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Type II collagen in the early embryonic chick cornea and vitreous: Immunoradiochemical evidence
hkp. Eye Rrs. (1983) 34. 371-379
Type II Collagen Vitreous: THOMAS
in the Early Embryonic Immunoradiochemical
F. LINSENMAYER,EILEEN
GIBNEY
Chick Cornea Evidence
AND CHARLES
and
D. LITTLE
The Developmental Biology Laboratory, Departments of Medicine and Anatomy, Harvard Medkal School and the Massachusetts General Hospital, Boston, MA 02114. U.S.A. (Received 30 January
1981~ ,Vew York)
The relative proportion of collagen types I and II was examined in early chick corneas and vitrous bodies by radioimmunoprecipitation. Embryos (4-5 days of incubation) were labelled in ova with 3H-proline. After an additional l-2 days, corneas and vitreous bodies were removed and the collagen was extracted and treated by limited pepsinization. Then, the labelled collagens were reacted with affinity purified antibodies against collagen types I and II, and the immune complexes precipitated with protein A-containing Staphylococc~e aureu8. In some cases, the precipitated collagen was further analysed by SDS-polyacrylamide gel electrophoresis. The results for the vitreous showed that greater than 99% of the labelled material precipitated by the antibodies was in type II. In the cornea1 samples, on the other hand, both types of antibodies precipitated appreciable labelled collagen with somewhat less than half being type II (3&46 %). These results are in agreement with our earlier biochemical observations on the relative content of type II collagen in these extracellular matrix-rich structures from embryonic avian eyes, Key words: cornea1 stroma; immune precipitation; collagen antibody: vitreous collagen; neural retina; collagen types.
1. Introduction In the vertebrate eye, both the cornea and the vitreous body owe their transparency in part to the orderly deposition of collagen into fibrils with a uniform diameter. In t’he developing chick embryo, the collagenous components within each of these structures are synthesized in two discrete stages and at each stage, a different cell population is responsible for synthesis. The earliest cornea1 matrix, termed the primary stroma, is synthesized by the cornea1 epithelium and is initially devoid of cells (for review see Hay, Linsenmayer, Trelstad and von der Mark, 1979). At about 5-6 days of development, this acellular stroma becomes populated by mesenchymal cells which subsequently differentiate into the cornea1 fibroblasts responsible for the synthesis of the mature cornea1 stroma. Likewise, for the vitreous the earliest collagen is also synthesized by an adjacent epithelium - in this case the primitive neural retina (Newsome, Linsenmayer and Trelstad, 1976). Subsequently the neural retina loses this capacity and by 12 days of development the vitreous collagen is largely synthesized by a cell population within t’he vitreous itself - possibly the hyalocytes. Biochemical analysis of in vitro labelled material has suggested that type II collagen, or a type II-like molecules is a major component of both the primary cornea1 stroma and the embryonic vitreous. Organ cultures of 5-6 day embryonic cornea1 epithelia, devoid of mesenchymal cells, synthesize a collagenous matrix which, in the electron microscope, appears similar to the primary cornea1 stroma as seen in vitro (see Hay et al., 1979). When incubated with H-proline, these cultures synthesize two major types of labelled collagens which by a number of criteria, are indistinguishable (K)l~-1835/82/030371+09 $01.00/O
0 1982 Academic Press Inc. (London) Limited
372
T. F. I~IESEh’MAYEK.
ET AL
from types 1 and 11 (Linsenmayer, Smith and Hay, 1977). Likewise. using biochemic*al criteria, in vitro cultures of 6-7 day whole embryonic neural retinas (Smith. cultures of neural retina cells Linsenmayer and Newsome, 1976) or monolayer (Linsenmayer and Little, 1978), synthesize type II as their predominant collagen type. In the present investigation we have used immunoradiochemical criteria to further analyse the synthesis of collagens within the eyes of embryos labelled in ovo. The results confirm the presence of a type II collagen molecule in both the early embryonic cornea and vitreous. Also in this study, since the embryos were labelled in ova, these results show that the data previously obtained by in vitro labelling of isolated tissues do indeed reflect the situation in the developing embryo.
