THE ANATOMICAL RECORD 228:151-162 (1990)

Changes in Glycoconjugates Revealed by Lectin Staining in the Developing Airways of Syrian Golden Hamsters TAKAAKI ITO, CARNELL NEWKIRK, JUDY M. STRUM, AND ELIZABETH M. McDOWELL Departments of Anatomy (T.I., J.M.S.) and Pathology (C.N., E.M.M.), University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT Lectin binding was studied in the developing airways of Syrian golden hamsters on gestational days 11-16 (day 16 is the day of birth). The trachea and lungs were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol; embedded in paraffin; and stained with eight lectin-horseradish peroxidase conjugates: Triticum vulgare (WGA), Dolichos biflorus (DBA), Helix pomatia (HPA), Maclura pornifera (MPA), Griffonia simplicifolia I-B4 (GSA I-B4), Arachis hypogaea (PNA), Ulex europeus I (UEA I), and Limulus polyphemus (LPA). Each lectin yielded a characteristic staining pattern, which modulated throughout development. In general, changes in staining characteristics of the tracheal epithelium preceded similar changes in the lobar bronchus, bronchiole, and alveolus. In the case of UEA I, MPA, WGA, and HPA, staining increased with time uniformly over the luminal surface of all epithelial cells. However, in the case of PNA, GSA I-B4, and LPA, after the differentiation of ciliated and secretory cells, the apical surfaces of the ciliated cells stained more intensely than the apical surfaces of the secretory cells. Neuraminidase pretreatment enhanced PNA and GSA I-B4 staining in both cell types. In the case of PNA, these light microscopic observations were confirmed by ultrastructural study. Unlike the other lectins, the pattern of staining with DBA was unusual. Staining was moderate a t first, then decreased (days 13 and 141, then increased at all airway levels. This study shows that different glycoconjugates modulate in airway epithelial cells throughout fetal development. In the respiratory system, the developing epithelium lining the conducting airways shows dramatic alterations associated with cellular differentiation and functional maturation. Development in the respiratory tree proceeds in a proximal to distal direction, starting in the trachea and progressing to include the lobar bronchi, bronchioles, and alveoli (Sorokin, 1965; Jeffery and Reid, 1977; Sturgess, 1985; Cutz, 1987). Most studies of lung development have focused on the alveolar lining cells, especially as they relate to neonatal respiratory distress syndrome. Although these investigations have provided important information relevant to many areas of pulmonary physiology and pathology, far less is known about the development of the airconducting system and of the lining epithelial cells. The present study employing lectins is a n effort in that direction. Lectins are sugar-binding proteins or glycoproteins of nonimmune origin that precipitate glycoconjugates (Goldstein et al., 1980). Glycoconjugates on the surfaces of cells are thought to play important roles in many biological phenomena (Kornfeld and Kornfeld, 1980; Spicer et al., 1981; Ivatt, 1984). The chemical nature and distribution of cell surface glycoconjugates appear to reflect changes in states of cellular maturation and function that have been observed in many c:

1990 WILEY-LISS, INC

differentiating systems (Sharon, 1983; Damjanov, 1987; Muramatsu, 1988; Kimber, 1989). For this reason, lectins have been used to demonstrate changes in epithelial cell surface carbohydrate composition in various organs during development (Maylie-Pfenninger and Jamieson, 1980; Muresan et al., 1982; Nakai et al., 1985; Colony and Steely, 1987; Holthofer and Virtanen, 1987; Laitinen et al., 1987; Caldero e t al., 1988; Shiojiri and Katayama, 1988). The distribution and characteristics of glycoconjugates on the surfaces of normal adult airway epithelial cells have been studied using lectins in several animal species, including Syrian golden hamsters (Mazzuca e t al., 1982; Schulte and Spicer, 1983, 1985; Spicer et al., 1983; Christensen et al., 1985; Ito et al., 1985;Flint et al., 1986; Geleff et al., 1986; Honda et al., 1986; Hennigar et al., 1987; Mariassy et al., 1988). However, lectin binding patterns have not been studied during fetal airway development. Therefore, in the present study, we have used a battery of lectin-peroxidase conjugates to characterize changes in cell glycoconjugates in the epithelium lining airways in developing hamsters. We

Received April 25, 1989; accepted January 12, 1990

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TABLE 1. Lectins and hapten sugars used Lectin (acronym) Triticum vulgare (WGA) Dolichos biflorus (DBA) Helix pomatia ( HPA ) Maclura pomifera (MPA) Griffonia simplicifolia I-B4 (GSA LB4) Arachis hypogaea (PNA) Ulex europeus I (UEA I) Limulus polyphemus (LPA)

Nominal specificity'.' GlcNAc (fi1,4GlcNAc),-,> fiGlcNAc>Neu5Ac GalNAcul,SGalNAc>> aGalNAc GalNAca1,3GalNAc> aGalNAc cuGalNAc>uGal aGal Galpl,SGalNAc>a and PGal aL-Fuc Neu5Ador Gc)cr2,6GalNAc> Neu5Ac

Concentration of lectin-HRP Hapten conjugate" ( ~ g i m l ) sugar"' 50 GlcNAc 50

GalNAc

10

GalNAc

50

Melibiose

50

Gal

100

Gal

50

aL-Fuc

100

NeuNAc

'Carbohydrate specificities of lectins a r e according to Goldstein and Poretz ( 1986). 'Abbreviations: GlcNAc, N-acetyl-glucosamine; Neu5Ac, 5-acetyl-neuraminic acid; GalNAc, N-acetyl-galactosamine; Gal, galactose; Fuc, fucose; Neu5Gc, 5-glycolyl-neuraminicacid; NeuNAc, N-acetyl-neuraminic acid. "Lectin-HRP conjugates were dissolved in 0.1 M phosphate buffer solution (pH 7.2) containing 0.1 mM CaCl,, MgCl,, and MnC1,. 'Concentration of hapten sugars 0.2 M.

chose the Syrian golden hamster as our experimental model because changes occur in the epithelium on a daily basis, a t the different airway levels (trachea, lobar bronchus, bronchiole), during the latter part of pregnancy (McDowell et al., 1985; Sarikas e t al., 1985). MATERIALS AND METHODS

