ELSEVIER SCIENCE IRELAND Mechanisms of Development 45 (1994) 193-201

Expression of collagen al(IV), laminin and nidogen genes in the embryonic mouse lung: implications for branching morphogenesis

Tim Thomas, Marie Dziadek *

Centre for Early Human Development, Institute of Reproduction and Development, Monash Medical Centre, Clayton, Vic. 3168, Australia

(Received 12 May 1993; revised 15 October 1993; accepted 9 November 1993)

Abstract

The patterns of laminin A, B1, B2, nidogen and collagen al(IV) gene expression in the embryonic mouse lung were determined using in situ hybridization histochemistry at a stage when branching morphogenesis is taking place. Collagen al(IV), laminin B1 and B2 genes were expressed throughout the mesenchyme and epithelium. Nidogen gene expression was uniform throughout the mesenchyme but was not detected in epithelial cells. Laminin A mRNA was localized to cells closely associated with a basement membrane at the epithelial-mesenchymal interface. However, expression of the laminin A gene was limited to the mesenchymal cells in bronchial regions and to epithelial cells in distal terminal lobules. We propose that the pattern of laminin A gene expression in different regions of the developing lung will influence the structure of the basement membrane at the epithelial-mesenchymal interface and thus have a role in branching morphogenesis.

Key words: Embryonic lung; Branching morphogenesis; Basement membrane; Laminin; Nidogen; Collagen IV

1. Introduction

Basement membranes are specialized extracellular matrices composed of a network of collagen IV com- plexed with laminin and other glycoproteins and pro- teoglycans (Timpl, 1989; Inoue, 1989; Inoue and Leblond, 1988) which underlie all epithelial and en- dothelial cells (Timpl and Dziadek, 1986). Basement membranes have been shown to have a profound effect on cell polarization, proliferation, multicellular organi- zation and gene expression in a variety of tissues (Blum et al., 1987; Schuetz et al., 1988; Streuli et al., 1991; Hadley et al., 1985; Thomas et al., 1992). For most systems it is not yet clear to what extent these effects are mediated by direct interactions with extracellular matrix components or indirectly via growth factors bound to the matrix (Pavalbar et al., 1991; Vukicevic et al., 1992; Ruoslahti and Yamaguchi, 1991).

The extracellular matrix, including the basement membrane, is important in regulating branching mor- phogenesis of the embryonic lung and salivary gland (Alescio, 1973; Banerjee et al., 1977; Spooner and

* Corresponding author. Tel: (03) 550 5478. Fax: (03) 550 5554.

Faubion, 1980; Spooner et al., 1985; Bernfield et al., 1984). At an early stage of development the lung consists of an undifferentiated epithelium surrounded by mesenchyme, which undergoes a process of re- peated branching into multiple epithelial buds which ultimately form the bronchial tree. Branching morpho- genesis is a highly ordered process which is controlled by epithelial-mesenchymal interactions (Alescio and Cassini, 1962; Wessells, 1970; Hilfer et al., 1985; Taka- hashi and Nogawa, 1991) mediated at least in part by the basement membrane at the epithelial-mesenchymal interface (Grobstein, 1954; Bernfield et al., 1984). Re- cent experiments have shown that lung morphogenesis in vitro is perturbed by anti-laminin antibodies (Schuger et al., 1990) and by peptides containing the integrin- binding RG D sequence (Roman et al., 1991). In the salivary gland the morphogenetic effect of the mes- enchyme can be duplicated in vitro by a basement membrane substrate containing epidermal growth fac- tor (Nogawa and Takahashi, 1991). It appears that quantitative changes in basement membrane composi- tion are involved in branching morphogenesis since increased proliferation of epithelial cells in the distal bud regions correlates with decreased amounts of base- ment membrane components in these regions (Bern-

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194 Z Thomas, M. Dziadek / Mechanisms of DeL,elopment 45 (1994) 193-201

