Matrix Vol. 1111991, pp. 412-427 © 1991 by Gustav Fischer Verlag, Stuttgart

Generation of a Monoclonal Antibody Against Avian Small Dermatan Sulfate Proteoglycan: Immunolocalization and Tissue Distribution of PG-II (Decorin) in Embryonic Tissues DONALD P. LENNON, DAVID A. CARRINO, MARILYN A. BABER and ARNOLD I. CAPLAN Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

Abstract

Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small derma tan sulfate proteoglycans and small heparan sulfate proteoglycans. A monoclonal antibody has been raised which recognizes the small derma tan sulfate proteoglycan. Immunblot analysis of a partially purified preparation of skeletal muscle proteoglycans indicates that the antibody reacts with a molecule which migrates with an estimated Mr of 100000. Prior treatment of the proteoglycans with chondroitinase results in immunostaining of a species of estimated Mr 45000. These values for the intact proteoglycan and its core protein suggest that the antibody is directed against a proteoglycan of the PG-II or decorin class. Immunohistochemistry indicates a widespread distribution of the proteoglycan, which is localized in connective tissue septa of skeletal and cardiac muscle, dermis, tendon, bone, perichondrium and cornea. Immunoblot analysis of the proteoglycan core proteins from these tissues demonstrates that the antibody recognizes the same 45 OOO-dalton band in each tissue. The widespread tissue distribution is also consistent with the antibody being directed against an epitope of PG-II. Neither the glycosaminoglycan chains nor N-linked oligosaccharides are required for reactivity and the antibody cross-reacts with other avian material, but not mammalian. This antibody, which has been designated CB-1, reveals developmental stage-specific changes in the deposition of PG-II in embryonic limb bud and skeletal muscle. Key words: chick embryonic skeletal muscle, chondroitin sulfate proteoglycans, derma tan sulfate proteoglycans, heparan sulfate proteoglycans. Introduction

Small derma tan sulfate proteoglycans have been identified and characterized within a broad range of tissues, including sclera (Coster and Fransson, 1981), tendon (Vogel and Heinegard, 1985), skin (Fujii and Nagai, 1981; Damle et aI., 1982), cartilage (Rosenberg et aI., 1985; Roughley and White, 1989), smooth muscle (Chang et aI., 1983; Rauch et aI., 1986), ovarian granulosa cells (Yanagishita and Hascall, 1983), endothelial cells (Kinsella

and Wight, 1988), cornea (Midura and Hascall, 1989), fetal membranes (Brennan et aI., 1984) and temporomandibular joint disc (Scott et aI., 1989). One class of these proteoglycans consists of a core protein with a Mr of approximately 45 000 and a single dermatan sulfate chain (Choi et al.; 1986) attached to the serine at residue 4 of the core protein (Chopra et aI., 1985). The core proteins of these proteoglycans have much structural similarity (Heinegard et aI., 1985; Vogel and Fisher, 1986) and are thought to represent the product of a single gene (Krusius

Avian PG-II (Decorin) in Embryonic Tissues and Ruoslahti, 1986). However, the dermatan sulfate of these proteoglycans shows subtle tissue-specific variations, such as differences in the length of the derma tan sulfate chain and in the content of iduronic acid (Larjava et aI., 1986; Rauch et aI., 1986). Bone contains a small proteoglycan with apparently the same core protein (Day et aI., 1987), but with chondroitin sulfate rather than derma tan sulfate as the attached glycosaminoglycan (Carrino and Caplan, 1985; Fisher et aI., 1983; Sato et aI., 1985). Proteoglycans of this type have been designated DSPG-II (or PG-II) because of the existence of another class of small dermatan sulfate proteoglycan, DSPG-I or PG-I (Rosenberg et aI., 1985), which has a distinct core protein (Fisher et aI., 1989; Neame et aI., 1989). PG-I is larger than PG-II with an estimated Mr of 165000-285000 compared to 87900-120000 (Rosenberg et aI., 1985), although the molecular weights of their core proteins are similar (Fisher et aI., 1989; Neame et aI., 1989). A further difference between these molecules is the presence of two glycosaminoglycans in PG-I which has led to its being termed biglycan (Fisher et aI., 1989; Neame et aI., 1989). PG-I has the ability to self-associate (Rosenberg et aI., 1985) but, unlike several large proteoglycans, neither of the small proteoglycans can interact with hyaluronic acid (Damle et aI., 1982; Fransson et aI., 1982). PG-II, but not PG-I, has been shown to bind to collagen fibers (Brown and Vogel, 1989) and thereby inhibit in vitro collagen fibrillogenesis (Brown and Vogel, 1989; Vogel et aI., 1984; Vogel and Trotter, 1987). The binding to collagen has been localized to the d and e bands of the fiber (Scott and Haigh, 1985; Pringle and Dodd, 1990), which is different than the site of binding of the small corneal keratan sulfate proteoglycan (Scott and Haigh, 1985). Because of its ability to associate with collagen fibers, PG-II has also been called decorin (Ruoslahti, 1988). The binding to collagen is mediated by the core protein with no requirement for the dermatan sulfate of the proteoglycan (Vogel et aI., 1984; 1987). Proteoglycans synthesized by skeletal muscle and its associated connective tissues have been characterized by this laboratory (Carrino and Caplan, 1982; 1984; 1989; Young et aI., 1988). In the analysis of the proteoglycans synthesized by muscle-associated fibrogenic cells, dermatan sulfate proteoglycans were found to represent a significant component (Carrino and Caplan, 1982; Young et aI., 1988). Indeed, a small dermatan sulfate proteoglycan with size characteristics similar to those produced in other tissues represents a major component of the proteoglycans synthesized in normal mature skeletal muscle tissue (Carrino et aI., 1988), although whether this proteoglycan is produced by the muscle cells themselves or by the associated fibrogenic cells was not determined. In the course of preparing monoclonal antibodies to chick embryonic skeletal muscle proteoglycans, we generated a hybridoma which reacts with a proteoglycan with characteristics of PG-II. While the antibody can recognize the intact proteo-

413

glycan, the epitope appears to reside on the core protein. The proteoglycan recognized by this antibody, which has been designated CB-1, is present in numerous tissues. In developing skeletal muscle, immunolocalization with CB-1 indicates that the proteoglycan is deposited, at least initially, by the fibrogenic cells, which suggests that the skeletal muscle cells themselves are not responsible for the deposition of PG-II in this tissue.

