ABSTRACT
The formation of the nasal passages involves complex morphogenesis and their lining develops a spatially ordered pattern of differentiation, with distinct domains of olfactory and respiratory epithelium. Using antibodies to the neural cell adhesion molecule (N-CAM), keratan sulphate and heparan sulphate proteoglycan (HSPG) and a panel of lectins (agglutinins of Canavalia ensiformis (ConA), Dolichos biflorus (DBA), peanut (PNA), Ricinis communis (RCA1), soybean (SBA), Ulex europaeus (UEA1), and wheatgerm (WGA)), we have documented cell surface characteristics of each epithelial domain. Binding of antibodies to N-CAM and to keratan sulphate, and the lectins ConA, PNA, RCA1, SBA and WGA marks the olfactory epithelial domain only. The restriction of N-CAM to the sensory region of the epithelium has also been reported in the developing ear. This striking similarity is consistent with the idea that N-CAM may be involved in the division of functionally and histologically distinct cell groups within an epithelium.
We traced the olfactory-specific cell markers during development to gain insights into the origin of the epithelial lining of the nasal passages. AU reagents bind at early stages to the thickened nasal placode and surrounding head ectoderm and then become progressively restricted to the olfactory domain. The expression of these characteristics appears to be modulated during development rather than being cell autonomous.
The distribution of keratan sulphate was compared with collagen type II in relation to the specification of the chondrocranium. Keratan sulphate and collagen type II are only colocalized at the epithelial-mesenchymal interface during early nasal development. At later stages, only collagen type II is expressed at the interface throughout the nasal passages, whereas keratan sulphate is absent beneath the respiratory epithelium.
Introduction
The lining of the nose exhibits strikingly different epithelial domains, with the sensory olfactory epithelium in the deeper regions and the nonsensory respiratory epithelium more distally. The development of these two different types of tissue adjacent to each other poses questions about how they are specified and how the boundary between them is defined. This patterned epithelium develops following extensive morphogenesis in the region of the nasal placodes, which are thickenings in the head ectoderm. We wish to understand how the form of the nasal passages arises and, using chick embryos, experimentally determine the mechanisms that lead to epithelial patterning. As a first step towards this long-term goal, we used antibodies to several extracellular and cell surface molecules and a panel of lectins to characterize the epithelial domains in the chick nasal passages.
N-CAM is a cell adhesion molecule that shows interesting spatial and temporal patterns of expression both in early embryos and during organogenesis (reviewed by Edelman, 1985). In the adult, the olfactory epithelium of both mice (Miragall et al. 1988) and chickens (Chuong & Edelman, 1984) express N-CAM. During development of the ear, which like the nasal system arises from an ectodermal placode, N-CAM becomes restricted to the sensory regions of the auditory epithelium (Richardson et al. 1987). Lectins can also be used to investigate cell surface characteristics. Lectins bind to specific sugars, such as those that are part of membrane-bound glycolipids or of glycoproteins or proteoglycans. In a number of different animals including rodents (Hempstead & Morgan, 1983; Key & Giorgi, 1986b) and frogs (Chen et al. 1986; Key & Giorgi, 1986a), there is specific binding of lectins within the adult olfactory system. The role of proteoglycans in lectin binding can be evaluated using MZ15, an antibody to keratan sulphate (Smith & Watt, 1985; Zanetti et al. 1985) and an antibody to heparan sulphate proteoglycan (Bayne et al. 1984).
The distribution of MZ15 staining is also interesting in relation to the ‘Fly-paper model ‘(Thorogood et al. 1986; Thorogood, 1988). This model suggests that transient expression of cartilage-specific molecules at epithelial-mesenchymal interfaces in the embryonic head signals the development of the chondrocranium and sensory capsules. The monoclonal antibody, MZ15, recognizes keratan sulphate in both cartilagespecific and noncartilage-specific proteoglycans (Smith & Watt, 1985; Zanetti et al. 1985; Mehmet et al. 1986). In the chick otic vesicle, an early stage in ear development, there is some degree of colocalization of staining with MZ15 and cartilage-specific type II collagen antibodies (Thorogood, 1988; Heath & Thorogood, 1989). Therefore, we have extended our investigation to include a comparison of the distribution of keratan sulphate and collagen type II during nasal placode development.
