Study of differential collagen synthesis during development of the chick embryo by immunofluorescence

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BIOLOGY

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(19761

Study of Differential Collagen Synthesis during Development Chick Embryo by lmmunofluorescence I. Preparation

of Collagen Application

HELGA

Type I and Type II Specific to Early Stages

VON DER MARK,

Max-Planck-lnstitut

fiir

KLAUS

Biochemie,

Abt.

Accepted

of the Chick

VON DER MARK, Kiihn,

8033 Martinsried

September

Antibodies

of the

and Their

Embryo AND STEFFEN bei Miinchen,

GAY German?

17, 1975

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, puritied by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos. the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.

INTRODUCTION

Four genetically distinct types of collagen, occurring in different connective tissues, have been described (for review see Miller and Matukas, 1974a). Type I collagen is a major constituent of skin, bone, tendon, and dentin, while type II collagen has been found only in hyaline cartilage (Miller, 1971a) and in the notochord (Linsenmayer et al., 1973a; Miller and Matthews, 1974b). Type III collagen has been detected in aorta, skin, lyomyoma, and other tissues (Miller et al., 1971b; Epstein, 1974). Type IV collagen is the collagenous component of basement membranes (Kefalides, 1971). Type I collagen molecules are built up from two al(I)-chains and one (~2chain, coiled into a triple helical molecule lal(I)l,a2. Type II, III, and IV collagen molecules are each composed of three identical chains and have molecular formulae 1al(II)13, 1(~1(111)1,,or [al(IV)13. crl(I), a2, ol(II), cxl(III), and (xl(IV) are genetically 237 Copyright All rights

cl 1976 by Academic Press, of reproduction in any form

Inc. reserved.

distinct chains and differ in amino acid composition and sequence and carbohydrate content, although largely sequence homologies between type I, II, and III collagen have been reported (Fietzek and Kuhn, 1975). It is unclear yet whether type specific structural features are related to different functions in the tissue. To elucidate the function of different collagen types in various tissues, it will be useful to describe their spatial and temporal appearance in the developing and adult tissue. The first collagen synthesized in the chick embryo is apparently basement membrane (type IV) collagen underneath the epiblast during gastrulation (Trelstad et al., 1967). The neural tube at stage 12-15 (Hamburger and Hamilton, 1951) synthesizes a collagen which consists of one type of o-chains only (Trelstad et al., 1973). Cornea1 epithelium at stage 22 produces two types of collagen, one forming basal lamina and another

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forming striated fibrils of the cornea1 stroma (for review see Hay, 1973). The first cartilage collagen (type II) was found in the 2Vz day old (stage 17?) notochord (Linsenmayer et al., 1973a). In the limb bud, collagen type I is synthesized by the mesenthyme at stage 23-24 prior to cartilage matrix deposition (Linsenmayer et al., 1973b). At stage 26, an increase of the al:a2 ratio of the collagen synthesized in the core mesenchyme was found, indicating an initiation of cartilage collagen synthesis. The (a&-type collagen produced by osteogenic cartilage was later proven to be type II collagen (Linsenmayer et al., 1973c; Linsenmayer, 1974). These examples demonstrate that the synthesis of distinct collagens during embryonic development seems to follow a precise temporal and spatial outline. All four collagen types can be distinguished by biochemical methods such as ion exchange chromatography, amino acid analysis, and the peptide pattern obtained after cleavage with cyanogen bromide and separation on CM-cellulose.’ A precise morphological characterization of collagen type distribution in tissues, however, requires a histological approach, e.g., by using collagen type specific antibodies. A number of investigations, employing immunofluorescence technique, on the localization of collagen in tissues, unspecified in regard to the collagen type, have been reported. More recent immunochemical studies on the structure of antigens of the collagen molecules have provided the chemical background for preparing purified collagen antibodies better characterized in terms of specificity (for reviews, see et al., Timpl et al., 1973a; Furthmayr 1975). Immunofluarescence studies using specific antibodies to dermatosparactic procollagen and to calf and rat skin collagen were undertaken to localize these antigens 1 Abbreviations used: CM, carboxymethyl; CFA, complete Freund’s adjuvant; EDTA, ethylene diamine tetraacetate; FITC, fluorescein isothiocyanate; TRITC, trimethylrhodamine isothiocyanate.