2. Materials
and Methods
Labelling of embryos and extraction of collqens Groups of 60 embryos were labelled in ovo during +6 or 5-a days of development. Eggs were injected with 50 ,uCi of 3H-proline (New England Nuclear, NET-323) plus 300 pg of P-aminopropionitrile (BAPN) in a total volume of 100 pl. The /3-aminopropionitrile prevents crosslinking, thus rendering the collagen soluble. The eggs were returned to the incubator and about l$ days later embryos were removed and the corneas and vitreous were cleanly dissected into physiological saline. In some cases, to produce collagens with a higher specific activity, B-day corneas were labelled in vitro for 24 hr. The labelling medium consisted of Dulbecco Modified Eagle’s Medium supplemented with 50,~g/ml BAPN, lOO,~g/ml ascorbic acid and 50,&i/ml 3H-proline. All tissues were extracted for 24 hr in @4 ionic strength potassium phosphate buffer, pH 7.6 at 4%. From previous experiments we know this extraction removes the majority of the collagen from these two tissues. The extracts were clarified by centrifugation and the supernatants dialysed exhaustively against the same buffer to remove unincorporated counts. In most experiments to furt,her remove non-collagenous material, the extracts were dialysed into 0~5 M-HAc and treated with pepsin (2OO,~g/ml at 4% for 24 hr). The pepsin was inactivated by neutralizing the solution and any proteolytically released material was removed by dialysis against potassium phosphate buffer. Preparation of antibodies Highly purified collagens were used both as immunogens and to test the purity of the antibodies. The type I collagen was a neutral salt soluble fraction extracted from 3-week-old lathyritic chicken skins. The type II was extracted from adult sternal cartilages and was extracted by limited pepsin digestion at 4’C (Miller, 1972). All collagens were purified by numerous salt precipitation steps from both neutral and acid solutions and low ionic strength precipitations at neutral pH. In addition, ethanol precipitations (14 %) were performed and the type II collagen was separated from type I by fractional salt precipitations (Trelstad, Kang, Toole and Gross, 1972). Antibodies against these two types of collagens were produced in rabbits according to von der Mark, von der Mark and Gay (1976). Five milligram quantities of antigens were administered subcutaneously with the initial injection emulsified in complete Freunds adjuvant and subsequent boosters in incomplete adjuvant. Serum was collected and the IgG fraction isolated by precipitation with 45 “/b ammonium sulfate followed by DEAE-cellulose fractionation. Finally, the antibodies were purified by affinity chromatography using immunoabsorbants of the collagens bound to CNBr-activated sepharose 4B. Cross reactive anticollagen antibodies were removed by passing the IgG over an affinity column of the heterologous collagen type until, by passive hemagglutination. (Beil, Furthmayer and Timpl, 1972), all detectable antitype I activity was removed from the type II preparation and vice versa. Then any non-collagen antibodies present were removed by subsequent passage of the preparations over affinity columns of the homologous collagen type. In the latter purification
TYPE
II COLLAGEN
373
step the antibodies which bound to the column were eluted with 3 M-KSCN and were immediately dialyzed against 605 M-Tris pH 7.5.0.15 M-NaCl. The final antibody concentrations were adjusted to 64 mg/ml. Immunoprecipitation of labelled collagens Immunoprecipitations of radiolabelled collagens were performed using killed S. aureu.3 (Staph A) as a solid phase immunoprecipitating agent (Kessler, 1975). Lyophilized Staph A was obtained from the Enzyme Center (Tufts University, Boston, MA) and was precpcled through 95 y0 Triton X-100 according to their directions. Antibody reactions were performed in buffer A which consisted of 605 M-T+ pH 55. 915 M-NaCl. 0005 M-EDTA and O.l”/” Triton X-100. Each incubation consisted of 100 yl of labelled antigen, a volume of antibody (IgG content 94 mg/ml) which varied from 5 to 506 ~1, an amount of buffer A to bring the volume to 6OOg1, plus 6yl of bovine serum albumin (100 mg/ml) and sodium azide to a final concentration of 601~~. The tubes were incubated overnight at 4OC and then 100 ,el of a 25 “/o suspension of Staph A in buffer A was added to each t,ube and the tubes were placed on a rocking table at room temperature for 1 hr. The Staph A antibody complex was collected by centrifugation, washed once with buffer A and the supernatants pooled. The pellet was extracted with 2 94 SDS in 606 M-Tris pH 6.8 at 1OOY’ for 10 min; the Staph A was removed by centrifugation, washed once with the 2 o/oSDS and the supernatants pooled. The supernatants were counted in 10 ml of Hydroflour (National Diagnostics) in a Beckman Scintillation Counter. Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions using 6 y0 gels (Laemmeli, 1970) and processed for fluorography using X-ray film (Royal X-OMAT) that had been prefogged (Bonner and Laskey. 1974; Laskry and Mills. 1975). They were exposed at -9O’C. OH-proline determination8 To determine the relative proportions of proline to 4-hydroxyproline, some fractions were hydrolized in 6 N-HCl for 24 hr and the two amino acids were separated on a Jelco automated amino acid analyser with a portion of the effluent being diverted by stream splitting. The effluent fractions were counted in 10 ml of hydrofluor in a Beckman scintillation counter.
3. Results Antibody
specificity
in immunoprecipitation
The purity of the antibodies was monitored at each step of their purification by passive hemagglutination. The final titers for type I antibody preparations, expressed as twofold serial dilutions, ranged from 8 to 10 ( -logz); and those of type II preparations ranged from 11 to 13 (-log,). No preparations had detectible reactivity to the heterologous collagen type as judged by this assay procedure. The antibodies were also tested for specificity and ability to interact with purified 3H-proline labelled collagen types I and II in the solid phase immunoprecipitation assay described in Methods. The standard collagens were extracted from in vitro labelled l7-day embryonic calvaria and from 12-day embryonic sterna. Calvarium, being a membrane bone, is almost entirely type I and contains no type II, whereas the sternum is predominately type II but may contain a small amount of type I cont,ributed by the perichondrium (see Miller, 1976). When increasing amounts of antitype II antibody were added to the sternal collagen preparation, increasing radioactivity was precipitated [Fig. l(a)]. In different experiments the maximum concentration of antibody (250 yl) precipitated 55-62 “/o
374
---
\ 0 s ‘Z ” .a !
--+I
-- ___-------I I
4 I
3co(b) 250 200-
_&----+--
_----
- -----4
1%
Y-1 I loo ; 50:
0
o:z
: 100
200
xx,
400
, 500
Antlbody odded (pl)
FIG. 1. Lmmunoprecipitation calvaria
(b) using affinity
curves of radiolabelled collagens from chick sternal cartilage (a) and chick purified antibodies against collagen types I (--O- --) and II (-a-).
of the labelled hydroxyproline. The precipitate contained about 42 y0 of the total labelled imino acid as 4-hydroxyproline. In the same assay, the antitype I antibody precipitated less than 3 y0 of the counts, thus attesting to its lack of cross-reactivity with the type II collagen. Conversely, when the two antibody preparations were tested on the collagen extracted from calvaria [Fig. l(b)], in various experiments the curves obtained with the antitype I antibody plateaued when 68-80 y0 of the hydroxyproline counts were precipitated, whereas the antitype II antibody removed less than 1 ‘x of the counts. In the type I precipitates, 4-hydroxyproline accounted for 4244 T/bof the total proline. Antibody
precipitation
of in ovo labelled cornea and vitreous
Neutral salt soluble, 3H-proline labelled material was extracted from corneas and vitreous bodies that had been labelled in ovo during 4+6 or 5-g days of embryonic development (five dozen embryos per experiment). The material was also subjected to limited pepsin digestion t,o remove most noncollagenous proteins and t,hen assayed by immunoprecipitation. Labelled cornea1 material was precipitate by both the antitype I and the antitype II collagen antibodies [Fig. 2(b) and Table I]. In four different experiments, the labelled collagen precipitated by the two antibodies, when added together, amounted to 46-75 y0 of the total CPM in the samples. Of the collagen precipitated, 3M6 V0 was accounted for by the antitype II antibodies. Radiolabelled material from the vitreous bodies reacted only with the antitype II collagen antibody [Fig. 2(a) and Table I]. in these preparations, the antitype I and type II antibodies together precipitated 41-42 y0 of the total counts and greater than 90% was due to the type II antibody.
