Epithelial-mesenchymal interactions are critical for the branching and differentiation of the lung, but the mechanisms involved are still unclear. To investigate this problem in mouse embryonic lung, we have studied the temporal and spatial expression of genes implicated in the morpho-genesis of other organs. At 11.5 days p.c., hepatocyte nuclear factor-3β (Hnf-3β) is expressed uniformly through-out the epithelium, while Wnt-2 expression is confined to the distal mesenchyme. Sonic hedgehog (Shh) trancripts are found throughout the epithelium, with high levels in the distal tips of the terminal buds, while bone morphogenetic protein-4 (Bmp-4) transcripts are localized at high levels in the distal tips of the epithelium, with lower levels in the adjacent mesenchyme. Epithelial expression is also seen for Bmp-7, but transcripts are less dramatically upregulated at the distal tips. The Type I Bone morphogenetic protein receptor gene(Bmpr/Tfr-11/Brk-1) is expressed at low levels in the epithelium and in the distal mesenchyme. To investigate the role of Bmp-4 in lung development, we have mis-expressed the gene throughout the distal epithelium of transgenic lungs using a surfactant protein C enhancer/promoter. From 15.5 days p.c., transgenic lungs are smaller than normal, with grossly distended terminal buds and, at birth, contain large air-filled sacs which do not support normal lung function. Labeling with BrdU reveals an inhibition of epithelial proliferation in 15.5 days p.c. transgenic lungs. A small but significant stimulation of proliferation of mesenchymal cells is also observed, but this is accompanied by an increase in cell death. In situ hybridization with riboprobes for the proximal airway marker, CC10, and the distal airway marker, SP-C, shows normal differentiation of bronchiolar Clara cells but a reduction in the number of differentiated Type II cells in transgenic lungs. A model is proposed for the role of BMP4 and other signalling molecules in embryonic lung morphogenesis.

Morphogenesis of the embryonic lung involves interactions between the epithelium and the surrounding mesenchyme and extracellular matrix (Alescio and Cassini, 1962; Wessels, 1970; Roman et al., 1991; Minno and King, 1994). In the mouse, these interactions start as early as 9.5 days p.c. with the outgrowth from the ventral foregut of a lung bud consisting of endodermal epithelium surrounded by splanchnic mesoderm. During the pseudoglandular stage (9.5 to 16.0 days of gestation; Ten Have-Opbroek, 1991), the epithelial tissue pro-liferates and branches to form the prospective bronchial and respiratory systems. By the beginning of the saccular stage at 16.0 days p.c., an abrupt demarcation develops between the columnar cells of the prospective bronchial system and the cuboidal cells of the future terminal air spaces. Epithelial-mesenchymal interactions have been studied in the pseudoglandu-lar stage. Transplantation of distal mesenchyme to an area of tracheal epithelium denuded of most of its mesenchyme leads to the formation of supernumerary buds that grow, branch and differentiate like normal pulmonary epithelium (Alescio and Cassini, 1962; Wessels, 1970; Goldin and Wessells, 1979; Shannon, 1994).

Epithelial-mesenchymal interactions and branching mor-phogenesis in the lung are likely to involve polypeptide signalling molecules and transmembrane receptors known to be required for the development of other organ systems. Potentially important secreted growth and differentiation factors include members of the fibroblast growth factor (FGF), trans-forming growth factor-beta (TGF-β), bone morphogenetic protein (BMP), and epidermal growth factor (EGF) families, as well as those related to mammalian WNT and hedgehog (HH) proteins (for reviews see Carpenter, 1993; Mason, 1994; Kingsley, 1994; Parr and McMahon, 1994; Ingham, 1995; Hogan, 1995).

Evidence for a crucial role for FGFReceptor-2 (FGFR2) in branching morphogenesis has recently been obtained using transgenic embryos. FGFR2 is expressed in the epithelium from the early embryonic bud stages through late fetal lung development. Targeted expression of a dominant negative form of the receptor in the developing epithelium using a surfactant protein C (SP-C) promoter/enhancer (Wert et al., 1993; Peters et al., 1994) leads to abnormal development; transgenic mice have two undifferentiated epithelial tubes that extend from the bifurcation of the trachea down to the diaphragm. In other studies, acidic FGF and basement membrane matrix can induce branching of lung epithelium in mesenchyme-free cultures (Nogawa and Takaaki, 1995). Taken together, these results establish a role for FGFs in branching morphogenesis, although the precise ligand that interacts with FGFR-2 in vivo is not yet known.

Several approaches have been used to investigate the role of TGF-β1 in lung development. Around 15.5 days p.c. transcripts are confined to the mesenchyme, whereas TGF-β1 protein is localized to the interface between the epithelial cells and the mesenchyme, in particular around the bronchiolar ducts and in the clefts of the epithelial branches, in association with matrix components such as collagen, fibronectin and pro-teoglycan (Lehnert and Akhurst, 1988; Heine et al., 1990; Pelton et al., 1991). In an embryonic lung culture system, TGF-β1 inhibits branching morphogenesis (Serra et al., 1994). More recently, it has been shown that overexpression of constitutively active TGF-β1 in the distal epithelial cells with the SPC promoter/enhancer delays embryonic lung development. At 18.5 days p.c. transgenic lungs are the same size as controls but their development is arrested at the pseudoglandular stage (Zhou et al., 1996).

Transcripts for bone morphogenetic protein-5 (BMP5), a TGF-β-related signalling molecule, are expressed throughout the mesenchyme of the embryonic mouse lung (but not around the trachea) from 10.5 days p.c. through at least 16.5 days p.c. (King et al., 1994). BMP5 is encoded by the short ear (se) locus and most mice homozygous for a null mutation are viable and have normal lungs. However, on some genetic back-grounds, newborn homozygous and heterozygous mutants have fluid-filled cysts in the lungs, although the etiology of this abnormality is not understood (Green, 1968).

To investigate the role of other signalling molecules in lung development, we have analyzed the temporal and spatial expression of genes encoding BMP4 and BMP7, a type 1 BMP receptor (BMPR/TFR-II/BRK-1), WNT2 and Sonic hedgehog (SHH). From 11.5 days p.c., Shh is expressed throughout the epithelium but with highest levels in the distal tips of epithelial buds. Similarly, Bmp-4 expression is high in the epithelium at the tips of the distal buds, but expression is also seen in the adjacent mesenchyme. This very localized expression of Bmp-4 at lung bud sites led us to examine the effect of overexpression of the gene throughout the distal epithelium of transgenic lungs using the SP-C promoter/enhancer. The resulting trans-genic lungs are significantly smaller than normal and show greatly distended terminal buds with a reduced proportion of type II alveolar cells at 16.5 and 18.5 days p.c.. These results suggest that BMP4 normally plays a key role in lung development. A model is presented for the potential interaction of BMP4 and other signalling molecules in regulating embryonic lung growth and differentiation.

Plasmid construction

The SP-C-human Bmp-4 vector was constructed using a human Bmp-4 cDNA (gift from John Wozney, Genetics Institute) modified to reduce the amount of 5′ and to eliminate the 3′ noncoding sequences. The 1232 bp cDNA including only 6 bp of 5′ noncoding sequence was inserted into a vector containing the 3.7 kb human SP-C promoter region (Korfhagen et al., 1990). An SV40 small T intron and a 0.4 kb sequence containing a poly(A) addition site with stop codons in all three reading frames were present at the 3′-end of the cDNA. The expression cassette was excised with NdeI and NotI.

