surfactant protein C was investigated. As seen in Fig. 5, C and F, staining for pro-surfactant protein C was evident in the distal, future alveolar region only in both Cebpa–/– mice and wild-type littermates. This indicates that proximodistal differentiation of the lung epithelium was intact. No phenotype was observed in heterozygous Cebpa+/– mice. Thus the lungs of Sftpc-Cebpa transgenic embryos and Cebpa–/– knockout embryos display a similar phenotype. This shows that both ectopic C/EBP{alpha} expression and loss of C/EBP{alpha} expression affect the later stages of lung development. Together, this underlines the need for C/EBP{alpha} to be expressed at the right level, location, and time during lung development and demonstrates a role for C/EBP{alpha} in the later stages of lung development.


Figure 5
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  		Fig. 5. Histology and immunohistochemistry of lungs from Cebpa–/– mice. A, B, D, and E: hematoxylin-eosin staining of E16.5 (A and D) and E17.5 (B and E) lungs from Cebpa–/– mice and WT littermates. C and F: Immunohistochemical staining (DAB, brown) for pro-SP-C as a marker for distal differentiation (sections were slightly counterstained with hematoxylin). Bars indicate magnification. G: quantitative morphometry of lungs from Cebpa–/– mice. Numbers and mean areas of the developing epithelial tubules in E17.5 Cebpa–/– and WT littermate lungs were determined in hematoxylin-eosin-stained sections as described in MATERIALS AND METHODS. Values are means ± SD; n = 3–5. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This study shows that the transcription factor C/EBP{alpha} exhibits a dynamic expression pattern during lung development. Expression is first detected during the late pseudoglandular stage in a subset of cells in the distal tubules, an area that actively grows and branches during this period, and expression is confined to nonproliferating cells. To examine the role of C/EBP{alpha} in lung development, we generated and studied mice ectopically expressing C/EBP{alpha} in the lung epithelium. At the late pseudoglandular stage, lungs from these transgenic mice were characterized by fewer and larger growing epithelial tubules. However, there were no signs of disturbed differentiation or overall proliferation. When we studied lung development in knockout mice lacking C/EBP{alpha}, we found a similar phenotype as in the Sftpc-Cebpa transgenic mice with fewer and larger developing airways. Together, these results indicate a role for C/EBP{alpha} in the later stages of lung development.

C/EBP{alpha} stimulates the transcription of many genes expressed in a tissue-specific and differentiation-dependent manner (5, 47). In addition, it has a role in regulating growth by inhibiting proliferation (34). This inhibition of proliferation appears independent of the transactivating functions of C/EBP{alpha}. The cell cycle inhibition is instead mediated via direct protein interactions with cell cycle regulators at several levels, for instance, by interacting with the cdk inhibitor p21 (55) and by binding to the cyclin-dependent kinases cdk2 and 4 (58) as well as to the E2F transcription factor (44). Findings indicating C/EBP{alpha} as a regulator of cell growth in the lung include observations that C/EBP{alpha} is absent in many cancer forms including lung cancers, suggesting that CEBP{alpha} is a potential tumour suppressor gene (21, 40). In this study, we found that C/EBP{alpha} is not normally expressed in proliferating cells, supporting a role for this transcription factor in the control of proliferation in the lung epithelium. However, overexpression of C/EBP{alpha} in the lung epithelium did not seem to have any inhibitory effect on overall proliferation. This indicates that forced expression of C/EBP{alpha} is not enough to cause complete block of proliferation on its own. Rather, it seems as if C/EBP{alpha} needs to act in concert with other factors that may not be expressed at these developmental stages. A possible such factor could be the cdk inhibitor p21. During lung development, p21 expression in the distal lung is first detected in the late pseudoglandular and canalicular stages (25). Because p21 is involved in mediating the antiproliferative effects of C/EBP{alpha} (56), overexpression of C/EBP{alpha} will not be able to arrest proliferation at earlier time points when p21 is absent, explaining why we failed to see an overall growth inhibition in Sftpc-Cebpa transgenic mice. Another possible explanation for the lack of growth inhibition could be that overexpression of C/EBP{alpha} causes the upregulation of other factors supporting proliferation, such as, for instance, C/EBPbeta and C/EBP{delta} (7, 34). The opposing effect of C/EBPbeta and C/EBP{delta} on proliferation could thus balance the growth inhibitory effect of C/EBP{alpha}, resulting in unchanged overall proliferation. However, we were unable to detect any upregulation of C/EBPbeta and C/EBP{delta} in Sftpc-Cebpa transgenic lungs (data not shown), indicating that this mechanism does not explain the lack of growth inhibition in Sftpc-Cebpa transgenic mice.