TYPE
0
loo
IT (‘OLI ,.l(:ES
200
MO
400
500
Antibody added (pl)
FIG. 1. Immunoprecipitation c,hivk (‘ornea (h) using affinity
curves of in ova-labelled collagens from chick embryonic vitreous (a) and purified antibodies against’cnllagen types I (- -0 --- ) and II (-0 -- -).
TAHI,F: 1
Prwipitntions Tisnur ROWW (lahelling periods of individual rxperiments)
Total c/min sample
SIM- PAGE analysis
with antibodies c/min Precipitated by antitype I (% of total c/min)
of antibody
agtimt
rollayen
c/min Precipitated by antitgpe II ( “‘. of total r/min)
types I md
OOPrecipitated counts in type II
I1
oo Total sample c/min precipitated by both antibodies
precipitates
Due to the small amounts of labelled collagen obtainable from in ovo precipit,ations. we were unable to further analyse the antibody precipitates and supernatants. Thus. to generate collagen with enough radioactivity for subsequent analysis by SDS-PACE, we used in vitro labelled &day corneas as the source of labelled material. In this case. after precipitation with antibodies, the labelled material remaining in the supernatants and the material stripped from the precipitates with boiling SDS were analyzed by SDS-PAGE fluorography. As can be seen in Fig. 3, the cornea1 material precipitated by t.hc antitype 1 antibody
376 (bl
(0) Type I! P
Front -
_“\
s
TYPO ’ P
S
-
-
FIG. 3. Fluorogram of immunoprecipitates (P) and supernatents (S) of radiolabelled collagens after SDS-PAGE. Collagens obtained from in vitro-labelled 5-day embryonic corneas. (b) results obtained with antitype I collagen antibody. (a) results obtained with antitype II collagen antibody. (type I, P) contained bands corresponding to al and a2 chains in the 2 : 1 ratio expected for a type I collagen. The material remaining in the supernatant (type I, S) had a strong al-chain band and a much lighter one corresponding to a2 chain. This result would be expected if the type I antibody specifically precipitated most of the type I collagen leaving the type II plus a small amount of type I in the supernatant. Analysis of antitype II antibody precipitate (type II, P) by SDS-PAGE revealed a single band which correspond to an al chain. The antitype II supernatant (type II, S) contained both al and a2 chain bands. It can also be seen in Fig. 3, that the antitype II supernatant (type II, S) contains several extremely light bands migrating slower than the al chain. Some of these bands. at times could also be detected in antibody precipitates. Their identities are unknown, however they may represent P-chain dimers and y-chain trimers of both collagen types and possibily grace amounts of some other collagens.
