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Am J Physiol Lung Cell Mol Physiol 288: L1033-L1039, 2005. First published January 28, 2005; doi:10.1152/ajplung.00277.2004
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Smad1 expression and function during mouse embryonic lung branching morphogenesis

Cheng Chen,1,3 Hui Chen,2 Jianping Sun,1 Pablo Bringas, Jr.,2 Yuhua Chen,3 David Warburton,1 and Wei Shi1,2

1Developmental Biology Program, The Saban Research Institute of Childrens Hospital Los Angeles; 2Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California; and 3Department of Developmental Biology, China Medical University, Shenyang, People's Republic of China

Submitted 21 July 2004 ; accepted in final form 21 January 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells ({alpha}-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing ~20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.

lung development; bone morphogenetic protein 4

MOUSE LUNG ORIGINATES from ventral foregut endoderm at embryonic day 9.5 (E9.5). It emerges as the laryngo-tracheal groove and evaginates into the surrounding splanchnic mesenchyme as a pair of primary buds (7). Then, the epithelium grows, extends, and divides repeatedly to form the respiratory tree, a process called branching morphogenesis (9, 10). The stereotypic pattern of early lung budding and branching is retained in vitro when the embryonic lung explants are cultured in a chemical defined medium (12), which makes early embryonic lung organ culture a very useful model for studying the basic mechanisms of lung development.

Lung branching morphogenesis is regulated by many peptide growth factors, including bone morphogenetic protein 4 (BMP4). BMP4 is a member of the transforming growth factor (TGF)-{beta}/BMP superfamily that is involved in many embryonic patterning events (8). Previous studies have shown that addition of BMP4 into the embryonic lung explant culture medium dramatically stimulates lung branching (2, 22), whereas an inhibitory effect of BMP4 on the growth of isolated epithelium has been reported (26). Therefore, although BMP4 seems to be one of the key growth factors essential for early embryonic lung development, the precise mechanism by which BMP4-mediated intracellular signaling regulates embryonic lung morphogenesis is unclear.

The signal transduction pathways of BMPs have been extensively studied in cultured cells. Upon BMP ligand and BMP receptor binding on the cell surface, the activated receptor protein kinases phosphorylate and activate downstream receptor-regulated Smads including Smad1. The phosphorylated Smads further form protein complexes with the common mediator Smad4 and translocate to the nucleus, where they modulate specific target gene expression (16). This BMP canonical signal transduction pathway is fine-tuned at several molecular levels, including ligand, receptor, and Smads. For example, BMP receptor-mediated Smad1 phosphorylation and activation can be blocked by inhibitory Smad6. Smad1 activation can also be coordinately regulated by other growth factor pathways, such as IGF2 as well as FGF8, which were shown to be involved in dorsalization and neural induction in the Xenopus embryo (17). Our previous studies found that overexpression of Smurf1, a specific ubiquitin ligase for BMP-specific downstream Smad proteins, inhibited mouse lung branching morphogenesis concomitantly with degradation of Smad1 (21, 29). However, the biological role of Smad1 in lung development has not been directly studied.

Although much is known about the biochemistry of Smad1 signal transduction, the role of Smad1 in lung development and maturation is still unclear. Smad1 has been detected in early embryonic mouse lung epithelium by in situ hybridization (6). By immunohistochemistry, Huang et al. (11) also found that Smad1 expressed in adult mouse bronchial epithelial cells and smooth muscle. Recently, Jeffery et al. (13) reported that Smad1 was a major signal transducer in BMP4-mediated effects of antiproliferation of cultured human fibroblast cells and prodifferentiation of these cells into smooth muscle cells. Because null mutation of Smad1 is lethal early in gestation (E9.5) with failure in establishing chorioallantoic circulation (15, 23), the role of Smad1 in lung development in vivo could not be evaluated in this mouse model. To determine the function of BMP-Smad1 and the related molecular mechanisms of BMP signaling in lung development, we studied the gene expression pattern and the function of Smad1 using the mouse embryonic lung culture approach, which is a simple and well-established ex vivo system to study early mouse embryonic lung development (25). We found that Smad1 expression changed dynamically during embryonic lung development and that reduction of Smad1 expression by an antisense oligonucleotide approach significantly inhibited lung branching morphogenesis in cultured lung explants.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse lung samples. Swiss-Webster mice were purchased from Simonsen Laboratories (Gilroy, CA). To study the embryonic lungs at different developmental stages, timed-pregnant mice were killed at the desired stages, and the embryos were removed by cesarean section. E11.5 mouse embryonic lung was excised en bloc and processed for organ culture, fixation, or RNA isolation.