In adipose cells as well as in cells of the myeloid lineages, liver, and lung, the induction of C/EBP{alpha} has been demonstrated to be sufficient to induce differentiation and/or to commit cells to differentiate into a predestined fate (19, 21, 23, 26, 29, 46, 57). Lack of C/EBP{alpha} has previously also been found to cause failure of complete differentiation of alveolar type II cells that start to overgrow the alveolar region in Cebpa–/– mice (18). The period of extensive cellular differentiation occurring after the pseudoglandular-canalicular transition in the lung (9, 12, 35, 45) also corresponds well to the point when expression of C/EBP{alpha} increases and exhibits a more widespread pattern in the developing lung epithelium. We therefore examined at the ability of forced C/EBP{alpha} expression to promote differentiation of the lung epithelium. We stained for several markers for lung epithelial differentiation, including TTF-1, surfactant protein C, cytokeratin 8, surfactant protein A, and secretoglobin1a1/CCSP. However, we could not detect any differences with regard to the expression pattern of these molecular differentiation markers. This indicates that C/EBP{alpha} cannot induce differentiation on its own in the lung. The absence of stimulatory effects on surfactant protein A and secretoglobin1a1/CCSP was especially surprising because both of these genes have been demonstrated to be regulated directly by C/EBP{alpha} (13, 49), further underscoring that C/EBP{alpha} needs to act together with additional factors to promote cellular differentiation in the lung epithelium. Recently, the results of lung-specific inactivation of C/EBP{alpha} in mice have been reported (1, 31). The resulting phenotype is characterized by neonatal lethality from respiratory failure and impaired lung maturation with a block of alveolar type II cell differentiation, decreased levels of surfactant proteins and lipids, and increased epithelial cell number in the alveolar region. Thus these loss of function models clearly show that C/EBP{alpha} is necessary for cellular differentiation of the distal lung epithelium. However, the present results, providing the corresponding gain of function model, indicate that even though C/EBP{alpha} is necessary for differentiation, it is not sufficient for lung epithelial differentiation.

Several mechanisms could underlie the structural phenotype caused by ectopic expression of C/EBP{alpha} with fewer and larger developing epithelial tubules. A general inhibition of growth, or an altered rate of apoptosis, seems not to be the underlying cause because no changes in overall proliferation, apoptosis, or lung size were observed. An alternative explanation for the decreased number of epithelial branches could be that the ectopic expression of C/EBP{alpha} interferes with the branching process. The restricted expression of C/EBP{alpha} close to the tips of the growing epithelial tubules, i.e., in locations of active branching, along with the abnormal pattern of fibronectin deposition, indicate that C/EBP{alpha} might have a role in the processes of branching. Because C/EBP{alpha} was found in nonproliferating cells, it is possible that C/EBP{alpha}, due to its antiproliferative activities, regulates the balance between dividing and nondividing cells in the distal parts of the growing epithelial tubules. The balance of cellular proliferation in the tubules is likely to be important for the branching process. Ectopic expression of C/EBP{alpha} could then be expected to disturb the balance between dividing cells and nondividing cells and result in a less controlled, more diffuse growth of the epithelium. This would lead to the establishment of fewer branches and the observed phenotype with fewer and larger epithelial tubules. Similarly, the lack of C/EBP{alpha}, as in Cebpa–/– mice, would also be expected to disturb the balance of proliferation in the epithelial tubules, causing insufficient formation of branches and diffuse growth of the epithelium, subsequently leading to fewer and larger airways. This hypothesis thus provides a possible explanation for the similar phenotypes of the Sftpc-Cebpa transgenic gain of function model and the Cebpa–/– loss of function model. However, even though the present results are consistent with a hypothesis along these lines, further studies will be needed to establish the specific function for C/EBP{alpha} in lung branching morphogenesis.