Antibodies to N-CAM and keratan sulphate and the lectins ConA, PNA, RCA1, SBA and WGA al) recognize the olfactory epithelial domain. These reagents bind to the placode and then become progressively restricted during development.
Materials and methods
Antibodies and lectins
Anti-type-II collagen is an affinity-purified rabbit antibody, produced and kindly donated by Charles Archer (The Institute of Orthopaedics, Stanmore, Middlesex). MZ15 is a mouse monoclonal antibody raised against pig chondrocytes that has been shown to be specific for keratan sulphate (Smith & Watt, 1985; Zanetti et al. 1985; Mehmet et al. 1986), and was a kind gift from Fiona Watt (Imperial Cancer Research Fund, Lincoln ‘s Inn Fields, London). Anti-heparan sulphate is a monoclonal antibody to heparan sulphate proteoglycan (HSPG) raised against chick embryo leg muscle, from the Developmental Studies Hybridoma Bank, Maryland (NICHT) contract number: NO1-HD-6–2915). Biotinylated lectins were obtained from Vector Laboratories. Lectins may bind to any exposed sugar residues in the tissue, such as in glycoproteins, glycolipids or the glycosaminoglycan chains (GAGs) of proteoglycans. However, the density of sugars in such glycoproteins or glycolipids is far lower compared to that in GAGs, which consist of alternating sugar residues; therefore, one can surmise that the lectins are mainly binding to GAGs (Gallagher, 1986). The binding specificities of these lectins and the GAGs to which they are predicted to bind are shown in Table 1. The anti-sera to N-CAM is an Fab fragment of a polyclonal rabbit antibody to N-CAM, which was a generous gift to Mark Noble from Urs Rutishauser (Case Western Reserve School of Medicine, Cleveland, Ohio).
Preparation of tissue
Recently laid, fertilized chicken eggs were incubated in a humidified incubator at 38(±1)°C. Embryos were staged according to Hamburger & Hamilton (1951). Heads were removed in cold phosphate-buffered saline (PBS, 10 mm-sodium phosphate, 0·9% saline, pH7·5).
Tissue for staining with anti-type-II collagen, anti-HSPG and MZ15, was allowed to sink in 30% sucrose in PBS, embedded in precooled OCT compound (Lab-Tek Products) and frozen in liquid nitrogen. Blocks were cut immediately at –20°C on a Reichert-Jung cryostat. 3–10 μm frontal cryosections were placed on gelatin-coated slides and stored at –20°C until required for immunostaining within 48h. Tissue for N-CAM staining was prefixed in 2·5 % paraformaldehyde in PBS and then handled as above. Lectins were applied to both prefixed and nonfixed cryosections.
MZ15 was applied to wax sections for greater histological detail. The tissue was fixed in precooled 96% methanol overnight at 4°C, then dehydrated, cleared and embedded in low melting-point paraffin wax according to the method of Sainte-Marie (1962). Blocks were stored at 4°C until required for sectioning. 5–10 μm sections were placed on gelatin-coated slides, allowed to air dry and stored at 4°C prior to staining. Tissue for general histology was fixed overnight in freshly prepared half-strength Karnovsky ‘s fixative at 4 °C (Kamovsky, 1965). Specimens were rinsed in 0·1 M-cacodylate buffer, dehydrated and embedded in Araldite resin. 1μm sections were mounted on glass slides and stained with toluidine blue.
Staining methods
Anti-type-II collagen, anti-HSPG, MZ15 and anti-N-CAM antibodies were applied to cryosections and observed using fluorescein-isothiocyanate (FITC)-conjugated secondary antibodies. The Sainte-Marie-prepared wax sections were stained with MZ15 and visualized with FITC-conjugated secondary antibodies or the peroxidase anti-peroxidase system (PAP, Amersham).