4. Discussion The present investigation both confirms and extends the results obtained in our previous biochemical characterizations of the collagens synthesized by in vitro organ
TYPE
II
COLLAGES
377
cultures of cornea1 epithelium (Linsenmayer et.al., 1977 j, and of organ and cell cultures of the neural retina (Smith et al., 1976; Linsenmayer and Little, 1978). In the present invest,igation we deemed it desirable to use in ovo labelled material, since it has been shown that under certain in vitro conditions, some connective tissue cells. including those of the cornea1 stroma, can change their biosynthetic patterns (Conrad, Dessau and von der Mark, 1980). Compared to in vitro labelled tissue, however, the amount of proline labelled collagen obtained from in ovo labelled tissue is much less. The antibodies showed the required specificity when tested in the immunoprecipit’ation assay using labelled sternal and calvaria collagen standards. although as stated they did not precipitate all of the homologous labelled collagen leaving 20-40 ?b in the supernatant fractions. Thus the data presented here, are only semiquantitative. It is not known why the antibody preparations did not precipitate all of the labelled collagens. However, different antibody preparations tended to behave similarly, both with respect to the shape of the precipitation curves obtained with increasing amounts of antibody and the total percentages of collagen precipitated. One possibility is that certain antigenic sites within collagen molecules can be destroyed by various proteolytic cleavages during extraction and other artifactual antigenic sights can be created, thus rendering a percentage of the molecules antigenically different. Alternatively, what is considered to be a single, homogenous collagen type may represent more than one gene product. Also, various post-translational modifications may be incomplete (Balian, Click, Hermodson and Bornstein, 1972), changing the antigenicity of a portion of the molecules. Giving credence to these latter two possibilities is the observation that the monoclonal antibodies produced against collagen types I (Linsenmayer, Hendrix and Little, 1979) and II (Linsenmayer and Hendrix, 1980), which have the narrowest specificity, precipitate a considerably smaller proportion of the respective collagen types than do the conventional antibodies*. The in ovo-labelled primary cornea1 stroma had 3(r47 “I0 of its antibody-precipitable collagen as type II. This fits well with our previous chromatographic analyses of the collagen+ synthesized in culture by isolated cornea1 epithelia (Linsenmayer et al.. 1977). In that study, the al :a2 ratios of chains obtained by carboxymethyl-cellulose chromatography suggested a type II content of 33-42 y/o. That the antibody precipitations showed the vitreous at this stage to be predominantly type II is also consistent with previous biochemical investigations. Newsome et al. (1976) showed that the collagen within the vitreous at this stage contained only al type chains, and also showed that this collagen was probably synthesized by the neural retina which then secreted it into the vitreous space. Subsequently, Smith et al. (1976) showed that the major CNBr peptides of this in vitro synthesized retina collagen are homologous t,o those of type II. The only other collagen type that has been detected in neural retina cell cultures is a series of procollagen forms which are probably related to type V (AB) (Linsenmayer and Little, 1978). These type V precursors account for less than 10 96 of the collagen synthesized in vitro, and in ovo they may not even be secreted along with the type II into the vitreous space. Instead they may remain associated with the neural retina cell layer. It is now apparent that the type II collagen of the cornea and vitreous are similar if not’ identical to the type II of hyaline cartilage by a number of criteria including the immunological cross-reactivity shown here and in the immunohistochemical study of von der Mark et al. (1977). * Since the monoclonal antibodies of labelled collagens. we used affinity
against collagen types purified. conventional
I and II precipitate only a small percentage antibodies in the present investigation.
37x
7’. F. I~Ih’SESMAl’EK
ET AI,
(in press) have In addition, Hendrix, Hay. von der ,Mark and Linsenmagrr performed an immunohistochemical study of t’he developing cornea at’ hot11 the fluorescence and electron microscopical levels. using monoclonal antibodies aga,inst collagens type 1 (Linsenmayer et al.. 1979) and type II (Linsenmayrr and Hendrix. 1980). In that study we observed by immunofluorescence that in the primary st,roma of the early embryonic !-day chick, equivalent to those used here. the type I and 11 are uniformly distributed throughout’ the stroma. lmmunoferritin labelling showed both collagens occur within the striated fibrils. After fihroblast invasion and during subsequent development of the secondary, mature stroma t,he immunofluorescence staining for type II collagen disappears from all but t’he anterior (epithelial) and posterior (endothelial) regions. At all stages the stromas retain uniform staining for type I with either staining procedure. Eventually, the type I1 staining disappears from the anterior portion of the adult stromas, but remains within t*hr posterior region forming part of the chick Descemet’s membrane. The fate, of the type II in the vitreous is different. Here the type I1 remains as the only detectable collagen, even into the adult (Swann. Constable and Harper. 1972). During development, however, the t,issue responsible for its synthesis changes from the neural retina which is responsible for early synthesis, to a cell population within the vitreous itself (possibly hyalocytes) which is responsible for later synthesis (Newsome et’ al., 1976). The function of type II collagen in the avian stroma is still not known, which is not surprising since it is not yet possible to correlate molecular properties of any of the collagen types with a specific function. However, the most striking features of this extracellular matrix is the precise orthogonal arrangement of the collagen layers and the uniform diameters of the fibrils which compose these layers (see Hay et al.. 1979). Possibly the type I and II molecules interact to determine fibril diameter, orthogonality or both. We are currently examining the possibility that fibril diameter is controlled by the two types of molecules acting in consort.