Materials and Methods Materials

Ultrapure guanidinium chloride, cesium chloride and urea were purchased from Schwarz/Mann. Carrier-free 35 [ Slsulfate and chondroitinase ABC and AC II were obtained from ICN. Protease inhibitors for proteoglycan isolation and enzymatic partial deglycosylation were from Aldrich Chemical Co. or Sigma Chemical Co. Resins for column chromatography were from Pharmacia LKB Biotechnology. Reagents for electrophoresis were purchased from Schwarz/Mann or Bio-Rad. Immobilon membrane was obtained from Millipore and nitrocellulose (0.45-[1m pore size) from Bio-Rad. Mice were purchased from Jackson Laboratories, Bar Harbor, ME. Tissue culture reagents obtained from Gibco included fetal bovine serum (qualified), powdered media, L-glutamine, 8-azaguanine, and complete and incomplete Freund's adjuvants. Gentamycin and NCTC-109 hyridoma growth supplement were procured from M. A. Whittaker; HAT and HT concentrates and polyethylene glycol 1500 were from Boehringer Mannheim. Plasticware used in tissue culture was obtained from Falcon or Costar. Chemicals, including oxaloacetic acid, insulin, sodium pyruvate, alkaline phosphatase substrate for immunohistochemistry and pristane were purchased from Sigma Chemical Co.; pphenylenediamine was from Kodak. The antibody isotyping kit was from Amersham and Tissue-Tek O. C. T. compound was from Miles Laboratories. Secondary antibodies for immunohistochemistry and ELISA (fluorescein isothiocyanate-conjugated goat antimouse and alkaline phosphatase-conjugated goat antimouse immunoglobulins) were obtained from the Organon Teknika Corporation. Secondary antibody for dot-blots and immunoblots and the substrates for color development were from Promega and were purchased from Fisher Scientific. Unlabeled goat anti-mouse immunoglobulin was from Fisher Scientific, Orangeburg, NY. Human articular cartilage dermatan sulfate proteoglycans I and II were generously provided by Dr. Peter J. Roughley, McGill University, Montreal, Quebec, Canada and bovine cartilage dermatan sulfate proteoglycan I and bovine skin derma tan sulfate proteoglycan II were the kind gifts of Dr. Lawrence C. Rosenberg, Shriners Hospital for Crippled Children, Tampa, FL, USA. Antibody MY -17 4 was graciously pro-

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vided by Dr. Koji Kimata, Aichi Medical University, Aichi, Japan and antibody II6 B3 (15A 4 ) was generously contributed by Dr. Thomas Linsenmayer, Tufts University School of Medicine, Boston, MA.

Preparation of the immunogen Leg muscle was dissected from approximately 15-dozen stage 40 chick embryos, frozen in dry ice-ethanol and stored at - 70°C. This tissue was supplemented with similar material dissected from 8 embryos that were first labeled for 6 h with [35 S]sulfate (100 !lCi per embryo) as previously performed (Carrino and Caplan, 1984). Subsequently, the tissue was extracted by stirring overnight at 4°C in 4 M guanidinium chloride containing 0.5% 3-([3-cholamidopropyl] dimethylammonio)-2-hydroxy-1-propane-sulfonate (CHAPS), protease inhibitors and sodium acetate, pH5.8 (Carrino et aI., 1988). For each extraction, tissue from

CSase:

approximately one-third of the embryos was extracted in a volume of 60 ml and then clarified by centrifugation. The proteoglycans were isolated by transfer of the macromolecular material into a solution consisting of 8 M urea, 0.15 M NaCl, 0.5% CHAPS, 0.05 M sodium acetate, pH 7.0; this was effected by chromatography of the extract as small aliquots on disposable columns of Sephadex G-50 (Chang et aI., 1983). The sample was then applied to a 6-ml (15 X 0.7 em) column of diethylaminoethyl (DEAE)Sephacel and the proteoglycans eluted with a linear 0.25 M -0.6 M gradient of NaCl in the same urea solution described above (Carrino et aI., 1988). Effluent fractions of 0.45 ml were collected and aliquots removed for scintillation spectrometry. Appropriate fractions were pooled, dialyzed against distilled water at 4°C and lyophilized to dryness. For immunization, a weighed aliquot was dissolved at 1 mg/ml in 0.1 M Tris, 0.1 M sodium acetate, pH 7.0 containing protease inhibitors (Haynesworth et aI., 1987) and chondroitinase AC II was added from a stock solution of 20 units/ml to a final concentration of 0.2 unit/mg of

+

CSaS8

AC ABC

kD

-

kO

97~ 66~

97~ 66~

43~

43~

31~

31~

22~

22~

Fig. 1. Effect of chondroitinase on the mobility of the molecule recognized by antibody CB-l. Equal aliquots of day 14 leg muscle proteoglycans were electrophoresed on a gradient gel either without or after prior treatment with chondroitinase AC II and then electrotransferred and immunostained with monoclonal antibody CB-l. The mobilities of molecular weight markers are shown to the left of the immunoblot (kD, kilodaltons; CSase, chondroitinase). Other experimental details are in Materials and Methods.

Fig. 2. Comparison of the effect of chondroitinase ABC and AC II on the mobility of the molecule recognized by antibody CB-l. Equal aliquots of the proteoglycan preparation from day 14 embryonic leg muscle were treated with either chondroitinase ABC or AC II, electrophoresed on a gradient gel, electrotransferred and immunostained with monoclonal antibody CB-l. Other details are in the legend for Fig. 1 and in Materials and Methods.

Avian PG-II (Decorin) in Embryonic Tissues

kD

C8-1 286 c M c M

415

E ~

U Q)

U

Q)

E

kD

66~ 97~ 66~ 43~

Fig. 3. Immunoblot of intact muscle and low buoyant density cartilage protoeglycans. The proteoglycans isolated by anionexchange chromatography from day 14 embryonic leg muscle and in the low buoyant density region of a CsCI gradient from hatchling epiphysis were electrophoresed on a gradient polyacrylamide gel and electrotransferred. Half of the immunoblot was treated with chondroitinase ABC to generate the 2B6 epitope (Caterson et aI., 1987) and then immunostained with this antibody. The other half of the immunoblot was immunostained with CB-l. The arrowheads to the right of the immunoblot indicate the bands stained by 2B6. Other details are in the legend for Fig. 1 and in Materials and Methods.

lyophilized material. After incubation at 37 DC for 1 h, the sample was split into 100 [11 aliquots, frozen in dry iceethanol and stored at - 70 DC. Aliquots were thawed as needed for immunization and for assay of antibody; thawed samples were stored at 4 DC, if necessary. The samples extracted from other tissues (stage 37 and 43 leg and pectoral muscle and stage 43 heart, skin, tendon and tibial diaphysis) were prepared in the same way, except that instead of a linear NaCl gradient, the material was stepeluted from a 2-ml DEAE-Sephacel column with 10 ml of 0.25 M NaCI in the urea solution, 10 ml of 1.0 M NaCl in the urea solution and 10 ml of 8 M guanidinium chloride; approximately 70% of the incorporated [35 Sl sulfate was recovered in the 1.0 M NaCI wash, with most of the remainder in the unbound fraction. For these samples, tissues were obtained from approximately 60 stage 37 and 15 stage 43 embryos and extracted in a volume of 20 ml.

31~

22~

Fig. 4. Immunoblot of muscle cell culture proteoglycans with antibody CB-l. Proteoglycans were isolated by ion-exchange chromatography from the cell layer and medium of day 3 chick leg muscle cell cultures. These samples as well as proteoglycans isolated from day 14 chick embryonic leg muscle were treated with chondroitinase AC II, electrophoresed on a gradient gel, electrotransferred and immunostained with monoclonal antibody CB-1. Other details are in the legend for Fig. 1 and in Materials and Methods.

