Airway branching patterns and cytodifferentiation in cultured fetal hamster lung

TISSUE AND CELL. 1992 24 (6) 853-868 0 IY92 Longman Group UK Ltd.

ANDREA

M. DESANTI”,

AIRWAY BRANCHING CYTODIFFERENTIATION HAMSTER LUNG Keywords:

Culture,

airways,

ELIZABETH

M. MCDOWELL*, and JUDY M. STRUMt

PATTERNS AND IN CULTURED

FETAL

branching,

lung

cytodiffercntiation,

hamster,

ABSTRACT. Intact fetal hamster lungs were taken for culture on gestational day 12. when only lobar bronchi and primary bronchioles are established and the epithelial cells arc undifferentiated. Explants were maintained on Transwell collagen membranes for 2 and 4 days in BGJb medium alone, with 5% FBS, or with the following additives: insulin. transfcrrin. hydrocortisone, cholera toxin. EGF, and vitamin A. Development of the respiratory tree was affected differently by each medium formulation. BGJb medium with 5% FBS permitted near normal branching of airways and presumptive alveoli. In contrast, BGJb medium alone permitted only limited branching of these structures. BGJb medium with additives permitted branching but markedly altered normal development. The differentiation of endocrine and secretory cells was monitored by immunolabcling for serotonin and calcitonin gene-related peptide. and Clara cell protein. respectively. Ciliated cells were identified by morphology. All medium formulations supported the timely differcntintion of endocrine. secretory. and ciliated cells. The ultimate goal of our studies is to characterize factors that influence alway branchmg and cytodifferentiation during fetal lung development. This study showed that near normal airway branching and cytodifferentiation were supported m trim by BGJb medium with 5% FBS. Although cytodifferentiation occurred with the two other formulations. airway development was impaired.

Introduction

Mammalian lung development is a complex process, but it offers the opportunity to follow the differentiation of a wide variety of cell types. In order to analyze various stages in this process, rodent animal models have been extremely useful. Until recently, most investigators have concentrated on the development of alveoli, since many respiratory diseases in infants are directly related to alterations in these peripheral structures. However, airways (bronchi and bronchioles) are also vitally important for proper respiratory function, and for the past several * Departments of Pathology and tAnatomy, University of Maryland School of Medicine, 655 West Baltimore St, Baltimore, MD 21201, USA. Correspondence to: Dr Judy M. Strum. Received 12 May 1992 Revised 24 June 1992

years. the development of the airway epithelium has been the focus of study in our laboratory. The Syrian golden hamster is our animal model and it offers some distinct advantages. The gestational period (just shy of 16 days) is shorter than that of other laboratory rodents, and the trachea and lung develop rapidly during the last 6 days in utero. Moreover. our studies of airway epithelial cell differentiation in viva, using various markers, have demonstrated well-defined structural and functional events, occurring at different airway levels on a daily basis, throughout the latter half of gestation (Ito et ul., 1990a.b: Strum et al., 1990a,b; McDowell et al., 1990). To our knowledge, airway development in fetal hamster lung has not been studied ill vitro. The purpose of the present study was to develop a culture method that would support airway development and permit normal air-

DESANTI

way branching patterns, as well as promote cytodifferentiation of the airway epithelial cells in fetal hamster lung. This would make it possible to compare lung development in vivo and in vitro. We believe our explant culture method and the different medium formulations will prove useful for future studies of hamster airway development, and in particular for identifying some of the specific factors that regulate it. Materials and Methods

Tissue culture 15 timed-bred Syrian golden hamsters were purchased from Charles River (Newfield, NJ, USA). The morning after mating was counted as the first day of gestation, and the animals arrived at our facility on gestational day 9. They were housed individually, given unlimited food and water, and maintained under standard laboratory conditions in a 12 hr light/l2 hr dark cycle. At gestational day 12 (Gd 12) each pregnant hamster was anesthetized with methoxyflurane (Metaphane, Pittman-Moore, Inc., Washington Crossing, NJ, USA), the gravid uterus was excised, all fetuses were removed and their lungs taken for culture. Using a stereo microscope and aseptic technique, the fetal lungs were carefully dissected free of surrounding tissues. 62 pairs of lungs with tracheas attached were placed on Transwell collagen membranes, 0.4 pm pore size, (Costar, Cambridge, MA, USA). Culture times were for either 2 days (Gd 12+2) or 4 days (Gd 12+4), in a 95% air-5% COz environment, at 37°C. The medium in each well contained the following antibiotics, 50 pg/ml gentamicin sulfate and 625 ng/ml amphotericin B, and was replaced every 2 days. BGJb culture medium (Gibco/BRL, Grand Island, NY, USA) was selected because it supported growth of fetal mouse lungs in culture (Jaskoll et al., 1988; Slavkin et al., 1989) and proved to be more satisfactory for fetal hamster lung than other commercial media that we tested in preliminary studies. This culture medium was utilized in three formulations: (1) alone; (2) with 5% fetal bovine serum (FBS); or (3) with defined additives, selected from a formulation developed by Wu et al. (1985), that supported mucociliary differentiation of hamster tracheal epithelial cells in culture. These defined