ACKNOWLEDGMENTS We thank Dr Jerome Gross for helpful suggestions throughout the course of this work. This is publication number 871 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. This study was supported by NIH grants EY02261 and AM03564.
Thomas
F. Linsenmayer
Award, AMO0031. Charles D. Little
was recipient
of an NIH
Research
was supported by Training
Career
Development
Grant, HD07092.
REFERENCES
Balian, G., Click, E. M., Hermodson,
M. A. and Bornstein,
P. (1972). Structure
of rat skin
collagen al-CB8. Amino acid sequence of the hydroxylamine-produced fragment HA 2. Biochemistry 11, 3798-806. Beil. W., Furthermayer, H. and Timpl, R. (1972). Chicken antibodies to soluble rat collagen. 1. Characterization of the immune response by precipitation and agglutination methods. Immunochemistry 9, 779-88. Bonner. W. M. and Laskey, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-8. Conrad, G. W., Dessau, W. and von der Mark, Ii. (1980). Synthesis of type III collagen by fibroblasts from the embryonic chick cornea. J. Cell Biol. 84, 501-12.
TYPE
IT (‘ol,LA(:ES
379
Hay, E.. Linsenmayer, T. F‘., Trelstad, R. L. and van der Mark, K. (1979). Origin and distribution of collagens in the developing avian cornea. In C’urrent Topics In Zhy Rasearch. Vol. 1 (Eds Zadunaisky. J. and Davson. H.). Pp. l-35. Academic Press. New \;ork. Hendrix. M. ,J. C’.. Hay, E. D., von der Mark. K. and Linsenmayer, T. F. (In press). lmmunohistochemical localization of collagen types I and II in the developing chick cornea by electron microscopy. Invest Ophthulmol. Kessler. S. W. (1975). Rapid isolation of antigens from cells with a staphylococcal protein .I-antibody ahsorhent.: parameters of the interaction of antihotly-ant)igen complexes with Protein 4. J. Zmmunol. 115. 1617-24. Laskry. R. A. and Mills, A. D. (1975). Q uantitative tilm detection of 3H and *Y’ in polyacrylamide gels by Auorography. Ew. .I. Hiorhrlrl. 56. X35-41. Laemmli, C’. K. (1970). Cleavage of struct,ural proteins during the assembly of the head of bacteriophage T4. Nu~ure 227. 680-5. Linsenmayer. T. F. and Hendrix, M. J. C’. (1980). M onoclonal antibodies to connective tissue macromolecules: type II collagen. Biochem. Biophys. Kes. C’ommun. 92, 440-6. Linsenmayer. T. F.. Hendrix. M. J. (‘. and Litt,l*b. C. I). (1979). Produt+on and characterization of monclonal antibody against chicken Type 1 c.ollagen. I’mc. Sut. .Icc~rl.Sri. f’.S.=l. 76, 3703-7. Linsenmayer. T. F. and Little. C. I). (1978). Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of Type II plus a new genetic type. I’roc. :Vrrl. Acad. Sri. I’.S.A. 75. 3235-9. Linsenmayer. T. F.. Smith. 0. N. and Hay, E. (1977). Svnthesis of two collagen types by rmhryonic: chick cornea1 epithelium in vitro. Proc. :$ut. .&ad. Sri. I~,S.A. 74, 3%43. Miller, E. J. (1972). Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11. 4903-n. Miller, E. J. (1976). Biochemical characteristics and biological signifit*ancr of the geneticallydistinct collagens. Mol. Cell Biochpm. 13, 165-92. Newsome. D. A.. Linsenmayer, T. F. and Trelstad. R. L. (1976). Vitreous body collagen : Evidence for a dual origin from the neural retina and hyaloctyes. J. Cell Biol. 71. 59-67. Smith, G. X.. *Jr. Linsrntnayer, T. F. and Nrwsome. 1). A. (1976). Synthesis of Type 11 collagen in vitro by emhryonir chick neural retina tissue. f’roc. Nut. Acnd. Sri. I’.h’.A. 73. 448&X Swann. D. A.. (!onstable. I. ,J. and Harper, E. (1972). Vitreous structure III. Oomposition of bovine vitreous collagen. Invest. Ophthulmol. 11. 73558. Trelstad, R. L.. Kang. A. H., Toole, B. P. and ($ross. J. (1972). Collagen heterogeneity. High resolution separation of native [a 1(I)], at? and (al (II)], and their component a chains. .J. Hiol.