Twenty-two timed-bred Syrian golden hamsters were purchased from Charles River (Newfield, NJ). The hamsters arrived a t our animal facility on the second gestational day (the first gestational day was taken to be the day after mating). Each pregnant hamster was caged individually, given food and water ad libitum, and maintained under standard laboratory conditions in a room with a 12-hr lightidark cycle. On gestational days 11, 12, 13, 14, and 15, three pregnant hamsters were anesthetized with methoxyflurane (Metaphane; Pittman-Moore Inc., Washington Crossing, NJ). This was done a t 9:30 AM EST each day. The abdomen was opened, and six fetuses were taken rapidly from the uterus of each pregnant hamster (18 fetuses per day; 90 fetuses total). Three fixatives were used for the light microscopic study: 1) 4% formaldehyde-1% glutaraldehyde in a 200 mOsm phosphate buffer (4FIG) (McDowell and Trump, 1976),2)6% mercuric chloride-1% sodium acetate-O.1V glutaraldehyde solution (HgCl,-G) (Schulte and Spicer, 1983), and 3) 95%)ethanol. On the specified gestational days, two fetuses from each mother were fixed by immersion in each of the three fixatives. On gestational days 11 and 12, fixation was achieved by immersion of the undamaged fetus; on days 13 and 14, the fetal abdomen was opened prior to immersion; and, on day 15, the abdomen was opened and a small volume of fixative was also injected into the oral cavity. The developing trachea and lung were also studied on gestational day 16, the day of birth. For this part of the study, 21 hamster pups were taken from three different mothers. Of

these, nine fetuses were taken just before birth and 12 neonates were taken shortly after birth; seven fetuses/ neonates were fixed in each of the three fixatives, a s described above, after being anesthetized with methoxyflurane. The abdomen was opened, and the head above the mandible was removed prior to immersing the animal in the fixative. Four additional pregnant hamsters were used to provide fetuses for a n electron microscopic study of the airways on gestational days 12, 13, 14, and 15. The fetuses were fixed by immersion in 4FIG, as described above. Light Microscopy

All specimens were fixed for 6 h r a t room temperature. Tissues fixed in 4FIG were then placed in phosphate buffer, and those fixed in 95% ethanol or HgC1,G were placed in 70% ethanol and stored for 2 days a t 4°C before embedment in paraffin. Some specimens were embedded longitudinally in order to obtain longitudinal sections ( 4 fJ-mthick) of the trachea, lobar bronchus, and lung. Others were embedded transversely to obtain transverse sections of midtrachea and lung at the level of the hilum. Before staining with each lectin conjugate, unstained slides from every fetus in the study were screened under low power and selected to ensure that all anatomical levels (trachea, lobar bronchus, bronchioles, primitive alveoli) were present. Information regarding the lectin-horseradish peroxidase (HRP)conjugates used, their concentrations, and their nominal specificities are listed in Table 1. The sources of the conjugates were a s follows: Triticuni uulgare (WGA), Dolichos biflorus (DBA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Ulex europeus I (UEA I), and L i m u l u s polyphemus (LPA) from E-Y Laboratories, Inc. (San Mateo, CA) and Helix pomatia (HPA) and Griffonia simplicifolia I-B4 (GSA LB4) from Sigma Chemical Co. (St. Louis, MO).

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I X C T I N S T A I N I N G OF DEVE1,OI’ING AIRWAYS

The tissue sections were deparaffinized, and those fixed in HgCI,-G were placed in Lugol’s iodine solution for 5 min. Endogenous peroxidase was blocked by a 15 min immersion of sections in 1%’H,O, in methanol. After rinsing in phosphate-buffered saline (pH 7.2), the sections were incubated with a lectin-HRP conjugate for 2 h r a t room temperature. Each conjugate was diluted with 0.1 M phosphate buffer (pH 7.2) containing 0.1 mM each of CaCl,, MgCl,, and MnC1,. After exposure to the conjugate, the sections were rinsed again, incubated in 0.1% 3-3‘-diaminobenzidine-H,02substrate (pH 7.6) for 8 min, and counterstained with 1% methyl green. In addition, sections stained by HRP conjugates of DBA, GSA I-B4, PNA, and LPA were incubated, prior to lectin staining, in neuraminidase type X from Clostridium perfringens (Sigma) a t a concentration of 0.1 Uiml in acetate buffer solution containing 0.04 M CaC1, (pH 5.25) for 18 h r a t 37°C (Warren and Spicer, 1961). At each developmental stage, the variation in stain intensity was absent or minimal within a particular airway level in the same or different animals a s long as they were fixed and stained in exactly the same way. Staining intensity was assigned a t each airway level on a subjectively estimated scale ranging from 0 (unreactive) to 4 (most reactive). Evaluations were made on sections from six fetuses on each gestational day for each of the three fixatives used (18 fetuses per day). For corroborative evidence of specific binding for all the lectins, control sections were incubated in parallel for 2 h r a t room temperature in a mixture of the lectinHRP conjugate and the appropriate hapten sugar (Table 1)a t a concentration of 0.2 M. The hapten sugars were purchased from Sigma. Additional controls were made on sections from a gestational day 14 fetus, according to the following procedures: 1)substitution of unconjugated lectins (WGA, DBA, MPA, PNA, UEA 1, and LPA from E-Y Laboratories and HPA and GSA I-B4 from Sigma, a t a concentration of 50 IJ-giml)for lectin-HRP conjugates, 2) exposure to HRP (type VI, Sigma) and media without lectins, and 3) exposure to 3-3’-diaminobenzidine-H202 substrate (pH 7.6) without previous lectin-HRP conjugates. Additional sections were stained with hematoxylin and eosin and by the alcian blue (pH 2.5)-periodic acid-Schiff method, with and without diastase digestion, to identify glycogen. The distribution and the changes in storage of glycogen that occur during development of the airways are reported elsewhere (It0 et al., 1990). Electron Microscopy

