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Development 109, 29-36 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 29 Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis* URSULA I. HEINE't, ELIANA F. MUNOZ 1 , KATHLEEN C. FLANDERS 2 , ANITA B. ROBERTS 2 and MICHAEL B. SPORN 2 ' Biological Carcinogenesis and Development Program, Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701, USA 2 Laboratory of Chemoprevention, National Cancer Institute, Bethesda, MD 20892 *This project has been funded at least in part with Federal funds from the Department of Health and Human Services under contract number NO1-CO-74102. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government tTo whom correspondence should be sent Summary The possible in vivo role of TGF-beta 1 in regulating various proteins of the extracellular matrix, including fibronectin, collagen I and III, and glycosaminoglycans, was examined by immunohistochemical methods during critical stages of lung morphogenesis in the 11- to 18- day-old mouse embryo. Sections of Bouin-nxed, paraf- fin-embedded whole embryos were exposed to polyclonal antibodies specific to synthetic peptides present in the precursor part of TGF-beta 1 (pro-TGF-beta 1), in the processed TGF-beta 1 (antibody CC), collagen J and m , fibronectin, followed by the PAP or ABC technique to visualize the location of the antibody. GAG were stained with Alcian Blue 8GX. Our results indicate colocaliza- tion of TGF-beta 1 expression and that of matrix proteins in the developing lung when branching morpho- genesis (cleft formation) and tissue stabilization occur. The presence of TGF-beta 1 at the epithelial- mesenchymal interfaces of stalks and clefts at a time when matrix proteins can first be visualized in these areas, suggests a direct participation of the growth factor in the development of the basic architecture of the lung. Key words: mouse embryo, lung morphogenesis, TGF-beta 1, extracellular matrix. Introduction Early development of the mammalian lung is character- ized by the growth of endodermal tubules of columnar cells into splanchnic mesoderm and by branching of these tubules to form the bronchial tree. This early stage is followed by further ramification of the tubules and by their differentiation into two morphologically defined entities: a proximal part composed of .columnar cells that will develop into the bronchial portion of the adult lung and a distal part consisting of cuboidal cells that will develop into tubules and terminal sacs. After undergoing further differentiation, tubules and ter- minal sacs, together with blood capillaries, nervous and connective tissue, will form the basis for the adult respiratory system. Endodermal branching, i.e. cleft formation, is one of the major events in lung morphogenesis. It, as well as branching processes in other developing organs such as kidney, salivary and mammary glands, are known to depend on the interaction of the epithelium with the mesenchyme and on the formation of an extracellular matrix (ECM) at the interface of these tissues; it has been shown that branching does not occur in the absence of either the mesenchyme (Rudnick, 1933) or the ECM (Bernfield etal. 1973; Bernfield, 1981; Grob- stein, 1954; Wessels and Cohen, 1968). Among the components of the ECM that have been implicated as being necessary for branching are the collagens, es- pecially collagen III which is provided by the mesen- chyme (Fukuda et al. 1988; Nakanishi et al. 1988), collagen IV associated with the basement membrane of the epithelium (Chen and Little, 1987; Hashimoto and Hoshino, 1988), and the sulfated glycosaminoglycans (S-GAG), which are major components of mammalian mesenchyme and epithelial matrices (Silberstein and Daniel, 1982; Toole etal. 1977). Not only are the ECM components necessary for cleft formation per se, they are also of importance for the stabilization and main- tenance of the newly formed tissue structures (Bern- field et al. 1972; Blum et al. 1987). ECM components interact with cell surfaces via

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Development 109, 29-36 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

29

Colocalization of TGF-beta 1 and collagen I and III, fibronectin and

glycosaminoglycans during lung branching morphogenesis*

URSULA I. HEINE't , ELIANA F. MUNOZ1, KATHLEEN C. FLANDERS2, ANITA B. ROBERTS2 and

MICHAEL B. SPORN2

' Biological Carcinogenesis and Development Program, Program Resources, Inc., National Cancer Institute, Frederick Cancer ResearchFacility, Frederick, Maryland 21701, USA2 Laboratory of Chemoprevention, National Cancer Institute, Bethesda, MD 20892

*This project has been funded at least in part with Federal funds from the Department of Health and Human Services under contractnumber NO1-CO-74102. The content of this publication does not necessarily reflect the views or policies of the Department of Health andHuman Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US GovernmenttTo whom correspondence should be sent