ET AL.

additives, listed as final concentrations, were: insulin (5 pg/ml), transferrin (5 pg/ml), hydrocortisone (1 PM), cholera toxin (40 ng/ml), epidermal growth factor (EGF, 25 ng/ml), and a vitamin A derivative [either retinol acetate (0.1 PM) or retinoic acid (0.1 PM)]. A thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) (Sigma Chemical Company, St. Louis, MO, USA) was added to all of the cultures 24 hr prior to fixation at a final concentration of 10m4M (Morstyn et al., 1986), to monitor proliferation of the cells. Morphology Gross analysis

Phase contrast micrographs were taken of all the explants immediately following their placement on the Transwell membranes at Gd 12 (see Fig. 2). At least 11 explants per BGJb medium formulation were photographed by phase microscopy after 2 and 4 days in culture. These gross images made it possible to compare changes observed in the same lung explant after culture for 2 and 4 days, and to compare the effects of the three different medium formulations on airway and lung development. Histology

At least four explants from each BGJb medium formulation were studied histologically after 2 and 4 days in culture. The lung explants (attached to the Transwell membranes) were fixed by immersion in 4% paraformaldehyde, 0.1% glutaraldehyde (Strum et al., 1990b) for 2 days at room temperature. They were then washed in 0.2 M cacodylate buffer (pH 7.3) at 4” C for up to 5 days. During dehydration in alcohols and paraffin embedding, extra care was taken to avoid the loss of these small tissues. The explants were flat-embedded in paraffin blocks and were cut serially en face, at 5 ,um, through their entire thickness. Sections were mounted on chrome alum-coated glass slides to prevent their loss during the pepsin digestion that was required for some of the immunochemical procedures (see below). To demonstrate glycogen within the developing airways, some sections were stained with Alcian blue, pH 2.5/periodic acid-Schiff (AB-PAS) (Mowry and Winkler, 1956). For high resolution light microscopy, a few explants were flat embedded in glycol meth-

AlRWAY

BRANCHING

PATTERNS

acrylate (GMA) and sectioned en face at a thickness of 2 pm. These GMA sections were stained with Alcian Blue, pH 2.5/PAS/ lead hematoxylin (AB- PAS-PbH)(McDowell et al.. 1985). Immunochemistry Laminin immunolabeling was used to delineate the basement membrane of the airways and presumptive alveoli and was very useful for monitoring the extent of branching. Antibodies against the amine, serotonin, and calcitonin gene-related peptide (CGRP) were used to detect the differentiation of endocrine cells. Immunolabeling for Clara cell protein was used to identify functionally differentiated secretory epithelial cells. lmmunoprroxidase procedure for laminin, serotonin, CGRP, and Clara cell protein Paraffin sections were hydrated and endogenous peroxidase activity was blocked by incubation in 1% HIOz in methanol for 20 min at room temperature. After washing three times in 0.1 M phosphate buffered saline (PBS) (pH 7.4). each section was encircled with a PAP pen (Kiyota International. Elk Grove Village, IL, USA) to isolate it for treatment with a specific antiserum. Pepsin digestion, prior to exposure to the primary antibody, was necessary to demonstrate laminin and serotonin immunoreactivity. Selected sections were incubated with pepsin (Sigma) at a final concentration of 1.0 mg/ml in 0.01 M HCI for 15 min (sero tonin) or 60 min (laminin). The slides were placed on a covered slide warmer in a humidified atmosphere at 37” C, and a drop containing pepsin was placed over selected sections. The other sections on the slide (not requiring pepsin digestion) were covered with drops of PBS. All slides were then rinsed three times in PBS, and incubated in 10% normal goat serum (NGS) in PBS for 20 min at room temperature. This was done to block non-specific binding of the biotinylated antiserum to the tissues. The NGS was then removed and the sections were incubated overnight (1620 hr) at room temperature with a primary antiserum. The primary antiserum was either: (a) rabbit anti-serotonin IgG (INCstar Corporation, Stillwater, MN, USA) diluted I:1000 in PBS/lo% NGS/ 0.1% BSA; (b) rabbit anti-CGRP IgG

(Amersham Corporation, Arlington Heights, IL. USA) diluted 1:lOOO in PBS/ 10% NGS/O.l% BSA; (c) rabbit anti-hamster Clara cell protein IgG (gift of Dr G. Singh and Dr S. L. Katyal, Pittsburgh, PA. USA) diluted 1:lOOO in PBS (Strum er al., 1990b): or (d) rabbit anti-laminin IgG (E-Y Laboratories, Inc., San Mateo. CA, USA) diluted I:500 in PBS. After three washes in PBS. all sections were incubated for 45 min at room temperature in biotinyiated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 in PBS. They were washed again in PBS and then incubated in the ABC complex (Vector) for 60 min at room temperature. After three final washes in PBS, the sections were incubated in 0.01% diaminobenzidine.4 HCI (DAB) and 0.02% HZ02 in 0.05 M Tris buffer (pH 7.6) for 8 min. They were subsequently counterstained in 1% methyl green in sodium acetate/ barbital sodium buffer (pH 4.0) for 2 hr, then dehydrated and mounted in Entellan (VWR Scientific, Bridgeport. NJ, USA). Immunoperoxidase

procedure for BrdU.