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in normal and dermatosparactic skin and in kidney tissue (Timpl et al., 1973b; Wick et al., 1975). Collagen type specific antibodies have been raised in rats against native calf collagen type I, which do not crossreact with native calf collagen type II (Hahn et al., 19741, and antibodies specific to al(H) chains from cartilage have been described (Hahn et al., 1975). Rabbit antibodies, specific for types I, II, and III collagen, from calf and human tissues (Gay, Adelmann, Remberger, in preparation) were employed to identify collagen types I, II, and III in normal and pathological tissues using immunofluorescence techniques (Gay et al., 1975). Reported here are the preparation and purification of antibodies specific for chick types I and II collagen and their application to problems of differential collagen biosynthesis during embryonic development. MATERIALS

AND

METHODS

Preparation of chick collagen types I and ZZ. Type I collagen was obtained from cranial bones of 17 day old chick embryos by extraction with 0.5 M acetic acid. The collagen was precipitated first with 5% NaCl and then twice by dialysis against 0.2 M Na2HP04. The purity of the collagen was checked by amino acid analysis and gel electrophoresis. Type II collagen was isolated from sternal cartilage of 6 week old chickens by solubilization with pepsin (Worthington Biochemical Corp.) following the procedure of Miller (1971a) with minor modifications. The sterna were carefully separated from the perichondrium, homogenized with a steel blade homogenizer (Ultraturrax, Germany), and extracted extensively with 1 M NaCl, 0.05 M Tris-HCl (pH 7.5) in order to remove proteoglycans, followed by 0.5 M acetic acid. The insoluble material was treated with pepsin without preceding lyophilization. The pepsin extracted type II collagen was purified by precipitation with 5% NaCl; by the criteria of SDS-electrophoresis

VON

DER

MARK

et al.

Antibodies

there were traces of a2 chains derived from perichondrial type I collagen present in this preparation. Immunization. Four rabbits were immunized intramuscularly with 5 mg of type I or type II collagen mixed with complete Freund’s adjuvant (CFA). Booster injections were given with 10 mg of antigen without CFA, intraperitoneally after 2 weeks, and with 5 mg subcutaneously after 4 weeks. Two groups of 20 rats received initial injections of 0.5 mg of collagen I or II, mixed with CFA, subcutaneously at two sites in the dorsal skin. Booster injections of 1 mg of antigen were given intraperitoneally after 3 weeks without CFA. Blood was collected by cardiac puncture after 6 weeks. Two groups of 10 guinea pigs each received initial injections of 0.75 mg of antigen mixed with incomplete Freund’s adjuvant into two foot pads. The animals were given booster injections and bled as described above for rats. Zmmunoabsorbents. One hundred milligrams of type I and type II collagens were each coupled to 50 ml of Sepharose 4B (Pharmacia), employing the procedure of Cuatrecasas (1970). The Sepharose 4B was activated with 200 mg of cyanogen bromide/ml of Sepharose, and coupled to collagen in 0.15 M NaCl, 0.05 M Tris-HCl (pH 8.0) at 4°C with a coupling efficiency greater than 90%. After coupling the Sepharose was washed with 0.15 M NaCl, 0.05 M Tris, followed by 3 M KSCN, 0.05 M potassium phosphate (pH 6.0). Immunoabsorption. After the sera of each animal were tested individually by passive hemagglutination, sera with comparable titres were pooled and purified by affinity chromatography. To remove crossreacting antibodies, the antisera to type I collagen were first passed through a collagen type II absorbent, equilibrated with 0.15 M NaCl, Tris-HCl, pH 7.5. When necessary, absorption was repeated until the antiserum which passed through failed