Shortly after immersion fixation of the fetuses, the left lungs were isolated and fixed for a n additional 24 h r a t room temperature in 4FIG. After rinsing thoroughly in 0.1 M phosphate buffer (pH 7.2), each lung was cut transversely, and small tissue blocks were incubated with ferritin-conjugated PNA (E-Y Laboratories) for 2 h r a t room temperature. The conjugate was dissolved a t a concentration of 100 IJ-giml, in 0.1 M phosphate buffer (pH 7.2) containing 0.1 mM each of CaCl,, MgCl, and MnC1,. Control tissue blocks were incubated in ferritin-conjugated PNA plus the hapten sugar galactose (0.2 M ) for 2 h r a t room temperature. Additional tissue blocks were incubated a t room tem-

perature in neuraminidase type X from Clostridium perfringens (0.1 Uiml in acetate buffer, pH 5.25) for 20 h r prior to incubation in the PNA-ferritin conjugate. After rinsing in 0.1 M phosphate buffer (pH 7.2), all tissues were postfixed with 1% OsO, for 1 h r and embedded in Epon. Thin sections were stained with lead citrate and uranyl acetate, and examined with a Jeol 1200 EX microscope. RESULTS Lectin Staining of Developing Airway and Alveolar Epithelium

Each lectin yielded a characteristic staining pattern throughout development (Table 2, Fig. 1).In general, changes in staining characteristics of the tracheal epithelium preceded similar changes in the lobar bronchus and bronchioles by 1and 2 days, respectively. The staining patterns of the developing terminal epithelium (corresponding to future alveoli) was generally similar to that of the secretory cell component of the developing bronchioles. Staining was blocked by the various histochemical control measures that were employed. None of the staining patterns was altered fundamentally by the different fixatives used, but in some cases subtle differences were seen. For example, in the case of UEA I, HPA, and DBA, the developmental changes were most obvious in tissues fixed in HgCI2-G. In the case of PNA, GSA I-B4, and LPA, the staining intensity was similar with all fixatives, but morphological preservation was best with 4FIG. For this reason, the divergent staining of the luminal surface of ciliated cells and secretory cells (seen in the latter part of gestation) was most obvious in 4FIG-fixed specimens. Staining intensities and developmental patterns of MPA and WGA were similar with all the fixatives used (4FIG, HgCI,-G, and ethanol). At each airway level, staining of the apical surfaces of the airway epithelial cells increased progressively during development with seven of the eight lectins studied. In the case of UEA I, MPA, WGA, and HPA, staining increased uniformly over the surface of all epithelial cells a t all anatomical levels, throughout the period of study. The progressive increase in staining intensity was most marked with UEA I (Fig. 1).In the case of PNA, GSA I-B4, and LPA, the staining intensity increased uniformly over the surfaces of the undifferentiated epithelial cells early in development, but later, after the differentiation of ciliated cells and secretory cells, the staining intensity was obviously increased only on the ciliated cell surfaces. In the case of GSA I-B4 and LPA, staining of the secretory cell surfaces was equivalent to staining of the undifferentiated cells. However, in the case of PNA, staining of the surface of the secretory cells decreased markedly compared with the undifferentiated state (Fig. 1).The pattern of staining with DBA was unique. With this lectin, the surfaces of the undifferentiated epithelial cells stained moderately. The stain then weakened, reaching its lowest intensity on gestational day 14 a t all airway levels. Staining then increased on days 15 and 16 (Fig. 1). Groups of endocrine cells in the form of neuroepithelial bodies appeared in the trachea on gestational day 12, in the lobar bronchus on gestational day 13, and in

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TABLE 2. Changes in lectin staining of luminal surface of developing airway' Lectin UEA I

MPA

WGA

HPA

PNA

GSA I-B4

LPA

DBA

Airway level Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus Trachea Bronchus Bronchiole Alveolus

-

11 1 1

2-3 2-3

3 3 2 2 2 2 1-2 1-2 1 1 2-3 2-3

12 2 1-2 1-2 1-2 3 3 3 3 3 3 3 3 2-3 2-3 2-3 2-3 2-3 2-3 2-3 2-3 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 2 2 2-3

13 3 2 2 2 3 3 3 3 3-4 3-4 3-4 3-4 3 2-3 3 3 2-3 2-3 2-3 2-3 2 2 2 2 1-2 1-2 1-2 1-2 1 0-1 1

2-3

1

14 3-4 2-3 3 3 3-4 3-4 3-4 3-4 4 3-4 3-4 3-4 3-4 3 3-4 4 2(3-4)' 2-3 3 3 2-3 2-3 2-3 2-3 2 2 2 2 0-1 0-1 0-1 0-1

15 4 3 3-4 3-4 3-4 3-4 3-4 3-4 4 4 4 4 4 3 3-4 4 1-2(4)' 1-2(4)2 3 2-3 2-3(4)2 2-3(4)' 2-3 3 2(4)' 2(4)' 2 2-3 2 1-2 1-2 2

16 4

3 3-4 3-4 3-4 3-4 3-4 3-4 4

4 4 4 4 3-4 3-4 4 1-2(412 1-2(4)' 1-2(4)2 1-2 2-3(4)' 2-3(4)' 2-3(4)' 3 2(4)' %4)' 2(4)" 2-3 3 2 2 3

'Data for UEA I, HPA, and DBA were derived from tissues fixed in HgCl,-G. Data for MPA, WGA, PNA, GSA I-B4, and LPA were derived from tissues fixed in 4FIG. See legend to Figure 1 for further information. 'Secretory cell (ciliated cell).

the bronchioles on gestational day 14. A characterization of the lectin staining of the apical surfaces of these endocrine cells was not possible; most of their apical surfaces were covered by neighboring columnar cells. UEA I (Figs. 2-5)

Staining of the apical cell surfaces was very low in the primitive epithelia (Figs. 2,4) but gained markedly in intensity throughout development, until it was moderate to strong a t all airway levels (Figs. 3, 5). MPA and WGA (Figs. 6-9)

Staining of the apical epithelial cell surfaces was moderate in the primitive epithelia a t all airway levels (Figs. 6, €31, and i t increased slightly throughout development (Figs. 7, 9).