field et al., 1984). Since it has been generally assumed that the mesenchyme does not contribute to the syn- thesis of basement membrane components, the role of the mesenchyme has been proposed to be in remod- elling of the subepithelial basement membrane, by production of enzymes which degrade glycosaminogly- cans and other matrix components at specific sites (Bernfield and Banerjee, 1982; Bernfield et al., 1984) and deposition of interstitial collagens in non-branch- ing regions which stabilize the basement membrane in these regions (David and Bernfield, 1981; Bernfield et al., 1984). While the presence of collagen I in the mesenchyme is not necessary for branching morpho- genesis of the embryonic lung and salivary gland (Kratochwil et al., 1986), differential localization of other interstitial collagens such as collagen III may be involved (Nakanishi et al., 1988). Recent studies have clearly demonstrated that both epithelial and mes- enchymal cells in embryonic tissues synthesize base- ment membrane components (Klein et al., 1990; Kuch- erer-Ehret et al., 1990; Simon-Assmann et al., 1988, 1989; Thomas and Dziadek, 1993a), and that the mes- enchyme is the only source of nidogen (entactin) which is deposited in the basement membrane of many em- bryonic tissues (Thomas and Dziadek, 1993a). Thus, regional differences in basement membrane production by the mesenchyme could play a role in the regulation of basement membrane assembly and deposition in the embryonic lung, which could then influence epithelial proliferation.

The aim of the present study was to establish the pattern of collagen al(IV), laminin A, B1, B2 and nidogen gene expression in the developing lung during the period when branching morphogenesis is occurring, but before epithelial differentiation, in order to deter- mine the respective roles of the epithelium and the mesenchyme in basement membrane production in dif- ferent regions of the lung, and to determine whether differential synthesis of basement membrane compo- nents might in part be responsible for variations in basement membrane structure in the developing lung.

2. Results

2.1. Northern gel analysis

The levels of laminin A, B1, B2, nidogen and colla- gen al(IV) mRNA was assayed by Northern gel analy-

A

[11

R E Lw LE LM Li R

EI2

N

C

Fig. 1. Northern gel analysis of total RNA isolated from 12.5 day embryonic tissues. The levels of mRNA coding for laminin A (A), laminin B1 (B1), laminin B2 (B2), nidogen (N) and collagen cd(IV) (C) are shown for Reicherrs membrane (R), the whole lung (Lw) compared to the whole embryo (E), separated lung epithelium (Le) and lung mesenchyme (Lm), and adult liver (Li) as a negative control. Nidogen mRNA can be detected in all embryonic RNA samples except for lung epithelium while all other mRNAs are present in each embryonic sample. The Reicherrs membrane sample contains 1 /xg of total RNA, and other lanes contain 7/zg of total RNA.

sis in 12.5 day gestation lung epithelium and lung mesenchyme. The mRNA levels in these compartments were compared to whole lung, whole embryo, Reichert's membrane and adult liver. One filter was probed se- quentially with cDNA probes for nidogen, laminin A, laminin B2 and laminin B1, and an identical filter was probed with a collagen al(IV) cDNA probe. This allowed direct comparisons in the relative amounts of the five mRNA species in these tissues. Using the mRNA levels in Reichert's membrane as a basis for comparison it can be seen that laminin A mRNA levels are substantially lower in the lung than nidogen, laminin B1, laminin B2 and collagen al(IV) mRNAs (Fig. 1). However, laminin A mRNA, relative to total RNA, was more abundant in the lung than in the whole embryo, and was synthesized in similar amounts by both epithelium and mesenchyme (Fig. 1). Relative to total RNA there appeared to be little difference in the amount of laminin B1, laminin B2 and collagen al(IV) mRNA levels in epithelial and mesenchymal compart-

Fig. 2. In situ hybridization histochemistry showing the distribution of mRNA coding for collagen IV (A), laminin A (B), laminin B1 (C), laminin B2 (D), and nidogen (E). The same section is shown in light-field on the left and in dark-field on the right. Laminin B1, B2 and collagen al(IV) genes are expressed in the mesenchyme (M) and epithelium (E), while nidogen mRNA is present only in the mesenchyme. The expression of laminin A in the mesenchyme stops abruptly at minor branch points (arrow in B) although it is continuous at the major bronchial bifurcation. In the lobules laminin A is expressed only by the epithelium (arrowhead in B). Scale bar = 100 izm.

I

196 T. Thomas, M. Dziadek /Mechanisms of Deuelopment 45 (1994) 193-201

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T. Thomas, M. Dziadek / Mechanisms of Del'elopment 45 (1994) 193-201 197

ments. These three mRNAs were present at higher levels per total RNA in the lung than in the whole embryo, but at lower levels than in parietal endoderm cells of Reichert 's membrane. In all tissues two colla- gen a l ( IV) mRNA bands were observed, the higher molecular weight band being more intense than the lower as has been reported previously (Kurkinen et al., 1985). The ratio between the two bands appeared to be similar in all tissues examined (Fig. 1). Collagen a l ( IV) has 63% homology in the NC1 region with collagen a2(IV) (Muthukumaran et al., 1989) but the molecular weight of collagen a2(IV) mRNA is intermediate be- tween the two species of collagen a l ( IV) mRNA (Kurkinen et al., 1985). No significant cross reactivity between the two collagen sequences was detected un- der these conditions. Nidogen differed from all other mRNAs assayed in that it was not detected in epithe- lial RNA, but was present in the mesenchyme at levels equivalent to the whole embryo (Fig. 1).