Paraffin sections were hydrated and endogenous peroxidase was blocked by incubation in 1% H1O, in methanol for 20 min at room temperature. After three washc\ in PBS, the sections were incubated in pepsin (Sigma; 0.4 mg/ml in 0.1 M HCI) for 25 min at 37” C. Next, they were washed twice in 0.5% Tween 20 (Sigma) in PBS buffer, and hydrolyzed in 4.0 M HCI for 20 min at room temperature. Following two washes in 0, 1 M borax buffer (pH 8.5) and three washes in PBS, the sections were incubated overnight at room temperature with monoclonal antiBrdU antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA. USA) diluted 1:40 in PBS. After the sections were washed in PBS, they were incubated for 45 min at room temperature with bioinylated horse anti-mouse IgG (Vector). They were rinsed again in PBS, and incubated in ABC complex (Vector) for 6U mln at room temperature. Antibody localization was achieved by incubation in 0.01 r/r DAB and 0.02% H202 in 0.05 M Tris buffer for 8 min. The sections were then counterstained with Gill’s No. 1 hematoxylin (Sigma) for two sec. prior to being quickly dehydrated and mounted in Entellan.

AIRWAY

BKAN(‘I(ING

PATTERNS

Airway terminology

The terminology used to describe clearly recognizable anatomical levels of the conducting airways in the developing hamster lung is that of Sarikas et al. (1985a, b). Results Lung at gestational

day 12, (Figs 1, 2)

Gestational day 12 (Gd 12) was selected as the day to begin the culture of fetal hamster lung because the lobar bronchi and divisional distributing bronchioles are established at this time (Sarikas er al., 1985a), but none of the epithelial cells lining the intrapulmonary airways are differentiated (McDowell et al.. 1990; Ito et al., 1990a). The airways are supported by an embryonic connective tissue

Ftgs I. 2. Lung at gestational Fig. I. dwisional terminal cellular.

that is more cellular adjacent to the airways than distant from them (Fig. 1). Grossly, the explants could be divided into concentric zones that served as landmarks for the airways. The central zone contained the main and lobar bronchi and the intermediate zone contained the divisional distributing bronchioles. Each bronchiole divided into a Yshaped structure that terminated in two terminal buds at the periphery of the explant (Fig. 2). Lung explants at gestational day 12 + 2 days in culture (Gd 12+2) BGJb medium with 5% fetal bovine .serum,

(Table 1, Figs 3, 9-11) After 2 days of culture in the presence of

day 12 (Gd 12)

Lett and infracardiac lobes in longitudinal section. The lohar bronchus (Lb) and the distributing bronchioles (h) are established. A bronchiole divides (arrow) into two buds. The embryonic connective tissue immediately surrounding the airways is very x50.

Fig. 2. Explant at start of culture. Central zone (C) and intermediate zone dlstrtbuting bronchiole divides mto a Y-shaped tubular structure (arrows) Trachea (T). x30. Figs 3. 4. Explants

cultured

in BGJb medium

(I). Each

diwrional at the periphery.

with 5% FBS.

Fig. 3. Two days of culture (Gd 12+2). The intermediate zone (I) and the periphery of the explant are characterized by a delicate lacy network which extends to the pleurn. For histological comparison set Figure 9. Trachea (T). x30. Fig. 4. Four days of culture (Gd 12+4). Forked bronchioles (b) in the intermediate zone. successively divide into smaller and smaller airways (arrows). The periphery is characterized ix a very hne. delicate meshwork. For histological comparison see Figure 19. Trachea (T). * 30. Figs 5. 6. Explants

cultured

in BGJb medium

alone.

Fig. 5. Gd 12+2. In the intermediate zone (I), the bronchioles have a knobby tubular appearence (large arrowhead). They divide and terminate in the periphery as blunted Y-shaped structures (arrows). For histological comparison see Figure 12. Trachea (T). x.10. Fig. h. Gd 12+4 In the wide intermediate Lone (I). the bronchioles show a knobby tubular pattern. Large cyst-like sacs fill the periphery of the explant. For histological compariwn see Figure 33 Trachea (T). x30. Figs 7. 8. Explants

cultured

in BGJb

medium

with defined

additives.

Fig. 7. Gd 12+2. Bronchioles appear coarse and irregular within the mtcrmediate LOX (I). They terminate in wide club-shaped profiles (arrow) or as small Y-shaped tubules (arrowhead). At the perimeter of the explant, there is a translucent flange (F). For histological comparison set: Figure 16. Trachea (T). x30. Fig. X. Gd 12+4. Structures within the explant are indistinct. The translucent flange (F) I:, wder than on Gd 12+2 (compare with Fig. 7). For histological comparison see Fig. 29. Trachea (7) X3(1

1.