Immunization and fusion Generation of antibody CB-l was initiated by intraperitoneal injection of the immunogen (100 [1g prior to chondroitinase digestion) mixed with an equal volume of complete Freund's adjuvant into a female CB6FlI] mouse. A second injection of the antigen mixed with incomplete Freund's adjuvant was administered seven days after the initial inoculation and additional injections of this antigen in PBS were given 15 and 22 days after the first injection. Four days after the final injection, blood was collected from the tail vein. Serum derived from this blood indicated a high titer of antibody to the immunogen in an ELISA; three days thereafter the mouse was sacrificed according to NIH guidelines for animal welfare.

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Table I. Comparison of the localization of decorin antibodies in chicken and human tissues. Tissue

Chicken

Human a

Skin Skeletal Muscle

Dermal Matrix Connective Tissue Sheaths Sclera, Cornea Connective Tissue Sheaths Perisinusoidal Connective Tissue Fibers parallel and oblique to long axis of tendon, tendon sheath Not examined, but reactivity observed in adventitia of large arteries Interstitial Connective Tissue Serosa, Muscle Sheaths, Connective Tissue Core of Villi Septa between tertiary bronchi, septa between respiratory atria Pericardium, fibers within myocardium, endocardium

Dermal Matrix Connective Tissue Sheaths Sclera, Cornea Connective Tissue Sheaths Perisinusoidal Connective Tissue

Eye

Peripheral Nerves Liver Tendon Aorta Kidney Small Intestine Lung Heart a

+

Adventitia, Media Interstitial Connective Tissue Not Reported "Large" and "Small" Interstitium Subpericardium

Bianco et al. (1990)

Cells liberated from the spleen were fused with SP 2/0 Ag14 mouse myeloma cells in a 3: 1 ratio (25 X 10 6 spleen cells to 8.3 X 106 myeloma cells) in the presence of 50% polyethylene glycol 1500. The fusion, cell selection and cloning procedures were similar to those described by Kaprielian and Fambrough (1987) and Bruder and Caplan (1989). After fusion, the cells were resuspended in 100 ml of Super HAT medium (Dulbecco's modified Eagle's medium with 4.5 gm/l glucose and 20% fetal bovine serum, 10% NCTC109 medium, 1 % gentamycin, 1 % L-glutamine, 0.2 units/ ml insulin, 0.5 mM sodium pyruvate, 1.0 mM oxaloacetic acid, 44mM sodium bicarbonate, and 2% SOX HAT medium supplement) for hybridoma selection. Aliquots of 0.1 ml of this suspension were added to wells of ten 96-well cluster dishes, to which an identical volume of murine peritoneal macrophage cells (1 X 105 cells per ml in Super HAT medium) had been added the previous day as a feeder layer. Dishes thus seeded were placed in a 37°C incubator with an atmosphere of 5% CO 2 and 95% air. Medium was changed 3 and 5 days after seeding. Colony formation was evident in some wells after several days, and additional colonies were noted up to 14 days after fusion.

Hybridoma screening and cloning Developing colonies were initially tested for the presence of mouse immunoglobulins. A 50-f-I,l aliquot of medium from wells containing colonies was added to individual wells of ELISA plates previously coated with unconjugated goat anti-mouse immunoglobulin and blocked with 1 % bovine serum albumin in phosphate-buffered saline (BPBS). After addition of the culture supernatant, the plates were incubated for 1 h at 25°C, rinsed with B-PBS and then exposed to 50 f-I,l of alkaline phosphatase-conjugated goat anti-mouse immunoglobulin at a 1:250 dilution in B-PBS. Following a I-hour incubation at 25°C, the plates were rinsed again and p-nitrophenyl phosphate substrate in

50 mM glycine, 1 mM MgCI 2 , pH 10.5 was added to each well. Positive and negative controls were tested in conjunction with each group of culture supernatants thus screened. Cells in microtiter wells testing positively for mouse immunoglobulins were continued in culture and tested for reactivity to the immunogen, which was bound to ELISA plates at a concentration of 40 f-I,g!ml in 0.1 M sodium carbonate, pH 9.4. Additional incubations and rinses were identical to those utilized in the assay for immunoglobulin. Colonies which reacted positively in both assays were switched to Super HT medium (identical to Super HAT, but lacking aminopterin), cloned several times by limiting dilutions, expanded into culture in large vessels (1 OO-mm dishes or 75 cm 2 flasks), frozen in an insulated container in dry ice and stored in liquid nitrogen.

Immunoblotting and dot-blotting Samples to be analyzed on an immunoblot were electrophoresed on a sodium dodecyl sulfate polyacrylamide gel by standard procedures (Laemmli, 1970). A Hoefer Mighty Small II slab gel unit was used. Gels were either 10% or 5 -17.5% acrylamide; in either case, the stacking gel consisted of 5% acrylamide. Samples were reduced with 2mercaptoethanol and heated to 100°C prior to electrophoresis. After electrophoresis, the samples were transferred to Immobilon (polyvinylidene fluoride) membranes; transfer was accomplished with a Hoefer Transphor (TE 42) electrotransfer apparatus operated at 0.5 -1 amp for 1-3 h with a buffer consisting of 25 mM T ris, 192 mM glycine, pH 8.3 containing 20% (v/v) methanol (Towbin et aI., 1979). After transfer, the membrane was blocked with 3% bovine serum albumin in TBS-T (10mM Tris, pH7.5, 140mM NaCl, 0.05% Tween-20). The membrane was subsequently incubated with primary antibody diluted in the same solution as that used for blocking, rinsed three

Avian PG-II (Decorin) in Embryonic Tissues times with TBS-T and then incubated in secondary antibody diluted in TBS-T. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG. After three rinses in TBS-T to remove the secondary antibody, the membrane was exposed to alkaline phosphatase substrate (0.033% nitro blue tetrazolium and 0.016% 5bromo-4-chloro-3-indolyl phosphate) in 0.1 M Tris, pH9.5, 0.1 M NaCl, 5 mM MgCh and then rinsed extensively with distilled water and allowed to dry. Dot-blots were performed with a Bio-Rad dot-blot apparatus. The samples to be tested were diluted with TBS (TBS-T without Tween-20); generally 10, 1 and O.lllg, based on dry weight of starting proteoglycan material, were applied in 100 III of TBS. For dot-blots, 0.451lm nitrocellulose was used to immobilize the samples. After the samples were drawn onto the nitrocellulose by vacuum, the wells were rinsed once with TBS and the membrane was processed through blocking, first antibody, rinses, second antibody, rinses and color development exactly as for the immunoblots. Immunofluorescence