Total RNA isolation and reverse transcription. The total RNA was isolated from lung tissues using a Qiagen RNeasy kit (Qiagen, Santa Clarita, CA), followed by DNase treatment to remove any potentially contaminated genomic DNA. The quality of isolated RNA was checked by formaldehyde agarose gel electrophoresis before the reverse transcription (RT) reaction. One microgram of total RNA was added to the RT reaction mixture (iScript; Bio-Rad Laboratories, Hercules, CA) and incubated at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min. The product of RT was diluted 10-fold and applied to polymerase chain reaction (PCR) analysis.

Primers and real-time PCR. Real-time quantitative PCR analysis was performed on iCycler-iQ system (Bio-Rad) by SYBR Green I dye detection as published previously (20). Briefly, 25 µl of reaction mixture contains 1x iQ SYBR Green Supermix (Bio-Rad), 200 nM forward and reverse primers, and the cDNA template from the sample. The PCR conditions are 3 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at annealing temperature, and 30 s at 72°C. The specificity of PCR was verified by measuring the melting curve of the PCR product at the end of reaction. Direct addition of isolated RNA in the PCR reaction was used to eliminate any interference caused by genomic DNA contamination in RNA sample. The relative gene mRNA level was calculated using {Delta}{Delta}Ct (18). The PCR primer sequences for mouse surfactant protein (SP)-C and Clara cell 10-kDa protein (CC10) were previously published (21). GAPDH was used as reference control to normalize equal loading of template cDNA.

E11.5 embryonic lung culture. The procedure for lung organ culture was previously described (22). Briefly, the intact E11.5 mouse embryonic lung explants were isolated under a dissecting microscope and placed on 0.80-µm MF-Millipore filters that were supported by a stainless-steel grid in a Grobstein culture dish. An appropriate amount of BGJb serum-free medium (Invitrogen) was added to each dish to establish an air-fluid interface at the level of the explants. Smad1 antisense DNA oligonucleotide, with the sequence of CTG GTC ACA TTC ATA GCG, was added to the medium at a final concentration of 40 µM. Sense DNA oligonucleotide, with the complementary sequence of the antisense oligonucleotide, was always used in parallel as a control. The E11.5 embryonic lungs were cultured for 4 days in 100% humidity and 95% air-5% CO2. The medium containing related oligonucleotide was changed every 2 days. Branching morphogenesis was measured as the number of epithelial sacs visible around the periphery of the lung explants under the microscope and presented as the percentage of the terminal branches of lung explants grown in contemporaneous medium control to minimize the variation in each individual experiment. At least 10 lung explants were used for each condition, and individual experiments were repeated at least three times. More than six cultured lungs were pooled and processed for RNA isolation and protein lysate preparation.

Immunostaining of lung tissue sections. Proliferating cell nuclear antigen (PCNA) immunostaining was performed using a kit from Zymed Laboratories (South San Francisco, CA). PCNA-positive cells in peripheral epithelium, which lines peripheral small airways, were counted. The PCNA index was calculated as the percentage of the PCNA-positive epithelial cells out of the total counted of the same type of cells. Smad1 protein was detected using a polyclonal antibody from Santa Cruz at a dilution of 1:50 after antigen recovery and an overnight incubation at 4°C. FITC and Texas red-labeled secondary antibody were purchased from Molecular Probes. Incubation with normal serum instead of primary antibody was used as a negative control.