The biotinylated lectins were applied to cryosections (Rittman & Mackenzie, 1983) and observed with the application of a biotin-streptavidin complex conjugated with the red fluorescent phycobiliprotein, Phycoerythrin (PE-SA, Amersham). Lectins are metalloproteins, therefore PBS was replaced with Tris-buffered saline (TBS, 100mm-Trizma base (Sigma), 100 mm-sodium acetate, pH 7·3) to maintain the correct balance of ions necessary for efficient saccharide binding.
(a) Cryosections
Sections were moistened with PBS (N-CAM) or TBS (other) in a humidified air chamber. To prevent masking by other matrix components, sections to be treated with MZ15 or antibodies to collagen type II and HSPG were pretreated with chondroitinase (0·25 i.u. ml-1, Sigma), and Streptomyces hyaluronidase (1·45 i.u. ml-1, Sigma), for 30min, then washed (PBS/TBS, twice 10min). Similarly, half of the sections to be stained with lectins were pretreated with neuraminadase to remove sialic acid, in order to assess the extent of masking by such components.
Primary solutions were applied for 45–60 min (anti-type-II collagen 1:20, anti-HSPG hybridoma supernatant 1:1000 and MZ15 1:1000 in TBS/0·1% BSA/0·01% azide; lectins 10 μg ml-1 in 10mm-Hepes, 0·15m-NaCl, 0·1mm-Ca2+, 0·04% azide, pH7·5) or overnight (anti-N-CAM 1:1000 in PBS/0·1% BSA/0×01% azide) then washed (PBS/TBS, twice 10 min).
Secondary solutions were incubated on the sections for 30 min (FITC-conjugated swine anti-rabbit IgG at 1:50 in TBS/0·1% BSA/0·01% azide for anti-type-II collagen; and FITC-conjugated rabbit anti-mouse IgG at 1:50 in TBS/0·1 % BSA/0·01% azide for anti-HSPG and MZ15; FITC-conjugated swine anti-rabbit IgG at 1:50 in PBS/0·1% azide for anti-N-CAM; and PE-SA for lectins.
Sections were given a final rinse (PBS/TBS, twice 10min) and mounted in glycerol/PBS (1:9 v/v), containing DABCO (Sigma), to preserve the fluorescence during subsequent microscopy (Johnson et al. 1982). Photographs were taken on a Zeiss photomicroscope with an epifluorescence attachment using HP5 35 mm film (Ilford).
Controls were processed either without the primary solution or with the relevant non-immune serum. The control sections were enzyme pre treated where applicable, for example control sections for lectins were also pretreated with neuraminidase and control sections for MZ15 or collagen type-II were pretreated with chondroitinase and hyaluronidase.
(b) Paraffin wax sections
Sections were dewaxed, hydrated and stained as above with MZ15 and with FITC-conjugated secondary antibodies. Controls were as for cryosections.
Results
The staining patterns produced by antibodies to collagen type II, keratan sulphate, N-CAM and the lectin binding in the nasal placodes and passages are summarized in Table 2 and details indicated in Tables 3, 4, 5. The lectin-binding specificities and the glycosaminoglycans that contain these sugar residues are shown in Table 1.
Characteristics of epithelial domains within the nasal passages
The binding patterns of several antibodies and lectins distinguish between the olfactory epithelium and respiratory epithelium. Antibodies to N-CAM and keratan sulphate, and the lectins ConA, PNA, RCA1, SBA and WGA all specifically bind to regions of the olfactory epithelium (Table 2). Fig. 1 shows their distribution in the nasal passages of the chick embryo at 10 days of development. The olfactory epithelium covers the superior concha, the roof of the nasal cavity and adjacent nasal septum; and the respiratory epithelium lines the remainder, which includes both the middle and the more distal vestibular conchae.
N-CAM expression is confined to the olfactory epithelium and adjacent olfactory nerve bundles (Fig. 2A,B,E). In comparison, the respiratory epithelium, which covers the middle concha, is negative for N-CAM staining (Fig. 2C,D) and this nonreactivity of the respiratory epithelium is made all the more striking by the expression of N-CAM in the surrounding mesenchyme (Fig. 2C,D). There is an abrupt limit in N-CAM expression corresponding to the histological boundary between the olfactory and respiratory epithelial types.