(Figs. 11-13). A regional difference was noted at all times in the trachea; the surfaces of cells in the cranial part stained stronger than in the caudal part. In tissues fixed in HgC1,-G or ethanol, stain was also seen in the apical cytoplasm of some tracheal columnar cells on gestational day 16 (Fig. 13). In contrast, staining of the cell surfaces was mild to moderate with DBA in the primitive epithelia (Fig. 14). It then decreased totally, or to trace amounts, on gestational days 13 and 14 (Fig. 15) but increased in intensity on gestational days 15 and 16 (Fig. 16) a t all anatomical levels. This pattern was the same regardless of the type of fixative used. Neuraminidase treatment did not enhance the staining intensity a t any time. As with HPA, a regional difference was noted in the trachea, and throughout development staining of the cell surfaces was stronger in the cranial part.

HPA and DBA (Figs. 10- 16)

The staining results for HPA and DBA are described together because their nominal sugar specificities are very similar (Table 1).However, the patterns of staining in the developing airway epithelia were quite different. With HPA, staining of the apical epithelial cell surfaces was mild to moderate at first (Fig. 10) but became stronger later in development a t each anatomical level

PNA (Figs. 17-22)

Prior to the differentiation of ciliated cells and secretory cells, the surfaces of the undifferentiated epithelial cells were mildly to moderately stained (Fig. 17). As ciliated and secretory cells became established, the staining of the apical surfaces of the ciliated cells increased, whereas that of the secretory cells decreased (Figs. 18, 19A). Neuraminidase treatment generally

4 .

j 2 1 :

UEA I

Trachea

T Bronchus

4 .

:::/ 1rn 1

I

m , *

I

1

A

I

. Bronchiole

,

i3; r

-

C >2 l

--

. Bronchiole

4

7

3 . 2 .

1 .

C

. Alveolus

1

.

Alveolus

-

7

3.

2:

1 1 1 . 12. 13. 14. Gestatlonal

.

.

16

15

Gestational

Days

C

~

Days

Gestational

Days

Gestational

Days

GSA 1-84

Trachea

2

S

l t Bronchus

l t 4

. Bronchiole ~~

32. 1.

t

A'veoius

l t

l t )

*

11

.

12

.

13

.

14

Gestattonal

a

L

.

15 Days

16

a

11

.

12

.

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13

.

14

Gestational

.

15

16

Days

Fig. 1. Lectin staining of the luminal surfaces of epithelial cells in the trachea, bronchus, bronchiole, and alveolus in fetal Syrian golden hamsters on gestational days 11-16 (day 16 is the day of birth). Staining intensity was assigned on a subjectively estimated scale ranging from 0 (unreactlve)to 4 (most reactive). At each developmental stage, variation in stain intensity a t comparable airway levels was minimal

LL 1 1 12 13 14 15 16 11 12 13 14 15 16 Gestatlonal

Days

Gestational

Days

in the same or different animals, assuming they were fixed and stained in exactly the same way. Patterns for UEA I, HPA, and DBA were derived from tissues fixed in HgC1,-G; those for MPA, WGA, PNA, GSA I-B4, and LPA were derived from tissues fixed in 4FIG. C, ciliated cells; S, secretory cells.

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T. IT0 ET AL.

Fig. 2. UEA I staining. Transverse section of trachea on gestational day 11. Trace staining is seen in the epithelium. HgCl,-G fixation. Counterstained with methyl green. x 170. Fig. 3. UEA I staining. Longitudinal section of trachea on gesta-

tional day 15. Strong staining is seen in the epithelium. Arrow, esophagus. HgCl,-G fixation. Counterstained with methyl green. X 170. Fig. 4. UEA I staining. Longitudinal section of left lung on gestational day 12. Trace staining is seen in the developing epithelium. LB, lobar bronchus; t, terminal buds. HgC1,-G fixation. Counterstained with methyl green. x 170. Fig. 5. UEA I staining. Transverse section of left lung on gestational day 15. Moderate staining is seen in lobar bronchus (LB);note neuroepithelial body a t arrow. Staining IS moderate to strong in the primitive respiratory saccules ( s ) .HgCl,-G fixation. Counterstained with methyl green. x 170. Fig. 6. MPA staining. Transverse section of trachea (TI and esophagus (arrow) on gestational day 11. Moderate staining is seen a t the

enhanced the intensity of PNA staining, and in particular staining was restored to the surfaces of the secretory cells (Fig. 19B). In the terminal (alveolar) epithelium, the surfaces of the epithelial cells were mildly to moderately stained through gestational day 14. As the terminal epithelium became attenuated to form the primitive respiratory saccules on gestational day 15, staining of the epithelial surfaces decreased. Neuraminidase treatment also enhanced the staining intensity in this site. The observations made by light microscopy on PNA staining were confirmed by transmission electron microscopy. Prior to cellular differentiation, the surfaces of the undifferentiated cells showed ferritin labeling (Fig. 20). Preciliated and ciliated cells first appeared in the lobar bronchi and bronchioles on gestational days 14 and 15, respectively, and ferritin labeling was increased on the apical surfaces of these cells, both on the microvilli and on the cilia (Fig. 21). At the same time, ferritin labeling was reduced on the apical surfaces of the presecretory cells (Fig. 21) compared with the amount of labeling on the surfaces of the undifferenti-

luminal surface of the trachea. 4FIG fixation. Counterstained with methyl green. x 170. Fig. 7. MPA staining. Longitudinal section of trachea (Ti and esophagus (arrow) on gestational day 15. Strong staining is seen a t the luminal surface of the trachea. 4FIG fixation. Counterstained with methyl green. x 170. Fig. 8. WGA staining. Transverse section of left lung on gestational day 11. Moderate staining is seen a t the luminal surface of the primitive epithelium. Note that vascular endothelium (arrows) and pleural surface (arrowheads) are also stained. 4FIG fixation. Counterstained with methyl green. x 170. Fig. 9. WGA staining. Longitudinal section of left lung on gestational day 15. Strong staining is seen a t the luminal surface of the lobar bronchus (LB), bronchiole (b), and primitive respiratory saccules ( s ) . Note neuroepithelial body in the lobar bronchus (arrowhead). The vascular endothelium is also stained (arrows).4FIG fixation. Counterstained with methyl green. x 170.