Protein detection by Western blot. The method for detecting proteins of embryonic lung tissues was published previously (28). Briefly, embryonic lung tissues were lysed on ice in radioimmunoprecipitation assay buffer with freshly added 1 mM PMSF, 1x protease inhibitor, and 1 mM sodium orthovanadate. The protein concentrations were measured by the Bradford method using a commercial kit (Bio-Rad). Total tissue lysate proteins (40 µg) were loaded onto NuPAGE Novex gels (Invitrogen) and separated by electrophoresis. Proteins were then transferred to Immobilon-P membrane (Millipore), incubated with related primary antibody, and detected by the enhanced chemiluminescence method. The same Smad1 antibody, as mentioned above, was used. The intensities of protein bands were quantified by Scion Image software (Scion, Frederick, MD) and normalized by the loading control.

Data presentation and statistical analysis. All experiments were repeated at least three times, with similar results obtained within repetitive experiments. All data were expressed as means ± SD. A Student's t-test was used for comparison of statistical difference between experimental groups, and P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Temporal-spatial expression pattern of Smad1 in mouse lung development. BMP4 is an important growth factor in regulating lung development (24), and Smad1 is a major intracellular signal transducer in the BMP canonical pathway. Therefore, determination of Smad1 dynamic expression pattern in lung development would be essential to understand its biological function. Smad1 protein temporal-spatial expression in mouse embryonic lung at different developmental stages was determined by immunohistochemistry (Fig. 1). In E12.5 embryonic lung, the majority of Smad1 protein was detected in the peripheral airway epithelial cells, whereas only a few Smad1-positive cells were scattered among the mesenchyme. This number of Smad1 positively stained airway epithelial cells was reduced when the lung developed into E14.5. Meanwhile, Smad1-positive mesenchymal cells were increased in mid-late gestation. In particular, Smad1 was detected in the cells surrounding the large airways and blood vessels in late embryonic lungs (E16.5–E18.5, Figs. 1 and 2).



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  		Fig. 1. Cellular localizations of Smad1 protein at different developmental stages were determined by immunohistochemistry. The majority of Smad1-positive cells were localized in the airway epithelium of embryonic day 12.5 (E12.5) lung, as indicated by arrows. Only a few Smad1 mesenchymal cells were sparsely distributed (arrowheads). The Smad1-positive epithelial cells were reduced when the embryonic lung further developed (E14.5–E18.5). Meanwhile the Smad1-positive mesenchymal cells were increased, and some of them were localized underneath the lining epithelial cells and endothelial cells of large airways and the accompanying blood vessels (E18.5). Normal serum was used as a negative control (Neg Ctrl).

 


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  		Fig. 2. Colocalization of Smad1 with {alpha}-smooth muscle actin (SMA) in some of the mesenchymal cells of E16.5 mouse embryo lung. Smad1 (green signals) and SMA (red signals) were detected on the same E16.5 lung tissue section by coimmunofluorescence staining (A and B). SMA, as a marker for smooth muscle cells, was specifically stained only in the cells surrounding the large airways and blood vessels (arrows), whereas Smad1 was stained broadly in epithelial cells (arrowheads) as well as mesenchymal cells (arrows). With merged images, part of Smad1 signals in mesenchymal cells localized in the walls of large airways (A) and blood vessels (V) were overlapped with SMA signals, indicating that these Smad1-positive cells were smooth muscle cells. Normal serum was used as a negative control. However, Smad1 and SMA expression did not overlap in E12.5 early mouse embryonic lung (C). Smad1 was mainly expressed in the epithelial cells of peripheral small airways, whereas SMA was detected in mesenchymal cells surrounding larger airways. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue color).

 
Colocalization of Smad1 protein and {alpha}-smooth muscle actin in large airways and blood vessels. To determine the type of mesenchymal cells with Smad1 expression in large airways and accompanied pulmonary blood vessels, Smad1 and {alpha}-smooth muscle actin (SMA), a marker for smooth muscle cell and myofibroblast cells, were localized by coimmunofluorescence staining. In E16.5 lung, Smad1 protein was detected in both epithelial cells of peripheral airways and the mesenchymal cells surrounding the large airways and blood vessels(Fig. 2, A and B), whereas SMA was detected only in smooth muscle cells in large airways and accompanied blood vessels. These two proteins are colocalized in the same cells as a circular pattern surrounding the lining of large airways and blood vessels, indicating that Smad1 was expressed in smooth muscle cells in these locations. This colocalization pattern was not seen in early embryonic lung (E12.5, Fig. 2) and 4-day cultured E11.5 lung explants (data not shown). Therefore, Smad1 expression in these smooth muscle cells suggests that Smad1-mediated signaling may be required for smooth muscle cell growth or maintenance at a particular developmental stage (E16.5).