A construct containing the SP-C enhancer/promoter and bacterial lacZ gene was generated to monitor the sites of transgenic expression. The lacZ gene with a poly(A) addition sequence was removed from pPD1.27 (Fire et al., 1990) with BamHI and NotI and cloned into a SP-C vector modified by BamHI and NotI digestion to remove the SV40 small T intron and the poly(A) addition fragment. The expression cassette was excised by HindIII and NotI.

Generation and identification of transgenic mice

Transgenic mice were generated as described by Hogan et al. (1994). DNA was purified by low melting point agarose gel electrophoresis (GIBCO, Life Technologies Inc.) followed by gelase digestion (Epicentre Technology), phenol-chloroform extraction, ethanol pre-cipitation and passage through a Qiaquik column (Qiagen). The trans-genes were injected independently into the pronuclei of (C57BL/6×DBA)F2 mouse eggs at a concentration of 3 ng/μl. Noon of the day of injection is 0.5 days p.c.

The genotype of embryos was determined by PCR analysis of genomic DNA extracted from embryonic liver. The primers used were 5′-AGGAACAAACAGGCTTCAAA-3′ (SP-C-primer for 5′) and 5′-AATGTTTATACGGTGGAAGC-3′ (human BMP-4 for 3′) or lacZ internal primers (5′-TCTGCTTCAATCAGCGTGCC-3′ for 5′ and 5′-GCCGTCTGAATTTGACCTGA-3′ for 3′). The cDNA was amplified for 27 cycles at 61°C. The genotypes of PCR-positive embryos showing no abnormal phenotypes were verified by Southern blot analysis.

Of a total of 106 embryos obtained between 11.5 days p.c. and 19.5 days of development after SP-C-Bmp-4 injection of eggs, 28 were transgenic. The number of transgenics obtained at each stage and the number showing an abnormal phenotype (respectively in parentheses) were as follows: 11.5 days p.c. (3;0); 12.5 days p.c. (1;0); 15.5 days p.c. (3;2); 16.5 days p.c. (10;5); 17.5 days p.c. (2;0); 18.5 days p.c. (8;4) and newborn (1;1).

In situ hybridization

The whole-mount in situ hybridization protocol was based on one used previously (Winnier et al., 1995). The following murine cDNAs were used as templates for synthesizing digoxigenin-labeled riboprobes: 1.5 kb HNF3β cDNA (Sasaki and Hogan, 1993); 630 bpWnt-2 (kindly provided by Dr Andrew McMahon); 642 bp Shh (Dr Andrew McMahon); 654 bp Bmpr (also known as Brk-1 or TFR-II) (Drs Yuji Mishina and Naoto Ueno); 1.2 kb mouse Bmp-2 (Blessing et al., 1993); a 1.5 kb full-length mouse Bmp-4 (Winnier et al., 1995) or a 1.0 kb mouse Bmp-4 cDNA (Jones et al., 1991). Both Bmp-4 probes gave identical results.

The nonradioactive in situ hybridization of tissue sections was based on a protocol used previously (Sasaki and Hogan, 1993). Digoxigenin-labeled antisense RNA probes were prepared for mouse surfactant protein C (758 bp) and rat CC-10 (450 bp) (Dr Jeffrey Whitsett). Radioactive in situ hybridization of lung sections was carried out essentially as described by Zhao et al. (1993).

Analysis of lung proliferation in vivo

Females at 16.5 and 18.5 days of pregnancy were injected intraperitoneally with a mixture of 5-bromo 2′-deoxyuridine (BrdU) and 5-fluoro 2′-deoxyuridine (FUdr; Sigma) (50 and 10 mg per kg body weight, respectively) in 0.5 ml of sterile PBS. After 2 hours, lungs were collected, fixed in 4% paraformaldehyde for 2 hours, washed 3 times with PBS, dehydrated in 25%, 50%, 75% and 100% ethanol (5 minutes each), embedded in paraffin and sectioned (7 μm). Sections were then dewaxed and stained as described previously (Sakai et al., 1994). Briefly, staining of trypsinized sections was carried out with a rat anti-BrdU antibody (Accurate Chemical, Westbury, New York) diluted 1:400 in PBS. Sections were then treated with peroxidase-coupled rabbit anti-rat IgG (Vector) diluted 1:100 in PBS using 3-amino,9-ethyl carbazole as the chromogen. All cells in 12 photo-micrographs at magnification of ×40 of random portions of a section of a control and transgenic lung were counted and scored as labeled or unlabeled. At 16.5 days p.c., it was possible to distinguish clearly between epithelial and mesenchymal cells. At 18.5 days, this distinc-tion was not possible, so that only the overall percentages of labelled nuclei were compared between control and transgenic. Paired groups of data were analysed by Student’s t test using the Systat program. Results were determined to be significant if P<0.05. Similar results were obtained when single photomicrographs from 12 different sections of the same transgenic and control lungs were analyzed.

Analysis of cell death

Cell death was initially measured using the ApopTag detection kit (Oncor) following the manufacturer’s instructions. Briefly, sections were dewaxed and washed in PBS, followed by proteinase K digestion for 15 minutes at room temperature. Endogenous peroxidase activity was then quenched in 2.0% hydrogen peroxide, and the sections incubated for 1 hour at 37°C with terminal deoxynucleotide transferase (TdT) and digoxigenin-labelled dUTP. After incubation, incor-porated nucleotide was detected using anti-digoxigenin-peroxidase coupled antibody with diaminobenzidine (Sigma Fast, Sigma Immunochemicals) as substrate. Sections were counterstained with methyl green. As a control for specificity, samples were incubated without TdT. Sections of adult mouse testis were used as a positive control. Identical results were obtained with the nick-translation method (Gold et al., 1993) using DNA polymerase, digoxigenin-labelled dUTP, rabbit anti-digoxigenin-alkaline phosphatase coupled antibody and NBT substrate from Boehringer Mannheim. To quantitate results, all of the cells were counted and scored in 12 photo-micrographs of a section of a single transgenic and control lung at 16.5 days as described for BrdU labeling above.

Gene expression in embryonic lung

Whole-mount in situ hybridization was used to study gene expression in embryonic mouse lung at 11.5 days p.c., when the organ consists of a single tracheal tube and branching epithelium, surrounded by a layer of mesenchyme. At this stage, hepatocyte nuclear factor3B (Hnf-3β), which encodes a winged-helix transcription factor (Sasaki and Hogan, 1993), is expressed uniformly throughout the endoderm-derived epithelium (Fig. 1A). In contrast, Wnt-2 expression is confined to the mesenchyme around the branching epithelium, as shown previously at 16.5 to 18.5 days p.c. (Levay-Young and Navre, 1992), and is absent from the tracheal mesenchyme (Fig. 1B). This distribution is confirmed by sectioning after whole-mount in situ hybridization (Fig. 1C).

Fig. 1.

Gene expression in embryonic mouse lung. Whole-mount in situ hybridization followed by sectioning was used to examine gene expression. (A) Hnf-3β is expressed uniformly throughout the epithelium of the 11.5 days p.c. lung from the trachea (t) to the terminal buds (tb). (B) Wnt-2 expression is confined to the mesenchyme around the terminal buds. In this sample, a portion of esophagus obscures the trachea but, in other cases, no expression was seen in tracheal mesenchyme. (C) Longitudinal section through a 13.5 days p.c. lung showingWnt-2 signal in mesenchyme adjacent to the terminal buds. (D) Bmp-4 expression is highest in the terminal buds. Arrowhead notes a terminal bud similar to one sectioned in E and F. (E,F) Section at two magnifications showing Bmp-4 expression in the distal epithelium of the terminal bud (ep) as well as in the adjacent mesenchyme (m). (G) Shh expression is highest in the terminal buds but extends proximally. Arrowhead notes a terminal bud similar to the one sectioned in H and I. (H,I) Section showing that the highest level of Shh expression is localized to the distal epithelium. Scale bar, A,B,D,E,G,H, 200 μm; C, 100 μm; F,I, 50 μm.