to Chicken

Type I and

T,pe

II Collagens

239

to cross-react with type II collagen in the hemagglutination test. Antisera to type II collagen were cross-absorbed on a type I absorbent in a manner similar to that described for type I collagen antisera. The antisera which passed through the heterologous collagen absorbent were concentrated by ultrafiltration and absorbed on a homologous collagen absorbent, using 5 ml of collagen-Sepharose per milliliter of serum. After thorough washing, the antibodies bound to the absorbent were eluted with 3 M KSCN-0.05 M phosphate, pH 6.0, at 4°C. The eluate was dialyzed immediately into 0.15 M NaCl, 0.05 M TrisHCl, pH 7.5 and then concentrated. In some cases the antibodies were eluated with 1 M acetic acid, followed by 0.05 M HCl-0.15 M NaCl (Beil et al., 1973). The eluates were combined, neutralized with 4 M Tris-HCl, pH 9.0, and dialyzed into Tris-buffered saline. Serologic techniques. The antisera were tested by passive hemagglutination using human erythrocytes, coated with chicken type I or type II collagen by glutaraldehyde coupling, according to Beil et al. (1972). Purified antibody solutions were also tested by hemagglutination inhibition (Timpl et al., 1970). Immunofluorescence. For indirect immunofluorescence 4-6 Frn frozen sections were cut from 19 day old embryonic chick tibia and sterna, and from 3-7 day old whole chick embryos. Tibiae were decalcified for 2 days with EDTA before sectioning. In some cases, sections of sterna and tibiae were treated for 30 min with 2% testicular hyaluronidase (Serva, Heidelberg, Germany) before application of antibodies. The 3-7 day old embryos were rinsed in Simm’s balanced salt solution, staged according to Hamburger and Hamilton (1951), prefixed in 4% formaldehydesaline for 24 hr, and soaked in 30% sucrose. The air-dried sections were treated for 30 min with collagen antibodies in serial dilutions, rinsed with saline, and stained with fluorescein (FITC) conjugated

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goat anti-rabbit y-globulin or rabbit antiguinea pig y-globulin (Behring Werke, Marburg, Germany), diluted l:lO, at room temperature. The lowest concentration of the collagen antibodies giving rise to specific fluorescence with embryonic bones and sterna was applied routinely on embryonic sections. Negative controls were carried out with nonimmune IgG or with FITC-anti yglobulins only. Double labeling technique. Limb bud sections were labeled consecutively with type II collagen antibodies from rabbit, type I collagen antibodies from guinea pig, rhodamine-conjugated goat anti-rabbit yglobulin (Nordic Pharmaceuticals, Tilburg, Netherlands, 1:lO dilution), and finally with FITC-swine anti-guinea pig yglobulin (Behring Werke, 1:lO dilution). The sections were incubated for 30 min with each antibody and rinsed with saline in between each step. The stained sections were sealed with glycerol-saline (9:l) under a coverslip and observed and photographed with a Zeiss Standard 19 microscope, equipped with fluorescent light incident condenser IV F, overhead-light from an Osram HBO SOW/oc lamp, KP 490/500 filter for FITC-

TITRES

OF RABBIT,

AGAINST

NATIVE

RAT,

AND

CHICKEN

1976

fluorescence and BP 54619 filter mine fluorescence.

for rhoda-

RESULTS

Preparation of antibodies. The immune response of rabbits, rats, and guinea pigs to native chicken type I and type II collagens was measured by passive hemagglutination. The mean titres of rabbit and rat antisera to native, pepsin extracted type II collagen were low as compared with type I collagen (Table 1). There was no measurable type I agglutinating activity present in rat and rabbit type II collagen antisera, whereas all type I collagen antisera showed a cross reaction with type II collagen. The immune response to type II collagen in guinea pigs greatly exceeded that in rabbits and rats, but cross-reactivity with type I collagen was observed in these antisera. Cross-reacting antibodies from all sera could be removed by absorption on the heterologous collagen type. Specific antibodies were obtained by affinity chromatography of the cross-absorbed sera on the homologous collagen type. Type II collagen antisera from rabbits and rats which did not show a cross reaction with type I collagen in the hemagglutination test were nevertheless cross-absrobed in order to re-

TABLE HEMAGGLUTINATION

48,

1 GUINEA

TYPE

PIG

I AND

ANTISERA

AND

TYPE

II COLLAGENS

titres

(-log,)

PURIFIED

ANTIBODIES

Animal

Immunogen lagen

COI-

Hemagglutination

? SD against

collagen

antisera0

antibodies*

Type

-

Rabbit Rat Guinea

Pig

‘Ihe Type ‘be Type Type Type

11.3 * -Cl 9.2 + Cl 16.6 r 7.9 ‘-

1 II 1 II 1 II

Type

I’

3.1 1.9 1.2 2.5

6.6 5.0 7.0 6.5 14.5 14.1

IF’

k 2 5 e + +

Type

2.8 1.0 1.4 2.4 1.1 2.3

I’

Type

IId

7


6

<1

3


4 -Cl 6


8 Cl

a Titres are average pigs. b Titres of antibodies ’ Human erythrocytes d Human erythrocytes

values

of 4 antisera

from

rabbits,

20 antisera

after purification of pooled antisera. coated with native chicken type I collagen. coated with native chicken type II collagen.

from

rats,

and 10 antisera

from

guinea

VON

DER

MARK

et al.