Tissues from chick embryos, hatchlings and adult chickens were dissected in PBS, either left unfixed or fixed in 10% buffered formalin or 70% ethanol for 30 min, rinsed in PBS, embedded in Tissue-Tek O. C. T. compound, frozen in liquid nitrogen and stored at - 20 DC. In most instances, tissues were prepared without fixation, although immunofluorescence was not qualitatively different in fixed specimens. Frozen sections were cut at a thickness of 61lm with an 1. E. C. model minitome/microtome cryostat, placed on glass slides and stored at - 20 DC. Tissue sections were incubated with primary antibody for 60 min at 25 DC. Culture supernatant was used undiluted, while ammonium sulfate-precipitated antibody and ascites fluid were used at a dilution of 1 :200. Slides were rinsed three times in B-PBS following incubation with primary antibody and then incubated for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody diluted 1:100 in B-PBS. Slides were again rinsed in BPBS and coverslipped in a medium consisting of glycerol: PBS (9:1), pH 8.5 and 0.011 M p-phenylenediamine. Observation and photography of these slides were carried out on an Olympus BH-2 epifluorescent/phase photomicroscope. Chondroitinase digestion and immunofluorescence control

Prior to incubation with primary antibody, tissue sections were treated with either PBS or chondroitinase ABC, 0.2 units/ml in PBS with protease inhibitors, for 60 min at 25 DC. As a control for immunofluorescence, identical samples were incubated with SH-2, a species-specific mono-

417

clonal antibody which reacts with an unidentified cell surface antigen on human marrow mesenchymal cells (Haynesworth et a1., 1991). Additionally, as a control for edgeeffect artifactual staining, embryonic chick limb buds and hatchling intestine were stained with monoclonal antibodies MY-174 and II6B3 (15A4), respectively; MY-174 reacts with PG-M, a proteoglycan found in early chick limb bud mesenchyme (Shinomura et a1., 1990) and II6B3 (15A 4 ) reacts with type II collagen (Linsenmayer and Hendrix, 1980). Production ofascites fluid

In order to suppress their immune systems, CB6FlI] female mice were injected intraperitoneally with 0.5 ml of pristane (2,6,10, 14-tetramethylpentadecane). After one week, they were similarly injected with 3- 5 X 106 hybridoma cells. After 7 to 14 days, ascites fluid was collected, centrifuged at 250 X g for 10 min to remove debris and stored at - 70 DC in small aliquots. Precipitation of culture supernatant with ammonium sulfate

Hybridoma culture supernatant was collected, clarified by centrifugation at 200 X g, filtered through a O.22-llm Nalgene filter to remove cells and debris and stored at 4 DC. An equal volume of saturated ammonium sulfate was added dropwise with stirring. Stirring was continued for an additional 30 min and the mixture was then centrifuged at 4000 X g for 20 min. The supernatant was discarded and the pellet washed twice with 50% ammonium sulfate and solubilized in a volume of PBS equal to 10% of the original medium volume. Resuspended material was dialyzed at 4 DC against PBS in dialysis tubing with a nominal molecular weight cut-off of 3500; the dialyzed material was clarified by brief centrifugation and stored at - 70 DC in small aliquots. Isolation of the small derma tan sulfate proteoglycans from cartilage

The epiphyses of hatchling chicks, 1-2 weeks of age, were dissected free, minced into small pieces and extracted as described above for the muscle tissue. After clarification by centrifugation, solid CsCI was added to a density of 1.50 gm/ml and the extract was centrifuged to equilibrium (Carrino and Caplan, 1982), except that the gradients were partitioned into six fractions. The low buoyant density fractions comprising D3 - D5, which contain the small dermatan sulfate proteoglycans (Rosenberg et a1., 1985) were pooled, dialyzed against water at 4 DC and lyophilized to dryness. Electrophoresis and staining with toluidine blue (Rosenberg et a1., 1985) confirmed the presence of the small proteoglycans.

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Results Characterization of the antigen

Initially, culture supernatants from hybridomas which gave a positive reaction in the ELISA assays were rescreened in a dot-blot assay against serial dilutions of the immunogen (chondroitinase AC II-treated day 14 chick embryo leg muscle proteoglycans). Several different culture supernatants, from wells which were clustered in the same region of the micro titer plate, showed reactivities which were qualitatively and quantitatively indistinguishable in the dot-blot assay (not shown). It is not clear if all of these hybridomas recognize the same epitope; however, all of the hybridomas are equivalent, as far as can be determined, in each screen that was performed (ELISA, dot-blot, immunoblot and immunohistochemistry). Each of the hybridomas was recloned by limit dilution and the resultant clones rescreened with no differences in reactivity. The isotype was determined for the antibody secreted by each of the hybridomas and each was found to be IgG 2b , which further suggests identity of all of the hybridomas. Because of this as well as the similarity in growth properties and reactivity in all of the screens, one of the hybridomas was selected and expanded. This hybridoma has been designated CB-l. Further characterization of the antibody by dot-blot analysis (not shown) indicates that it does not bind to the core protein preparation of the large cartilage proteoglycan synthesized by embryonic chick limb bud chondrocytes in culture (Hascall et ai., 1976). This suggests that the antibody is not simply binding non-specifically. Moreover, if primary antibody is omitted in a dot-blot assay, the staining obtained with the muscle proteoglycan preparation (i. e., the immunogen) is equal to background (not shown). Subsequent analysis indicates that the molecule recognized by CB-1 is a small proteoglycan. This is shown by immunoblot of the muscle proteoglycan preparation without or after treatment with chondroitinase AC II. Without chondroitinase treatment, the antibody recognizes a band with a molecular weight greater than 100000, while after chondroitinase, the antibody reacts with a 45000-dalton band (Fig. 1). In actuality, the core protein band recognized by CB-1 is a doublet, the resolution of which requires an appropriate combination of the amount of sample and the length of incubation in alkaline phosphatase substrate. The doublet nature of this band is more clearly seen in results presented below. Treatment of the muscle proteoglycan preparation with either chondroitinase AC II or ABC results in an identical immunoblot pattern (Fig. 2). Although these data could be interpreted to indicate that the proteoglycan recognized by the antibody is a small chondroitin sulfate proteoglycan, it has previously been shown that the small dermatan sulfate proteoglycan of cartilage (PG-II) gives rise to the same size core protein after treatment with chondroitinase AC II or ABC (Rosenberg et ai., 1985); this suggests that the gly-

cosaminoglycan chains contain glucuronic acid residues sufficiently close to the linkage region so that both chondroitinases remove approximately equal amounts of the glycosaminoglycan. Because previous analysis of the proteoglycans synthesized by skeletal muscle indicates that small dermatan sulfate proteoglycans, but not small chondroitin sulfate proteoglycans, are produced in this tissue (Carrino and Caplan, 1982; Carrino et ai., 1988), it is likely that the molecule recognized by CB-1 is a derma tan sulfate proteoglycan. To test the species specificity of the antibody, dot-blots were performed with chondroitinase-generated core proteins from a mixture of human articular cartilage derma tan sulfate proteoglycans I and II and also from bovine cartilage derma tan sulfate proteoglycan I and bovine skin derma tan sulfate proteoglycan II. In all cases,

CSase G) (I)

as as

c

t)

>-

C!) I

Z

G)

rn

as as c

0

>-

C!) I

Z

+

Fig. 5. Effect ofN-glycanase on the mobility of the molecule recognized by antibody CB-1. Proteoglycans isolated by ion-exchange chromatography from day 14 embryonic chick leg muscle were treated with chondroitinase AC II and split into three aliquots. One aliquot was not treated further, one aliquot was treated with N-glycanase and one aliquot was subjected to the N-glycanase incubation without added enzyme. All three samples were electrophoresed on a 10% gel, electrotransferred and immunostained with monoclonal antibody CB-1. Other details are in the legend for Fig. 1 and in Materials and Methods.