Fig. 1.

Gene expression in embryonic mouse lung. Whole-mount in situ hybridization followed by sectioning was used to examine gene expression. (A) Hnf-3β is expressed uniformly throughout the epithelium of the 11.5 days p.c. lung from the trachea (t) to the terminal buds (tb). (B) Wnt-2 expression is confined to the mesenchyme around the terminal buds. In this sample, a portion of esophagus obscures the trachea but, in other cases, no expression was seen in tracheal mesenchyme. (C) Longitudinal section through a 13.5 days p.c. lung showingWnt-2 signal in mesenchyme adjacent to the terminal buds. (D) Bmp-4 expression is highest in the terminal buds. Arrowhead notes a terminal bud similar to one sectioned in E and F. (E,F) Section at two magnifications showing Bmp-4 expression in the distal epithelium of the terminal bud (ep) as well as in the adjacent mesenchyme (m). (G) Shh expression is highest in the terminal buds but extends proximally. Arrowhead notes a terminal bud similar to the one sectioned in H and I. (H,I) Section showing that the highest level of Shh expression is localized to the distal epithelium. Scale bar, A,B,D,E,G,H, 200 μm; C, 100 μm; F,I, 50 μm.

While HNF3β is expressed uniformly throughout the epithelium, this is clearly not the case for Shh, which is expressed at high levels in the distal tips and only weakly in the proximal airways (Fig. 1G). This pattern was confirmed histologically (Fig. 1H,I). A very similar expression pattern is seen for Bmp-4 but, in this case, transcripts are also present in the mesenchyme adjacent to the most distal epithelium (Fig. 1D-F). This pattern of Bmp-4 expression continues through 12.5 and 15.5 days p.c. (Fig. 3A) but declines by 18.5 days p.c. (data not shown). Bmp-7 transcripts are seen throughout the epithelium and are also increased somewhat at the tips of the buds, but not as dramatically as Shh (Fig. 2A). Bmpr is expressed at low levels throughout the lung epithelium and in the distal tip mesenchyme (Fig. 2C). Finally, at the sensitivity of this technique, no expression at all was seen for Bmp-2 (Fig. 2D).

Fig. 2.

Expression of Bmp-7, Bmpr and Bmp-2 in 11.5 days p.c. embryonic mouse lung. (A) Bmp-7 is expressed uniformly throughout the epithelium with increased signal in the terminal buds. (B) Sense Bmp-7 probe shows no signal. (C) Bmpr expression is mostly epithelial with some signal in the mesenchyme of the terminal buds. (D) Bmp-2 is not expressed in the embryonic lung at levels that can be detected by whole-mount in situ hybridization. Scale bar, A-D, 160 μm.

Fig. 2.

Expression of Bmp-7, Bmpr and Bmp-2 in 11.5 days p.c. embryonic mouse lung. (A) Bmp-7 is expressed uniformly throughout the epithelium with increased signal in the terminal buds. (B) Sense Bmp-7 probe shows no signal. (C) Bmpr expression is mostly epithelial with some signal in the mesenchyme of the terminal buds. (D) Bmp-2 is not expressed in the embryonic lung at levels that can be detected by whole-mount in situ hybridization. Scale bar, A-D, 160 μm.

Fig. 3.

Bmp-4 expression in control and transgenic lung as revealed by in situ hybridization of sections with either digoxigenin-labelled antisense mouse Bmp-4 riboprobe (A-C) or antisense small T 3′ untranslated region probe (D). (A) In control 15.5 days p.c. lung, Bmp-4 is expressed in localized regions of the distal epithelium, presumably corresponding to the distal tips. Color reaction time 40 hours. With non-radioactive in situ hybridization, expression in the mesenchyme cannot be detected efficiently. (B) High levels of Bmp-4 expression are seen throughout the distal epithelium of transgenic 15.5 days p.c. lung. Color reaction time 16 hours. (C) In the 16.5 days p.c. transgenic lung, high levels of Bmp-4 RNA are seen in the distal epithelium, with a rather more patchy distribution than in B. Color reaction time 16 hours. (D) Expression of transgene specific RNA at 16.5 days p.c. Color reaction time 16 hours. Scale bar, A,B, 200 μm; C,D, 50 μm.

Fig. 3.

Bmp-4 expression in control and transgenic lung as revealed by in situ hybridization of sections with either digoxigenin-labelled antisense mouse Bmp-4 riboprobe (A-C) or antisense small T 3′ untranslated region probe (D). (A) In control 15.5 days p.c. lung, Bmp-4 is expressed in localized regions of the distal epithelium, presumably corresponding to the distal tips. Color reaction time 40 hours. With non-radioactive in situ hybridization, expression in the mesenchyme cannot be detected efficiently. (B) High levels of Bmp-4 expression are seen throughout the distal epithelium of transgenic 15.5 days p.c. lung. Color reaction time 16 hours. (C) In the 16.5 days p.c. transgenic lung, high levels of Bmp-4 RNA are seen in the distal epithelium, with a rather more patchy distribution than in B. Color reaction time 16 hours. (D) Expression of transgene specific RNA at 16.5 days p.c. Color reaction time 16 hours. Scale bar, A,B, 200 μm; C,D, 50 μm.

Expression of SP-C-Bmp-4 in transgenic lungs

As expected from the studies of Wert et al. (1993) using an SP-C-CAT transgene, the SP-C enhancer/promoter drives expression of Bmp-4 throughout the distal epithelium at 15.5 days p.c, but not in the more proximal airways (Fig. 3B and data not shown). This was confirmed using an SP-C-lacZ reporter transgene (data not shown). Levels of Bmp-4 RNA are significantly higher in the distal epithelium of transgenic lungs compared with controls (see legend to Fig. 3). At 16.5 days p.c., Bmp-4 expression is seen in most distal epithelial cells, but with a somewhat more patchy distribution than a day earlier (Fig. 3C). Transgene expression at this time was confirmed using a transgene-specific in situ hybridization probe (Fig. 3D).

Abnormal development of SPC-Bmp-4 transgenic lungs

As shown in Figs 1D-F and 3A, there is normally tight localization of endogenous Bmp-4 RNA to the distal tips of the epithelial buds and the adjacent mesenchyme. In contrast, the SP-C enhancer/promoter drives transgene expression in epithelial cells throughout the distal airways (Wert et al., 1993; Fig. 3 and data not shown). We therefore used this promoter to investigate the effect of misexpressing Bmp-4 in a larger domain of the distal epithelium than normal. At 11.5 and 12.5 days p.c., embryonic lungs identified as transgenic by PCR had no altered phenotype, presumably because the level of transgene expresssion is low at this time (Wert et al., 1993). However, by 15.5 and 16.5 days, the transgenic lungs are dramatically affected. First, they are significantly smaller than normal (Figs 4A, 5A-H) and about half the wet weight (for example, 6.2 mg compared with 12.9 mg at 15.5 days p.c., 7.3 mg compared with 16.0 mg at 16.5 days p.c. and 17.2 mg compared with 30.1 mg at 18.5 days p.c.). Second, the epithelium shows less extensive branching with fewer, grossly dilated, terminal buds separated by abundant mesenchyme (Fig. 5A-H). Finally, analysis of adjacent sections through the lungs showed that the dilated buds are not closed cysts, but are continuous with the bronchi and trachea (for example, Fig. 5F,H). By 18.5 days, the transgenic phenotype is even more pro-nounced and alveolar development is clearly abnormal (Fig. 5I-L). The reduced branching in the transgenic lungs at this time was confirmed by radial alveolar counts (see legend to Fig. 5I,J). One transgenic pup was born but soon died. The lungs were composed of grossly dilated, air-filled sacs (Fig. 4B,C) and histological analysis showed large dilated sacs, and a dramatic absence of alveolar septation characteristic of normal newborn lung (Fig. 5M-P). Death was probably due, at least in part, to emphysema secondary to the decreased type II alveocytes and decreased surfactant levels (see later).