In addition to the striking localization of N-CAM, the lectins ConA, PNA, RCA1, SBA and WGA all bind to the surface of the olfactory epithelium but not to the respiratory epithelium. Fixation enhances the binding of ConA (Fig. 3B vs. 3A), RCA1 (Fig. 3E), SBA (Fig. 3G vs. 3H) and WGA (Fig. 3F). UEA1 does not bind significantly under any conditions (Fig. 3C,D). The binding of ConA, RCA1 and WGA to the surface of the olfactory epithelium in fixed tissue is irrespective of neuraminidase pretreatment. In contrast, SBA only binds to the olfactory epithelium of fixed tissue that has been enzyme pretreated (Fig. 3H vs. 3I).
Lectins not only bind to the cell surface but also bind to the basement membrane zone. Both DBA and RCA1 bind to the basement membrane zone under both the olfactory and respiratory epithelia in unfixed material (Fig. 3E,J), and PNA binds to the basement membrane zones of both olfactory and respiratory epithelia in the absence of neuraminidase pretreatment (Fig. 3K,L,M). RCA1 and PNA are predicted to bind to the sugars present in heparan sulphate (Table 1), and an antibody to heparan sulphate proteoglycan (HSPG) stains the basement membrane zone under both nasal epithelia (not shown).
The olfactory epithelium, which is marked by the binding patterns of PNA, RCA1, SBA and WGA, is also positively stained by the antibody MZ15, which recognizes keratan sulphate. Therefore, these lectins may be recognizing keratan-sulphate-containing proteoglycans (see Table 1) in the olfactory epithelium. MZ15 stains only the thickened olfactory epithelium and its continuation with the lining of the maxillary sinus (Fig. 4D). The respiratory epithelium, lining the middle concha and lower nasal septum, does not significantly stain with MZ15 (Fig. 4D). As the nasal epithelium changes from the thick olfactory epithelium to thinner respiratory epithelium, there is an abrupt decrease in MZ15 staining (Fig. 4E). It is the surface of the olfactory epithelium that exhibits the strongest staining, with weaker staining within the olfactory epithelium itself (Fig. 4D,E). MZ15 also stains the nasal capsule cartilage (Fig. 4E).
Temporal and spatial changes in epithelial characteristics during development
The stages of chick nasal development have been well documented (Bancroft & Bellairs, 1977; Yee & Abbott, 1978; Will & Meller, 1981; Tamarin et al. 1984) and are summarized in Fig. 5. The olfactory placodes first become visible after 2–2·5 days of development, as shallow depressions on either side of the presumptive face (Fig. 5A,B). With further development the nasal pits invaginate to form shallow grooves (Fig. 5C,D), with the thickened epithelium becoming localized distally. The nasal cavities become further elaborated by outgrowths, or conchae, from the lateral wall of the nasal passages (Fig. 5E,F,G,H). In the definitive nasal passages, the olfactory epithelium lines the superior concha, the respiratory epithelium covers the middle concha (Fig. 5G,H).
All the markers of olfactory cells are found spread throughout the ectoderm of the placode and surrounding head of the early embryo but become restricted to the olfactory epithelium during development. For example, in the early embryo, stage 15, N-CAM is present in the thickened placodal epithelium and general head ectoderm (Fig. 6A,B), but it is confined to the olfactory epithelium in the mature nasal passage (Fig. 2A,B,E). In the early embryo, N-CAM is also expressed within a cell mass in the mesenchyme immediately beneath the nasal placode (Fig. 6A). This cell mass may represent epithelioid cells which are known to migrate out of the nasal placode towards the telencephalon (Robecchi, 1972; Cushieri & Bannister, 1975a,b; and Mendoza et al. 1982). As the nasal placode invaginates to form the nasal pit, stages 24–28, N-CAM staining becomes localized to the deeper regions of the pit within the presumptive olfactory epithelium and is lost from the presumptive respiratory epithelium.