ated cells (Fig. 20). Treatment with neuraminidase exposed PNA-binding sites on the apical surfaces of the secretory cells and enhanced binding to the surfaces of the ciliated cells (Fig. 22). Secretory granules were first seen in the apical cytoplasm of the presecretory and preciliated cells of the lobar bronchi on gestational day 15 (Figs. 21, 22). GSA 1-84 and LPA (Figs. 23-25)

The staining patterns of these lectins were similar (Fig. 1). Before differentiation of the ciliated and secretory cells, the apices of the primitive airway epithelial cells showed mild to moderate staining (Fig. 23), which increased with time. After the ciliated cells appeared, their apical surfaces stained more strongly than those of the secretory cells (Figs. 24, 25). Neuraminidase digestion enhanced GSA I-B4 staining of both cell types, and the differential staining between ciliated and secretory cells was diminished. In contrast, neuraminidase digestion reduced staining with LPA but failed to abolish the stain completely.

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LECTIN STAINING O F DEVELOPING AIRWAYS

Fig. 10. HPA staining. Longitudinal section of lungs on gestational day 11. Mild staining is seen at the luminal surface of the primitive epithelium. HgCl,-G fixation. Counterstained with methyl green. x 60.

Fig. 14. DBA staining. Longitudinal section of lungs on gestational day 11(adjacent to section in Fig. 10).Moderate staining is seen in the primitive epithelium. HgC1,-G fixation. Counterstained with methyl green. ~ 6 0 .

Fig. 11. HPA staining. Transverse section of left lung on gestational day 14. Moderate staining is seen in the lobar bronchus (LB) and strong staining is seen in the developing terminal sacs. HgC1,-G fixation. Counterstained with methyl green. x 60.

Fig. 15. DBA staining. Transverse section of left lung on gestational day 14 (adjacent to section in Fig. 11).No stain is seen in the conducting airways and developing terminal sacs. LB, lobar bronchus; b, bronchiole. HgC1,-G fixation. Counterstained with methyl green. x 60.

Fig. 12. HPA staining. Longitudinal section of left lung on gestational day 16. Moderate to strong staining is seen in the lobar bronchus (LB), bronchiole (b), and alveoli. HgCl,-G fixation. Counterstained with methyl green. x 60. Fig. 13. HPA staining. Transverse section of trachea on gestational day 16. The apical surface and supranuclear cytoplasm stain strongly. HgC1,-G fixation. Counterstained with methyl green. x 360.

Additional Observations

The endothelial cells of arteries, veins, and capillaries were moderately stained by WGA (Figs. 8, 9) and GSA I-B4 (Fig. 23) and mildly stained by MPA, PNA, and LPA a t all the times studied. The endothelial cells showed no staining with UEA I, HPA, or DBA. Pleural mesothelial cells stained moderately with WGA (Fig. 8 ) , PNA, and DBA and mildly with MPA, HPA, and LPA. Mesothelial cells failed to stain with UEA I and GSA LB4. The trachealis muscle and the smooth muscle cells that form a sheath supporting the larger intrapulmonary airways were stained with MPA only. The stain was mild to moderate on gestational days 11-14 (Fig. 6) and trace to mild on days 15 and 16. (The stain was most obvious in tissues fixed in HgC1,-G.) Condensations of mesenchymal cells along the ventral aspect of the trachea were first seen on gestational day 12. These primordial cartilage rings began to stain with alcian blue (pH 2.5) on gestational day 14. The cartilage rings revealed a trace amount of staining with MPA (Fig. 7 ) , WGA, HPA, DBA, and PNA

Fig. 16. DBA staining. Longitudinal section of left lung on gestational day 16 (adjacent to section in Fig. 12). Mild to moderate staining is seen a t the surface of the lobar bronchus (LB) and bronchiole (b).The surface of the alveoli is stained moderately. HgC1,-G fixation. Counterstained with methyl green. x 60.

throughout their development but failed to stain with UEA I (Fig. 3 ) , GSA I-B4, or LPA. DISCUSSION

Our study shows that different glycoconjugates are modulated in airway epithelial cells throughout fetal development. Three patterns of staining were seen. 1) Staining increased with time throughout development with UEA I, MPA, WGA, and HPA, and, after the ciliated and secretory cells became differentiated, their apical cell surfaces stained with similar intensity. 2) Staining also increased over time with PNA, GSA I-B4, and LPA, but after cell differentiation, the apical surfaces of the ciliated cells stained more intensely than the apical surfaces of secretory cells. 3 ) With DBA, the staining was moderate a t first and then it fell to trace levels, but it became more intense later in development. Moreover, with DBA, these changes occurred at all airway levels, simultaneously, but with the other lectins, staining intensity in the trachea generally preceded similar staining in the lobar bronchus, bronchi-

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Fig. 17. PNA staining. Transverse section of left lung on gestational day 13. Mild to moderate stain is uniform over the luminal surface of the lobar bronchus. 4FIG fixation. Counterstained with methyl green. x 450. Fig. 18.PNA staining. Longitudinal section of lobar bronchus of left lung on gestational day 15. The luminal surface of the ciliated cells (arrowheads) stains strongly and the surface of the secretory cells