Still, the findings that both overexpressing C/EBP{alpha} and losing C/EBP{alpha} expression disturbed branching indicate that C/EBP{alpha} needs to be expressed at the correct level, location, and time, underscoring the need for an exact and fine-tuned regulation of C/EBP{alpha} during lung development. This raises the question of which upstream regulators regulate C/EBP{alpha} during lung development. In the past few years, significant progress has been made in understanding the genetic and molecular control of lung branching (reviewed, for instance, in Refs. 9, 14, 24, 35, 52, and 60). Several key players that mediate epithelial-mesenchymal interactions have been identified, including members of the hedgehog (2, 30, 41), fibroblast growth factor (Fgf) (3, 43), and bone morphogenetic protein families (4, 61). The studies indicating a critical role for these factors have primarily focused on the earlier branching processes. Less is known about the function and expression pattern of these molecules during the branching morphogenesis occurring during the late pseudoglandular and canalicular stages. This branching also appears morphologically different from the branching morphogenesis of the preceding stages (45) and results in the formation of terminal sacs. Still, it is well possible that these factors continue to have a role throughout lung branching and therefore could be upstream regulators of C/EBP{alpha} expression. Alternatively, other factors could be of importance during the later stages of branching. Several members of the Wnt family of signaling molecules have been demonstrated to be important for lung development (28, 37, 53), and, using Wnt signaling reporter mice, dynamic Wnt signaling was recently demonstrated in the developing lung epithelium (39). Most notably, at E15.5–E18.5, Wnt signaling decreases specifically in the distal epithelium, in striking correlation with the onset of C/EBP{alpha} expression observed in the present study. In mice overexpressing constitutively active beta-catenin, the downstream effector of canonical Wnt signaling, in the lung, C/EBP{alpha} expression was downregulated (39). Also, in preadipocytes, Wnt signaling has been shown to inhibit C/EBP{alpha} expression, stopping differentiation into mature adipocytes (50). Together this indicates that decreased Wnt signaling to the epithelium could act as an upstream signal to allow C/EBP{alpha} expression. In addition, we observed that deposition of the extracellular matrix protein fibronectin was altered in transgenic mice overexpressing C/EBP{alpha}. Fibronectin is important for branching morphogenesis (51) and is suggested to be downstream of Wnt in the developing lung (16), further indicating an interplay between C/EBP{alpha} and Wnt signaling in embryonal lung development. As discussed above, Fgfs have a central role in lung development. Of the Fgfs expressed in the lung, Fgf7 exhibits a potentially relevant expression pattern; it is expressed from E14.5 in the pulmonary mesenchyme (32), closely correlating with the onset of C/EBP{alpha} expression in the epithelium. Moreover, the addition of Fgf7 has been demonstrated to upregulate C/EBP{alpha} expression in isolated lung cells (33). Together, this suggests that Fgf7 expression could act as a stimulatory signal and be an important inducer of C/EBP{alpha} expression during lung development. Thus a combination of spatiotemporally restricted stimulatory Fgf7 signals and inhibitory Wnt signals could underlie the specific expression pattern of C/EBP{alpha} during late lung development. Further studies will be needed to elucidate the upstream regulators of the finely tuned expression pattern of C/EBP{alpha} necessary for a complete branching to occur during the later stages of lung development.

In conclusion, our results show a role for C/EBP{alpha} in lung development and suggest a function for this intracellular factor in the later stages of lung branching morphogenesis, and we speculate that this is achieved through the localized regulation of proliferation in the developing epithelium. A role for C/EBP{alpha} in branching could have possible general implications in mammalian development because this intracellular factor is expressed in several organs that develop through branching morphogenesis, such as the mammary gland and liver (12, 47). Because our present results are centered on intracellular regulatory mechanisms, future studies mechanistically integrating these results with previous models of branching morphogenesis focused on extracellular signaling molecules are of central importance to further understand this fascinating developmental process.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This work was supported by Swedish Research Council of Medicine Grants 14677, 14678, and 14794; Swedish Heart-Lung Foundation Grant 20050443; and grants from the Jeansson, Åke Wiberg, Magnus Bergvall, and Konsul Th. C. Bergh Research Foundations, General Maternity Hospital Foundation, and Karolinska Institute.