Fig. 4.

Comparison of SP-C-Bmp-4 transgenic and control lungs. (A) Transgenic lung at 15.5 days p.c. (right) is smaller than control, with grossly dilated terminal buds. (B) The newborn transgenic lung (right) has more exaggerated cystic changes than at 15.5 days p.c. The portion of lung enclosed by the red box is magnified in C. (C) Dramatic dilations of distal respiratory epithelium in newborn lung are filled with air.

Fig. 4.

Comparison of SP-C-Bmp-4 transgenic and control lungs. (A) Transgenic lung at 15.5 days p.c. (right) is smaller than control, with grossly dilated terminal buds. (B) The newborn transgenic lung (right) has more exaggerated cystic changes than at 15.5 days p.c. The portion of lung enclosed by the red box is magnified in C. (C) Dramatic dilations of distal respiratory epithelium in newborn lung are filled with air.

Fig. 5.

Comparison of SP-C-Bmp-4 transgenic and control lungs at 15.5, 16.5, 18.5 days p.c. and newborn. (A) Section of 15.5 days p.c. control lung. (B) Section of 15.5 days p.c. transgenic lung. (C) Magnification of boxed portion of control lung in A shows multiple, small terminal buds and distal epithelium (de) among mesenchymal (m) cells. (D) Magnification of boxed portion of transgenic lung in B shows dilated airways lined by distal epithelium. The mesenchyme appears thicker than control lung. (E) Section of control lung at 16.5 days p.c. reveals portion of trachea and bronchus. (F) Section of transgenic lung at 16.5 days p.c. shows trachea and its connection with a primary bronchus. (G)mesenchyme. (H) Magnification of boxed portion of transgenic lung in F shows distal, dilated secondary bronchus connecting with dilated terminal buds, and thicker mesenchyme. (I)Section of 18.5 days p.c. control lung with labelled bronchiole (bl) and blood vessel (bv). The mesenchyme has thinned and septation appears to be progressing. In this section, ten terminal bronchioles are distinguishable. The number of alveoli crossed by a perpendicular line dropped from each terminal bronchiole to the nearest septum or parenchymal edge (the radial alveolar count) is 7,6,2,5,2,2,6,3,2,3 (average 3.8). (J) Section of 18.5 days p.c. transgenic lung showing continuity between trachea and primary bronchus. In this section, five terminal bronchioles are present and the radial alveolar count is 2,3,1,2,4 (average 2.4). (K) Magnification of boxed portion of control lung in I shows developing alveoli (a). (L) Magnification of boxed portion of transgenic lung in J shows abnormal alveolar development. (M) Section of newborn control lung shows a bronchiole, blood vessel and normal septation. (N) Section of newborn transgenic lung shows abnormal septation. (O)Magnification of control lung in M shows normal septation. (P) Magnification of transgenic lung in N shows abnormal septation. Scale bar, A,B,E,F,I,J,M,N, 500 μm; C,D,G,H,O,P, 100 μm; K,L, 50 μm.

Fig. 5.

Comparison of SP-C-Bmp-4 transgenic and control lungs at 15.5, 16.5, 18.5 days p.c. and newborn. (A) Section of 15.5 days p.c. control lung. (B) Section of 15.5 days p.c. transgenic lung. (C) Magnification of boxed portion of control lung in A shows multiple, small terminal buds and distal epithelium (de) among mesenchymal (m) cells. (D) Magnification of boxed portion of transgenic lung in B shows dilated airways lined by distal epithelium. The mesenchyme appears thicker than control lung. (E) Section of control lung at 16.5 days p.c. reveals portion of trachea and bronchus. (F) Section of transgenic lung at 16.5 days p.c. shows trachea and its connection with a primary bronchus. (G)mesenchyme. (H) Magnification of boxed portion of transgenic lung in F shows distal, dilated secondary bronchus connecting with dilated terminal buds, and thicker mesenchyme. (I)Section of 18.5 days p.c. control lung with labelled bronchiole (bl) and blood vessel (bv). The mesenchyme has thinned and septation appears to be progressing. In this section, ten terminal bronchioles are distinguishable. The number of alveoli crossed by a perpendicular line dropped from each terminal bronchiole to the nearest septum or parenchymal edge (the radial alveolar count) is 7,6,2,5,2,2,6,3,2,3 (average 3.8). (J) Section of 18.5 days p.c. transgenic lung showing continuity between trachea and primary bronchus. In this section, five terminal bronchioles are present and the radial alveolar count is 2,3,1,2,4 (average 2.4). (K) Magnification of boxed portion of control lung in I shows developing alveoli (a). (L) Magnification of boxed portion of transgenic lung in J shows abnormal alveolar development. (M) Section of newborn control lung shows a bronchiole, blood vessel and normal septation. (N) Section of newborn transgenic lung shows abnormal septation. (O)Magnification of control lung in M shows normal septation. (P) Magnification of transgenic lung in N shows abnormal septation. Scale bar, A,B,E,F,I,J,M,N, 500 μm; C,D,G,H,O,P, 100 μm; K,L, 50 μm.

Bmp-4 overexpression inhibits lung epithelial cell proliferation and induces cell death in the mesenchyme

Transgenic lungs are about half the size and wet weight of normal (Figs 4, 5). Cell proliferation was therefore examined by exposing 16.5 and 18.5 days p.c. embryos in utero to BrdU and FUdr for 2 hours. At 16.5 days p.c., the number of nuclei in the transgenic epithelium that had incorporated labelled nucleotide is reduced compared to normal (26% versus 34% of cells labelled, P=0.032 by Student’s t-test) (Figs 6A,B, 7A). In contrast, when the mesenchymal cells are compared, there is a small but statistically significant increase in the proportion of nuclei labelled in the transgenic lung compared to normal (15% versus 11%, P=0.029 by Student’s t-test) (Fig. 7B).

Fig. 6.

Cell proliferation and cell death in transgenic and control lungs. (A-D) Pregnant females were injected intraperitoneally with BrdU and FUdr, and 2 hours later embryonic lungs were collected, processed for histology, and the proportion of labelled cells determined as described. (A)Normal lung at 16.5 days p.c.. As shown graphically in Fig. 5A, a higher proportion of the epithelial cells are labelled compared to the transgenic. (B) Transgenic lung at 16.5 days p.c. shows dilated airways with less BrdU labelling of epithelial cells. However, the proportion of labelled mesenchyme cells is increased compared with the control, as shown graphically in Fig. 5B. (C) Normal lung at 18.5 days p.c. A smaller proportion of cells are labelled compared to the transgenic, as shown graphically in Fig. 5C. (D) Transgenic lung at 18.5 days p.c. with more labelling of cells than in the control, as shown graphically in Fig. 5C. (E-H) Cell death in normal and transgenic lung at 16.5 days p.c. (E) Section of normal lung showing a single cell stained by the TUNEL method (arrow). (F) Transgenic lung showing an increase in the number of individual labelled cells in the mesenchyme (arrows). (G) Normal lung with a single apototic cell revealed by the ISNT method (H) Transgenic lung showing individual apoptotic cells. Scale bar, A-D, 40 μm; E-H, 24 μm.