oles, and terminal epithelium (presumptive alveoli) (see Fig. 1). An increase in the number and variety of terminal sugar residues on cells, a s revealed by lectin histochemistry, has been reported during the embryonic differentiation of various epithelial tissues (MayliePfenninger and Jamieson, 1980; Nakai et al., 1985; Holthofer and Virtanen, 1987; Laitinen et al., 1987; Caldero et al., 1988) and during cellular maturation of the epidermis (Reano et al., 1982; Zieske and Bernstein, 1982; Nemanic et al., 1983). In the present study, increased staining over time was most obvious with UEA I. Since this lectin recognizes fucosyl residues (Pereira e t al., 1978), our study suggests that fucosyltransferase activity (a key enzyme in the formation of fucose residues) andior synthesis of endogenous acceptors for fucose increase in the airway epithelial cells throughout the developmental period. In the mouse, UEA I receptors could not be detected in embryos between days 5 and 13 but were detected in various epithelia, including the airways, later in fetal development (Sato e t al., 1986). Although MPA and WGA do not share common sugar specificities (Table l ) , the staining patterns of the fetal airway epithelium were similar. The stain increased gradually during development (Fig. 1) and did not show any special affinity for secretory cells after they had differentiated. Staining with HPA was quite similar to t h a t with WGA and MPA. This is consistent with the observation of Geleff et al. (19861, who reported HPA staining a t the apical surface of both ciliated and secretory cells in the airways of adult hamsters. Our observations of fetal hamsters are a t variance with those of Wasano et al. (19881, who found HPA staining to be specific only for secretory cells in airways of adult hamsters. The sugar specificities of HPA and DBA are similar (Table 1; Etzler and Kabat, 1970; Hammarstrom, 1972). However, in our study, the staining pattern of the airway epithelium by these two lectins during fetal development was very different (Fig. 1).We have no explanation for this, but other studies have shown that lectins with similar biochemically defined carbohy-

stains mildly. Note neuroepithelial body in the lobar bronchus (arrow).4FIG fixation. Counterstained with methyl green. x 450.

Fig. 19. PNA staining without (A) and with (B) neuraminidase pretreatment. Transverse serial sections of trachea on gestational day 16. A: Luminal surface of ciliated cells is stained strongly, but secretory cell surface shows only trace staining. B: Strong staining is distributed uniformly over the surfaces of all the cells. 4FIG fixation. Counterstained with methyl green. x 300.

drate binding affinities may differ in their staining patterns of the same tissue sections (Schulte and Spicer, 1983; Damjanov, 1987; Holthofer and Virtanen, 1987). Furthermore, we cannot explain the decrease followed by a n increase in DBA binding to the airway epithelial cells during fetal development. This occurred in all the specimens examined regardless of the fixative used. The decreased binding of DBA could be the result of a deposition of new sugars over the DBAbinding sites, changes in the steric arrangement of the terminal sugars, decreased activity of a glycosyltransferase, or increased activity of a glycosidase. We know from morphological studies that cell differentiation in the tracheal epithelium (McDowell et al., 1985) precedes that in the lobar bronchus and bronchioles by 1 and 2 days, respectively (Sarikas e t al., 1985). Therefore, whatever is responsible for the unusual DBA staining pattern, it seems unrelated to differentiation of the epithelium per se; the changes in staining occurred simultaneously at all airway levels (Fig. 1). Staining with the lectins PNA, GSA I-B4, and LPA increased over time, but after differentiation of the

Fig. 20. PNA staining. Transmission electron micrograph of undifferentiated cells of lobar bronchus on gestational day 13. Labeling with ferritin particles (arrows) is seen a t the luminal surface. x 34,000. Fig. 21. PNA staining. Transmission electron micrograph of lobar bronchial epithelium on gestational day 15. The microvilli of the preciliated cell (PC) are heavily labeled with ferritin particles. In contrast, the luminal surface of the presecretory cell (PS) is not labeled. Note a secretory granule (sg) in the apical cytoplasm of the preciliated cell and glycogen deposits (G) in the cytoplasm of the presecretory cell. x 27,000. Inset: Cross section through cilia and microvilli of a ciliated cell on gestational day 15. The surfaces of the cilia and microvilli are labeled with ferritin particles. x 30,000. Flg.22. PNA staining after neuraminidase digestion. Transmission electron micrograph of lobar bronchial epithelium on gestational day 15. The apical surfaces of the ciliated (C) and secretory (S)cells are heavily labelled with ferritin particles (arrows).Note secretory granule ( s g ) and glycogen (GI in the cytoplasm of the secretory cell. x 30,000.

IXCTIN STAINING O F DEVELOPING AIRWAYS

Figs. 20-22.

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Fig. 23. GSA I-B4 staining. Longitudinal section of left lung on gestational day 14. Moderate uniform staining is seen a t the luminal surface ofthe lobar bronchus (LB).Endothelium ofartery ( A ) and vein ( V ) is stained. Neuroepithelial body (arrow).Developing terminal sac ( s ) . 4FIG fixation. Counterstained with methyl green. x 450.

heads) stains stronger than that of the secretory cells in the lobarbronchus (LB).Arrow, neuroepithelial body. 4FIG fixation. Counterstained with methyl green. x 450.

Fig. 24. GSA I-B4 staining. Longitudinal section of left lung on gestational day 15. The lurninal surface of the ciliated cells (arrow-

Fig. 25. LPA staining. Longitudinal section of trachea (T)and esophagus (arrow)on gestational day 16. The luminal surface of the ciliated cells (arrowheads)stains more strongly than that of the secretory cells. 4FIG fixation. Counterstained with methyl green. x 450.

ciliated and secretory cells in the trachea, bronchus, and bronchioles, the apical surfaces of the ciliated cells were stained more heavily than those of the secretory cells (Fig. 1).Since the apical surface area of ciliated cells is much greater than that of secretory cells, increased staining with a lectin a t the light microscopic level might reflect merely morphological differences between these two cell types rather than a n actual increase in lectin binding to the apical cell membrane. The apical surface of ciliated cells is modified not only by cilia but also by many long, thin microvilli (Breeze and Wheeldon, 1977; Becci et al., 1978). In contrast, the apical surface of secretory cells bears fewer, wider, shorter microvilli (Andrews, 1974; Breeze and Wheeldon, 1977; Becci et al., 1978). Furthermore, as secretory cells become filled with secretions, they swell, their apical surface bulges and becomes smooth in contour, and their microvilli disappear (Andrews, 1974; Mariassy et al., 1975), all of which lead to a decrease in surface area. To verify a distinct labeling difference between these two cell types, we investigated this issue ultrastructurally using a PNA-ferritin conjugate that recognizes galactosyl residues (Pereira et al., 1976). Our results show that more ferritin particles were seen associated with the cilia and microvilli of the ciliated cells than with the membrane of the secretory cells. Moreover, this difference was manifest even in the immature preciliated cells (Fig. 21) before the cilia had fully developed. Earlier studies by Schulte and Spicer (1985)on PNA binding in the tracheal epithelium of adult hamsters reported that only a few ciliated cells in the cranial region were stained, but most cells (ciliated and secretory) were unstained. We applied the PNA-HRP conjugate to the adult airways (unpublished observation) and found that, a s in the fetus, ciliated cells and secretory cells stained, but the ciliated cells stained more intensely. This staining discrepancy appears to result from differences in the concentration of the PNA-HRP conjugates used. We used 100 pgiml, and Schulte and Spicer (1985)used 10-20 pgiml. When we treated fetal or adult tissues with the lower concentrations, staining intensity decreased markedly, and most cells appeared to be unstained.