Summary

The possible in vivo role of TGF-beta 1 in regulatingvarious proteins of the extracellular matrix, includingfibronectin, collagen I and III, and glycosaminoglycans,was examined by immunohistochemical methods duringcritical stages of lung morphogenesis in the 11- to 18-day-old mouse embryo. Sections of Bouin-nxed, paraf-fin-embedded whole embryos were exposed to polyclonalantibodies specific to synthetic peptides present in theprecursor part of TGF-beta 1 (pro-TGF-beta 1), in theprocessed TGF-beta 1 (antibody CC), collagen J and m ,fibronectin, followed by the PAP or ABC technique tovisualize the location of the antibody. GAG were stainedwith Alcian Blue 8GX. Our results indicate colocaliza-

tion of TGF-beta 1 expression and that of matrixproteins in the developing lung when branching morpho-genesis (cleft formation) and tissue stabilization occur.The presence of TGF-beta 1 at the epithelial-mesenchymal interfaces of stalks and clefts at a timewhen matrix proteins can first be visualized in theseareas, suggests a direct participation of the growthfactor in the development of the basic architecture of thelung.

Key words: mouse embryo, lung morphogenesis, TGF-beta1, extracellular matrix.

Introduction

Early development of the mammalian lung is character-ized by the growth of endodermal tubules of columnarcells into splanchnic mesoderm and by branching ofthese tubules to form the bronchial tree. This earlystage is followed by further ramification of the tubulesand by their differentiation into two morphologicallydefined entities: a proximal part composed of .columnarcells that will develop into the bronchial portion of theadult lung and a distal part consisting of cuboidal cellsthat will develop into tubules and terminal sacs. Afterundergoing further differentiation, tubules and ter-minal sacs, together with blood capillaries, nervous andconnective tissue, will form the basis for the adultrespiratory system.

Endodermal branching, i.e. cleft formation, is one ofthe major events in lung morphogenesis. It, as well asbranching processes in other developing organs such askidney, salivary and mammary glands, are known todepend on the interaction of the epithelium with the

mesenchyme and on the formation of an extracellularmatrix (ECM) at the interface of these tissues; it hasbeen shown that branching does not occur in theabsence of either the mesenchyme (Rudnick, 1933) orthe ECM (Bernfield etal. 1973; Bernfield, 1981; Grob-stein, 1954; Wessels and Cohen, 1968). Among thecomponents of the ECM that have been implicated asbeing necessary for branching are the collagens, es-pecially collagen III which is provided by the mesen-chyme (Fukuda et al. 1988; Nakanishi et al. 1988),collagen IV associated with the basement membrane ofthe epithelium (Chen and Little, 1987; Hashimoto andHoshino, 1988), and the sulfated glycosaminoglycans(S-GAG), which are major components of mammalianmesenchyme and epithelial matrices (Silberstein andDaniel, 1982; Toole etal. 1977). Not only are the ECMcomponents necessary for cleft formation per se, theyare also of importance for the stabilization and main-tenance of the newly formed tissue structures (Bern-field et al. 1972; Blum et al. 1987).

ECM components interact with cell surfaces via

30 U. I. Heine and others

specific receptor complexes. One of these, a newlyisolated cell surface receptor and a member of theintegrin superfamily, has been implicated in the regu-lation of cell-matrix interactions. This receptor bindsfibronectin to human lung fibroblasts and colocalizeswith pericellular fibronectin during morphogenesis andcytodifferentiation of the chick lung (Chen et al. 1986).Likewise, a receptor for the extracellular matrix glyco-protein tenascin has been described (Bourdon andRuoslahti, 1989). Tenascin has been found in smoothmuscle cells and extracellular spaces that surround thebronchial ducts of the developing chicken lung (Crossinet al. 1986). Such bindings are thought to mediateadhesion of cells to the ECM; conversely, lack of suchbinding could result in loss of stabilization andenhanced cell movement. Thus, in the case of thedeveloping lung, deposition of fibronectin and tenascinand their binding to parenchymal cells may also help instabilizing the developing bronchial tree and facilitateits development. The importance of fibronectin in theECM is further highlighted by its capacity, due to itsmultiple binding sites, to act as a cross-linking agentbetween collagen and proteoglycans.