Antibodies

move cross-reacting antibodies not detectable by passive hemagglutination. From 20 ml of antiserum of any species, between 3.0 and 5.7 mg of immunoglobulins were recovered by KSCN elution. When the acid elution procedure (Beil et al., 1973) was used, between 0.6 and 0.8 mg immunoglobulins were obtained. Although 3 M KSCN is a denaturing agent, the immunoabsorbents prepared from native collagens did not lose their capacity to bind antibodies against native collagen after repeated use with 3 M KSCN at 4°C. Specificity of the antibodies to type Z and type II collagens. The hemagglutination titres of the purified and concentrated antibodies from all three species are given in Table 1. A titre decrease was observed after subjecting the ant,isera to the immune absorption procedure, the purified antibodies being concentrated to less than half of the original serum volume (Table 1). All antibodies were specific in the direct hemagglutination test. Purified type I collagen antibodies did not cross react with type II collagen and vice versa. In the he-

IWY

la

n

150 I5 RABBI

60

15 110 I1 1

to Chicken

Type I and

Type II Collagens

241

magglutination inhibition test, however, type I collagen antibodies were still inhibited by type II collagen (Figs. la, lc, and le), but the activity was tenfold lower than that of type I collagen. This cross inhibition was probably due to small amounts of type I collagen present in the type II collagen inhibitor preparation (see Discussion). All antibodies against type II collagen could be inhibited effectively by type II collagen, but not with type I collagen even in tenfold higher concentrations (Figs. lb, Id, and 10. The specificity of the antibodies was further tested by indirect immunofluorescence staining of embryonic chicken tibiae and sternal cartilage, known to contain type I and type II collagens. Sections of embryonic chicken sterna were stained with rabbit-type I collagen antibodies (Fig. 2a) and rabbit-type II collagen antibodies (Figs. 2b and 2~). The inner cartilage “capsules” of the lacunar wall of sternal chondrocytes shows fluorescence with type II collagen antibodies (Fig. 2b), whereas the perichondrium was stained with type I collagen antibodies (Fig. 2a).

110 11 150 15 GUINEA

15 0 rlG

J-t I I 5 110 II

1I I

110 II

IIn

III11

150 15 150 15 110 I1 R A T

FIG. 1. Hemagglutination-inhibition of purified rabbit, rat, and guinea pig antibodies to native chick type I and type II collagens. Human erythrocytes were coated with native chick type I and type II collagen; the same collagen preparations were used as inhibitors. (a) Rabbit antibodies to type I collagen, red cells coated with type I collagen; (b) rabbit antibodies to type II collagen, red cells coated with type II collagen; (cl guinea pig antibodies to type I collagen, red cells coated with type I collagen; td) guinea pig antibodies to type II collagen, red cells coated with type II collagen; (e) rat antibodies to type I collagen, red cells coated with type I collagen; (f7 rat antibodies to type II collagen, red cells coated with type II collagen

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The intercellular space distal from the cells failed to react with type II collagen antibodies although it contains cartilage collagen. By treatment with testicular hyaluronidase proteoglycans which may have masked the collagen were removed, and specific fluorescence of the cartilage matrix was obtained, particular along the chondrogenic zone (z) of the sterna (Fig. 2c). Sections of 19 day embryonic tibiae, decalcified with EDTA, were treated with hyaluronidase, followed by type I collagen antibodies (Figs. 2d and 2e) and type II collagen antibodies (Fig. 2f)). In Figs. 2d and 2e bone trabeculae (B) give rise to strong fluorescence with type I collagen antibodies. Figs. 2e and 2f show sagittal sections of the epiphyseal cartilage-bone border. No fluorescence of the cartilage matrix (0 was obtained with type I collagen antibodies in Fig. 2e. Type II collagen antibodies, on the contrary, labeled only cartilage matrix (C) (Fig. 2f), but not bone and bone marrow (Ml. In Figure 2f, the photograph was printed with low contrast in order that the location of the immunonegative bone (B) be appreciated; the flourescent cartilage (C) is green. These results confirm the specificity of the purified type I and type II collagen antibodies determined by the hemagglutination test. Identification of type I and type II collagen in chick embryos at stage 1731. In sections of stage 19 embryos stained with type I collagen antibodies, type I collagen was mainly located along with the dermatomal-myotomal plate, presumptive der-

to Chicken

Type I and

il:vpr

II Collngen.v

243

mis, somatic mesoderm, around the notochord, and at the dorsal half of the neural tube (Fig. 3, arrows). Sections of stage 15-31 embryos labeled with type II collagen antibodies showed fluorescence of the notochord sheath only