No

Yes No

Complex delicate network; terminal sacs extend to pleura

Gd 12+2

Gd 12+4

Yes

Yes Yes

Very complex delicate network; respiratory saccules extend to pleura

Orderly

BGJb Medium with 5% FBS

* Laminin immunolabeling was used to delineate the basement membrane monitoring the extent of branching. 1- Immunolabeling for serotonin. $ Immunolabeling for Clara cell protein. 0 Identified by their distinctive morphology in GMA (2 pm) sections.

Ciliateds

DIFFERENTIATION OF AIRWAY EPITHELIAL CELLS Endocrinet Secretory+

BRANCHING* Airways Prealveolar Structures

Table

of the airways

No

No

Yes

Simple network; terminal sacs extend to pleura

Orderly

Gd 12+2

BGJb Gd 12+4

and the prealveolar

Yes

Yes Yes

Simple network; dilated cyst-like sacs extend to pleura

but limited

Medium alone

structures

and was useful

Yes Yes (few cells) No

for qualitatively

Yes

Yes Yes

Complex network; irregular distorted finger-like tubules confined to intermediate zone; a peripheral flange of loose connective tissue is devoid of epithelial structures

Disorderly

BGJb Medium with defined additives Gd 12+2 Gd 12+4

AIRWAY

BRANCHING

PATTERNS

serum (Gd 12+2), the explants in this group appeared similar grossly. The intermediate zone and the periphery were characterized by a delicate lacy network which extended to the pleura (Fig. 3). This network was in sharp contrast to the simple tubular character of the intermediate zone and the Y-shaped terminations observed on Gd 12 (compare Figs 2 and 3). Histological sections revealed that the delicate network was composed of branched bronchioles and developing terminal sacs, whose basement membrane was clearly delineated by laminin immunolabeling. The terminal sacs completely filled the peripheral zone and abutted the pleura (Fig. 9). Glycogen was abundant in the epithelial cells lining the airways in the central and intermediate zones and lining the terminal sacs at the periphery (Fig. 10). Cell growth was demonstrated by widespread BrdU labeling, which was extensive in epithelial and connective tissue cells throughout the explants. However, label was absent from scattered clusters of epithelial cells seen in the large airways of the central and intermediate zones. Cells within these clusters showed marked labeling for serotonin (Fig. 11) and very mild labeling for CGRP (not shown), demonstrating functional differentiation of endocrine cells and the formation of presumptive neuroepithelial bodies (NEBs). Clara cell protein was not detected by immunocytochemistry in any of the epithelial cells at this time. BGJB medium alone (Table 1, Figs 5. 12-

14) After 2 days of culture in medium alone (Gd 12+2), the explants in this group showed little or no variation grossly. The intermediate zone and the periphery had a simple appearance compared with explants grown in medium with serum. The bronchioles in the intermediate zone had a knobby tubular appearance. In the periphery, the Y-shaped structures were still recognizable but were blunted in comparison with Gd 12 architecture (compare Figs. 2 and 5). The histology was consistent with diminished branching (Fig. 12). The supporting connective tissue appeared to be homogeneous and free of glycogen, whereas epithelial cells of the airways and developing terminal sacs were rich in glycogen (Fig. 13).

Despite a simplified branching pattern, NEBs had developed in the large airways of the central and intermediate zones. Serotonin labeling was intense in the endocrine cells (Fig. 14), but CGRP was not detected. BrdU labeling was heavy in cells of the connective tissue and airway epithelium, but was absent from the endocrine cells in NEBs. Clara cell protein was not detected in any of the airway epithehal cells. BGJb medium with defined additives

(Table 1. Figs 7, 15-18) After 2 days in culture in medium with defined additives (Gd 12+2), the gross appearance of the explants in this group was very similar. However, they differed markedly from explants grown in medium plus serum. The bronchioles of the intermediate zone appeared coarse and irregular. They terminated in wide, blunt, club-shaped profiles or in Y-shaped structures, neither of which extended to the pleura, because a translucent flange (free of epithelial structures) had formed at the perimeter of the explant (Fig. 7). The histology was consistent with the gross picture. Small distorted bronchioles and finger-like tubules branched from larger bronchioles and extended towards the periphery (Fig. 16). Cross-sections of the distorted bronchioles and tubules displayed cloud-like profiles and small rosettes (both with lumens) that were outlined by crenated basement membranes (Figs 16, 17). The epithelial cells forming these structures were rich in glycogen (Fig. 18). The connective tissue had become partitioned. In the intermediate zone, where it supported the airways and finger-like tubules (epithelial structures), the connective tissue was cell-rich and compact. At the periphery. a fibrous flange of relatively cell-poor. loose connective tissue was present (Figs 16. 17). Cells in both regions of connective tissue were free of glycogen (Fig. 18). The epithehal structures extended to the interface between the two regions of connective tissue but failed to penetrate the peripheral flange (Figs 16. 18). Despite a disorganized developmental pattern, numerous NEBs had formed in the large airways of the central and intermediate zones. The endocrine ceils were strongly labeled for serotonin (Fig. 15) and CGRP (not

AIRWAY

BRANCHING

PATTERNS

Y,,

shown). Moreover, Clara cell protein was detected in a few secretory cells that lined airways in the intermediate zone (Fig. 1.5, inset). BrdU labeling was highly variable in epithelial cells lining the airways and irregular finger-like tubules. However, it was consistently very heavy in cells of the connective tissue in the peripheral flange.