TGF-beta 1, originally discovered based on its abilityto phenotypically transform normal fibroblasts, is nowknown to be the prototype of a large superfamily ofproteins that control various aspects of differentiation.TGF-beta 1 is distinctive in the multifunctional natureof its actions; and it can either stimulate or inhibitcellular proliferation, differentiation or function(Roberts, A.B. et al. 1988; Sporn et al. 1987). Many ofthe actions of TGF-beta 1 are related to its ability toregulate the formation of ECM, especially collagen (I,III and V), fibronectin and GAG. The mechanisms bywhich TGF-beta 1 acts to control the ECM are variedand include: (a) an increase in synthesis of the majorcomponents of ECM (Ignotz et al. 1987; Ignotz andMassague", 1986; Ignotz and Massagu6, 1987; Roberts,CJ. et al. 1988); (b) control of their proteolytic degra-dation (Edwards et al. 1987; Keski-Oja et al. 1988;Laiho et al. 1986); and (c) modulation of synthesis ofintegrin receptors (Heino et al. 1989; Ignotz and Massa-gu6, 1987; Roberts et al. 1988). Taking into account theknown role of the ECM in branching morphogenesis(and in morphogenesis and cytodifferentiation in gen-eral) and the striking enhancement of the ECM byTGF-beta 1, it should not be surprising to find a role forthe growth factor in control of many aspects of embryo-genesis via modulation of the ECM.

In this study, we have investigated in vivo, in a time-dependent manner, the relationship of the localizationof TGF-beta 1 to collagens I and III, fibronectin,hyaluronate and S-GAG during branching morphogen-esis of the embryonal mouse lung. We focused ourattention especially around the time of lung develop-ment when potential alveoli (respiratory tubules andterminal sacs) were forming (between day 14 and 15).However, earlier developmental stages (day 11 and 13)were also included in this study. We have used specificantibodies and immunohistochemical staining tech-niques for the visualization of these proteins. We

provide evidence for the colocalization of TGF-beta 1with ECM components during cleft formation andstabilization of the newly formed structures and thussuggest a special role of the growth factor in this event.

Materials and methods

EmbryosNIH/Swiss mouse embryos of 11, 13, and 15 days of gestationwere used. After death of the mothers by cervical dislocation,the embryos were excised for immediate fixation. Embryonalage was determined by measuring the crown-rump length andby evaluating the developmental stages of the digits of frontand hind feet (Rugh, 1968).

AntibodiesAntibodies to TGF-beta 1

Two different antibodies recognizing distinct epitopes inTGF-beta 1 were utilized in this study. One, a polyclonalantibody raised to a synthetic peptide corresponding to aminoacids 267-278 that are present in the precursor part of theTGF-beta 1 molecule, is referred to here as pro-TGF-beta 1antibody. Its staining pattern is exclusively intracellular andmay indicate the location of TGF-beta 1 synthesis. The other,a polyclonal antibody to TGF-beta 1 made in rabbits to asynthetic peptide corresponding to the amino-terminal 30amino acids of mature, processed TGF-beta 1 was a generousgift of L. R. Ellingsworth, Collagen Corporation, Palo Alto,California and is referred to as antibody CC (Flanders et al.1989). This antibody specifically detects TGF-beta 1 and doesnot recognize TGF-beta 2. Its purification and specificity(Ellingsworth et al. 1986) and its staining pattern in mam-malian tissues (Flanders et al. 1989; Heine et al. 1987;Thompson et al. 1989) have been described previously and it isknown to preferentially localize TGF-beta 1 extracellularly.The antibodies were used at a concentration of 20^gml~'.

Antibodies to collagen I and III and fibronectinRabbit anti-collagen 1 polyclonal antiserum was purchasedfrom Chemicon International, Inc., El Segundo, California. Itwas used at a dilution of 1:500. Affinity-purified goat antibodyagainst type III collagen was obtained from Southern Biotech-nology Associates, Inc., Birmingham, Alabama, and wasused at a dilution of 1:100. Rabbit anti-human fibronectinantiserum purchased from Collaborative Research, Inc.,Bedford, Massachusetts, was used at a dilution of 1:30.

Immunohistochemical stainingThe distribution of TGF-beta 1, collagen 1 and III, andfibronectin was evaluated by immunohistochemical staining ofsagittal sections prepared from whole mouse embryos. Em-bryos were fixed in Bouin's solution, dehydrated through agraded series of ethanol solutions, and embedded in paraffinat a temperature not exceeding 60°C. Sections of 5fimthickness were deparaffinized, stained with Harris's hematox-ylin, and subjected to immunohistochemical staining as de-scribed previously (Heine et al. 1987). The location of theantibody was visualized using the avidin-biotin-peroxidasedetection system (Vector Laboratories, Burlingame, Califor-nia) or the peroxidase-antiperoxidase technique using chemi-cals from Cooper Biomedical, Inc., Malvern, Pennsylvania.