Avian PG-II (Decorin) in Embryonic Tissues

419

Fig. 6. Longitudinal cryosections of stage 40 (day 14) embryonic chick leg. Panel A shows a region close to the bone and panel B shows a region near the skin. Indirect immunofluorescence reveals CB-llocalization within bone (B), notably on the surfaces of trabeculae (T) and lacunae (L) and in muscle-associated connective tissues, including epimysium (EP) and perimysium (PE). Reactivity is also seen in the dermis (D) and subcutaneous connective tissue (5). Included in the latter is a small vascular bundle; CB-l immunostaining is seen in the tunica adventitia of the artery (A) and vein (V). All figures illustrating CB-l immunostaining depict antibody localization in undigested tissue sections.

Fig. 7. Cross sections ofcryo-preserved embryonic chick legs at stage 35 (day 9) (A) and stage 38 (day 12) (B) and of chick thigh at stage43 (day 17) (C) and at 20 days after hatching (D). At stage35, only the epimysium (EP) is reactive among muscle-associated connective tissues whereas the perimysium (PE) is also reactive by stage 38. At stage 43, a more intricate pattern of perimysial reactivity is observed. Although Fig. 7 C depicts stage 43 thigh, reactivity in leg muscle at the same stage is comparable. At 20 days after hatching, localization of CB-l in the endomysium (EN) is seen; a similar staining pattern is noted as early as stage 46. Immunolocalization of CB-l in tendon (T) can be seen at stage 31 and thereafter (A and B). Slight cell surface reactivity is associated with chondrocytes in the cartilaginous tibial core (C) seen in panel A. In the stacked cell layer (SC), fibers in the matrix are reactive, whereas the presumptive osteoblastic round cell layer (R) is largely non-reactive. The neurovascular bundle in panel B reveals CB-llocalization in the peripheral nerve (N) endoneurium (reactivity in the perineurium is not prominent in this section) and in the tunica adventitia of the artery (A). F, fibula. Other abbreviations are as in Fig. 6.

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D. P. Lennon et aI.

Fig. 8. Cryosections of stage 24 (A) and stage 25 (B) leg limb buds immunostained with CB-l. In each case the mesoderm (M) is nonreactive. Reactivity throughout the ectodermal (E) matrix is seen in panel A, whereas reactivity is primarily restricted to an intensely stained superficial area (arrows) in panel B. While these patterns are not necessarily stage-specific, ectoderm in limb buds at stages 22 - 24 more typically displays the diffuse staining pattern seen in panel A, while more distinctly fibrous ectodermal elements (as in panel B) are noted with greater frequency at stages 25 - 27.

no reactivity was observed with the mammalian samples under conditions where strong reactivity was seen with the chick muscle preparations (not shown). The characteristics of the molecule recognized by CB-1 are similar to those of the small proteoglycan called dermatan sulfate proteoglycan-II (Rosenberg et aI., 1985) or decor in (Fisher et aI., 1989; Neame et aI., 1989). Because the core protein of this proteoglycan shares much homology with the core protein of derma tan sulfate proteoglycanlor biglycan (Fisher et aI., 1989; Neame et aI., 1989), it was important to ascertain if CB-1 also recognizes the core protein of biglycan. For this purpose, the intact proteoglycans extracted from hatchling chick epiphysis and isolated in the low buoyant density region of a CsCI gradient were subjected to an immunoblot with CB-l. The different proteoglycan species present were detected both by staining with toluidine blue or with monoclonal antibody 2B6, which recognizes chondroitin-4-sulfate (Caterson et aI., 1987). Although only PG-.II is detected with 2B6 in the muscle preparation, both of the small proteoglycans are observed in the cartilage sample after staining with 2B6 (Fig. 3) or toluidine blue (not shown). Indeed, PG-I is more abundant in this sample, which is expected for the cartilage of a young animal (Melching and Roughley, 1989). (The 2B6-reactive material at the top of the gel is low buoyant density large proteoglycan, which has been observed previously in the CsCI gradient fractions which contain PG-I and PG-II (Rosenberg et aI., 1985).) In contrast to 2B6, CB-1 immunostains only the band at approximately 100000 daltons (i. e., PG-II), while the band at approximately 200000 daltons (PG-I) is not recognized by CB-1 (Fig. 3). This is observed even if the immunoblot is digested extensively with chondroitinase ABC prior to immunostaining, so that any steric hindrance by the glycosaminoglycans of PG-I can be alleviated. Thus, in spite of the extensive homology between the core proteins of PG-I (biglycan) and

PG-II (decorin), the CB-1 epitope appears to be present only on the latter. CB-1 also reacts with material isolated from embryonic leg muscle cell cultures. For this analysis, the proteoglycans were isolated from both the cell layer and the culture medium, as previously reported (Carrino et aI., 1988) and then treated with chondroitinase AC II prior to immunoblot with CB-l. The antibody detects material in both the cell layer and the medium, but the exact pattern of reactivity is somewhat different from that of the day 14 embryo leg muscle sample (Fig.4). In the 14-day leg muscle material, CB-1 recognizes a closely spaced doublet, the faster migrating member of which is more prominent. However, with the cell culture sample, the band with slower mobility is more prominent. This is more evident in the cell layer sample, because it appears that the medium sample contains less of the faster migrating band relative to the other band. The reason for this heterogeneity is unknown, but similar heterogeneity has been observed in other systems for the core proteins of small derma tan sulfate proteoglycans (Brennan et aI., 1984; Giossl et aI., 1984; Kinsella and Wight, 1988; Rauch et aI., 1986). It may result from differences in the number of N-linked oligosaccharides (Scott and Dodd, 1990) or may be due to catabolism. If the latter process is the cause, then this catabolism is more prevalent in vivo, and the molecules in the culture medium are spared relative to those in the cell layer. The reactivity of CB-1 with core protein bands prepared from proteoglycans isolated from muscle cell cultures is further evidence that the antibody reacts with a small derma tan sulfate, rather than chondroitin sulfate, proteoglycan, because small chondroitin sulfate proteoglycans are not synthesized in these cultures, but small derma tan sulfate proteoglycans are (Carrino et aI., manuscript in preparation). While the data presented above are consistent with the antibody recognizing an epitope on the derma tan sulfate