Figs 9-18. Histology Figs %I

I.

Explants

of lung explants cultured

in BGJh

Lung explants at gestational day 12 + 4 days in culture (Gd 12+4) BGJb medium wirh 5% FBS (Table I. Figs 4, 19-22) After 4 days of culture in medium with serum (Gd 12+4), distinct changes had occurred in the explants (Figs 4. 19). Within the intermediate zone. forked branches of airw;rv\

after 2 days of culture medium

(Gd 12+21

with 5% FBS.

Fig. 9. The bronchioles (b) branch into smaller airways which end m terminal sacs (arrow). The terminal sacs completely fill the periphery of the explant and abut the pleura Bawncnt membranes stained for laminin: methyl green counterstain. x 130. Fig. IO. Glycogcn is abundant in epithelium lining the small bronchiole\ and terminal The supporting connective tissue is free of glycogcn. AB-PAS stain. x I(H). Fig. I I. Serotonm labeling is strong m endocrine cclla of NEB\ Immunopcroxidase labeling: methyl green counterstain. x2(X). f-lga 12-14. Explants

cultured

in BGJb

medium

m the larger

wc\.

an,:~y\.

alone.

Fig. 12. The bronchioles (b) divide and terminate in developing termmal sacs (arrow ) The extent of branching is less than that obscrvcd after culture with swum (compare with Fig 9). Basement memhrancs stained for laminin: methyl green counterstain. x 130. Fig. 13. The cpithelium glycogen. The supportmg

lining the small bronchioles and dewloping terminal sac\ IS rich in connective tissue is free of glycogen. AB-PAS stain x i(X)

Fig. 14. Scrotonin labeling is intense in the NEBs of the large airways labeling; methyl green counterstain. x 130. Figs 15-1X. Explants

cultured

in BGJh

medium

with defined

Immunopcrl,xltl;t\c

additives.

Fig. IS. Serotonin labeling is strong in NEBs in the large airways. Inset: Clara cell protcm is detected in some secretory cells lining a small airway in the intcrmediaic ~onc Immunopcroxidasc labeling: methyl green counterstain. x200: Inset x350. Fig. 16. A dilated bronchiole (b) branches successively (arrows) into smaller hronchiolc\ and finger-like tubules. The basement membrane surrounding the bronchwlcs and tubules ib crenated. The eplthelial structures arc irregular. as shown in cross-sectton (lower left) The connective tissue that supports the airways and finger-like tubules is cell-rich and tightly-packed. whereas that of the peripheral flange (F) is loose and fibrous. Note that the eplthclial structure5 do not penetrate the peripheral flange. Basement membranes stained for laminin: methyl srccn countcntain. X 130. Fig. 17. Cross-section of the irregular epithelial structures. Cloud-like protiles and small rosettes are outlined hy crenated basement membranes. The connective tissue wpportmg the epithelial structures in the intermediate zone is very cellular and condensed, in contrast to the loore and fibrous penphcral Range (F). Basement membranes stained for laminin: methyl green counterstain. X370. Fig. 18. The cells comprising epithelial structures. similar to those observed m Figure 17. are rich in glycogen. The highly cellular connective tissue that supports the cpithelium. and the loose connective tissue of the peripheral Range (F) are free of glycogen. The abrupt interface bctwcen the inner cell-rich and the outer fibrous connective tissue is shown at the arrows. Glycol methacrylatc section. AB-PAS-PbH stain. x370.

.

032

AIKWAY

BRANCHING

Figs 19-32. Histology Figs 19-22. Explants

PATTERNS

of lung explants cultured

in BGJb

after 4 days of culture medium

(Gd 12+4)

with 5% FBS

Fig. 19. In the intermediate zone. forked bronchioles (b) succcssivcly diwde mto smaller branches (arrows) that extend into the peripheral zone. The periphery is filled with a network of tiny respiratory saccules (rs) that abuts the pleura. Basement membranes \taincd for laminin: methyl green counterstain. X.50. Fig. 20. A ciliated cell (arrowhead) and NEB (arrows) arc present in an anvay 01 the intermediate zone. The NEB and the adjacent secretory cells contain modcrate amounts ot glycogen. Glycol methacrylate section. AB-PAS-PbH stain. xX00. Fig. 21. Labeling for CGRP is weak in endocrine cells of NEBs in a large airway noperoxidase labcling: methyl green counterstain. X260.

Immu-

Fig. 22. Labeling for Clara cell protein is intense in secretory cells of an airway in the intermediate zone. The NEB (arrow) is not labeled. Immunopcroxidasc labeling: methyl preen counterstain. x400. Figs 2>27.