Histochemical localization of GAGBouin's-fixed sections (see previous paragraph) of 5 fan thick-

TGF-beta 1 and ECM during lung morphogenesis 31

ness were stained with Alcian Blue 8GX as previouslydescribed by Silberstein and Daniel (1982). To demonstrateboth hyaluronate and sulfated GAG's, the dye solutioncontained 0.1 % Alcian Blue in 0.025 M sodium acetate buffer(pH5.8) with 0.3M MgCI2. Staining of hyaluronate wasselectively blocked by adjusting the solution to pHl.O toobtain specific staining of sulfated GAG.

Results

Distribution of TCF-beta 1, collagen I and III, andfibronectin at day 11To investigate the involvement of TGF-beta 1 in thedevelopment of the embryonal mouse lung, we madeuse of two polyclonal antibodies. One of them, raised toa synthetic peptide corresponding to amino acids267-278 of the TGF-beta 1 precursor, has been shownto recognize the intracellularly located pro-TGF-beta 1(Flanders et al. 1989). The other was raised to asynthetic peptide corresponding to the M-terminal 30amino acids of mature TGF-beta 1 and has beendemonstrated to detect principally extracellularTGF-beta 1 (Flanders et al. 1989). Our immunohisto-chemical study indicated that, as early as day 11, pro-TGF-beta 1 was detectable in the cytoplasm of the twomajor cell types constituting the developing lung;namely, stromal cells and epithelial cells of the primor-dial ducts (Fig. 1A). This precursor to TGF-beta 1 waslocalized in distinct granules that were distributeduniformly throughout the cytoplasm. In contrast, extra-cellular TGF-beta 1 recognized by antibody CC wasdetectable only in low concentrations throughout thestroma (Fig. 2A). However, on closer examination, itcould be seen that staining was enhanced in closeproximity to primordial ducts (Fig. 2B). Staining forcollagen I (not shown) and III (Fig. 2C) and fibronectin(Fig. 2D) colocalized with that of the TGF-beta 1 sinceall these ECM proteins surrounded the ducts forming adistinct, yet weakly stained, sleeve. Fibronectin alsooutlined the capillaries (Fig. 2D).

Distribution of TGF-beta 1, collagen I and III, andfibronectin at day 13The staining pattern of the antibody to pro-TGF-beta 1indicated the presence of the growth factor precursor inthe cytoplasm of epithelial and stromal cells with agranular distribution similar to that found on day 11(Fig. IB). In contrast, staining with the antibody CC,which recognizes the extracellular growth factor, wasmarkedly intensified, compared to day 11; at this stateof lung development the entire stroma was deeplystained (Fig. 2E, F). Although difficult to discern dueto the intensity of the stromal staining, the stainingpattern suggested that TGF-beta 1 preferentially ac-cumulated at two distinct locations, namely, in the cleftsof branching ducts and around ducts of high columnarepithelium (Fig. 2F, arrow). Collagen I (not shown)and 111 were found at the epithelial-mesenchymalinterface as continuous sheets around such ducts(Fig. 2G, arrow). They were also distributed in a spottyfashion adjacent to proliferating ducts of cuboidal

epithelium (Fig. 2G, arrowhead). The distribution offibronectin was similar to that at day 11, being charac-terized by a light staining throughout the mesenchymeand, in addition, by more intense staining in clefts ofbranching ducts and at basement membranes of suchducts (Fig. 2H).