The sugar residues to which the lectins bind are not only present on the olfactory epithelium, but are also found on the nasal placode and head ectoderm (Table 5). The lectins ConA (Fig. 7A,B), DBA (Fig. 7C,D), SBA (Fig. 7E,F), RCA1 (Fig. 7G,H), WGA (Fig. 7J) and PNA (Fig. 7K,L), all bind to the placodal epithelium, particularly at the surface and basement membrane zone, but the lectin UEA1 does not give significant staining of the nasal placode and adjacent head structures at this stage of development (Fig. 7I). Fixation enhances the binding of the lectins ConA (Fig. 7B,vs. 7A), DBA (Fig. 7D vs. 7C), SBA (Fig. 7F,vs. 7E) and RCA1 (Fig. 7H vs. 7G) to the basement membrane zone underlying the placode and adjacent head ectoderm. None of the binding patterns produced by these lectins are significantly altered by pretreatment of the cryosections with neuraminidase, except for PNA, which has a bold pattern of binding to the basement membrane zone under both the placode and telencephalon (Fig. 7K) and loses this striking localization after neuraminidase pretreatment (Fig. 7L).
The lectins PNA, RCA1, SBA and WGA bind to sugars contained in keratan sulphate proteoglycan (Table 1). Consistent with the lectin-binding patterns, keratan sulphate is present from the earliest stages of nasal development during placode formation. At stage 17, keratan sulphate is present in the placode and head ectoderm, particularly at the surface and basement membrane zone (Fig. 4A,B). During subsequent development, the presumptive olfactory ‘epithelium becomes localized to the upper and deeper regions of the nasal cavities, and keratan sulphate staining becomes progressively restricted to this thickened sensory epithelium (Fig. 4C). First, there remains some surface staining over the middle concha, which is lined by respiratory epithelium (Fig. 4C), but no staining is present within this epithelium itself. Keratan sulphate, at this stage, is also detected in the cartilage of the nasal septum (Fig. 4C, arrows). Then keratan sulphate is further restricted to the surface of the olfactory epithelium only and the respiratory epithelium does not exhibit significant MZ15 staining (Fig. 4D). Keratan sulphate is distributed particularly along the surface of the olfactory epithelium (Fig. 4A), but there is also some staining within the epithelium itself. In the stage 17 embryo, anti-HSPG stains only the basement membrane zone of the general head ectoderm, placode and telencephalon (not shown).
Comparison of collagen type II and keratan sulphate expression during nasal development
Collagen type II is present at the epithelialmesenchymal interface of the nasal placode and continues to be expressed throughout nasal development up to the time when overt chondrogenesis takes place within the perinasal mesenchyme. Therefore there is no regional-specific pattern of collagen type II expression within the chick nasal passages. Collagen type II can be detected beneath the nasal placode (Fig. 8A), nasal pit (Fig. 8B), and whole nasal cavity (Fig. 8C,D,E,F). In the early embryo, stage 17, there is also positive staining of type-II collagen at the base of both the general head ectoderm (Fig. 8A), and telencephalon. This contrasts with the expression of keratan sulphate at the epithelial-mesenchymal interface, which, although pronounced at early stages can only be detected in olfactory regions later in development (Fig. 4D,E).
Discussion
This survey of extracellular matrix and cell surface molecules has identified a number of markers for the domains of olfactory epithelium in the chick nasal passages. Antibodies to N-CAM and keratan sulphate, plus a number of lectins all bind to parts of the olfactory epithelium but not to the respiratory epithelial domain, and their binding abruptly stops at the boundary between these two epithelial cell types. The olfactory markers are present in the nasal placode and become progressively restricted to the olfactory domain as development proceeds. A comparison of the distribution of keratan sulphate and collagen type II shows that there is colocalization at early stages in nasal development but unlike keratan sulphate, collagen type II is not restricted to the olfactory domain at later stages of development.
Epithelial domains in the nasal passages
The olfactory epithelial domain of the nasal lining can be distinguished from the respiratory domain by the binding patterns of a number of antibodies and lectins.