Reduction of Smad1 expression significantly inhibited branching morphogenesis of the cultured embryonic lung explants. To determine the function of Smad1 in early embryonic lung development, loss of function experiments were performed by reducing Smad1 expression in mouse embryonic lung organ culture. A Smad1 specific antisense DNA oligonucleotide or a complementary sense DNA oligonucleotide was added into the culture medium, and the effects of these DNA oligonucleotides on Smad1 expression were determined by Smad1 protein detection using Western blot (Fig. 3). About 50–60% reduction of Smad1 protein expression in the 4-day cultured lung explants was detected in the samples treated with the antisense DNA oligonucleotide, whereas Smad1 protein was not reduced in the presence of the sense DNA oligonucleotide control compared with the lungs cultured with medium control. Consistent with the changes of Smad1 protein expression, lung branching morphogenesis was significantly inhibited in the presence of the antisense DNA oligonucleotide. The lung explants grew less than the control groups, and about a 20% reduction (P < 0.01) in terminal branches was observed (Fig. 3). These data indicate that Smad1 is essential to promote embryonic lung development in the early stages.



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  		Fig. 3. Lung branching morphogenesis is significantly reduced when endogenous Smad1 expression in embryonic lung explants was inhibited by Smad1 antisense (AS) oligonucleotide. A: Western blot detection of Smad1 expression in 4-day cultured lung explants when Smad1-specific AS oligonucleotide was added into the culture medium. The complementary sense (SE) oligonucleotide and medium-only culture (MC) were used as controls. {beta}-Actin detection was used as a protein lysate loading control. B: we quantified the amount of Smad1 protein detected in the Western blot by measuring the protein band intensity using Scion Image software. We detected ~40% reduction of Smad1 expression in the cultured lungs treated with Smad1 AS oligonucleotide. C: size and terminal branches of the 4-day cultured lung explants were photographed under the same magnification (scale bar: 250 µm). D: changes of the cultured lung terminal branches were compared and presented as the relative percentage of the branching number in MC. There was no difference between MC and SE groups (100 ± 7.91 vs. 100.5 ± 13.43%). However, significant reduction of the terminal branches was observed in the lung explants treated with AS oligonucleotide (82.28 ± 7.56%, *P < 0.01). Figures are representative of 3 independent experiments.

 
Reduced Smad1 expression in cultured lung explants inhibited cell proliferation and differentiation. Reduced growth of embryonic lungs might be caused by reduced cell proliferation. Thus lung cell proliferation was evaluated by measuring the PCNA, a marker for mitotic cells. After PCNA immunostaining (Fig. 4), the percentage of PCNA-positive cells was quantified and expressed as PCNA index to estimate the rate of cell growth. In the group of cultured lungs treated with Smad1 antisense DNA oligonucleotide, the PCNA index in airway epithelial cells was only 23.7 ± 2.26%. Compared with the PCNA index in the control groups (35.45 ± 3.75%), airway epithelial cell proliferation in the lung explants treated with Smad1 antisense DNA oligonucleotide was reduced ~31%. Consistent with PCNA immunostaining, the PCNA protein level in total protein lysate, as detected by Western blot, decreased by 18–29% in Smad1 antisense-treated lung explants compared with controls (data not shown). Furthermore, differentiation of lung epithelial cells was also evaluated based on the changes of mRNA expression for the differentiation markers of epithelial cells, SP-C, and CC10. By real-time quantitative PCR, SP-C mRNA and CC10 mRNA levels were found to be significantly reduced by 43 and 60%, respectively, in the cultured lungs treated with the Smad1 antisense oligonucleotide, compared with medium control group (Fig. 5). No significant changes in the expression of these genes were observed in the sense oligonucleotide control group, indicating the specific effect of Smad1 antisense oligonucleotide. In addition, no changes of SMA protein expression, as detected by immunostaining, were observed in the lung explants treated with antisense oligonucleotide (data not shown).