Distribution of TGF-beta 1, collagen I and III, andfibronectin at day 15A major change in the distribution of TGF-beta 1 tookplace between days 13 and 15, coinciding with both thedifferentiation of the ducts into their proximal bron-chiolar and distal alveolar components and theincreased branching of the latter. A comparison withday 13 revealed a reduction of the overall mesenchymalstaining which was seen for both intracellular pro-TGF-beta 1 and extracellular TGF-beta 1. At this stage ofdevelopment pro-TGF-beta 1 was detected only in thecytoplasm of the epithelial cells lining bronchial(Fig. 1C) and alveolar tubules (Fig. ID) and in smoothmuscle cells surrounding bronchial epithelium(Fig. 1C). Antibody CC revealed enhanced extracellu-lar staining principally in the clefts of the newly formedalveolar ducts (Figs. 3A, B, arrows). Furthermore, adistinct sleeve of TGF-beta 1-specific stain surroundedthose ducts that terminated in alveolar buds (Fig. 3C,arrow). The distribution of collagen III coincided withthat of TGF-beta 1; the epithelial-mesenchymal inter-faces of bronchiolar ducts (Figs 3D, E) and the clefts ofbranching bronchiolar and alveolar ducts (Fig. 3E,arrow) were stained. A similar, however, less pro-nounced, staining pattern was seen with anti-fibronec-tin antibody (Fig. 3F). As shown in Fig. 3G, the stain-ing pattern of collagen I was also, in part, similar to thatof TGF-beta 1; however, its distribution was morerestricted: the sleeve-like staining pattern of bronchio-lar ducts was prominent; yet, the V-shaped stainingpattern in the clefts of branching ducts, that was socharacteristic for the TGF-beta 1, collagen III, andfibronectin, was less distinct (Fig. 3G, H). In allsamples studied, the areas around terminal buds (com-pare Fig. 3B, C, E, F, and G) were devoid of stainingfor either the growth factor or the ECM proteins.

Distribution of TGF-beta 1 at day 18As illustrated in Fig. IE, pro-TGF-beta 1 was confinedat day 18 to the cytoplasm of the bronchiolar duct andthe adjacent smooth muscle cells. In contrast to ourobservation at day 15 (see Fig. ID), the alveolar paren-chyme was negative with respect to pro-TGF-beta 1.The distribution of the extracellular form of TGF-beta 1recognized by antibody CC coincided with that of pro-TGF-beta 1 as the antibody CC was localized adjacentto the bronchiolar ducts (Fig. IF). In addition, largeblood vessels stained positive with antibody CC.

Distribution of GAG between day 11 and 15At day 11, a light fibrillar staining surrounding indi-vidual mesenchymal cells of the developing lung indi-cated the presence of small amounts of GAG through-out the stroma (not shown). In addition, a more

32 U. I. Heine and others

pronounced staining at basement membranes of thebronchiolar ducts was evident. Throughout the periodstudied, staining was independent of the pH used, thusindicating the presence of both hyaluronate and sul-fated GAG. At day 13, the distribution of GAG wassimilar to that observed at day 11 (not shown). Fig. 31demonstrates the distribution of GAG at day 15.Intense staining at basement membranes of bronchiolarstalks and in clefts of forming branches indicated adense layer of GAG at both locations. In contrast,GAG were only poorly expressed around alveolar budsand in the stroma. The GAG staining pattern wastherefore identical to that described in preceding para-graphs for the extracellular TGF-beta 1, collagens I andIII, and fibronectin.

Discussion

This study, for the first time, provides evidence that theextracellular form of TGF-beta 1 is expressed in aspecific spatial and temporal pattern during branchingand cleft formation of the developing mouse lung andthat it colocalizes with collagens I and III, fibronectin,and proteoglycans. Although colocalization ofTGF-beta 1 and ECM proteins is found as early as day11, it is most pronounced between days 14 and 15 whendifferentiation of the primordial tubules into alveolarand bronchiolar ducts of the lung takes place and whenstabilization of the newly established pattern has beenshown to be paramount for organ formation. Earlier(Bernfield et al. 1973; Wessels and Cohen, 1968) andmore recent studies (Chen and Little, 1987; Fukuda etal. 1988; Hashimoto and Hoshino, 1988; Nakanishi etal. 1988) emphasize the importance of the ECM,especially collagens, in branching morphogenesis ofvarious organs. By using specific coUagenases, Naka-nishi and collaborators (1988) provide evidence sup-porting a central role of interstitial type III collagen incleft formation of the submandibular gland of themouse embryo. They also indicate that restricted ex-pression of type III collagen in spatiotemporally regu-lated fashion may contribute to pattern formation. Onthe other hand, active involvement of collagen I inpattern formation is less likely as lung morphogenesisproceeds normally until midgestation in a mutantmouse with a nonfunctional gene for collagenase type I<*-chains (Dziadek et al. 1987; Kratochwil et al. 1986).