Our study clearly shows that neuraminic acid increases in the developing airway epithelium during the latter part of pregnancy. The lectin LPA binds to Nacetyl-neuraminic acid (Roche et al., 1975), and, after cell differentiation, the ciliated cells stained more strongly than the secretory cells (Fig. 1).After treatment with neuraminidase, staining with LPA was decreased markedly, whereas staining with PNA and GSA I-B4 was increased (in particular, staining of the surfaces of the secretory cells was increased). This indicates that reduced binding of PNA and GSA I-B4 to the surfaces of secretory cells (Fig. 1) was due to the addition of neuraminic acid residues to their apical surfaces, around the time of birth. Our results for PNA were confirmed a t the ultrastructural level (Fig. 22). In adult animals, neuraminic acid residues cover the luminal surfaces of cells of the respiratory tract in several species (Spicer et al., 1983; Schulte and Spicer, 1985), and enhanced PNA-binding occurs in rodents following the removal of neuraminic acid by enzymatic digestion (Ito et al., 1985; Schulte and Spicer, 1985). Neuraminic acid residues terminate oligosaccharide side chains by linkage to penultimate galactose or galactosamine (Kornfeld and Korfeld, 1976, 1980; Montreuil, 1980). The function of neuraminic acid and the penultimate sugars is not clear, but they may act as receptor sites for exogenous macromolecules and may also keep the cilia separated from one another (Schulte and Spicer, 1985). The formation of neuraminic acid residues in the developing airway epithelium is clearly associated with the functional maturation of airway epithelial cells. Moreover, in the alveoli, the presence of neuraminic acid during the prenatal period in humans is regarded as a n index of maturation of the alveolar surface epithelium (Faraggiana et al., 1986; Barresi et al., 1987). In the present study, PNA staining decreased sharply in the developing alveoli around the time of birth (Fig. l),and neuraminidase digestion enhanced PNA staining. Similar results with PNA staining have been reported to occur in rats during the perinatal period (Honda et al., 1989). Our study has revealed that glycoconjugates modulate during airway development in fetal hamsters. These alterations in the expression of sugars relate to