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  		Fig. 4. Reduced Smad1 expression significantly inhibited epithelial cell proliferation of the cultured mouse embryonic lung explants. A: proliferating cells were detected by PCNA immunostaining (brown-colored nuclei) in the cultured lungs treated with Smad1 AS or SE oligonucleotide. MC was also included. B: PCNA index was measured based on the above PCNA immunostaining, as presented by the percentage of the PCNA-positive epithelial cells over the total counted epithelial cells. The PCNA index was significantly reduced to 23.7 ± 2.26% in AS-treated lungs vs. 36.05 ± 0.63% in SE-treated lungs or 35.45 ± 3.75% in MC lungs, respectively. *P < 0.05.

 


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  		Fig. 5. Reduced Smad1 expression inhibited airway epithelium cell differentiation in cultured mouse lung explants. The embryonic lung epithelial cell differentiation was evaluated by quantifying the mRNA expression of Clara cell protein 10 (CC10) and surfactant protein C (SP-C). In the cultured lung explants treated with Smad1 AS oligonucleotide, in which Smad1 expression was shown to be reduced, SP-C and CC10 mRNA levels were significantly decreased by 43 ± 4% and 60 ± 5% (*P < 0.01) compared with MC. However, there were not significant changes of SP-C (118 ± 22%) and CC10 (99 ± 3%) mRNA levels in the cultured lung explants treated with SE oligonucleotide control.

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Like many other growth factors, BMPs play an important regulatory role in embryonic organogenesis (8). Previous studies indicate that BMP4 is one of the important regulators in lung development. Misexpression of BMP4 in lung peripheral epithelial cells under the control of SP-C promoter resulted in abnormal growth of mouse embryonic lung (1). On the other hand, addition of BMP4 into the culture medium stimulated mouse embryonic lung branching morphogenesis in intact embryonic lung explant culture but inhibited the growth of isolated lung epithelial tubes in MatriGel (2, 22, 26). The molecular mechanisms for these phenotypic changes remain unknown. It is well known that BMP-activated Smads are the canonical intracellular signaling proteins that transduce the BMP signal from cell surface to nuclei, wherein gene expression is regulated (5). This signaling pathway is modulated by several molecular mechanisms, such as ligand inactivation by many antagonists on cell surface, the dominant-negative effect by pseudoreceptor sequestration, negative feedback by inhibitory Smads, as well as degradation of activated Smad by protein ubiquitination (16). Therefore, altered ligand expression does not always result in changes of the net activity of the signaling pathway. For example, an increase of BMP expression may just be a compensatory response to reduced BMP signaling activity. Therefore, it is very important to determine the direct role of BMP signaling activity at the level of intracellular Smads.