The exact mechanisms by which TGF-beta 1 mightregulate in vivo various proteins of the ECM aredifficult to establish due to the multifunctional actionsof the growth factor. Moreover, as yet, we cannotexclude that TGF-beta 1 accumulation may be second-ary to matrix accumulation. However, in vitro,TGF-beta 1 increases production of collagens I and IIIby fibroblasts (Fine and Goldstein, 1987) with corre-sponding increases in the steady-state level of themRNAs (Keski-Oja et al. 1988; Rossi et al. 1988; Vargaet al. 1987). Likewise, TGF-beta 1 is known to increasethe production of fibronectin in fibroblasts (Ignotz andMassagu6, 1986; Rhagow et al. 1987; Varga and Jime-

Fig. 1. Expression of pro-TGF-beta 1 during thedevelopment of the mouse lung. (A, B) Localization of pro-TGF-beta 1 in the primordial tubules of the embryonicmouse lung at days 11 (A) and 13 (B) of gestation using apolyclonal antibody raised to a synthetic peptidecorresponding to amino acids 267-278. Punctate staining forpro-TGF-beta 1 is seen in the cytoplasm of both epithelialand stromal cells. (C, D) Distribution of pro-TGF-beta 1 atday 15. Pro-TGF-beta 1 is localized in the cytoplasm of theductal epithelium, the adjacent smooth muscle cells (arrow)(C), and in the cytoplasm of the terminal bud epithelium(D). Stromal cells are negative. (E, F) Distribution of pro-TGF-beta 1 and extracellular TGF-beta 1 (visualized byantibody CC) at day 18. Both antibodies stain theepithelium of the bronchiolar duct, anti-pro-TGF-beta 1intracellularly (E) and anti-CC in the adjacent extracellularmatrix (F). In contrast, no staining of the respiratoryepithelium of the alveoli is observed with these twoantibodies. Magnification A-D: X500, E, F: x200.Fig. 2. Expression of TGF-beta 1 in the embryonal mouselung (day 11 and 13). (A, B) Extracellular TGF-beta 1visualized by staining with antibody CC is present in lowconcentration throughout the stroma of the developingmouse lung at day 11 (A) and accumulates around epithelialducts (B). (C, D) Collagen III (C) and fibronectin (D)colocalize with the growth factor. (E. F) Localization ofextracellular TGF-beta 1 at day 13 of gestation. Enhancedstromal staining and accumulation of TGF-beta 1 is seen atbranch points (F, arrow). (G, H) Antibodies to collagen III(G) and fibronectin (H) show specific staining of fibronectinat clefts of epithelial branches (H, arrow), intense stainingof collagen III surrounding bronchiolar duct (G, arrow),and spotty distribution adjacent to cuboidal epithelium (G,arrowhead). Magnification: X500.

nez, 1986). In this study, we have used an antibody(CC) that preferentially localizes TGF-beta 1 at extra-cellular sites (Flanders et al. 1989). The enhancedstaining of the antibody at those epithelial—mesenchymal interphases, i.e. clefts and ducts, wherethe adjacent stromal cells are actively producing col-lagens and fibronectin, to facilitate pattern formationand tissue stabilization, suggests an active role of thegrowth factor in these events. Such a hypothesis isconsistent with our observation that neither extracellu-lar TGF-beta 1 nor components of the ECM aredetectable immunohistochemically at the tips of ac-tively growing ducts, an area where accumulation ofECM would only hinder the growth of the expandingducts. The role of TGF-beta 1 may not be limited toenhancing the production of ECM; rather, it may alsocontribute to the enhancement of the ECM by regu-lation of those genes that control the degradation of thenewly synthesized matrix proteins. Such control can beachieved by decreased synthesis and secretion ofproteases and increased synthesis and secretion ofprotease inhibitors (for review, see Roberts and Sporn,1990). Furthermore, TGF-beta 1 is also known toregulate and elevate cell adhesion receptors and bydoing so mediates cell adhesion to extracellularmatrices including type I collagen, fibronectin, andlaminin (Heino et al. 1989).

'*.-Vv

G

Fig. 3. Localization of extracellular TGF-beta 1 in the embryonic mouse lung at day 15 of gestation. (A, B, C) Staining ofanti-CC is concentrated in V-shaped fashion at the crotches of epithelial branches (A, B, arrows) and forms a sleeve aroundbronchiolar ducts (C). The surface of terminal buds is not stained (C, arrowheads). (D-I). Collagen III colocalizes withTGF-beta 1 with staining surrounding bronchiolar epithelium (D, arrow) and accumulating in clefts of branching epithelium(E, arrow). Fibronectin (F), collagen I (G, H) and GAG (I) express similar patterns of distribution. Magnification: X500.