2.2. Pattern o f expression of laminin, nidogen and colla- gen a l (IV) genes in the lung

In situ hybridization histochemistry was used to identify cells expressing laminin, nidogen and collagen a l ( IV) genes in the 12.5 day lung. Laminin B1 and B2 genes were expressed uniformly throughout the mes- enchymal compartment (Fig. 2C,D). The levels of ex- pression of these genes appeared higher in the epithe- lial buds than in bronchial regions or surrounding mesenchyme, particularly the laminin B2 gene (Fig. 2D). Nidogen gene expression was found to be com- pletely uniform throughout the mesenchyme and ab- sent from the epithelium (Fig. 2E). Collagen a l ( IV) mRNA was found evenly throughout the epithelial layer and also throughout the mesenchyme. However, in the mesenchyme, there were groups of ceils contain- ing very high levels of this mRNA (arrow, Fig. 2A) with other mesenchymal ceils containing levels similar to the epithelium. In cross section, mesenchymal cells containing high levels of collagen a l ( IV) formed cords running parallel to the epithelial branches (Fig. 3D) and spaced at regular intervals around the epithelium. Laminin A was the only basement membrane compo- nent specifically found in cells adjacent to where a basement membrane can be demonstrated histologi- cally, separating epithelial and mesenchymal cells (Fig.

2B). Laminin A mRNA was present in both epithelial and mesenchymal layers, but was not distributed uni- formly. In bronchial regions this gene was expressed in the underlying mesenchyme and not by the epithelial cells (Fig. 3A,B) whereas in large epithelial buds it was expressed primarily in epithelial cells with only back- ground hybridization seen in the surrounding mes- enchyme (Fig. 3C). In intervening regions the level of expression was low, and in some places gaps could be seen between regions of high expression in the mes- enchyme which may correspond to places where ep- ithelial branching is being initiated. The mesenchymal cells expressing high levels of the laminin A gene appear to correspond to the cuboidal mesenchyme cells reported by Jaskoll and Slavkin (1984).

3. Discussion

In this paper we show that genes coding for base- ment membrane glycoproteins, laminin, nidogen and collagen IV are differentially expressed in the embry- onic lung. Laminin B1, B2 and collagen a l ( IV) mR- NAs are distributed throughout both epithelial and mesenchymal compartments of the lung, whereas nido- gen mRNA is synthesized only by the mesenchyme. No significant differences in the abundance of collagen al(IV), nidogen, and laminin B1 mRNAs were de- tected between the branching and the non-branching regions of the lung, while levels of laminin B2 mRNA appeared higher in distal bud epithelium than in other regions of epithelium and mesenchyme. The laminin A chain gene was the only gene studied whose expression pattern correlated with sites where a typical basement membrane is known to be present. Laminin A mRNA has an interesting distribution pattern, being expressed only in the mesenchyme surrounding bronchial tubes, and only in the epithelium in distal regions where buds are forming. In intermediate regions, only low levels of laminin A mRNA could be detected in either the mesenchyme or the epithelium.

Collagen IV forms the major structural element of basement membranes (Inoue, 1989; Timpl, 1989), and has been shown to be produced by a variety of epithe- lial and endothelial cells (Timpl and Dziadek, 1986). Deposition of collagen IV in mesenchymal layers of several embryonic tissues including the lung has been

Fig. 3. In situ hybridization histochemistry showing details of laminin A (A,B,C) and collagen al(IV) (D) gene expression in the lung. In bronchial regions laminin A is expressed only in the mesenchyme adjacent to the epithelial-mesenchymal interface (arrows in B), while expression in the mesenchyme ceases at branch points (arrowheads in A). In the terminal lobules expression is restricted to the epithelium (arrow in C). In the intervening regions the mRNA levels are low. Collagen al(IV) mRNA is not uniformly expressed in the mesenchyme, with groups of cells containing much higher levels than average (D). In a cross section through the epithelial tree it can be seen that these cells form cords running parallel to the epithelium (D). The dark-field picture in D has been exposed so that only cells containing the highest levels of mRNA can be seen clearly. Scale bars are 100 ~m in A and D, and 50 p~m in B and C.