   ACKNOWLEDGMENTS
 
The authors are most grateful to Drs. Per Flodby and Kleanthis G. Xanthopoulos for access to the Cebpa–/– mouse strain and to Drs. Jeffrey A. Whitsett and Stephan W. Glasser for the reagents needed for this study. The authors thank Johanna Fugelstad, Josefine Julin, and Lena Nordlund-Möller for the valuable assistance provided and Jenny L. Barton for critically reading the manuscript. The facilities of the Clinical Research Center at the Karolinska University Hospital-Huddinge and the Karolinska Institute Center for Transgene Technologies are also gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Nord, Div. for Respiratory Medicine, Dept. of Medicine, Karolinska Institute, Lung Research Laboratory L4:01, Karolinska Univ. Hospital-Solna, Stockholm SE171 76, Sweden (e-mail: magnus.nord{at}ki.se)

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.


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TOP
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 RESULTS
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  1. Basseres DS, Levantini E, Ji H, Monti S, Elf S, Dayaram T, Fenyus M, Kocher O, Golub T, Wong KK, Halmos B, and Tenen DG. Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBPalpha in mice. Mol Cell Biol 26: 1109–1123, 2006.[Abstract/Free Full Text]
  2. Bellusci S, Furuta Y, Rush MG, Henderson R, Winnier G, and Hogan BL. Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124: 53–63, 1997.[Abstract]
  3. Bellusci S, Grindley J, Emoto H, Itoh N, and Hogan B. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124: 4867–4878, 1997.[Abstract]
  4. Bellusci S, Henderson R, Winnier G, Oikawa T, and Hogan BL. Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122: 1693–1702, 1996.[Abstract]
  5. Birkenmeier EH, Gwynn B, Howard S, Jerry J, Gordon JI, Landschulz WH, and McKnight SL. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev 3: 1146–1156, 1989.[Abstract/Free Full Text]
  6. Boller K, Kemler R, Baribault H, and Doetschman T. Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies. Eur J Cell Biol 43: 459–468, 1987.[Web of Science][Medline]
  7. Buck M and Chojkier M. Signal transduction in the liver: C/EBPbeta modulates cell proliferation and survival. Hepatology 37: 731–738, 2003.[CrossRef][Web of Science]
  8. Cao Z, Umek RM, and McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev 5: 1538–1552, 1991.[Abstract/Free Full Text]
  9. Cardoso WV. Lung morphogenesis revisited: old facts, current ideas. Dev Dyn 219: 121–130, 2000.[CrossRef][Web of Science][Medline]
  10. Cassel TN, Berg T, Suske G, and Nord M. Synergistic transactivation of the differentiation-dependent lung gene CCSP (Scgb1a1) by the bZIP-factor C/EBPalpha and the homeodomain factor Nkx2.1/TTF-1. J Biol Chem 277: 36970–36977, 2002.[Abstract/Free Full Text]
  11. Cassel TN, Gustafsson JA, and Nord M. CYP2B1 is regulated by C/EBP alpha and C/EBP delta in lung epithelial cells. Mol Cell Biol Res Commun 3: 42–47, 2000.[CrossRef][Medline]
  12. Cassel TN and Nord M. C/EBP transcription factors in the lung epithelium. Am J Physiol Lung Cell Mol Physiol 285: L773–L781, 2003.[Abstract/Free Full Text]
  13. Cassel TN, Norlund-Möller L, Andersson O, Gustafsson JÅ, and Nord M. C/EBPalpha and C/EBPdelta activate the Clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites. Am J Respir Cell Mol Biol 22: 469–480, 2000.[Abstract/Free Full Text]
  14. Chuang PT and McMahon AP. Branching morphogenesis of the lung: new molecular insights into an old problem. Trends Cell Biol 13: 86–91, 2003.[CrossRef][Web of Science][Medline]
  15. Darlington GJ. Molecular mechanisms of liver development and differentiation. Curr Opin Cell Biol 11: 678–682, 1999.[CrossRef][Web of Science][Medline]
  16. De Langhe SP, Sala FG, Del Moral PM, Fairbanks TJ, Yamada KM, Warburton D, Burns RC, and Bellusci S. Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 277: 316–331, 2005.[CrossRef][Web of Science][Medline]
  17. Flodby P, Antonson P, Barlow C, Blanck A, Porsch-Hallstrom I, and Xanthopoulos KG. Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats. Exp Cell Res 208: 248–256, 1993.[CrossRef][Web of Science][Medline]