Antibodies to N-CAM and keratan sulphate, plus the lectins ConA, PNA, RCA1, SBA and WGA all characterize the olfactory domain. There is an abrupt change in epithelial character, as recognized by these antibodies and lectins, at the junction between olfactory and respiratory epithelia. The localization of N-CAM expression to the sensory olfactory epithelium of the nasal lining shows a striking similarity to the regionalization of N-CAM (Richardson et al. 1987) and A-CAM (Raphael et al. 1988) in the developing ear. In the ear,. the expression of N-CAM becomes localized to the ventral regions of the otocyst and invests only the sensory regions. The striking boundary in N-CAM expression at the junction between two distinct domains in both nasal and aural epithelia supports the idea that N-CAM may be involved in the production and maintenance of boundaries between histologically and functionally distinct cell types within an epithelium. The maintenance of such boundaries is of particular importance since human tumours, for example in the cervix, appear to arise at junctions between distinct epithelial domains (Richart, 1966).
In addition to the restriction of N-CAM expression to the olfactory epithelial domain, MZ15, an antibody to keratan sulphate also shows restricted binding to the olfactory epithelial domain of the nasal lining. The pattern of lectin binding shows that the olfactory domain is recognized by ConA, PNA, RCA1, SBA and WGA. From their binding specificities (Table 1), all of these lectins except ConA are predicted to recognize some of the sugar components that are contained in the glycosaminoglycan (GAG) keratan sulphate. Therefore, this could provide an explanation for the similarity of the binding patterns of PNA, RCA1, SBA and WGA compared with the antibody MZ15. However, because of the binding affinities of PNA and RCA1, we cannot discount the possibility that chondroitin and dermatan sulphates are also present. ConA, which is not predicted to recognize keratan sulphate, also binds specifically to the olfactory domain. ConA is predicted to bind to hyaluronate and the binding of WGA, which also recognizes sugars present in hyaluronate, is consistent with the presence of hyaluronate in the olfactory domain.
Previous investigations have shown that the olfactory epithelium in Xenopus specifically binds SBA, whereas PNA and WGA bind to all neurones of the central nervous system (Key & Giorgi, 1986a). In the adult rat and mouse, the olfactory epithelium has been shown to specifically bind ConA, PNA and SBA (Hempstead & Morgan, 1983; Key & Giorgi, 1986b). ConA binds particularly to the central layer of the olfactory epithelium (Rat: Hempstead & Morgan, 1983; Frog: Chen et al. 1986), which corresponds to the olfactory cells themselves. Here, ConA binds only to the surface epithelial cells of the olfactory domain although the reason for this difference in the embryo is not clear.
Tracing changes in epithelial characteristics to understand the origin of domains in the nasal passages
The development of the olfactory-specific characteristics during morphogenesis of the nasal placode may give clues to the origin of this domain. The reagents anti-N-CAM, MZ15 and ConA, PNA, RCA1, SBA and WGA all characterize the olfactory epithelium in the definitive nasal passages. When we investigated how their distribution changes during development, we found that all of these reagents bind to the nasal placode and then become progressively restricted to the olfactory domain. This pattern is consistent with the idea that the olfactory placode gives rise to the olfactory domain of the epithelium of the nasal passages. If this is the case, what is the origin of the respiratory epithelium? One possibility is that the respiratory epithelium is derived from the adjacent head ectoderm (Street, 1937; Romanoff, 1962). According to this idea, the ectoderm would be drawn into the developing nasal pits as the facial processes enlarge. However, the head ectoderm also binds anti-N-CAM, MZ15 and all of the lectins investigated except UEA1, and yet the respiratory epithelium which develops later is essentially negative for all of these reagents. Therefore, the characteristic patterns of antibody staining and lectin binding do not appear to be cell autonomous and cannot be used to trace the origin of cells. Instead, the changes in the expression of cell surface characteristics appear to be the result of modulation of expression during development. To further investigate the origins of these epithelial domains we require markers for the respiratory epithelium.
Functional significance of cell surface changes during nasal morphogenesis
Cell surface characteristics may be important in maintaining epithelial shape during folding of the nasal epithelia. It is striking that N-CAM and the cell surface glycoconjugates recognized by specific lectins are present in the early nasal placode and, only after extensive morphogenesis, become restricted to the olfactory epithelium. In mammalian nasal development, it has been found that the lectin ConA binds to the nasal placode, and later particularly to the prospective fusion zones of the nasal folds (Gaare & Langman, 1977; Smuts, 1977 and Burk et al. 1979). However, the distribution of the cell surface glycoconjugates found here suggests that they are not solely involved in regions of epithelial fusion and must play some other role.