Explants

cultured

in medium

alone

Fig. 23. Bronchioles (h) in the intermediate zone terminate in large cy\t-like Basement membranes stained for laminin; methyl green counterstain. X50.

sac> (cs)

Fig. 24. Preciliatcd cells (arrows) are present in an airway in the intermcdiatc zone. Adlaccnt secretory cells contain moderate to heavy amounts of glycogen. Glycol methacrylatc section. AB-PAS-PbH stain. x800. Fig. 25. The airway cpithelial cells show extensive label for BrdU (arrowhead>), hut cndocrme cclis within a NEB (arrow) arc unlabeled. BrdU immunochcmical stam: hcmatoxylin countentain. x250. I’ig. 26. Labeling for CGRP is strong in endocrine cells of a NEB (arrow) airway. Immunoperoxidase labeling; methyl green counterstain. x540. Fig. 27. Clara cell protem is demonstrated in secretory noperoxidase labeling; methyl green counterstain. x540. Figs 2X-32. Explants

cultured

in BGJh

medium

lining

a large

cells of a small airway.

Immu-

with defined

additives.

Fig. 28. Labeling for Clara cell protein is very strong within the secretory airway. Immunoperoxidase labeling; methyl green counterstain. x540.

cells of thi\ small

Fig. 20: Distorted bronchioles (h) and finger-like tubules (t) are seen in the Intermediate zone of the explant. The epithelial structures are supported by a cell-rich. compact connective tissue. The flange (F) of loose, fibrous connective tissue at the perimeter of the explant. IS free of epithelial structures. Basement membranes stained for laminin: methyl green counterstain. x50. Fig. 30. arc shown and fields structures membranes

Crenatcd basement membranes around distorted hronchiolcs and finger-like tubules clearly at high magnification. Cloud-like profiles (cross-sections of small hronchmles) of small rosettes (cross-sections of finger-like tubules) arc present. All cpithclial are supported by a cell-rich, compact connective tissue. Fibrous flange (F). Basement stained for laminin; methyl green counterstain. x 180.

Fig 31. Epithelial cells lining a distorted bronchiole epithelial rosettes (r) arc depleted of glycogen. Glycol stain. X 180.

(h) contain abundant methacrylate section.

Fig. 32. Endocrine cells of NEBs in a large airway are intensely Immunoperoxidase labeling: methyl green counterstain. x350.

labeled

glycogcn. hut AB-PAS-PhH

for CGRP.

DESANTI

were observed grossly (Fig. 4). These airways further subdivided into smaller and smaller branches which ended in a delicate meshwork (Fig. 19). This delicate meshwork was composed of many small respiratory saccules (presumptive alveoli) that filled the periphery and abutted the pleura (Fig. 19). In the majority of these explants, the epithelium lining the respiratory saccules was depleted of glycogen. However, the epithelial cells lining bronchioles of the intermediate zone, contained moderate to heavy amounts of glycogen (Fig. 20). BrdU labeling was especially high in epithelial cells lining the bronchioles and the respiratory saccules. Preciliated and ciliated cells could now be seen in airways of the central and intermediate zones, in GMA sections. The ciliated cells were identified by their palestained cytoplasm and cilia extending from their flat apical surfaces (Fig. 20). NEBs were prominent within the epithelium of the larger airways (Figs 20-22) and were also identified for the first time in the smallest airways. The NEBs were immunolabeled for CGRP (Fig. 21) and serotonin. Many epithelial cells were intensely stained for Clara cell protein. These labeled secretory cells were most common in airways of the intermediate zone (Fig. 22).

BGJb medium alone (Table 1, Figs 6, 2327) After 4 days of culture in medium alone (Gd 12+4), the intermediate zone had increased in size (i.e. it was, wider) but the airways still showed a knobby tubular pattern, rather similar to that seen on Gd 12+2 (compare Figs 5 and 6). The regular pattern of successive branching, so obvious in the explants grown in medium with serum (Fig. 4) was absent, and large cyst-like sacs extended towards the edge of the explants in the periphery (Figs 6, 23). The flattened epithelial cells that lined the cysts contained trace to moderate amounts of glycogen, whereas the columnar secretory cells lining the airways in the intermediate zone contained more glycogen (Fig. 24). In GMA sections, palestained preciliated and ciliated cells were now identified in airways of the central and intermediate zones (Fig. 24). Extensive BrdU labeling was noted in many of the airway epithelial cells but not

ET AL.

within endocrine cells of the NEBs (Fig. 25) which were now present even in the smallest airways. The endocrine cells showed strong immunolabeling for serotonin and CGRP (Fig. 26). Labeling for Clara cell protein was strong in secretory cells of the airway epithelium but was especially marked in cells that lined the small airways of the intermediate zone (Fig. 27). BGJb medium with defined additives