TGF-beta 1 and ECM during lung morphogenesis 33

Establishment of tissue architecture is known to bedependent on the coordinated interaction of parenchy-mal cells with the stroma. A special role in this processhas been established for proteoglycans due to theircapability to provide a linkage between the intracellular(actin) and extracellular (collagen) environment, thusanchoring parenchymal cells to their substrate andproviding structural stability (for review, see Iozzo,1988; Ruoslahti, 1989). Enhancement and modulationof GAG synthesis by TGF-beta 1 has been shown forchondrocytes (Hiraki et al. 1988), arterial smoothmuscle cells (Chen et al. 1987), lung epithelial cells andpreadipocytes (Bassols and Massague\ 1988), and fibro-blasts derived from patients with progressive systemicsclerosis (Falanga et al. 1987). In this study, we havedemonstrated the coexpression of GAG with extra-cellular TGF-beta 1, collagen I and III, and fibronectinat bronchiolar stalks and clefts implying that the growthfactor may be capable of inducing GAG synthesis instrategically located parenchymal cells. By interactingwith both the cells and elements of the ECM, GAG mayfurther contribute to the structural stability of theevolving lung tissue. This assumption is supported bythe observation by Roos et al. (1985) that filamentnetworks underlying H35 hepatoma cells are not ran-domly distributed but are localized to specific mem-brane domains containing cell surface glycoproteins.Furthermore, it has become apparent that the proteo-glycans of the ECM can function as reservoirs forvarious growth factors including fibroblast growth fac-tor, Schwann cell growth factor, retinal survival factorand TGF-beta 1, and thus they can be immediatelyavailable to cells when need arises (for review, seeRuoslahti, 1989).

Cellular sites of synthesis of TGF-beta 1 have beenidentified in this study by examining the distribution ofthe TGF-beta 1 precursor using an antibody raised to asynthetic peptide corresponding to amino acids 267-278of the TGF-beta 1 precursor (Flanders et al. 1989;Wakefield et al. 1988). In the early stages of lungdevelopment characterized by the growth of primordialtubules into undifferentiated and expanding mesen-chyme (growth phase, prior to day 13 of gestation), lowamounts of pro-TGF-beta 1 are detected in parenchy-mal and mesenchymal tissues. We would like to suggestthat during this time the growth factor acts mainly as agrowth initiating agent and inhibits differentiation ofparenchymal cells (see schema, Fig. 4). The ability ofTGF-beta 1 to suppress differentiation of various celltypes including myoblasts (Florini et al. 1986; Massagu6et al. 1986; Olson et al. 1986), chondrocytes (Rosen etal. 1988), and human mesothelial cells (Gabrielson etal.1988) is well known. After day 13, a major shift in thedevelopment of the ductal system takes place as evi-denced by cessation of growth and subsequent differen-tiation of the primordial tubules into proximal bron-chiolar and distal alveolar ducts (stage of patternformation) (see schema, Fig. 4). This phase of lungdevelopment is highly dependent on the extracellularskeleton formed by the ECM. Our in vivo datademonstrating colocalization of TGF-beta 1 and ECM

components together with in vitro data demonstrating acomplex and multifaceted role of TGF-beta 1 in controlof ECM (for review, see Roberts and Sporn, 1989)suggest that TGF-beta 1 may be a driving force in thisprocess. Only after completion of pattern formationdoes the development process continue to a third phasecharacterized by tissue differentiation. In the case ofthe developing lung, this third phase includes theproduction of cell-specific proteins, sudh as the surfac-tant-associated glycoproteins in the pulmonary type IIepithelial cells of the alveolar sacs. Ten Have-Opbroek(1981) has shown that this process starts at day 14/15 inthe mouse embryo. Our model is in good agreementwith the concept of pattern formation which states thatthe positional information that a cell receives deter-mines not only its position but is especially importantfor its future molecular differentiation (Saunders,1982).