Fig. 6.

Cell proliferation and cell death in transgenic and control lungs. (A-D) Pregnant females were injected intraperitoneally with BrdU and FUdr, and 2 hours later embryonic lungs were collected, processed for histology, and the proportion of labelled cells determined as described. (A)Normal lung at 16.5 days p.c.. As shown graphically in Fig. 5A, a higher proportion of the epithelial cells are labelled compared to the transgenic. (B) Transgenic lung at 16.5 days p.c. shows dilated airways with less BrdU labelling of epithelial cells. However, the proportion of labelled mesenchyme cells is increased compared with the control, as shown graphically in Fig. 5B. (C) Normal lung at 18.5 days p.c. A smaller proportion of cells are labelled compared to the transgenic, as shown graphically in Fig. 5C. (D) Transgenic lung at 18.5 days p.c. with more labelling of cells than in the control, as shown graphically in Fig. 5C. (E-H) Cell death in normal and transgenic lung at 16.5 days p.c. (E) Section of normal lung showing a single cell stained by the TUNEL method (arrow). (F) Transgenic lung showing an increase in the number of individual labelled cells in the mesenchyme (arrows). (G) Normal lung with a single apototic cell revealed by the ISNT method (H) Transgenic lung showing individual apoptotic cells. Scale bar, A-D, 40 μm; E-H, 24 μm.

Fig. 7.

Graphic representation of BrdU labelling. (A) Total and labelled epithelial cells counted in photomicrographs of sections of control and transgenic lungs at 16.5 days p.c. (B) Total and labelled mesenchymal cells at 16.5 days p.c. (C) Total and labelled cells at 18.5 days p.c. Samples were analysed as described in Materials and Methods and P values are for the Student’s t-test.

Fig. 7.

Graphic representation of BrdU labelling. (A) Total and labelled epithelial cells counted in photomicrographs of sections of control and transgenic lungs at 16.5 days p.c. (B) Total and labelled mesenchymal cells at 16.5 days p.c. (C) Total and labelled cells at 18.5 days p.c. Samples were analysed as described in Materials and Methods and P values are for the Student’s t-test.

At 18.5 days p.c., it was not possible to distinguish unambiguously between epithelial and mesenchymal cells, due to the flattened shape of the epithelial cells and their close apposition to the underlying mesenchyme (Fig. 6C,D). However, considering the cell population as a whole, there was a statistically significant increase in the proportion of cells labelled in the transgenic lungs compared with the controls (12% versus 6%, P<0.001 by Student’s t-test) (Fig. 7C).

Since transgenic lungs are significantly smaller than normal at 16.5 days p.c., even though BrdU labelling is increased in the mesenchyme, we investigated cell death, using both the terminal deoxyribonucleotide transferase-mediated digoxigenin-dUTP end labelling (TUNEL) technique, and the in situ nick translation (ISNT) method. Both gave essentially identical results, but the ISNT method allowed the number of positive cells to be quantitated (compare Fig. 6E,F with G,H). In a control lung at 16.5 days p.c., only 3 positive cells were seen in a total of 7,983 (approximately 0.04% apoptotic cells). By contrast, in a lung from a transgenic littermate, 76/6,823 positive cells were seen (approximately 1%).

Expression of SP-C and CC-10 in transgenic lungs

To evaluate epithelial cell differentiation, in situ hybridization was carried out using riboprobes for two marker genes, SP-C and CC-10. SP-C is normally expressed in all distal epithelial cells from 10.5 days p.c. (Wert et al., 1993) but as development proceeds transcription is gradually switched off in the precursors of type I cells and expression is confined to the mature type II cells in the alveolar sacs. CC-10 expression is not apparent until 17.5 days p.c (Singh et al., 1993) and is seen only in the epithelial Clara cells that line the terminal bronchioles.

At 16.5 days p.c., SP-C is expressed in most of the epithelial cells of the normal lung buds (Fig. 8A,B). By contrast, in the transgenic lung fewer cells in the grossly dilated terminal buds express SP-C (Fig. 8C,D). At 18.5 days, the same pattern of reduced SP-C expression is noted in the transgenic lung compared with the control (Fig. 8 E,F compared with G,H).

Fig. 8.

Expression of SP-C and CC-10 in control and transgenic lungs and lungs. In situ hybridization of tissue sections was performed with digoxigenin-labeled antisense RNA probes. (A,B) Sections at two magnifications of control lung at 15.5 days p.c. showing expression for SP-C in most distal epithelial cells (de) but not in bronchioles (br) or mesenchyme (m). (C,D) Sections at two magnifications of transgenic lung at 16.5 days p.c. showing grossly distended terminal airways with fewer SP-C-expressing cells than normal. (E,F) Section at two magnifications of peripheral region of a control lung at 18.5 days p.c. showing extensive expression of SP-C. Arrow marks the edge of the lung. (G,H) Section at two magnifications of a peripheral region of a transgenic lung at 18.5 days p.c. showing distorted septation with many fewer SP-C-expressing cells. (I) Section of control lung at 18.5 days p.c. with normal expression of CC-10 in Clara cells which line the terminal bronchioles. (J) Section of transgenic lung at 18.5 days p.c. also shows CC-10 expression in the terminal bronchioles. Scale bar, A,B,C,G, 80 μm; B,F,D,H, 40 μm; I,J, 160 μm.

Fig. 8.

Expression of SP-C and CC-10 in control and transgenic lungs and lungs. In situ hybridization of tissue sections was performed with digoxigenin-labeled antisense RNA probes. (A,B) Sections at two magnifications of control lung at 15.5 days p.c. showing expression for SP-C in most distal epithelial cells (de) but not in bronchioles (br) or mesenchyme (m). (C,D) Sections at two magnifications of transgenic lung at 16.5 days p.c. showing grossly distended terminal airways with fewer SP-C-expressing cells than normal. (E,F) Section at two magnifications of peripheral region of a control lung at 18.5 days p.c. showing extensive expression of SP-C. Arrow marks the edge of the lung. (G,H) Section at two magnifications of a peripheral region of a transgenic lung at 18.5 days p.c. showing distorted septation with many fewer SP-C-expressing cells. (I) Section of control lung at 18.5 days p.c. with normal expression of CC-10 in Clara cells which line the terminal bronchioles. (J) Section of transgenic lung at 18.5 days p.c. also shows CC-10 expression in the terminal bronchioles. Scale bar, A,B,C,G, 80 μm; B,F,D,H, 40 μm; I,J, 160 μm.

Because CC-10 expression is not detected in normal lung until 17.5 days p.c., the pattern of expression of CC-10 was only compared at 18.5 days p.c. between transgenic and control. There appears to be no difference in expression between the two samples (Fig. 8I,J).

Expression of HNF3β, Shh, Bmp-7 andWnt-2 in transgenic lungs

To determine if misexpression of Bmp-4 altered the expression of genes encoding other regulatory molecules, sections of control and transgenic lungs at 15.5 days p.c. were hybridized with riboprobes for HNF3β, Shh, Wnt-2 and Bmp-7. Initial studies using digoxigenin-labeled riboprobes showed no change in expression of these genes in trans-genic lungs. To increase the sensitivity of the analysis, 35S-labeled riboprobes were then used, in conjunction with autoradiography. However, even at the higher sensitivity of this technique, no change was seen in the level of HNF3β, Shh, Wnt-2 or Bmp-7 tran-scripts in transgenic lungs compared with controls (data not shown). As expected, using this technique, transgenic lungs did show an increase in hybridization with the mouse Bmp-4 riboprobe in the distal epithelium due to cross hybridization with transcripts from the human Bmp-4 transgene. However, no significant increase was seen in the level of endogenous RNA in the mesenchyme of the transgenic lung.