FIG. 3. Cross section of a stage 19 chick embryo. posterior half, labeled with rabbit anti chick type 1 collagen (0.12 mg/mlI and FITC-conjugated goat anti-rabbit y-globulin (diluted 1:lO). N 2 notochord. S = spinal cord, D = dermatomal myotomal plate, So = somatic mesoderm. Arrows indic;itc. *Itch of fluorescence. * 40.

FIG. 2. la-c) Indirect immunofluorescence staining of sternal cartilage from 17 day old chick embryos. labeled with rabbit antibodies against chicken collagens and FITCcojugated goat anti rabbit y-globulin. ias Rabbit anti-chick type I collagen 10.12 mg/ml), P = perichondrium. ib) Rabbit anti-chick type II collagen (0.14 mg!ml). (c) Rabbit anti-chick type II collagen (0.14 mg:mli, sections were pretreated with 2’, testicula hyaluronidase for 30 min, z = chondrogenic zone. Id-f, Sagittal sections of tibiae from 19 dav old chick embryos, decalcified with EDTA. Sections were treated with 2’; hyaluronidase before applying antibodles. td) Bone trabeculae, labeled with rabbit antibodies to chicken type I collagen (0.19 mg ml! and FI’I’Cconjugated goat anti rabbit y-globulin. (e) Epiphyseal bone-cartilage border, labeled like Fig. 2d but using TRITC-conjugated goat anti rabbit y-globulin, B = bone trabeculae, C = cartilage matrix. If1 Epiphyseal bone-cartilage border, labeled with guinea pig antibodies to chicken type II collagen ~0.15 mg’ml I and FITC conjugated rabbit anti-guinea pig y-globulin, M = bone marrow. i 160.

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FIG. 4. Indirect immunofluorescence staining of notochord and developing vertebral body with rabbit goat anti rabbit y-globulin (diluted 1:lO). (a) anti chick type II collagen (0.14 mg/ml) and FITC-co njugated Stage 17: Fluorescence is restricted to the notochord !sheath. The photograph is printed with low contrast so caused by type II collagen spreads the cells show; they are not fluorescent. x 160. (bl St: age 25: Fluorescence around into the adjacent sclerotome. Sp = spinal COI*d, N = notochord, S = sclerotome. x 80. (c) Stage 28: notochord and dorsal aorta (A). x Type II collagen of th’e’ventral spine of the vertebral bNody appears between 50. (d) Stage 31: Vertebral cartilage starts to enclose : the spinal cord. x 30.

(Fig. 4a). At stage 25, type II collagen starts to spread around the notochord into the adjacent sclerotome (Fig. 4b). The neural tube was not stained by type II collagen antibodies. Figs. 4c and 4d (stages

28 and 31) show the expansion of type II collagen outlining the shape of the develop ing vertebral body; the neural tube is not labeled, although the low contrast of the photograph might give that interpretation

VON

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Antibodies

FIG. 5. Presumptive dermis from a stage 31 limb bud, labeled with rabbit anti-chick type I collagen (0.12 mgiml) and FITC-conjugated goat anti-rabbit y-globulin (diluted 1:lO). E = ectoderm, M = mesenchyme. x 160.

in a black and white scheme. Type I collagen antibodies also label presumptive dermis of the limb at the mesodermal-ectodermal interface. At stage 24 fluorescence of interstitial type I collagen only extends approximately one cell layer from the basement membrane into the mesenchyme (Fig. 6, arrow), but spreads over several mesenchymal cell layers at stage 31 (Fig. 5). The ectoderm and the mesenthyme (E, M, Fig. 5) is not stained by type I collagen antibodies. The precartilage core of limb mesenchyme gives rise to weak fluorescence with type I collagen antibodies at stage 24 (Fig. 61, prior to the appearance of metachromatic straining of cartilage matrix at stage 26. The site of fluorescence is encircled by a dashed line (Fig. 6). From stage 26 on, limb cartilage shows fluorescence with type II collagen antibodies (Fig. 7). Treatment with testicular hyaluronidase does not enhance fluo-