  18. Flodby P, Barlow C, Kylefjord H, Ährlund-Richter L, and Xanthopoulos KG. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein {alpha}. J Biol Chem 271: 24753–24760, 1996.[Abstract/Free Full Text]
  19. Freytag SO, Paielli DL, and Gilbert JD. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev 8: 1654–1663, 1994.[Abstract/Free Full Text]
  20. Glasser SW, Korfhagen TR, Wert SE, Bruno MD, McWilliams KM, Vorbroker DK, and Whitsett JA. Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice. Am J Physiol Lung Cell Mol Physiol 261: L349–L356, 1991.[Abstract/Free Full Text]
  21. Halmos B, Huettner CS, Kocher O, Ferenczi K, Karp DD, and Tenen DG. Down-regulation and antiproliferative role of C/EBPalpha in lung cancer. Cancer Res 62: 528–534, 2002.[Abstract/Free Full Text]
  22. He Y and Crouch E. Surfactant protein D gene regulation. Interactions among the conserved CCAAT/enhancer-binding protein elements. J Biol Chem 277: 19530–19537, 2002.[Abstract/Free Full Text]
  23. Hendricks-Taylor LR and Darlington GJ. Inhibition of cell proliferation by C/EBP alpha occurs in many cell types, does not require the presence of p53 or Rb, and is not affected by large T-antigen. Nucleic Acids Res 23: 4726–4733, 1995.[Abstract/Free Full Text]
  24. Hogan BL. Morphogenesis. Cell 96: 225–233, 1999.[CrossRef][Web of Science][Medline]
  25. Ikoma T, Ito T, Okudela K, Hayashi H, Yazawa T, and Kitamura H. Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters. Cell Prolif 34: 233–241, 2001.[CrossRef][Web of Science][Medline]
  26. Jover R, Bort R, Gomez-Lechon MJ, and Castell JV. Re-expression of C/EBP alpha induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells. FEBS Lett 431: 227–230, 1998.[CrossRef][Web of Science][Medline]
  27. Lane MD, Tang QQ, and Jiang MS. Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation. Biochem Biophys Res Commun 266: 677–683, 1999.[CrossRef][Web of Science][Medline]
  28. Li C, Xiao J, Hormi K, Borok Z, and Minoo P. Wnt5a participates in distal lung morphogenesis. Dev Biol 248: 68–81, 2002.[CrossRef][Web of Science][Medline]
  29. Lin FT and Lane MD. CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci USA 91: 8757–8761, 1994.[Abstract/Free Full Text]
  30. Litingtung Y, Lei L, Westphal H, and Chiang C. Sonic hedgehog is essential to foregut development. Nat Genet 20: 58–61, 1998.[CrossRef][Web of Science][Medline]
  31. Martis PC, Whitsett JA, Xu Y, Perl AK, Wan H, and Ikegami M. C/EBP{alpha} is required for lung maturation at birth. Development 133: 1155–1164, 2006.[Abstract/Free Full Text]
  32. Mason IJ, Fuller-Pace F, Smith R, and Dickson C. FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech Dev 45: 15–30, 1994.[CrossRef][Web of Science][Medline]
  33. Mason RJ, Pan T, Edeen KE, Nielsen LD, Zhang F, Longphre M, Eckart MR, and Neben S. Keratinocyte growth factor and the transcription factors C/EBP alpha, C/EBP delta, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. J Clin Invest 112: 244–255, 2003.[CrossRef][Web of Science][Medline]
  34. McKnight S. McBindall–a better name for CCAAT/enhancer binding proteins? Cell 107: 259–261, 2001.[CrossRef][Web of Science][Medline]
  35. Mendelson CR. Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu Rev Physiol 62: 875–915, 2000.[CrossRef][Web of Science][Medline]
  36. Mischoulon D, Rana B, Bucher NL, and Farmer SR. Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture. Mol Cell Biol 12: 2553–2560, 1992.[Abstract/Free Full Text]
  37. Mucenski ML, Wert SE, Nation JM, Loudy DE, Huelsken J, Birchmeier W, Morrisey EE, and Whitsett JA. beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis. J Biol Chem 278: 40231–40238, 2003.[Abstract/Free Full Text]
  38. Nord M, Låg M, Cassel TN, Randmark M, Becher R, Barnes HJ, Schwarze PE, Gustafsson JÅ, and Lund J. Regulation of CCSP (PCB-BP/uteroglobin) expression in primary cultures of lung cells–involvement of C/EBP. DNA Cell Biol 17: 481–492, 1998.[Web of Science][Medline]
  39. Okubo T and Hogan BL. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. J Biol 3: 11, 2004.[CrossRef][Medline]