Avian PG-II (Decorin) in Embryonic Tissues proteoglycan core protein, the possibility exists that residual saccharide may at least contribute to, if not constitute, the epitope. This residual saccharide includes the glycosaminoglycan linkage region, which is not removed by chondroitinase treatment (Campbell et aI., 1990), as well as N-linked and O-linked oligosaccharides, which have been shown to be present on small derma tan sulfate proteoglycans (Yanagishita and Hascall, 1983; Midura and Hascall, 1989). To address the question of saccharide contribution to the epitope, muscle proteoglycan core protein preparations were treated with N-glycanase to remove N-linked oligosaccharides and then probed in an immunoblot. After incubation in N-glycanase buffer, with or without N-glycanase, the core protein is still recognized by the antibody (Fig. 5). After incubation without N-glycanase, the molecular weights of the bands recognized by the antibody are unchanged relative to treatment with only chondroitinase (Fig. 5). However, treatment with N-glycanase leads to a decrease in the molecular weights of the bands recognized by the antibody (Fig. 5), which suggests that the N-linked oligosaccharides have been removed. The mobility of the bands stained with CB-1 in the sample treated with Nglycanase are comparable to those reported for DSPG-II core protein devoid of N-linked oligosaccharides (Kinsella and Wight, 1988; Scott and Dodd, 1990). Silver staining of a gel similar to that used for the immunoblot indicates the presence of bands of the same mobility as those detected with CB-1; no other bands of lower molecular weight are observed in the sample treated with N-glycanase (data not shown). This indicates that there are no other lower molecular weight forms of the core protein which are not recognized by CB-l. Thus, it appears that the presence of N-linked oligosaccharides is not required for antibody reactivity and, therefore, that such oligosaccharides do not comprise or contribute to the epitope.

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rabbit; the absence of reactivity with sections of mammalian tissue correlates with the inability of CB-1 to react on dot-blots with preparations of mammalian PG-II. The staining patterns for the quail and turkey tissues examined (muscle, skin and bone) are identical to those for the chick tissues described in detail below. CB-1localization in muscle-associated connective tissue, one of the tissues stained most dramatically by this antibody, has its onset at stage35-36 (9-10days). At that time only the epimysium, the large septum partitioning groups of myotubes into a discrete muscle organ, is reactive (Fig. 7 A). The perimysium, the septum which compartmentalizes myotubes into individual fascicles, elicits a positive reaction by stage 38 (12 days) (Fig.7B). The pattern of perimysial staining becomes more intricate as development progresses and more fascicles become segregated (Fig. 7 C). At stage 43 (17 days), CB-1 immunostaining is noted in the endomysium, the connective tissue sheath surrounding individual muscle fibers. Endomysial reactivity is rather weak at that time, but becomes more prominent at stage 46 (21 days) and continues to be very strong at 3 weeks after hatching (Fig. 7D). Localization of CB-1 reactivity was also examined in legs from chick embryos of early stages. From stage 22 (3! days), the earliest stage examined, through stage 26 (5 days), immunostaining in the leg is observed only in the extracellular matrix in the ectoderm. Two different staining patterns are observed in this area. In some cases, the entire ectodermal extracellular zone is reactive without any discernible foci within the immunoreactive area (Fig. 8 A). This pattern is typically seen at stages22 and 23 (3!-4 days), but has been noted as late as stage 25 (4!- 5 days). In

Immunohistochemistry As indicated in the Methods section, each type of tissue described below was immunostained either without or following digestion with chondroitinase ABC. Antibody localization patterns were identical in either case in all tissues except bone and hypertrophic cartilage (see below); the intensity of staining was somewhat enhanced in digested specimens. Because the material utilized as the immunogen was isolated from stage 40 (14-day) chick embryo leg muscle, our preliminary efforts at ascertaining sites of antibody localization focused on immunostaining frozen sections of intact chick legs of the same age. Positive reactivity is most striking in three tissues: dermis (Fig. 6B), large and small intermuscular septa and bony trabeculae (Fig. 6 A); cell surfaces of chondrocytes and areas of loose connective tissue are reactive to a lesser degree (Fig. 7 A). It should be noted that CB-1 also immunostains tissue sections prepared from quail and turkey, but not human and

Fig. 9. Cross section of a stage 38 (day 12) chick leg. The dermis (D) stains intensely with CB-l, while the antibody does not localize in the epidermis (E). Only sparsely distributed fibers in the subcutaneous tissue (S) immediately deep to the dermis are reactive; deeper subcutaneous tissues are strongly reactive. Several feathers cut transversely exhibit CB-llocalization in the dermal pulp (DP) and in the dermal portion of the developing barb veins (BV). Two feather germs (FG) with immunoreactive dermal components are present (PE, perimysium; EP, epimysium).

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Fig. 10. A. Sagittal section of stage 24 (day 4~) heart. CB-] localizes within fibers in the developing myocardium (MC), in the endocardium (EC) and in a portion of the pericardium (PC). B. Coronal section of the heart at stage 43 (day 17). CB-1 continues to be localized in the endocardium (EC), myocardium (MC) and pericardium (not shown).

Fig. 11. Coronal section of a portion of a stage37 (day 11) embryonic chick eye immunostained with CB-1. Reactivity of the antibody is observed in the corneal stroma (CS) and in fibrous elements associated with the ciliary muscle (CM) and the iris (I). There is a slight degree of CB-1 localization in the capsule of the lens (L). While the corneal epithelium appears to be reactive in this section, localization of CB-l is variable in this tissue; staining is sometimes observed at the very periphery of the corneal epithelium but not in the central portion.

the second pattern observed at early stages (24-27), an intensely stained area is seen at the basal surface of the outermost and sometimes second-most superficial cell layer (Fig. 8 B). Diffuse reactivity within the ectodermal extracellular matrix may be associated with this pattern and in other instances short extracellular fibers coursing parallel and perpendicular to the surface of the limb are observed. Localization of MY-174, an antibody which reacts with mesenchymal proteoglycan, was not detected in ectoderm, which suggests that the ectodermal staining by CB-1 is not the result of an edge effect. At stage 27 (5! days), the same epithelial patterns are seen. Generally the depth of reactivity is greater proximally

Fig. 12. Cross section of the small intestine of a l-week-old chick hatchling. Connective tissue associated with smooth muscle (SM) deep to the serosa (S), as well as the serosa itself, stains with CB-1. Localization of the antibody is also seen in fibrous portions of the crypts of Lieberkuhn (L) and in the stroma of villi (V), some of which are cut longitudinally, while others are sectioned transversely or obliquely.

than distally in the limb. Additionally, localization of CB-1 in the central core of the limb is observed for the first time as the cell surfaces of chondrocytes are slightly reactive. The pattern of antibody staining at stage 29 (6 days) is essentially unaltered, except that dermal mesoderm reacts positively. Moreover, a non-reactive epidermal layer is observed superficial to the reactive epithelial structures noted earlier. Antibody localization in several additional structures is detected at stage 31 (7 days). Notably, the perichondrial lining of the cartilagenous long bone precursors is highly reactive. Chondrocyte cell surfaces continue to be slightly reactive. CB-1 also stains short fibers, which appear to be developing tendons. Matrix associated with the soft tissues between and adjacent to cartilaginous bone models at the site of developing joints is immunoreactive. Similar staining