Extracellular matrix components at the epithelial-mesenchymal interface of the developing nasal epithelium and chondrogenic patterning in the head
The spatial and temporal expression of cartilage-specific extracellular matrix components described here are relevant to the ‘Fly-paper model’ (Thorogood et al. 1986; Thorogood, 1988), which proposes that cranial epithelia (the brain and head ectoderm) act as templates to specify the patterning of chondrogenic differentiation of the neural crest. According to this model, the transient expression of cartilage-specific molecules, in particular collagen type II, at the epithelialmesenchymal interface acts as an autocatalytic signal to define where neural crest cells will arrest and subsequently differentiate into cartilage.
The distribution of collagen type II correlates spatially with the developing olfactory capsule. Collagen type II can be detected beneath both the olfactory placode and head ectoderm of the early embryo and continues to be associated with the nasal epithelium during development. Such a distribution of collagen type II beneath the chick nasal pit has recently been confirmed (Fitch et al. 1989). At later stages, collagen type II is found in the basement membrane zone throughout the nasal passages and therefore is associated with both the olfactory and respiratory epithelia. This spatial distribution correlates with the differentiation of the nasal capsule cartilage, which forms a discrete band that follows the contours of the nasal epithelia. The presence of collagen type II at epithelialmesenchymal interface of the developing nose is in agreement with its proposed action as a signal that triggers cartilage differentiation to form the nasal capsule, although the timing and duration of specification of perinasal cartilage differentiation is not known.
During chondrogenesis, cartilage-specific matrix components are coordinately expressed (Von der Mark et al. 1976; Oettinger et al. 1985, and Smith & Watt, 1985). Keratan sulphate is a GAG that is present in both cartilage-specific and noncartilage-specific proteoglycans. The monoclonal antibody, MZ15, recognizes keratan sulphate (Smith & Watt, 1985; Zanetti et al. 1985; Mehmet et al. 1986). In the chick otic vesicle, there is some degree of colocalization of staining with MZ15 and cartilage-specific collagen type II (Thorogood, 1988; Heath & Thorogood, 1989). Here we have found that in the developing nasal system keratan sulphate does not strictly colocalize with collagen type II. Since the MZ15 antibody recognizes keratan sulphate both in cartilage- and noncartilage-specific proteoglycans, the distribution of keratan sulphate can only be suggestive. For example, the MZ15-positive cell-associated staining probably represents noncartilage-specific proteoglycans (see also Heath & Thorogood, 1989); however, the extracellular staining probably represents cartilage-specific proteoglycans. Therefore, the colocalization of staining with MZ15 and collagen type II antibodies at the interface between the olfactory placode and underlying mesenchyme during early nasal development suggests that cartilage-specific molecules are coordinately expressed at this stage. However, as development proceeds, collagen type II is found beneath the respiratory epithelium, whereas keratan sulphate is absent here. Therefore, it seems unlikely that coordinate expression of these cartilage-specific molecules is required to signal the formation of the nasal capsule cartilage.
Conclusions
The results provide us with several markers to distinguish between respiratory and olfactory epithelial domains in the chick embryo. Antibodies to N-CAM and keratan sulphate, and the lectins ConA, PNA, RCA1, SBA and WGA specifically bind to the olfactory epithelium. Studies on the expression of a range of intermediate filament proteins have identified markers for the respiratory epithelial domain of the nasal lining (Croucher & Lane, unpublished data). We intend to use these markers to explore the mechanisms that determine epithelial patterning during development.
ACKNOWLEDGEMENTS
We wish to thank Dr Tony Leathern (Histopathology, The Middlesex Hospital) for advice on the use of lectins for histochemistry; Professor Lewis Wolpert and Dr Jim Ralphs for reading and commenting on the manuscript. We are indebted to Dr Mark Noble for his help and advice in the preparation of the manuscript. We also thank Lindsay Heath and Dr Peter Thorogood for sending us a copy of their manuscript. S.J.C. is supported by a Medical Research Council Studentship, and the MRC also funds the research of C.T.