198 T. Thomas, M. Dziadek / Mechanisms of Del~elopment 45 (1994) 193-201

demonstrated previously (Kimata et al., 1985; Kuhl et al., 1984; Simon-Assmann et al., 1988; 1990; Chen and Little, 1987), although it is not clear in some of these cases whether material detected by immunofluores- cence is synthesized by endothelial cells or mesenchy- real stroma[ ceils (Simon-Assmann et al., 1988). The results presented in this paper show that collagen ~I(IV) mRNA is produced by both mesenchymal and epithelial cell types in the lung. Particularly high levels were present in groups of mesenchymal cells which in cross section of lung tissue appeared to form cords running parallel to the axis of the epithelial tubes. These mesenchymal 'cords' are most likely to be pre- cursors of blood vessels. High levels of collagen al(IV) mRNA are present in endothelial cells in other embry- onic tissues (Thomas and Dziadek, 1993a) and placenta (Thomas and Dziadek, 1993b). Similar levels of colla- gen al(IV) mRNA were present in epithelial and mesenchymal cells of branching and non-branching regions of the lung. Previous immunohistochemical studies have shown that although the basement mem- brane around epithelial buds is discontinuous in the lung (Blueminck et al., 1976; Jaskoll and Slavkin, 1984), no differences in collagen IV deposition can be seen in branching and non-branching regions (Chen and Little, 1987; Jaskoll and Slavkin, 1984). While normal collagen production is required for lung morphogenesis (Alescio, 1973; Spooner and Faubion, 1980), the uniform pattern of expression of collagen al(IV) mRNA in the lung suggests that differential expression of collagen IV is unlikely to be a factor in control of basement mem- brane structure during branching morphogenesis in the lung.

Like collagen c~l(IV), laminin B1 and B2 chain mRNAs were distributed throughout both mesenchy- real and epithelial layers in the lung. Expression of the laminin B2 gene appeared to be higher in the distal bud regions than in other parts of the epithelium. However, nidogen mRNA was restricted to the mes- enchymal cells and was not found in lung epithelium, as was shown previously for several other embryonic tissues (Thomas and Dziadek, 1993a). Unlike epithelial cells, endothelial cells do express the nidogen gene (Thomas and Dziadek, 1993a,b), and the hybridization of nidogen probes to lung mesenchyma[ tissue is due to the presence of both endothelial and mesenchymal cells in this tissue layer. Nidogen forms stable com- plexes with laminin in basement membranes (Paulsson et al., 1987), and also interacts by a separate domain with collagen IV (Aumailley et al., 1989; Fox et al., 1991) and heparan sulfate proteoglycan (Battaglia et al., 1992). Nidogen is thus thought to have a major role in basement membrane stability by mediating these inter-component interactions important for basement membrane structure. Levels of nidogen expression are therefore expected to play an important role in regulat-

ing basement membrane assembly. This study demon- strates that no significant differences are found in the steady-state levels of nidogen mRNA in different re- gions of the lung, indicating that thinning and disconti- nuities in the basement membrane in regions of epithe- lial branching are not due to reduced expression of the nidogen gene in these regions.

In contrast, laminin A mRNA is produced by either epithelial or mesenchymal cells at the epithelial- mesenchymal interface where a basement membrane is deposited. In a previous survey of laminin A, B1, and B2 gene expression in the whole embryo we have shown that the laminin A gene was the only basement membrane component gene to be expressed exclusively by cells associated with the basement membrane (Thomas and Dziadek, 1993a). In the kidney, laminin A chain expression is restricted to epithelial cells (Klein et al., 1990; Ekblom et al., 1990; Thomas and Dziadek, 1993a), while in other tissues, including the gut, mes- enchymal cells immediately beneath the epithelial layer also synthesize laminin A mRNA (Thomas and Dzi- adek, 1993a). In the lung, both of these patterns are seen. Around the trachea and base of the bronchial tubes, laminin A gene expression is restricted to mes- enchymal cells beneath the epithelial layer, while in regions where new buds have formed, expression is restricted to epithelial cells. In intermediate, transition regions, laminin A mRNA levels are very low in both epithelial and mesenchymal cells. We have previously suggested that formation of complexes between nido- gen and laminin molecules composed of A, B1 and B2 chains are essential for the assembly of a stable base- ment membrane structure at the epithelial-mesen- chymal interface (Thomas and Dziadek, 1993a). Laminin which is localized within mesenchymal tissues lacks the A chain (Thomas and Dziadek, 1993a; Ek- blom et al., 1990) and is more soluble than laminin within basement membranes (Dziadek and Mitrangas, 1989; Kucherer-Ehret et al., 1990). The development of polarized epithelial cells and formation of a basement membrane in the embryonic kidney depends on laminin A chain production (Klein et al., 1988). In addition, studies of MDCK epithelial ceils have shown that laminin A chain secretion is necessary for the assembly of a basement membrane in culture (Ecay and Valen- tich, 1992). Possibly, the formation of a laminin het- erotrimer including a laminin A chain allows a greater range of associations between neighbouring molecules and so forms a more extensively cross-linked basement membrane matrix.