Although anti-pro-TGF-beta 1 detected synthesis ofTGF-beta 1 in both stromal and epithelial cells in theperiod between day 11 and 15, the distribution patternof the precursor changed dramatically prior to day 18.At day 18, staining of anti-pro-TGF-beta 1 is restrictedto cells of the bronchiolar ducts, whereas the alveolarcells are negative for TGF-beta 1. Whitsett and colla-borators (1987) found inhibition of synthesis of a

Day 11 Day 13 Day 16

Fig. 4. TGF-beta 1: A model to indicate the role of themultifunctional growth modulator during the developmentof the embryonal mouse lung. (Pro-TGF-beta 1 is expressedin punctated, extracellular TGF-beta 1 in dashed form.) I.Growth phase (day 11): Inhibition of differentiation in theepithelium of the advancing high columnar primordialtubules. II. At day 13 we recognize a growth phase withrespect to the prospective alveolar epithelium of the lung(B) and a stabilization phase with respect to the bronchiolarepithelium (A) as ECM and TGF-beta 1 begin to formsleeve-like envelopes around the latter at A (indicated bydashed arrows). Growth phase with respect to bloodcapillaries, nervous tissue, and lymphatics, invading themesenchyme. III. At day 15 we recognize a growth phasewith respect to the epithelium of the terminal buds (B) anda stabilization phase with respect to bronchiolar andproximal, prospective alveolar epithelium shown byextension of ECM and TGF-beta 1 in distal direction(indicated by solid arrows). Stabilization of branchingpoints is indicated at asterisk (*). Stabilization phase withrespect to various tissues in the mesenchyme.IV. Maintenance of stabilization at day 18 around ducts atA.

34 U. I. Heine and others

specific surfactant-associated glycoprotein (SAP-35) inorgan cultures of human fetal lung in the presence ofTGF-beta 1. Our findings are consistent with this reportsuggesting a role of TGF-beta 1 in suppression ofsurfactant production during early stages of lung devel-opment and with the report of Masui et al. (1986) thatTGF-beta 1 is the primary differentiation inducingserum factor for normal human bronchial epithelialcells.

There are two observations in this study that shouldnot go unnoticed. One concerns the enhanced presenceof the extracellular TGF-beta 1 in the mesenchyme atday 13. This may be related not only to regulation ofECM production, but also to the massive invasion ofblood vessels, lymphatics and nerve fibers that takesplace at this time. TGF-beta 1 is known to be angio-genic in various assay systems in vivo, including alocalized response at the site of injection (Roberts et al.1986) and the rabbit corneal pocket assay (Fiegel andKnighton, 1988). The other observation relates to thepresence of pro-TGF-beta 1 in the smooth muscle cellsthat underlie the high columnar ductal epithelium (seeFig. 1C). We defined these elongated cells as smoothmuscle cells in agreement with results of electronmicroscopic studies of the embryonal rat lung (Colletand Des Biens, 1974), sheep lung (Fukuda et al. 1983),and our own immunological studies in which we used anactin probe to localize muscle cells in the mouse embryo(Rehm and Heine, unpublished observations). The roleof TGF-beta 1 in these cells is as yet not known.However, recent investigations have given evidencethat TGF-beta 1 is capable of inducing the productionof tenascin (Pearson et al. 1988), a glial-mesenchymalextracellular matrix protein that is found in varioustissues of the developing chicken embryo, includingsmooth muscle cells and basement membranes thatsurround the developing bronchial ducts (Crossin et al.1986). The latter authors proposed that tenascin maynot only help in stabilizing tubular epithelia by anchor-ing cells to the ECM, but may also be of importance instabilizing interactions among different cell types. Asthe tissue distribution of TGF-beta 1 and tenascinappear to be strikingly similar in the developing lung, asshown by a comparison of the pattern in the chicken(Crossin et al. 1986) with that in the mouse (Heine, thisreport), a role of TGF-beta 1 in inducing tenascinproduction may be indicated during these developmen-tal events.

In this study, we have investigated the role ofTGF-beta 1 in the development of the mammalian lung.To date, four other isoforms of TGF-beta have beenidentified; of those, TGF-beta's 1, 2, and 3 appear to beimportant in mammalian development (Roberts andSporn, 1990). TGF-beta 1 and 2 are known to beinterchangeable in most biological assays; whereas dataregarding the biological activity of TGF-beta 3 are notyet available. In preliminary studies, TGF-beta 3 wasfound preferentially in cells of mesenchymal originincluding smooth muscle cells surrounding bronchiolarducts (K. Flanders, personal communication). Theantibody CC used in our study is specific for TGF-beta 1

and does not recognize TGF-beta 2 (Ellingsworth et al.1986). However, Pelton et al. (1989) and Miller et al.(1989) have shown that TGF-beta 2 mRNA is expressedin the submucosal layer of bronchioles and in arterialwalls of the 16.5-day-old mouse embryo lung, thusindicating that other TGF-beta isoforms may also beinvolved in lung morphogenesis.

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(Accepted 7 February 1990)