  40. Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, Behre G, Hiddemann W, and Tenen DG. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet 27: 263–270, 2001.[CrossRef][Web of Science][Medline]
  41. Pepicelli CV, Lewis PM, and McMahon AP. Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 8: 1083–1086, 1998.[CrossRef][Web of Science][Medline]
  42. Perl AK, Wert SE, Nagy A, Lobe CG, and Whitsett JA. Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci USA 99: 10482–10487, 2002.[Abstract/Free Full Text]
  43. Peters K, Werner S, Liao X, Wert S, Whitsett J, and Williams L. Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J 13: 3296–3301, 1994.[Web of Science][Medline]
  44. Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis-Hansen L, and Nerlov C. E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo. Cell 107: 247–258, 2001.[CrossRef][Web of Science][Medline]
  45. Prodhan P and Kinane TB. Developmental paradigms in terminal lung development. Bioessays 24: 1052–1059, 2002.[CrossRef][Web of Science][Medline]
  46. Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, and Tenen DG. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Mol Cell Biol 18: 4301–4314, 1998.[Abstract/Free Full Text]
  47. Ramji DP and Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 365: 561–575, 2002.[Web of Science][Medline]
  48. Rangwala SM and Lazar MA. Transcriptional control of adipogenesis. Annu Rev Nutr 20: 535–559, 2000.[CrossRef][Web of Science][Medline]
  49. Rosenberg E, Li F, Reisher SR, Wang M, Gonzales LW, Ewing JR, Malek S, Ballard PL, Notarfrancesco K, Shuman H, and Feinstein SI. Members of the C/EBP transcription factor family stimulate expression of the human and rat surfactant protein A (SP-A) genes. Biochim Biophys Acta 1575: 82–90, 2002.[Medline]
  50. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, and MacDougald OA. Inhibition of adipogenesis by Wnt signaling. Science 289: 950–953, 2000.[Abstract/Free Full Text]
  51. Sakai T, Larsen M, and Yamada KM. Fibronectin requirement in branching morphogenesis. Nature 423: 876–881, 2003.[CrossRef][Medline]
  52. Shannon JM and Hyatt BA. Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 66: 625–645, 2004.[CrossRef][Web of Science][Medline]
  53. Shu W, Jiang YQ, Lu MM, and Morrisey EE. Wnt7b regulates mesenchymal proliferation and vascular development in the lung. Development 129: 4831–4842, 2002.[Medline]
  54. Ten Have-Opbroek AA. The development of the lung in mammals: an analysis of concepts and findings. Am J Anat 162: 201–219, 1981.[CrossRef][Web of Science][Medline]
  55. Timchenko NA, Harris TE, Wilde M, Bilyeu TA, Burgess-Beusse BL, Finegold MJ, and Darlington GJ. CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice. Mol Cell Biol 17: 7353–7361, 1997.[Abstract]
  56. Timchenko NA, Wilde M, Nakanishi M, Smith JR, and Darlington GJ. CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev 10: 804–815, 1996.[Abstract/Free Full Text]
  57. Umek RM, Friedman AD, and McKnight SL. CCAAT-enhancer binding protein: a component of a differentiation switch. Science 251: 288–292, 1991.[Abstract/Free Full Text]
  58. Wang H, Iakova P, Wilde M, Welm A, Goode T, Roesler WJ, and Timchenko NA. C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4. Mol Cell 8: 817–828, 2001.[CrossRef][Web of Science][Medline]
  59. Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, Taylor LR, Wilson DR, and Darlington GJ. Impaired energy homeostasis in C/EBP alpha knockout mice. Science 269: 1108–1112, 1995.[Abstract/Free Full Text]
  60. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, and Cardoso WV. The molecular basis of lung morphogenesis. Mech Dev 92: 55–81, 2000.[CrossRef][Web of Science][Medline]
  61. Weaver M, Yingling JM, Dunn NR, Bellusci S, and Hogan BL. Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 126: 4005–4015, 1999.[Abstract]
  62. Wert SE, Glasser SW, Korfhagen TR, and Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156: 426–443, 1993.[CrossRef][Web of Science][Medline]
  63. Whitsett JA and Glasser SW. Regulation of surfactant protein gene transcription. Biochim Biophys Acta 1408: 303–311, 1998.[Medline]



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