Expression of Hnf-3β, Bmps and Shh during mouse lung development

Here, we report the expression in embryonic mouse lung of genes encoding signalling molecules known to play important roles in the morphogenesis of other organs. Of particular interest is the finding that Bmp-4 and Shh are both expressed at high levels in the distal epithelium of the terminal buds, apparently in very similar or overlapping domains (Fig. 1). This localization is not an artifact due to probe trapping. Under identical conditions, Hnf-3β antisense RNA gave uniform labelling of epithelial cells (Fig. 1A), while antisense Bmp-2 and sense Bmp-7 and Bmp-4 probes gave no hybridization above back-ground (Fig. 2B,D and data not shown). In addition, sectioning of the lungs after whole-mount hybridization confirmed the localization of transcripts seen in the intact organ (Fig. 1F,I). Although Bmp-4 and Shh are both expressed at high levels in the distal epithelium, some significant differences in the overall patterns were seen. In particular, Bmp-4 transcripts are also present in the adjacent mesenchyme, while Shh RNA appears to be confined to the epithelial cells.

Bmp-2, which is closely related to Bmp-4, is not transcribed in the embryonic lung, at least at a level detectable by whole-mount in situ hybridization (Fig. 2D). Bmp-7, in contrast, is expressed through-out the epithelium, with some increase in the terminal buds (Fig. 2A). This raises the possibility that Bmp-4 and Bmp-7 are co-expressed in the distal tips. Bmp-4 null mutant embryos die before the formation of lung primordia (Winnier et al., 1995) but no defects have been seen in the lungs of homozygous null Bmp-7 embryos, which survive to birth (A. Dudley and E. J. Robertson, personal communication) suggesting that any function of Bmp-7 homo- or heterodimers in lung development can be compensated for by other proteins.

The effect of misexpressing Bmp-4 in transgenic mouse lungs

To explore the role of BMP4 in lung development, we generated transgenic embryos in which human Bmp-4 is expressed through-out the distal epithelium under the control of the SP-C promoter/enhancer (Fig. 3). No abnormalities were seen in trans-genic lungs at 11.5 and 12.5 days p.c. However, at 15.5 and 16.5 days p.c., Bmp-4 misexpression leads to a dramatic effect on lung development (Figs 4A, 5A-H). In particular, trans-genic lungs are smaller than normal, are about half the wet weight (see text) and have fewer, greatly distended, epithelial terminal buds separated by abundant mesenchyme. By 18.5 days, the lung lobes contain huge, epithelial sacs apparently continuous with the bronchi and trachea and separated by mes-enchyme (Fig. 5I-L). At birth, these sacs fill with air (Fig. 4C), but they show a dramatic lack of normal alveolar seg-mentation (Fig. 5M-P) and do not sustain normal lung function.

The fact that transgenic lungs are smaller than normal raises the possibility that overexpression of Bmp-4 inhibits cell proliferation and/or promotes cell death. Indeed, both mechanisms do appear to be operating in transgenic lungs, suggesting that the effect of the ligand in vivo is also complex. BrdU labelling experiments at the pseudoglandular stage showed a 24% decrease in the labelling of transgenic epithelium, but a 26% increase in labelling of the mesenchyme compared with controls. At 18.5 days, there was a 49% increase in the labelling of cells in transgenic lungs, although it was not possible to distinguish between epithelial and mes-enchymal cells at this stage. At 16.5 days, analysis of lung sections using both the TUNEL and ISNT techniques revealed a very low level of cell death in the normal lung (approximately 0.04%), but a large increase in the number of labelled cells in the mesenchyme of transgenic lungs (to approximately 1.0%).

There are several possible, non-exclusive explanations for these findings. One is that ectopically expressed BMP4 has different effects on epithelial versus mesenchymal cells, inhibiting the proliferation and differentiation of the former, and stimulating either proliferation or cell death in the latter. To account for the effect on mesenchyme, it is possible that there are two different populations of cells, that respond differently to BMP4. For example, endothelial cells may proliferate and interstitial or stromal cells may die in response to the ligand. Alternatively, different levels of BMP4 may induce different responses in the mesenchyme, so that cells close to the epithelium (the source of ectopic BMP4) undergo cell death and those further away are stimulated to proliferate. Further studies will be needed to distinguish between these models. Meanwhile, it should be noted that BMP4 has been observed to promote apoptosis in neural crest cells from rhombomeres 3 and 5 in the chick embryo hindbrain (Graham et al., 1994). Does BMP4 affect cell differentiation in the embryonic lung? In situ hybridization reveals a reduction in the number of cells expressing SP-C in transgenic lungs versus controls, but no change in the pattern of expression of CC-10 (Fig. 8). This result shows that Bmp-4 overexpression has no effect on the differentiation of the CC-10-expressing epithelial Clara cells in the bronchioles and proximal airways, but does appear to affect the differ-entiation of the distal epithelial cells. Again, there are several, not mutually exclusive, mechanisms that may underlie this phenomenon. During the transition from the pseudoglandular to saccular stage of development, the SP-C-expressing, cuboidal, airway precursor cells begin to differentiate into either type I cells, which do not transcribe SP-C, or into mature type II cells, which upregulate and maintain SP-C expression (Ten Have-Opbroek, 1991; Wert et al., 1993). In the transgenic lung, BMP4 may either actively promote the differentiation of primordial cells into type I rather than type II cells, or selectively inhibit the terminal differ-entiation or survival of mature type II cells that maintain high levels of SP-C expression. Resolution between these models may require the identification of specific markers for type I cells.

Finally, misexpression of Bmp-4 throughout the distal epithelium in transgenic lungs has a dramatic effect on branching morphogenesis after about 15.5 days, resulting in the formation of fewer, grossly dilated terminal sacs compared with control lungs (Fig. 5). The fact that Bmp-4 overexpression inhibits epithelial cell proliferation raises the possibility that, during normal development, when Bmp-4 is expressed in a very restricted domain, high local levels of the protein accumulate at the tips of branches and gradually suppress distal epithelial growth. Any new outgrowth can then only occur from regions proximal and lateral to the distal tips, resulting in dichotomous branching. Unfortunately, no phenotype was observed in SP-C transgenic lungs at the early pseudoglandular stage (e.g. 11.5-

12.5 days), when the pattern of branching morphogenesis can be observed clearly. This may reflect a low level of BMP4 expression from the transgene at this time, so further experiments will be needed to test the hypothesis that BMP4 specifi-cally affects dichotomous branching morphogenesis in vivo. Meanwhile, it should be noted that, in the Drosophila larva, dpp expression in the anterior foregut epithelium regulates the formation of the proventriculus. In dpp null mutants, proventricular development is abnormal and, in some cases, ectopic outgrowths appear in the oesophagus, consistent with a normal role for the protein in suppressing local endodermal cell movement and morphogenesis (Pankratz and Hoch, 1995).

The phenotypic effect of overexpressing BMP4 in the developing mouse lung is significantly different from that of over-expressing TGF-β1, utilizing the same SP-C vector. Lungs transgenic for SP-C-TGF-β1 are the same size as controls at 16.5 days p.c. (Zhou et al., 1996). However, development from the pseudoglandular to canalicular and saccular stage is blocked, and in situ hybridization demonstrates a reduction in the level of both SP-C and CC-10 gene expression.