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Ito, T., N. Nagahara, T. Ogawa, Y. Inayama, and M. Kanisawa 1985 Lectin binding to the luminal surface of distal airway epithelial cells of rodents. J . Electron Microsc., 34t381-388. Ito, T., C. Newkirk, J.M. Strum, and E.M. McDowell 1990 Modulation of glycogen stores in epithelial cells during airway development in Syrian golden hamsters. A histochemical study comparing concanavalin A binding with the periodic acid-Schiff reaction. J. Histochem. Cytochem., in press. Ivatt, R.J. 1984 Role of glycoproteins during early mammalian embryogenesis. In: The Biology of Glycoprotein. R.J. Ivatt, ed. PleACKNOWLEDGMENTS num Press, New York, pp. 95-181. P.K., and L.M. Reid 1977 Ultrastructure of airway epithelium This work was supported by National Institutes of Jeffery, and submucosal gland during development. In: Lung Biology in Health grant HL 37640. This is contribution No. 2774 Health and Disease, Vol. 6. Development of the Lung. W.A. Hodson, ed. Marcel Dekker, New York, pp. 87-134. from the Pathobiology Laboratory, University of MaryKimber, S.J. 1989 Changes in cell-surface glycoconjugates during emland School of Medicine. bryonic development demonstrated using lectins and other probes. Biochem. SOC. Trans., 17t23-27. LITERATURE CITED Kornfeld, R., and S. Kornfeld 1976 Comparative aspects of glycoprotein structure. Annu. Rev. Biochem., 45217-237. Andrews, P.M. 1974 A scanning electron microscopic study of the Kornfeld, R., and S.Kornfeld 1980 Structure of glycoproteins and extrapulmonary respiratory tract. Am. J. Anat., 139t399-424. their oligosaccharide units. In: The Biochemistry of GlycoproBarresi, G., G. Tuccari, and F. Arena 1987 Peanut lectin binding to teins and Proteoglycans. W.J. Lennarz, ed. Plenum Press, New the alveolar lining layer in hyaline membrane disease. Virchows York, pp. 1-34. Arch., 411r157-160. Laitinen, L., I. Virtanen, and L. Saxen 1987 Changes in the glycosyBecci, P.J., E.M. McDowell, and B.F. Trump 1978 The respiratory lation pattern during embryonic development of mouse kidney as epithelium. 11. Hamster trachea, bronchus, and bronchioles. J. revealed with lectin conjugates. J . Histochem. Cytochem., 35t55Natl. Cancer Inst., 61t551-561. 65. Breeze, R.G., and E.B. Wheeldon 1977 State of the art. The cells of the Mariassy, A.T., C.G. Plopper, and D.L. Dungworth 1975 Characterispulmonary airways. Am. Rev. Respir. Dis., 116t705-777. tics of bovine lung as observed by scanning electron microscopy. Caldero, J., E. Campo, X. Calomarde, and M. Torra 1988 Distribution Anat. Rec., 183t13-26. and changes of glycoconjugates in rat colonic mucosa during development. A histochemical study using lectins. Histochemistry, Mariassy, AT., C.G. Plopper, J.A. St. George, and D.W. Wilson 1988 Tracheobronchial epithelium of the sheep. IV. Lectin histochem90t261-270. ical characterization of secretory epithelial cells. Anat. Rec., 222: Christensen, T.G., L.J. Hornstra, and G.L. 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A quantitative morphologic study tochemistry. Lab. Invest., 57t5-20. in the fetus. Anat. Rec., 213:429-447. Etzler, M.E., and E.A. Kabat 1970 Purification and characterization McDowell, E.M., and B.F. Trump 1976 Histologic fixatives suitable of a lectin (plant hemagglutinin) with blood group A specificity for diagnostic light and electron microscopy. Arch. Pathol. Lab. from Dolichos bzflorus. Biochemistry, 9t869-877. Med., I00t405 -414. Faraggiana, T., D. Villari, J. Jagirdar, and J. Patil 1986 Expression of sialic acid Montreuil, J. 1980 Primary structure of glycoprotein glycans. Basis _ ~ . on the alveolar surface of adult and fetal human lungs. for the molecular biology of glycoproteins. Adv. Carbohydrate J. Histochem. Cytochem., 34t811-816. Chem. Biochem., 37t157-223. Flint, F.F., B.A. Schulte, and S.S. Spicer 1986 Glycoconjugate with Muramatsu, T. 1988 Developmentally regulated expression of cell terminal 01 galactose. A property common to basal cells and a subpopulation of columnar cells of numerous epithelia in mouse surface carbohydrates during mouse embryogenesis. J. Cell. Biochem., 36:l-14. and rat. Histochemistry, 84t387-395. Muresan, V., M.P. Sarras, Jr., and J.D. Jamieson 1982 Distribution of Geleff, S., P. Bock, and L. Stockinger 1986 Lectin-binding affinities of sialoglycoconjugates on acinar cells of the mammalian pancreas. the epithelium in the respiratory tract. A light microscopical J. Histochem. Cytochem., 3Ot947-955. study of ciliated epithelium in rat, guinea pig, and hamster. Acta Nakai, M., Y. Tatemoto, H. Mori, and M. Mori 1985 Lectin-binding Histochem., 78t83-95. patterns in the developing tooth. Histochemistry, 83t455-463. Goldstein, I.J., R.C. Hughes, M. Monsigny, T. Osawa, and N. Sharon Nemanic, M.K., J.S. Whitehead, and P.M. Elias 1983 Alterations in 1980 What should be called a lectin? Nature 285t66. membrane sugars during epidermal differentiation: Visualization Goldstein, I.J., and R.D. Poretz 1986 Isolation, physiocochemical charwith lectins and role of glycosidases. J. Histochem. Cytochem., acterization, and carbohydrate-binding specificity of lectins. In: 31t887-897. The Lectins. Properties, Functions, and Applications in Biology Pereira, M.E.A., E.A. Kabat, R. Lotan, and N. Sharon 1976 Immunoand Medicine. I.E. Liener, N. Sharon, and I.J. Goldstein, eds. chemical studies on the specificity of the peanut (Aruchis hyAcademic Press, Orlando, Florida, pp. 33-247. pogaea I agglutinin. Carbohydrate Res.. 51 :107-118. Hammarstriim, S. 1972 Snail ( H d i r pornntia) hemagglutinin. In: Pereira, M.E.A., E.C. Kisailus, F. Gruezo, and E.A. Kabat 1978 ImMethods in Enzymology. V. Ginsburg, ed. Academic Press, New munochemical studies on the combining site of the blood group York, Vol. 28, pp. 368-383. H-specific lectin 1 from Ulrx ruropeus seeds. Arch. Biochem. 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differentiation of the trachea and lung in general and to the establishment of cell lineages and cellular maturation in particular. Understanding the mechanisms and significance of these modulations will require further study of the glycosyltransferases required to form the sugars related to development and differentiation, and of the core proteins that carry them.

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binding lectins in embryos and adult tissues of the mouse. Differentiation, 30t211-219. Schulte, B.A.. and S.S. Spicer 1983 Light microscopic histochemical detection of sugar residues in secretory glycoproteins of rodent and human tracheal d a n d s with lectin-horseradish ueroxidse conjugates and the galactose oxidase-Schiff sequence'. J. Histochem. Cytochem., 31t391-403. Schulte, B.A., and S.S. Spicer 1985 Histochemical methods for characterizing secretory and cell surface sialoglycoconjugates.J. Histochem. Cytochem., 33:427-438. Sharon, N. 1983 Lectin receptors as lymphocyte surface markers. Adv. Immunol., 34t213-298. Shiojiri, N., and H. Katayama 1988 Development of Dol~chosbiflorus agglutinin (DBA) binding sites in the bile duct of the embryonic mouse liver. Anat. Embryol., 178t15-20. Sorokin, S.1965 Recent work on developing lungs. In: Organogenesis. R.L. De Haan and H. Ursprung, eds. Holt, Reinhart and Winston, New York, pp. 467-491.

Spicer, S.S.,D.A. Baron, A. Sato, and B.A. Schulte 1981 Variability of cell surface glycoconjugates-Relation to differences in cell function. J . Histochem. Cytochem., 29t994-1002. Spicer, S.S.,B.A. Schulte, and G.N. Thomopoulos 1983 Histochemical properties of the respiratory tract epithelium in different species. Am. Rev. Respir. Dis., I28tS20-526. Sturgess, J.M. 1985 Ontogeny of the bronchial mucosa. Am. Rev. Respir. Dis., 131tS4S7. Warren, L., and S.S. Spicer 1961 Biochemical and histochemical identification of sialic acid containing mucins of rodent vagina and salivary glands. J. Histochem. Cytochem., 9t400-408. Wasano, K., K.C. Kim, R.M. Niles, and J.S. Brody 1988 Membrane differentiation markers of airway epithelial secretory cells. J. Histochem. Cytochem., 36t167-178. Zieske, J.D., and LA. Bernstein 1982 Modification of cell surface glycoprotein: Addition of fucosyl residues during epidermal differentiation. J. Cell Biol., 95t626-631.