The present study shows that the embryonic mouse lung can be divided into three regions based on the expression pattern of the laminin A gene (Fig. 4). In region A mesenchymal cells are totally responsible for synthesis of the laminin A chain and hence formation of the laminin-nidogen complex in the subepithelial

T. Thomas, M. Dziadek / Mechanisms of Decelopment 45 (1994) 193-201 199

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Fig. 4. Proposed model for generation of different basement mem- brane structures in the embryonic lung, based on patterns of laminin A mRNA ( • ) and nidogen mRNA ( [] ) localization in the epithelial (e) and mesenchymal (m) layers. Nidogen mRNA is uniformly dis- tributed throughout the mesenchyme. Laminin A mRNA is pro- duced at high levels by mesenchymal cells in the bronchial regions (A) and by the epithelial cells in distal lobules (C), and is present at low levels in the intermediate regions (C). Complexes between nido- gen and A chain-containing laminin would be produced by mes- enchymal cells in region A, and by an interaction between epithelial and mesenchymal cells in region C. It is predicted that such com- plexes are important for the structural integrity of the basement membrane at the epithelial-mesenchymal interface (solid line). Low levels of laminin A chain production, and hence laminin-nidogen complex production in region B is predicted to generate a thin or discontinuous basement membrane (broken line).

which synthesize Reichert's membrane (Thomas and Dziadek, 1993b), lens epithelial cells which produce the lens capsule (Dong and Chung, 1991), and mes- enchymal cells which produce the connective tissue of the meninges around the central nervous system (Thomas and Dziadek, 1993a). We predict that particu- larly stable basement membranes are produced by these cells, and by mesenchymal cells in the tracheal and bronchial regions of the developing lung, which provide a rigid structural support for the tissue and do not promote morphogenetic activity. We propose that the branching pattern of the epithelium in the lobular regions is influenced by differences in basement mem- brane structure resulting from regional differences in laminin A and B2 chain production by the epithelium. Since previous studies have demonstrated that mes- enchymal cells isolated from these regions are able to induce supernumerary bud formation in the epithelium (Hiller et al., 1985), we propose that mesenchymal cell populations in this region either differentially promote the expression of laminin A and B2 genes in the epithelium, or differentially inhibit expression. Further studies are needed to determine the actual influence of mesenchymally-derived factors on laminin production by the epithelium, and to correlate regional differences in basement membrane production with epithelial pro- liferation.

The data presented in this study indicate that re- gional differences in basement membrane production in the developing lung arise as a result of differences in epithelial-mesenchymal interactions. A previous model to explain regional differences in basement membrane structure was based on differential remodelling of the basement membrane (Bernfield et al., 1984). The rela- tionship between these two models awaits further in- vestigation.

basement membrane. In region C (Fig. 4) epithelial cells produce high levels of laminin A chain mRNA, and thus laminin-nidogen complexes in the basement membrane would be formed by an interaction between nidogen synthesized by mesenchymal cells and A chain-containing laminin synthesized by the epithe- lium. In the intermediate region B (Fig. 4) only low levels of laminin A mRNA are produced by both epithelial and mesenchymal cells, and we predict that a stable, intact basement membrane is not assembled in this region. We propose that differences in basement membrane structure in these three regions play an important role in influencing epithelial proliferation and establishing the branching pattern. Previous stud- ies have shown that very few cell types in the embryo express all four of the laminin A, B1, B2 and nidogen genes. These cells include parietal endoderm cells

4. Materials and methods

4.1. Animals

Random Swiss mice were kept with free access to food and water. They were allowed to mate overnight and pregnancy was detected by the presence of a vaginal plug the next morning, which was designated day 0.5 of gestation. Embryos were dissected from pregnant females on day 12.5 of gestation.