to Chicken

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245

rescence of type II collagen in limb cartilage until stage 28. Between stage 24 and stage 31, collagen type I gradually moves from the core of the cartilage blastema to the periphery, focusing to the perichondrial sleeve by stage 31 at the latest. Figures 8a and 8b show embryonic diaphysis midsections from stage 31 tibiae, labeled simultaneously with type I collagen (Fig. 8b) and type II collagen (Fig. 8a) antibodies using the double labeling technique described under 2.5 (one red, the other green fluorescence). Whereas the central cartilage matrix shows fluorescence with type II collagen antibodies (Fig. 8a), fluorescence with type I collagen antibodies is restricted to the perichondrium (Fig. 8b). The type I collagen specific fluorescence of the perichondrial sleeve overlaps partially with type II collagen specific fluorescence of the cartilage core, but light microscopic resolution does not show whether both types of collagen are synthesized by one cell. DISCUSSION

Differences in amino acid composition, sequence, and carbohydrate content and chain composition explain why type I and type II collagens gave rise to specific, noncross-reacting antibodies, together with cross-reacting antibodies which were removed by immunoabsorption. Purified antibodies to type II collagen from chicken, raised in rabbits, rats, and guinea pigs were specific in the passive hemagglutination test (Table 11, and hemagglutination inhibition tests (Figs. lb, Id, and 10, and failed to label type I bone collagen (Fig. 2b). In contrast, type I collagen antibodies were specific in the passive hemagglutination test but not in the hemagglutination inhibition test. This difference may be explained by contamination of the inhibitor (type II collagen from sternal cartilage) with type I collagen, possibly derived from remainders of the perichondrium. The fact that type I collagen antibodies gave rise to specific fluorescence with perichondrium (Fig. 2d), but not with cartilage matrix,

FIG. 6. Limb bud at stage 24 labeled with rabbit anti-chick type I collagen (0.12 mgimll and FITC conjugated goat anti-rabbit y-globulin (diluted 1:lOl. The arrow indicates site of fluorescence of presumptive dermis. Fluorescence of core mesenchyme is encircled by a dashed line. X 80. FIG. 7. Limb cartilage at stage 26 labeled with rabbit anti-chick type II collagen (0.14 mgimll and rhodamine conjugated goat anti-rabbit y-globulin (diluted 1:lOl. x 80. FIG. 8. Indirect immunofluorescent double staining of diaphysis mid sections from stage 31 chick leg primordia labeled consecutively with: rabbit anti-chick type II collagen (0.14 mgimll, guinea pig anti-type I collagen (0.09 mgiml), TRITC-conjugated goat anti-rabbit y-globulin and FITC-conjugated swine antiguinea pig y-globulin. (a) Red fluorescence (TRITC) of diaphysis cartilage with type II collagen antibodies, photographed with a BP 546/9 filter. (b) Green fluorescence (FITC) of diaphysis perichondrium with type I collagen antibodies, photographed with a KP 4901500 filter; identical section as in Sa. x 160. 246

VON

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supports this interpretation. Despite the specificity of the antibodies in immunofluorescence tests, conclusions drawn about the presence of a certain collagen type in tissues are limited in two respects: On the one hand, lack of fluorescence does not necessarily mean lack of antigen, since the antigen concentration may be too low to give rise to visible fluorescence, or the antigen may be masked by other compounds. Fluorescence of what seems to be older cartilage collagen was suppressed by substances, probably proteoglycans, which were removed by treatment with testicular hyaluronidase. On the other hand, specific fluorescence also may be created by other cross-reacting compounds, like non collageneous proteins or yet unknown collagen types. In the figures shown there is probably no fluorescence caused by type III collagen, since the purified type I and type II collagen antibodies failed to cross-react with chick skin type III collagen in the passive hemagglutination test (K. von der Mark, in preparation). Cross-reaction with basement membrane collagen (type IV) is also unlikely since purified type I and type II collagen antibodies failed to label glomerular basement membrane of adult chicken (not shown). Collagen type specific antibodies, nevertheless, have proved to be useful reagents for discriminating collagen types in normal and pathological tissues (Gay et al ., 1975), and in this paper it is shown that many current questions of collagen distribution during embryonic development may be answered by immunofluorescent techniques. Although a complete description of the development of type I and type II collagens in the chicken embryo would be far beyond the scope of this paper, a few examples of collagen morphology were given to illustrate the usefulness of the described antibodies. The specific fluorescence of the notochord sheath at stage 17 with type II collagen antibodies is in agreement with find-