Avian PC-II (Decorin) in Embryonic Tissues· patterns are seen at stage 32 (7! days); additionally, short fibers in the stacked cell layer (presumptive pre-osteogenic cells) of the tibia and femur demonstrate positive immunostaining. At stage 33 (7! days) and continuing at least through stage 41, cartilaginous matrix in long bones is not immunoreactive in undigested tissues. However, when these tissues are digested with chondroitinase ABC, immunolocalization of CB-1 is detected in matrix associated with hypertrophic chondrocytes, but not with resting or proliferating chondrocytes. As noted earlier, the development of an unreactive epidermallayer is first observed at stage 29. At that time and th ro ugh stage 36, this layer is only one- to two-cells thick. By stage 39 (13 days), however, the unreactive epidermis appears to be 4- to 5-cells deep. A dermal layer, approximately twice as thick as the epidermis, reacts with CB-l. The antibody localizes in dermal fibers running chiefly parallel to the limb surface, although some fibers run perpendicular to these. This netw ork of fibers within the dermis is very dense. Beneath it is an area of loose connective tissue which i s largely non-reactive. Deep to this non-reactive zone is another area of connective tissue which includes short but thick, fairly dense, positively staining wavy fibers. This latter area extends to the m uscle epimysium. Localiza- · tion of CB-1 within developing feathers is seen beginning on day 11. The mesodermal core and dermal portions of the longitudinal ridges (primodia of separate down feather barb vanes) are immunoreactive (Fig. 9) . Interestingly, the stain ing pattern for PC-II in the feather germs is different than th at previously reported for a large chondroitin sulfate proteoglycan synthesized in embryonic chick skin (Kitamura, 1987). Frozen sections of legs stained with CB-l at stages of first bone formation reveal a high degree of reactivity on the surfaces of bony trabeculae (Fig. 7A). The lining of bony lacun ae stains with CB-1, although osteocytes within these lacunae appear to be unreactive (Fig. 6A) . Bone matrix proper is not reactive in undigested samples, but in specimens digested with chondroitinase ABC a diffuse but moderately intense pattern of immuno localiza tion is noted in this tissue. These patterns of staining persist at least through stage 46 (2 1 da ys). Chondrocyte cell surfaces are weakly reactive (Fig. 7 A), while the periosteum stains very intensely, particularly in the outer stacked cell layer where numerous fibers paralleling the shaft of the bone are observed. The inner aspect of the periosteum, the presumptive osteoblastic round cell layer, is much less reactive (Fig. 7B and 7C). As ea rly as stage 25, CB-1 reacts with cardiac connective tissue, the endocardium and pericardium (Fig. 1OA). As development proceeds, reactivity in these tissues continues, with the most intense staining patterns paralleling those seen in skeletal muscle. That is, cardiac muscle itself is nonreactive, but the connective tissues partitioning muscle into ind ividu al bundles are positive. However, as evidenced by

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Fig. 13. Immunoblot of proteoglycans isolated from various tissues of chick embryos. Tissues were dissected from day (d.) 11 or 17 chick embryos and the proteoglycans isolated by anion exchange chromatography. Samples of equal amount, based on dry weight of lyophilized material, were treated with chondroitinase AC II, electrophoresed on a gradient gel, electrotransferred and immunostained with monoclonal antibody CB-l. Other details are in Materials and Methods (m use., muscle; pect., pectoral; heart, skin, tendon and diaph ysis were obtained from day 17 embryos).

staining with CB-l, the connective tissue partitioning within cardiac muscle is not as elaborate as that in skeletal muscle (Fig. lOB). Inter-chamber septa are also reactive as is the adventitia of arteries in and around the heart. In peripheral arteries, the tunica adventitia is also reactive (Fig. 6B and 7B). CB-l localizes in the connective tissu e sheath (epineurium) surrounding large peripheral nerves, in the perineurium encompassing individual nerve bundles or fascicles, and in the endoneurium aro und individual nerve fibers (Fig. 7B). While this localization is analagous to that fo und in muscle-associated connective tissue, the developmental onset of immunoreactivity of individual components of nerve-associated connective tissue has not been investigated.

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Structures associated with the embryonic eye at stages 34, 37, 40 and 42 (days 8, 11, 14 and 16, respectively) were examined for immunolocalization of CB-l. The antibody reacts with corneal stroma (Fig. 11), with the lining of the cartilaginous sclera, with connective tissues surrounding the eye and in the iris and ciliary muscle. The retina and lens are not reactive, although the capsule of the lens is slightly reactive. As in most other cartilage tissues examined, the cell surfaces of chondrocytes in the sclera are slightly reactive. Staining of connective tissues associated with the optic nerve is consistent with that seen in peripheral nerves. Localization of CB-l reactivity in embryonic chick leg tendon was examined at days 12,14 and 16. The antibody localizes in longitudinal and oblique fibers within tendons and in tissues immediately surrounding the tendon (sheath or epitendineum). Examination of sections of small and large intestine of one-week hatchling chicks reveals reactivity of CB-l in the connective tissue septa of longitudinal and circular smooth muscle groups in the muscularis externa. Thus, CB-1 localizes in septa of skeletal, cardiac and smooth muscle. Immunostaining also appears in the serosa and in the connective tissue stroma of villi and crypts of Lieberkuhn (Fig. 12). Similar patterns are noted in the large intestine. The mucosa and submucosa in both the small and large intestines are non-reactive. Control staining for possible edge effect is negative. Immunostaining of liver, kidney and lung in one-week hatchling chicks reveals a consistent picture of CB-l localization in related connective tissues. CB-l reacts with connective tissues found in the hexagonal boundary between adjacent tertiary bronchi in the lung. It is also detected immediately adjacent to respiratory epithelium in the atria of these bronchi. The antibody localizes in parasinusoidal connective tissues in the liver and intensely stains interstitial tissues surrounding tubular structures in the kidney. Because of the widespread distribution of the epitope, some of the tissues which were found by immunohistochemistry to contain reactive material were dissected from embryos and extracted and a proteoglycan-enriched fraction prepared by step elution from DEAE-Sephacel. These tissues include day 11 and 17 leg muscle, day 11 and 17 pectoral muscle and heart, skin, tendon and tibial diaphysis (bone) from day 17 embryos. These samples were subjected to immunoblot analysis after treatment with chondroitinase. In the extracts of all the tissues, one prominent band of approximately 45 000 daltons is recognized by the antibody (Fig. 13). This band is of the same molecular weight as that detected in the immunogen preparation, chondroitinase-treated embryonic day 14 leg muscle proteoglycans (Figs. 1-3). A band of the same mobility, though of lower intensity, is observed after chondroitinase treatment of material extracted from day 17 epiphysis and recovered in the low-buoyant density region of a CsCI gradient (not shown). For some of the tissue samples, the

material was also probed without prior chondroitinase digestion and, in all samples tested, the antibody reacted with a single major band of greater than 100000 daltons (not shown). The quantitative differences in reactivity may be meaningful (Carrino and Caplan, 1984) but may be due to likely variability in the degree of purity of the various preparations, since the amount applied to the gel was based on dry weight of lyophilized material.