(Table 1, Figs 8, 28-32) After 4 days of culture in medium with defined additives (Gd 12+4) (Fig. 8), the explants were quite similar in appearance to those observed 2 days earlier (compare with Fig. 7). However, the blunt club-shaped profiles were now less well-defined and the translucent peripheral flange was wider (Fig. 8). In histological sections, the partitioning of the connective tissue was even more obvious than at Gd 12+2. An inner region, comprised of densely-packed connective tissue cells, supported the distorted bronchioles and irregular finger-like tubules. This region was separated sharply from the loosely-knit, peripheral fibrous flange, which was free of all epithelial structures (Fig. 29). At higher magnification, crenated basement membranes were clearly visible surrounding the distorted bronchioles and finger-like tubules. Cloudlike profiles (cross-sections of small bronchioles), partially surrounded by fields of rosettes (cross-sections of the finger-like tubules), were commonplace in the cell-rich compact connective tissue (Fig. 30). In most explants, the epithelial cells of the small rosettes were depleted of glycogen. In contrast, the bronchiolar epithelium contained moderate to heavy amounts of glycogen (Fig. 31). Many NEBs were now present in both the large and small airways. Labeling for CGRP (Fig. 32) and serotonin was marked. In the large airways of the central and intermediate zones, preciliated and ciliated cells were identified. Clara cell protein (Fig. 28) was present within numerous secretory epithelial cells in large and small airways but was not seen in the epithelial cells of the small rosettes. Labeling for BrdU was low to moderate in cells throughout all regions of the explants, except within the outer flange of loosely-

AIRWAY

BRANCHING

PATTERNS

knit fibrous connective tissue, where large numbers of cells were labeled.

Discussion

We have successfully cultured fetal hamster lungs on Transwell membranes, and achieved cytodifferentiation of the airway epithelium in the three medium formulations used, namely BGJb medium alone, with 5% FBS, and with defined additives. However, these formulations clearly differed in their effects on airway development and branching. The branching was evaluated initially in phase contrast images of the whole explants, which provided an overview of the airway architecture and branching patterns. This greatly facilitated an interpretation of the histological sections (cut en face through the explants). BGJb medium with 5% FBS was remarkable for permitting a nearly normal pattern of airway development to occur in uitro. Virtually the entire explant was occupied by many complex branches. While in culture, prealveolar respiratory saccules developed at the periphery of the lung. Therefore, factors in serum were crucial for allowing a nearly normal pattern of successive and orderly airway branching to emerge, and to facilitate the development of respiratory saccules. We are unaware of any other culture method or medium that surpasses this one for studying intrapulmonary branching of fetal hamster airways in vitro. In contrast, in BGJb medium alone, branching was partially inhibited throughout the airways and prealveolar structures. After 4 days of culture in this serumless chemically defined medium, large cysts formed at the periphery. Although others have reported limited branching in embryonic mouse lung cultured in BGJb medium alone (Jaskoll et al.. 1988; Slavkin et al., 1989)) the branching was significantly less than that observed in comparable in vivo controls. This is in agreement with the present fetal hamster lung results. The cyst-like sacs that formed at the periphery of our explants cultured in medium similar to those alone, are somewhat observed in cultures of developing rat lung (Sorokin etaf., 1992). However, the rat lungs were maintained in 40% FBS on a solid medium.

BGJb medium with defined additives significantly altered development of the fetal hamster lung. The reason is probably related to the fact that the connective tissue partitioned itself into two forms, a cell-rich (condensed) region enveloping and supporting the distorted bronchioles and irregular finger-like tubules, and a loose fibrous flange at the perimeter. Epithelial structures did not penetrate the fibrous flange of connective tissue, but were confined to the intermediate and central zones of the explant. The branched epithelial structures were crowded together in a manner analogous to roots of a pot-bound plant being crowded together. As a result of the epithelial confinement, the development of bronchioles and prealveolar structures was abnormal. After 4 days of culture, the epithelial cells lining the distorted bronchioles retained glycogen. whereas those lining the terminal linger-like tubules were glycogen-depleted. Since alveoli lack glycogen at neonatal day one (Ito et al., 1990a), we believe the finger-like tubules represent structures that normally would have differentiated into prealveolar respiratory saccules, but under these culture conditions, they were prevented from doing so. The inability of the finger-like tubules to penetrate the fibrous flange of connective tissue underscores the importance of epithelial-mesenchymal interactions in airway morphogenesis (Alescio and Cassini. 1962; Spooner and Wessels. 1970; Masters. 1976: Goldin, 1980; Jaskoll and Slavkin, 1984: Hilfer et al.. 1985; Guzowski et al., 1990). Airway branching is a complex process that depends on cleft formation in a lung bud (which is the initial step in branching) and a splitting into two branches. Each bud branches repeatedly to form the primitive bronchial tree (Ten Have-Opbroek, 1981: Sarikas et al., 1985b). This branching depends on epithelial-mesenchymal interactions. because if the mesenchyme (connective tissue) is removed, the branching stops (Masters, 1976; Roman et al., 1990). Many years ago Grobstein (1953; 1967) proposed that organogenesis was dependent on a balance between the shaping forces of the mesenchyme and the expansion of the epithelium. In the lung, it has been proposed that epithelial expansion is prevented in areas where the mesenchyme condenses, and a cleft forms. The furrow deepens and the air-

DESANTI ET AL.