A model for the interaction between BMP4 and other polypeptide signalling molecules in lung morphogenesis

The patterns of expression of Bmp-4, Shh, Bmpr and Wnt-2 (Fig. 1) suggest a model for the role of these genes in lung mor-phogenesis (Fig. 9). First, we propose that SHH protein secreted by the distal tip epithelium induces Bmp-4 expression in the adjacent mesenchyme. This idea is based on the obser-vation that, in Drosophila, the Shh homolog, hedgehog (hh), induces the expression of the Bmp-2/4 homolog, decapenta-plegic (dpp) in adjacent cells (Herberlein et al., 1993; Diaz-Benjumea et al., 1994; Ingham, 1995, for review). The hh gene encodes a secreted glycoprotein which exists in both membrane-associated and soluble forms, and it is believed that the protein interacts with target cells via a membrane-associated protein, patched (ptc) (Ingham et al., 1991). A mouse ptc homolog has recently been cloned and is expressed in the mesenchyme of the lung (L. V. Goodrich, R. L. Johnson, L. Milenkovic and M. Scott, personal communication). This supports the idea that SHH secreted by the distal epithelium activates Bmp-4 in the adjacent mesenchyme. However, induction by SHH of Bmp-4 in epithelial cells independent of PTC cannot be ruled out. While the evidence that hh regulates dpp expression in Drosophila embryos is very strong, the data concerning Bmp expression in response to SHH in vertebrate embryos is still quite minimal. However, in the chick embryo, Bmp-2 is induced in limb bud anterior mesenchyme in response to ectopic SHH (Laufer et al., 1994) and ectopic SHH induces Bmp-4 in hindgut mesenchyme (Roberts et al., 1995).

Fig. 9.

Model for the inter-relationship of Shh, Bmp-4 and Wnt-2 in the embryonic mouse lung. For details, see text. It is proposed that SHH protein secreted (red arrow) by distal tip epithelial cells induces the expression of Bmp-4 and/or Wnt-2 in the adjacent mesenchyme. Bmp-4 expression in the distal epithelium may also be regulated by SHH. Alternatively, or in addition, expression of BMP4 in the epithelium may be autoinduced by protein made in the mesenchyme (blue arrows). WNT2 made by the mesenchyme (green arrows) may also affect gene expression in the epithelium.

Fig. 9.

Model for the inter-relationship of Shh, Bmp-4 and Wnt-2 in the embryonic mouse lung. For details, see text. It is proposed that SHH protein secreted (red arrow) by distal tip epithelial cells induces the expression of Bmp-4 and/or Wnt-2 in the adjacent mesenchyme. Bmp-4 expression in the distal epithelium may also be regulated by SHH. Alternatively, or in addition, expression of BMP4 in the epithelium may be autoinduced by protein made in the mesenchyme (blue arrows). WNT2 made by the mesenchyme (green arrows) may also affect gene expression in the epithelium.

In the Drosophila embryo, hh also regulates the expression of wingless (wg), which encodes a secreted glycoprotein related to the Wnt family of signalling molecules (Diaz-Benjumea et al., 1994). In addition, wg maintains hh expression (Lee et al., 1992). In the developing vertebrate limb bud, simultaneous Wnt-7a and Fgf4 expression are required to maintain Shh expression in the posterior mesenchyme expression (Yang and Niswander, 1995). This raises the possibility that SHH in the distal epithelium of the lung induces Wnt-2 in the surrounding mesenchyme, and that WNT2 (and possibly an FGF family member) is required to maintain Shh expression.

Finally, BMP4 protein made in the distal mesenchyme could act on the distal epithelium to activate Bmp-4 expression. This would establish an autoregulatory circuit similar to that proposed in the developing mouse tooth bud (Vainio et al., 1993). In order for BMP4 to induce its own expression, it is necessary that the target cells express appropriate receptors. We have shown here that the lung epithelium and distal mesenchyme express a Type I transmembrane serine-threonine kinase receptor which can bind both Bmp-2 and Bmp-4 (Bmpr/Trk-11/Brk-1) (Koenig et al., 1994). However, it is very likely that other Type I and Type II receptors that bind BMPs are also expressed. This, and other aspects of the model, are currently under investigation.

We thank Dr Jeffrey Whitsett for the SP-C vector, for in situ hybrid-ization probes and for generous advice and encouragement, Terry Johnson, Division of Neonatology, Vanderbilt Medical School, and Martin Offield, Department of Cell Biology, for help with graphics, Dr Yu Shyr, Department of Preventive Medicine for statistical analysis, and Dr Lillian Nanney and the Tissue Analysis Core laboratory (suppported by AR41943) for analysis of BrdU labelling. We also thank Drs Robert Cotton, Christopher Wright, Rosa Serra and laboratory colleagues for critical comments on the manuscript and Linda Hargett, Julie Blackwell, Yolanda McClain and Lorene Batts for skilled technical assistance. S. B. acknowledges support from The Philippe Foundation. This work was supported by NIH grants HD28955 and ST32HL07256. Brigid Hogan is an Investigator of the Howard Hughes Medical Institute.