Discussion This report describes the generation of a monoclonal antibody, CB-l, which appears to recognize an epitope on avian PG-II. That avian PG-II (decorin) is the molecule recognized by this antibody is indicated by the spectrum of reactivities in several assays with various samples. For example, on an immunoblot of the immunogen without prior treatment of the sample with chondroitinase, CB-l recognizes a molecule which migrates with an estimated molecular weight of approximately 100000, while after chondroitinase treatment of the sample, the material immunostained by CB-1 has an estimated molecular weight of approximately 45000. These values are in agreement with those reported for intact PG-II and its core protein (Kinsella and Wight, 1988; Rosenberg et aI., 1985). Other proteoglycan core proteins, such as those of the large cartilage proteoglycan or the large chondroitin sulfate proteoglycan of skeletal muscle, are not recognized by CB-l. Also characteristic of PG-II, the reactive material shows a wide tissue distribution, by both immunohistochemistry and by immunoblot analysis of partially purified preparations of proteoglycans. In immunoblots of intact material, the band recognized in the sample prepared from day 17 heart has lower mobility than that for the bands recognized in the other tissues, such as skeletal muscle (not shown). This correlates with the presence of larger glycosaminoglycans on the small derma tan sulfate proteoglycans isolated from embryonic chick heart (Carrino and Caplan, 1984). Tissuespecific differences in the size of the derma tan sulfate of PGII have been reported (Larjava et ai., 1986; Rauch et aI., 1986). The epitope recognized by CB-l is present in all avian species examined, but reactivity could not be demonstrated with mammalian samples, either immunohistochemically with tissue sections or in a dot-blot assay with isolated proteoglycans. It should be noted that, in spite of the similarity across species in the structure of the PG-II core protein, a probe for the human PG-Il core protein failed to hybridize with the mRNA for rat PG-Il core protein (Fisher et aI., 1989). Thus, if the core protein comprises at least part of the CB-l epitope, then the species selectivity may indicate a structural difference in the avian and mammalian PG-U core proteins. The derma tan sulfate does not appear to be required for reactivity, since treatment with chondroitinase

Avian PG-II (Decorin) in Embryonic Tissues ABC or AC II does not abolish or even diminish reactivity in a dot-blot or immunoblot. Treatment of the immunogen with N-glycanase to remove N-linked oligosaccharides also does not abolish antibody recognition. Consequently, the only saccharide which might contribute to the epitope, if any is so involved, is the derma tan sulfate linkage region or the O-linked oligosaccharides. Because the core protein appears to at least partly comprise the epitope of CB-1, this antibody is useful in immunolocalization analysis of PG-II. Such analysis indicates that PG-II is localized in the fibrous connective tissue septa of developing skeletal and cardiac muscle and is not initially deposited around the myogenic cells. A similar finding has been reported for fetal human muscle (Bianco et aI., 1990). These observations suggest that the muscle cells themselves do not elaborate the PG-II found within muscle, but instead that fibroblastic cells, which are located in the septa between the bundles of muscle cells, are responsible for the deposition of PG-II. PG-II is also located in the periosteally deposited bony collar, which represents the first bone formed in the developing tibia (Pechak et aI., 1986). This correlates with published results for fetal human bone (Bianco et aI., 1990). Thus, PG-II is present in bone even at the earliest stages of embryonic osteogenesis. Table I illustrates that the distribution of PG-II in chick embryos, as represented by immunolocalization of CB-1, correlates very closely with the distribution of human PG-II core protein reported by other investigators (Bianco et aI., 1990) in non-skeletal tissues. Localization of the respective antibodies in the two species is also similar in skeletal tissues, although there are important exceptions. Among the similarities are the localization of antibodies in bony matrix and on chondrocyte cell surfaces. In human femora, however, hypertrophic cartilaginous matrix did not stain with the PG-II-specific antibody (Bianco et aI., 1990) while staining of similar chick tissue with CB-1 was noted. However, in hypertrophic zones of metacarpal, phalangeal and vertebral human bones, localization of PG-II was observed. In human developing diarthrodial joints, an outer cap of prospective articular cartilage and articular soft tissues stained with antiserum to PG-I, but not with PG-II- specific antiserum (Bianco et aI., 1990). In contrast, the PG-IIspecific CB-1 was localized in similar tissues in chick embryos. Immunohistochemical analysis of these and other developing tissues confirms the widespread distribution of PG-II noted by others in more mature tissues (Poole et aI., 1986); Voss et aI., 1986) and extends these observations to show the presence of this proteoglycan within connective tissue during development. The lack of staining for PG-II previously reported for the subepidermal zone of the dermis of fetal calf skin (Pringle et aI., 1985) is not seen in embryonic chick skin. The localization of PG-II in early limb buds is of particular interest because the mesenchymal tissue appears to be devoid of this proteoglycan while the epider-

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mis stains intensely. This is in contrast to later stages when the epidermis is unstained, but the dermis and other connective tissues contain PG-II. The epidermal staining is also of interest in light of the recent report that the epidermis of mature skin contains epitopes present in chondroitin sulfate (Sorrell et aI., 1990). Because chick represents a useful system in which to study development, CB-1 has proven helpful in developmental studies of PG-II distribution and localization in embryogenesis.

Acknowledgements The authors are grateful for the technical assistance of Mrs. Carol Ingle and to Mrs. Adele Gandal and Mrs. Christine Kehoe for typing the manuscript. This work was supported by grants from NIH and the Muscular Dystrophy Association.

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by embryonic chick skeletal muscle and heart. ]. Bioi. Chern. 259: 12419~ 12430,1984. Carrino, D. A. and Caplan, A. I.: Isolation and characterization of proteoglycans synthesized in ova by embryonic chick cartilage and new bone.]. Bioi. Chern. 260: 122~ 127,1985. Carrino, D.A., Oron, U., Pechak, D. G. and Caplan, A.I.: Reinitiation of chondroitin sulfate proteoglycan synthesis in regenerating skeletal muscle. Develop. 103: 641 ~656, 1988. Carrino, D. A. and Caplan, A.I.: Structural characterization of chick embryonic skeletal muscle chondroitin sulfate proteoglycan. Conn. Tiss. Res. 19: 35~50, 1989. Caterson, B., Calabro, T. and Hampton, A.: Monoclonal antibodies as probes for elucidating proteoglycan structure and function. In: Biology of Proteoglycans, ed. by Wight, T. N. and Mecham, R. P., Academic Press, London, 1987, pp. 1 ~ 28. Chang, Y., Yanagishita, M., Hascall, V.c. and Wight, T.N.: Proteoglycans synthesized by smooth muscle cells derived from

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Generation of a monoclonal antibody against avian small dermatan sulfate proteoglycan: immunolocalization and tissue distribution of PG-II (decorin) in embryonic tissues.

Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small dermatan sulfate proteo...
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