to Chicken Type I and Type II Collagens

247

ings of Linsenmayer et al. (1973a) who reported that notochord of stage 17 in culture produces a collagen composed predominantly from al(H)-chains. Trace amounts of cy2-chains from type I collagen, found by chromatographic procedures, are consistent with our findings that notochord of stage 17 also gives rise to fluorescence with type I collagen antibodies. Notochord collagen was further identified as cartilage collagen by cyanogen bromide cleavage (Miller and Matthews 1974b). The (alJ3-type collagen which is secreted by the spinal cord as described by Trelstad et al. (1973) seems to be not identical with that synthesized by the notochord; no fluorescence of the spinal cord with type II collagen antibodies could be observed, using type II antibodies with a titre high enough to give strong fluorescence with notochord. However, our study detected type I collagen around the neural tube, whereas Trelstad et al. (1973) did not. The differentiation of sclerotome mesenthyme adjacent to the notochord into cartilage at stage 25 is indicated by appearance of collagen fibrils (Minor, 1973) and metachromatic staining material (Strudel, 1971). By labeling of axial regions with type II collagen antibodies, we found that sclerotome cells start to synthesize significant amounts of type II collagen at that stage (Figs. 4b-4d). Depending on the section plane, we also observed at stage 26 weak fluorescence of the sclerotome with type I collagen antibodies surrounding the type II collagen-specific fluorescence (not shown). But there was no distinct perichondrial sheath of type I collagen as in the perichondrium of long bones. The diaphyseal region of tibiae from stage 33 (8 days) chick embryos was reported to synthesize only cartilage collagen (type II) in culture (Linsenmayer et al., 1973c). No significant amounts of type I collagen could be detected prior to stage 33 (Linsenmayer et al., 1973b), although first appearance of perichondrial bone in midsections of diaphyses was described at

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stage 31(7 days) by histochemical methods by Lutfi (1971). Applying antibodies specific for type I and type II collagen to midsections of stage 31 diaphyses, it could be demonstrated that bone collagen (type 11is synthesized in the perichondrium as a thin layer immediately adjacent to the diaphysis cartilage as early as stage 31. The advantage of the histological method is evident in cases where different collagen types occur in close proximity and cannot be preparatively separated, as seen in Fig. 8a and 8b. However, it is also useful to relate immunohistologic results of collagen localization on biochemical data if accessable. For example, Linsenmayer et al. (1973b) reported that limb bud mesenthyme synthesizes type I collagen in culture at stage 23-24. In limb bud sections of stage 24, fluorescence with type I collagen antibodies was obtained in the core mesenthyme. It can be concluded that mesenchyma1 cells in the limb bud synthesize basal amounts of type I collagen in the blastema stage before they undergo chondrogenesis, which is accompanied by a steep rise in type II collagen synthesis. The fairly uniform distribution of fluoresence in the chondrogenic zones indicates a homogeneous change from type I to type II collagen synthesis by all core blastema cells. Why a mesenchymal cell synthesizes type I collagen before differentiating into a chondrocyte is not clear yet. We wish to thank Dr. Klaus Kuhn for generous support of this work, Dr. Rupert Timpl for valuable advice and discussions, and Miss Magdalena Grujic for skillful1 technical assistance. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ma 53412). REFERENCES BEIL, W., FURTHMAYR, H., and TIMPL, R. (1972) Chicken antibodies to soluble rat collagen. I. Zmmunochemistry 9, 779-788. BEIL, W., TIMPL, R., and FURTHMAYR, H. (1973) Conformation dependence of antigenic determinants on the collagen molecule. Immunology 24, 13-24. CIJATRECASAS, P. (1970) Protein purification by affinity chromatography. J. Biol. Chem. 245, 30593065.