Although Smad1, 5, and 8 are all BMP-specific downstream Smads that have >75% identity in their protein sequences, it is unlikely that the functions of these BMP-specific Smads are completely redundant. Smad1-null mutation in mice causes embryonic lethality (E9.5) with failure in establishing chorioallantoic circulation (15, 23). Smad5 knockout mice display not only embryonic lethality (E9.5–10.5) but also multiple defects, such as deficient angiogenesis, incorrect left-right asymmetry, craniofacial abnormalities, as well as induced mesenchymal apoptosis (3, 4, 14). The unique phenotypes of specific Smad-null mutations may result from their different expression patterns and/or their unique biochemical properties, or as was reported in the case of TGF-{beta}-specific Smad2 and Smad3, being activated by distinct receptor pools, possibly localized to different cellular subcompartments (19). Herein we selectively studied the biological function of Smad1 during mouse lung development. The temporospatial expression pattern of Smad1 in embryonic lungs was determined by immunohistochemistry. Although Smad1-positive cells were mainly detected in epithelial cells in E12.5 embryonic lungs, Smad1 protein was also detected in a few sparsely distributed mesenchymal cells. Interestingly, Smad1 expression in epithelial cells was gradually reduced, whereas Smad1 positively stained mesenchymal cells increased as the embryonic lung further developed. In particular, some of the Smad1-positive mesenchymal cells were localized adjacent to both the columnar epithelial cells of large airways and the flat endothelial cells of accompanying blood vessels at E16.5. The colocalization of SMA and Smad1 in these mesenchymal cells may suggest that Smad1 has important functions in either smooth muscle cell differentiation or maintenance of cell growth in this particular stage. Interestingly, a Smad1-negative regulator, Smurf1, was also found to express mainly in the epithelial cells in early embryonic lung and in the mesenchymal cells of late stage lung, but not in the mesenchymal cells in the walls of large airways and blood vessels (21). During preparation of this manuscript, Jeffery et al. (13) reported that Smad1-mediated BMP4 signaling is essential for myofibroblast differentiation into smooth muscle cells in vitro. Further experiments will be needed to determine the exact role of Smad1 in smooth muscle cell and/or myofibroblast cell differentiation in vivo.

The function of BMP4 and its signaling pathway in early embryonic lung development are controversial since contradictory results have been obtained from different experimental models. In cultures of isolated lung epithelial tubes, addition of BMP4 inhibited epithelial cell proliferation and migration (26). However, exogenous BMP4 stimulated early epithelial branching morphogenesis in intact lung explant culture (2, 22). Herein we have used the same lung explant culture model to study one of the BMP-specific Smads, Smad1. As published previously, addition of a gene-specific antisense DNA oligonucleotide to the organ culture medium can significantly knock down the endogenous gene expression of the embryonic lung explants, since oligonucleotides can penetrate easily and distribute evenly into the embryonic lung tissue (27). By the same approach, the endogenous Smad1 protein expression in cultured embryonic lung explants was reduced by ~50%. The growth and airway branching morphogenesis of these lung explants with reduced Smad1 expression were significantly inhibited. Further studies on the epithelial cell proliferation and differentiation found that decreased Smad1 expression reduced both cellular processes. These results suggest that Smad1-mediated BMP signaling has a positive regulatory effect on early mouse embryonic lung growth and maturation, therefore stimulating early embryonic lung development.

The observations described above are consistent with our previous finding regarding BMP4 signaling in early lung branching morphogenesis (21, 22). Addition of BMP4 into the medium of the cultured lung explants stimulated lung growth and terminal branching, and a similar growth phenotype was also observed in lung explants when expression of a BMP4 antagonist Gremlin was reduced by an antisense oligonucleotide (22). Recently, we also showed that overexpression of Smurf1 in the airway epithelial cells of the cultured lung explants inhibited lung branching (21). Smurf1 is a Smad1/5-specific ubiquitin ligase that promotes Smad degradation through the ubiquitination-proteasome pathway. Overexpression of Smurf1 resulted in reduction of Smad1/5 protein levels in the cultured lung explants. Although there are three BMP-specific downstream Smads (Smad1, 5, and 8), the combined data suggest that Smad1 is one of the essential downstream Smads in mediating BMP-stimulated cell proliferation and differentiation during early mouse embryonic lung development. However, the individual function of Smad1 in either epithelial cells or mesenchymal cells of the developing lung needs to be further determined using location-specific gene knockout approaches, such as Smad1 conditional knockout in genetically manipulated mice.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study is supported by National Heart, Lung, and Blood Institute Grants HL-68597 and HL-61286 (W. Shi), HL-44977, HL-44060, HL-60231, and HL-75773 (D. Warburton), an American Heart Association Grant-in-Aid (W. Shi), and a Childrens Hospital Los Angeles Research Career Development Award (W. Shi).


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Shi, Developmental Biology Program, Dept. of Surgery, Childrens Hospital Los Angeles, 4650 Sunset Blvd., MS 35, Los Angeles, CA 90027 (E-mail: wshi{at}chla.usc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 GRANTS
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