way divides (Hutchins et al., 1981; Roman et al., 1991). This process is repeated over and over as additional airway branches are formed. For reasons we do not understand, in the presence of the defined additives, the airways were confined to regions with cellrich ‘condensed’ mesenchyme. They were unable to migrate, or expand, into the loose fibrous connective tissue of the peripheral flange. It was surprising that the three formulations in BGJb medium caused such diverse developmental patterns in the fetal hamster lungs. In medium with FBS, airway branching was nearly normal and prealveolar structures extended to the pleura. In medium alone, branching was partially inhibited and cyst-like sacs formed at the periphery. BGJb medium with defined additives allowed airway branching to occur, but the epithelial structures were unable to migrate out into the peripheral flange of the fibrous connective tissue. The specific signals controlling the development of the respiratory tree remain to be determined, but epithelial-mesenchyma1 interactions are critical. Lung mesenchyme is composed of a variety of extracellular matrix (ECM) molecules, including glycoproteins (such as laminin, hyaluronic acid, elastin, proteolglycans) and different types of collagen (Hay, 1981) as well as glycosaminoglycans (Bernfield et al., 1973). The importance of ECM involvement, and especially the crucial role of collagen in airway branching is illustrated in numerous studies carried out for more than two decades (Wessels and Cohen, 1968; Alescio, 1973; Spooner and Faubion, 1980; Chen and Little, 1987). In cultured mouse lung, antibodies against laminin inhibited branching (Schugar et al., 1990), as well as growth. Fibronectin is also critical for normal airway branching to occur (Roman et al., 1990), and Heine and coworkers (1990) demonstrated that fibronectin, collagens I and III, and glycosaminoglycans were concentrated at areas of cleft formation in developing mouse lung. Integrins are membrane glycoproteins that link the ECM components with the cytoskeleton, thus mediating a variety of cell matrix interactions. An inhibitor of ligands that bind uiu the RGD (Arg-Gly-Asp) sequence to integrin receptors diminished cleft formation and branching in developing mouse lung (Roman et al., 1991). Without

question, many or perhaps all, of these ECM components are involved in airway branching in the fetal hamster lung. Functional differentiation of the intrapulmonary airway epithelial cells (endocrine, ciliated, and secretory) was observed in explants maintained in each of the three medium formulations. Endocrine cells are first identified in fetal hamster lungs in viuo in the lobar bronchi at Gd 13 and in the bronchioles at Gd 14 (Sarikas et al., 1985a; It0 et al. 1990a). Serotonin is immunolocalized in uiuo in endocrine cells of the lobar bronchi of fetal hamster lung at Gd 13, and in the bronchioles at Gd 14 (McDowell, unpublished data). In our study, the earliest immunochemical evaluation was made at Gd 12+2. At this time, explants in all medium formulations contained endocrine cells that were strongly labeled for serotonin in the bronchi and large bronchioles. Sorokin and coworkers (1992) have recently demonstrated endocrine cell differentiation by immunolocalization of PGP 9.5 and CGRP, in cultured fetal rat lungs. In fetal hamster lungs, preciliated and ciliated cells first appear irt uivo in the lobar bronchi at Gd 14 and in the bronchioles at Gd 1.5 (McDowell et al., 1990; Ito et al., 1990a). These cells were identified at Gd 12+4, in the bronchi and bronchioles of the fetal lung explants cultured in all medium formulations. Presecretory cells appear in the conducting airways of fetal hamster lungs at about the same time as the ciliated cells. Clara cell protein is not detected in airways of the fetal hamster lung in uivo until Gd 15 (Strum et al., 1990b). In the present study, the earliest detection of Clara cell protein was made at Gd 12+2, iii explants cultured in medium with defined additives. By Gd 12+4, secretory cells with strong labeling for Clara cell protein were present at all airway levels, in explants cultured in the three medium formulations. In the cultured explants, glycogen stores were abundant at Gd 12+2, in the epithelium lining the terminal sacs (medium with FBS and medium alone) and finger-like tubules (medium with defined additives), but at Gd 12+4, glycogen was depleted from the prealveolar structures. This pattern of glycogen depletion is similar to that which occurs in the alveoli of fetal hamster lung in uivo (Ito et al.. 1990a).

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We have followed the development and differentiation of fetal hamster airways in uitro. Our in vitro model permits functional differentiation of the airway epithelium to occur in a manner that closely parallels that observed in uivo, and demonstrates that cytodifferentiation takes place in BGJb medium alone. However, to achieve a nearly normal airway branching pattern, FBS is required. Our defined additives resulted in an unusual separation of the embryonic connective tissue which was related to a distorted branching pattern, and an exclusion of epithelial structures from the peripheral fibrous flange. The mechanisms causing these changes are unknown, but preliminary studies in our laboratory have shown that removal of both vitamin A and EGF from the defined additives prevents partitioning of

the connective tissue and allows migration of the epithelial structures to the pleura. Thus, our explant culture method should prove useful for elucidating the role that these (and other) factors play in the important process of intrapulmonary airway development.

Acknowledgements

This study was supported by USPHS Grant HL-37640. The authors would like to thank Carnell Newkirk for technical assistance with the GMA sections. This work was submitted by A. M. DeSanti in partial fulfilment of a Master of Science degree from the Department of Pathology, School of Medicine, Graduate School, University of Maryland. Baltimore.

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