VOLUME

48, 1976

EPSTEIN, E. H., JR. (1974). [al(III)]3 human skin collagen. Release by pepsin digestion and preponderance in fetal life. J. Biol. Chem. 249, 32253231. FIETZEK, P., and KUHN, K. (1975) Primary structure of collagen. Znt. Review of Connective Tissue Res. 7, in press. FURTHMAYR, H., and TIMPL, R. (1975) Immunochemistry of collagens and procollagens. Znt. Review of Connective Tissue Res. 7, in press. GAY, S., FIETZEK, P. P., REMBERGER, K., EDER, M., and KUHN, K. (1975) Liver cirrhosis: immunofluorescence and biochemical studies demonstrate two types of collagen. Klin. Wschr. 53, 205-208. HAHN, E., TIMPL, R., and MILLER, E. J. (1974) The production of specific antibodies to native collagens with the chain compositions [al(IIJs, [al(IIlJ~ and (al(Il),a2. J. Immunology 113, 421-423. HAHN, E., TIMPL, R., and MILLER, E. J. (1975). Demonstration of a unique antigenic specificity for collagen al(H)-chains from cartilaginous tissue. Immunology 28, 561-568. HAMBURGER, V., and HAMILTON, H. L. (19511. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAY, E. D. (1973). Origin and role of collagen in the embryo. Amer. 2001. 13, 1085-1107. KEFALIDES, N. A. (1971). Isolation of a collagen from basement membranes containing three identical a-chains. Biochem. Biophys. Res. Commun. 45, 226-235. LINSENMAYER, T. F., TRELSTAD, R. L., and GROSS, J. (1973a). The collagen of chick embryonic notochord. Biochem. Biophys. Res. Commun. 53, 3945. LINSENMAYER, T. F., TOOLE, B. P., ~~~TRELSTAD, R. L. (1973b). Temporal and spatial transitions in collagen types during embryonic chick limb development. Devlop. Biol. 35, 232-239. LINSENMAYER, T. F., TRELSTAD, R. L., TOOLE, B. P., and GROSS, J. (1973c). The collagen of osteogenic cartilage in the embryonic chick. Biochem. Biophys. Res. Commun. 52, 870-876. LINSENMAYER, T. F. (1974). Temporal and spatial transitions in collagen types during embryonic chick development. II. Develop. Biol. 40,372-377. LUTFI, A. M. (1971). The fate of chondrocytes during cartilage erosion in the growing tibiae in domestic fowl. Actu. Anat. 79, 27-35. MILLER, E. J. (1971a). Isolation and characterization of a collagen from chick cartilage containing three identical a-chains. Biochemistry 10, 1652-1659. MILLER, E. J., EPSTEIN, E. H., JR., and PIEZ, K. A. (1971b). Identification of three genetically distinct collagens by cyanogen bromide cleavage in insoluble human skin and cartilage collagen. Biochem. Biophys. Res. Commun. 42, 1024-1029. MILLER, E. J., and MATUKAS, V. A. (1974a). Biosynthesis of collagen. Fed. Proc. 33, 1197-1204.

VON

DER

MARK

et al.

Antibodies

E. J., and MATHEWS, M. B. (1974b). Characterization of notochord collagen as a cartilage type collagen. Biochem. Biophys. Res. Commun. 60, 424-430. MINOR, R. R. (1973). Somite chondrogenesis. J. Cell. Biol. 56, 27-50. STRUDEL, G. (1971). Materiel extracellulaire et chondrogenese vertebrale. C. R. Acad. Sci. Paris 272D 473-476. MILLER,

TIMPL,

R.,

FIETZEK,

P. P., FURTHMAYR,

H.,

MEIGEL,

W.. and KUHN, K. (1970). Evidence for two antigenie determinants in the C-terminal region of rat skin collagen. FEBS Letters 9, 11-14. TIMPL, R., FURTHMAYR, H., HAHN, E., BECKER, U., and STOLTZ, M. (1973a). Immunochemistry ofcollagen. Behring Inst. Mitt. 53, 66-79.

to Chicken

Type

I und

T-ype II Collagens

249

R., WICK, G., FURTHMAYR, H.. LAPI~RE, C. M., and K~~HN, K. (1973b). Immunochemical studies of procollagen from dermatosparactic calves. Eur. J. Biochem. 32, 584-591. TRELSTAD, R. L., HAY, E. D.. and REVEI,. J. P. (1967). Cell contact during early morphogenesis in the chick embryo. De[~elop. Blol. 16, 78-106. TRELSTAD, R. L., KANG, A. H., COHEN, A. M., and HAY, E. D. (1973). Collagen synthesis in vitro by embryonic spinal cord epithelium. Science 179. 295-297. WICK, G., FURTHMAYR, H., and TIMPL, R. (1975). Purified antibodies to collagen: an immunofluorescence study of their reaction with tissue collagen. Internat. Archs. Allergy Appl. Immun. 4X. 664.. 679. TIMPL,