4.2. Separation of lung epithelium and mesenchyme

Lung from 12.5 day embryos were dissected in phos- phate buffered saline (PBS) and then transferred to an ice cold solution of 2.5% pancreatin (Sigma), 0.5% trypsin (CSL, Melbourne) in phosphate buffered saline. Tissue was incubated at 4°C for 30 rains, then trans-

200 T. Thomas, M. Dziadek / Mechanisms of Development 45 (1994) 193-201

ferred to medium M2 (Quinn et al., 1982). Epithelial cell layers could then be separated from mesenchyme using tungsten needles. Lung tissue from 60 embryos was separated into epithelium and mesenchyme for RNA purification. In addition, RNA was purified from intact embryonic lungs, whole embryos and Reichert's membrane for comparison.

4.3. cDNA probes

The 1.36 kb SacI fragment of a 1.8 kb 3' laminin A cDNA clone (Deutzmann et al., 1988), and the 1.6 kb HindlII-SphI fragment of the laminin B1 cDNA clone (Oberbaumer, 1986) were subcloned into the pGEM4Z vector. The laminin B2 probe used was a 1.7 kb EcoRI-XbaI fragment (Sasaki and Yamada, 1987), and the nidogen probe was the 1.5 kb 6c clone (Mann et al., 1989), both in Bluescript vectors. A 1.9 kb BamHI/Pst I fragment of the NC1 region of collagen al(IV) (Muthukumaran et al., 1989) was subcloned into pGEM3zf - .

4.4. RNA analysis

in 2 xSSC/50% formamide for 1 h at 50°C then rinsed in 2 x SSC. Slides were treated with 150/zg/ml RNase A for 1 h at 37°C and rinsed again in 2 x SSC. Sections were then dehydrated through graded alco- hols and autoradiographed overnight using Kodak XAR film. Slides having low background signals were then coated with Amersham LM-1 liquid emulsion (diluted 50% with 2% glycerol in water) and exposed at 4°C for 1 week except for some experiments with laminin A where slides, including controls, were autoradio- graphed for 2 weeks. Sections were counterstained with either toluidine blue or haematoxylin. Every sixth section was incubated with sense RNA probes. In all experiments shown, the level of background labelling was only slightly higher than the background fogging of the emulsion, and was evenly distributed throughout the tissue section. In each experiment comparison could be made between the pattern produced by five anti- sense probes as well as sense probes. As a distinct pattern was produced in each case we conclude that specific mRNAs coding for collagen al(IV), laminin A, B1, B2 and nidogen can be specifically detected using this technique.

Total RNA was purified from Reichert's membrane and embryonic tissues essentially as described by Chirgwin et al. (1979). Northern gel analysis was per- formed as described by Maniatis et al. (1982). One Northern blot was hybridized sequentially with cDNA probes for nidogen, laminin A, laminin B2 and laminin B1, without stripping the filter between each hybridiza- tion. An identical filter was probed with the collagen al(IV) cDNA probe.

For in situ hybridization histochemistry freshly dis- sected embryos were rapidly frozen in Tissue Tek OCT embedding compound (Miles, Naperville, IL) cooled in isopentane on dry ice. Serial 8 ~m sections were cut using a Reichert Jung Frigocut 2800E cryostat, and after air-drying, were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 mins. at room tem- perature. After dehydration through graded alcohols slides were stored dessicated at 4°C before use. cRNA probes were generated by transcription of linearized DNA using T3, T7 or SP6 RNA polymerase incorpo- rating [3SS]-UTP (Amersham, 1000 Ci/mmol). Sections were then incubated under coverslips at 53°C, in 25/zl of a hybridization buffer consisting of 50% formamide, 0.3M NaC1/0.03M sodium citrate (2 x SSC), 50 mM phosphate pH 6.5, 0.02% ficoll, 0.02% polyvinylpyrroli- done, 0.02% bovine serum albumin, 10% dextran sul- phate, 50 mM dithiothreitol and 1 mg/ml yeast RNA, to which either 5 x l0 s cpm of either sense or anti- sense cDNA probe had been added. At least 27 sec- tions from ten different lungs were incubated with each probe. After overnight incubation slides were washed

5. Acknowledgments

This study was supported by the National Health and Medical Research Council. We thank I. Ober- baumer, R. Deutzmann, R. Timpl and Y. Yamada for providing the cDNA clones. We are grateful to Eliza- beth Stadler for expert photography, and Alison Couper and Anne Hocking for typing the manuscript.

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