Alescio
,
T.
and
Cassini
,
A.
(
1962
).
Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium
.
J. Exp. Zool
.
150
,
83
94
.
Blessing
,
M.
,
Nanney
,
L. B.
,
King
,
L. E.
,
Jones
,
C. M.
and
Hogan
,
B. L. M.
(
1993
).
Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles
.
Genes Dev
.
7
,
204
215
.
Carpenter
,
G.
(
1993
).
EGF : new tricks for an old growth factor
.
Current Opinion in Cell Biol
.
5
,
261
264
.
Diaz-Benjumea
,
F. J.
,
Cohen
,
B.
and
Cohen
,
S. M.
(
1994
).
Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs
.
Nature
372
,
175
179
.
Fire
,
A.
,
White Harrison
,
S.
and
Dixon
,
D.
(
1990
).
A modular set of lacZ fusion vectors for studying gene expression in C. elegans
.
Gene
93
,
189
198
.
Gold
,
R.
,
Schmied
,
M.
,
Rothe
,
G.
,
Zischler
,
H.
,
Breitschopf
,
H.
,
Wekerle
,
H.
and
Lassmann
,
H.
(
1993
)
Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections
.
J. Histochem. Cytochem
.
41
,
1023
1030
.
Goldin
,
G. V.
and
Wessels
,
N. K.
(
1979
).
Mammalian lung development: the possible role of cell proliferation in the formation of supernumerary tracheal buds and in branching morphogenesis
.
J. Exp. Zool
.
208
,
337
346
.
Graham
,
A.
,
Francis-West
,
P.
,
Brickell
,
P.
and
Lumsden
,
A.
(
1994
).
The signalling molecule BMP4 mediates apoptosis in the rhomobocephalic neural crest
.
Nature
372
,
684
686
.
Green
,
M. C.
(
1968
).
Mechanism of the pleiotropic effects of the short ear mutant gene in the mouse
.
J. Exp. Zool
.
167
,
129
150
.
Heine
,
U. I.
,
Munoz
,
E. F.
,
Flanders
,
K. C.
,
Roberts
,
A. B.
and
Sporn
,
M. B.
(
1990
).
Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis
.
Development
109
,
29
36
.
Herberlein
,
U.
,
Wolff
,
T.
and
Rubin
,
G. M.
(
1993
).
The TGFβ homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in Drosophila retina
.
Cell
75
,
913
926
.
Hogan
,
B. L. M.
(
1995
).
The TGFβ-related signalling system in mouse development
.
Seminar in Dev Biol
6
,
257
265
.
Hogan
,
B. L. M.
,
Beddington
,
R.
,
Constantini
,
F.
and
Lacy
,
E.
(
1994
).
Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor
,
New York
:
Cold Spring Harbor Publications
.
Ingham
,
P.W.
(
1995
).
Signalling by hedgehog family proteins in Drosophila and vertebrate development Curr. Op
.
Genetics Dev
.
5
,
492
498
.
Ingham
,
P. W.
,
Taylor
,
A. M.
and
Nakano
,
Y.
(
1991
).
Role of the Drosophila patched gene in positional signalling
.
Nature
353
,
184
187
.
Jones
,
C.M.
,
Lyons
,
K.M.
and
Hogan
,
B.L.M.
(
1991
).
Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse
.
Development
111
,
531
542
.
Korfhagen
,
T.
,
Glasser
,
S.
,
Wert
,
S.
,
Bruno
,
M.
,
Daugherty
,
C.
,
McNeish
,
J.
,
Stock
,
J.
,
Potter
,
S.
and
Whitsett
,
J.
(
1990
).
Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice
.
Proc. Natl. Acad. Sci. USA
87
,
6122
6126
.
King
,
J. A.
,
Marker
,
P. C.
,
Seung
,
K. J.
and
Kingsley
,
D. M.
(
1994
).
BMP5 and the molecular, skeletal, and soft-tissue alterations in short ear mice
.
Dev. Biol
.
166
,
112
122
.
Kingsley
,
D. M.
(
1994
).
What do BMPs do in mammals? Clues from the mouse short ear mutation
.
Trends in Genetics
10
,
16
22
.
Koenig
,
B. B.
,
Cook
,
J. S.
,
Wosling
,
D. H.
,
Ting
,
J.
,
Tiesman
,
J. P.
,
Correa
,
P. E.
,
Olson
,
C. A.
,
Pecquet
,
A. L.
,
Ventura
,
F.
,
Grant
,
R. A.
,
Chen
,
G.
,
Wrana
,
J. L.
,
Massague
,
J.
and
Rosenbaum
,
J. S.
(
1994
).
Characterization and cloning of a receptor for Bmp-2 and Bmp-4 from NIH 3T3 cells
.
Mol. Cell. Biol
.
14
,
5961
5974
.
Laufer
,
E.
,
Nelson
,
C. E.
,
Johnson
,
R. L.
,
Morgan
,
B. A.
and
Tabin
,
C.
(
1994
).
Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud
.
Cell
79
,
993
1003
.
Lee
,
J. J.
,
von Kessler
,
D. P.
,
Parks
,
S.
and
Beachy
,
P. A.
(
1992
).
Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog
.
Cell
71
,
33
50
.
Lehnert
,
S. A.
and
Akhurst
,
R. J.
(
1988
).
Embryonic expression pattern of TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of action
.
Development
104
,
263
273
.
Levay-Young
,
B. K.
and
Navre
,
M.
(
1992
).
Growth and developmental regulation of wnt-2 (irp) gene in mesenchymal cells of fetal lung
.
Am. J. Physiol
.
262
,
672
683
.
Mason
,
I. J.
(
1994
).
The Ins and Outs of fibroblast growth factors
.
Cell
78
,
547
552
.
Minno
,
P.
and
King
,
R. J.
(
1994
).
Epithelial-mesenchymal interactions in lung development
.
Ann. Rev. Physiol
.
56
,
13
45
.
Nogawa
,
H.
and
Takaaki
,
I.
(
1995
).
Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture
.
Development
121
,
1015
1022
.
Pankratz
,
M.
and
Hoch
,
M.
(
1995
).
Control of epithelial morphogenesis by cell signaling and integrin molecules in the Drosophila foregut
.
Development
121
,
1885
1898
.
Parr
,
B. A.
and
McMahon
,
A. P.
(
1994
).
Wnt genes and vertebrate development
.
Current Opinion Genetics and Development
4
,
523
528
.
Pelton
,
R. W.
,
Saxena
,
B.
,
Jones
,
M.
,
Moses
,
H. L.
and
Gold
,
L. I.
(
1991
).
Immunohistochemical localization of TGF-β1, TGF-β2, and TGF-β3 in the mouse embryo: expresion patterns suggest multiple roles during embryonic development
.
J. Cell Biol
.
115
,
1091
1105
.
Peters
,
K.
,
Werner
,
S.
,
Liao
,
X.
,
Wert
,
S.
,
Whitsett
,
J.
and
Williams
,
L.
(
1994
).
Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung
.
EMBO J
.
13
,
3296
3301
.
Roberts
,
D.J.
,
Johnson
,
R.L.
,
Burke
,
A.C.
,
Nelson
,
C.E.
,
Morgan
,
B.A.
and
Tabin
,
C.
(
1995
)
Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut
.
Development
121
,
3163
3174
.
Roman
,
J.
,
Little
,
C. W.
and
McDonald
,
J. A.
(
1991
).
Potential role of RGD-binding integrin in mammalian lung branching morphogenesis
.
Development
112
,
551
558
.
Sakai
,
Y.
,
Nelson
,
K. G.
,
Snedeter
,
S.
,
Bossert
,
N. L.
,
Walker
,
M. P.
,
McMachon
,
J.
and
DiAugustine
,
R. P.
(
1994
).
Expression of epidermal growth factor in suprabasal cells of stratified squamous epithelia: implications for a role in differentiation
.
Cell Growth Diff
.
5
,
527
535
.
Sasaki
,
H.
and
Hogan
,
B. L. M.
(
1993
).
Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo
.
Development
118
,
47
59
.
Serra
,
R.
,
Pelton
,
R. W.
and
Moses
,
H. L.
(
1994
).
TGFβ1 inhibits branching morphogenesis and n-myc expression in lung bud organ cultures
.
Development
120
,
2153
2161
.
Shannon
,
J. M.
(
1994
).
Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme
.
Dev.Biol
.
166
,
600
614
.
Singh
,
G.
,
Katyal
,
S. L.
,
Brown
,
W. E.
and
Kennedy
,
A. L.
(
1993
).
Mouse clara cell 10-kDa (CC10) protein: cDNA nucleotide sequence and molecular basis of variation in progesterone binding of CC10 from different species
.
Exp. Lung Research
19
,
67
75
.
Ten Have-Opbroek
,
A. A. W.
(
1991
).
Lung development in the mouse embryo
.
Exp. Lung Research
17
,
111
130
.
Vanio
,
S.
,
Karavanova
,
I.
,
Jowett
,
A.
and
Thesleff
,
I.
(
1993
).
Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development
.
Cell
75
,
45
58
.
Wert
,
S.
,
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
.
Wessels
,
N. K.
(
1970
).
Mammalian lung development: interaction in formation and morphogenesis of tracheal buds
.
J. Exp. Zool
.
175
,
455
466
.
Winnier
,
G.
,
Blessing
,
M.
,
Labosky
,
P. A.
and
Hogan
,
B.
(
1995
).
Bone Morphogenetic protein-4 (BMP-4) is required for mesoderm formation and patterning in the mouse
.
Genes Dev
.
9
,
2105
2116
.
Yang
,
Y.
and
Niswander
,
L.
(
1995
).
Interaction between the signaling molecules Wnt-7a and SHH during vertebrate limb development: dorsal signals regulate anteroposterior patterning
.
Cell
80
,
939
947
.
Zhao
,
G-Q.
,
Zhou
,
X.
,
Eberspaecher
,
H.
,
Solursh
,
M.
and
de Crombrugghe
,
B.
(
1993
).
Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes
.
Proc. Natl. Acad. Sci. USA
90
,
8633
8637
.
Zhou
,
L.
,
Dey
,
C. R.
,
Wert
,
S. E.
and
Whitsett
,
J. A.
(
1996
)
Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-β1 chimeric gene
.
Dev. Biol. (In press)