(‘hwn.
247, 646S73.
van der Mark. H.. von der Mark, K. and Gay, S. (1976). Study of differential collagen sivnthesistiuring development of the chick embryo by immunofluorescence. 1. preparation of collagen Type I and Type II specific antibodies and their application to early stages of t,he chick emhryo. Dev. Biol. 48, 237-49. van der Mark. Ii., vor, der Mark, H.. Timpl. R. and Trrlstad. R. L. (1977). Immunofluorescent localization of collagen Types I. II and III in the embryonic chick eye. I)rv. Biol. 59. -7~-85.
Type II Collagen Vitreous: THOMAS
in the Early Embryonic Immunoradiochemical
F. LINSENMAYER,EILEEN
GIBNEY
Chick Cornea Evidence
AND CHARLES
and
D. LITTLE
The Developmental Biology Laboratory, Departments of Medicine and Anatomy, Harvard Medkal School and the Massachusetts General Hospital, Boston, MA 02114. U.S.A. (Received 30 January
1981~ ,Vew York)
The relative proportion of collagen types I and II was examined in early chick corneas and vitrous bodies by radioimmunoprecipitation. Embryos (4-5 days of incubation) were labelled in ova with 3H-proline. After an additional l-2 days, corneas and vitreous bodies were removed and the collagen was extracted and treated by limited pepsinization. Then, the labelled collagens were reacted with affinity purified antibodies against collagen types I and II, and the immune complexes precipitated with protein A-containing Staphylococc~e aureu8. In some cases, the precipitated collagen was further analysed by SDS-polyacrylamide gel electrophoresis. The results for the vitreous showed that greater than 99% of the labelled material precipitated by the antibodies was in type II. In the cornea1 samples, on the other hand, both types of antibodies precipitated appreciable labelled collagen with somewhat less than half being type II (3&46 %). These results are in agreement with our earlier biochemical observations on the relative content of type II collagen in these extracellular matrix-rich structures from embryonic avian eyes, Key words: cornea1 stroma; immune precipitation; collagen antibody: vitreous collagen; neural retina; collagen types.
1. Introduction In the vertebrate eye, both the cornea and the vitreous body owe their transparency in part to the orderly deposition of collagen into fibrils with a uniform diameter. In t’he developing chick embryo, the collagenous components within each of these structures are synthesized in two discrete stages and at each stage, a different cell population is responsible for synthesis. The earliest cornea1 matrix, termed the primary stroma, is synthesized by the cornea1 epithelium and is initially devoid of cells (for review see Hay, Linsenmayer, Trelstad and von der Mark, 1979). At about 5-6 days of development, this acellular stroma becomes populated by mesenchymal cells which subsequently differentiate into the cornea1 fibroblasts responsible for the synthesis of the mature cornea1 stroma. Likewise, for the vitreous the earliest collagen is also synthesized by an adjacent epithelium - in this case the primitive neural retina (Newsome, Linsenmayer and Trelstad, 1976). Subsequently the neural retina loses this capacity and by 12 days of development the vitreous collagen is largely synthesized by a cell population within t’he vitreous itself - possibly the hyalocytes. Biochemical analysis of in vitro labelled material has suggested that type II collagen, or a type II-like molecules is a major component of both the primary cornea1 stroma and the embryonic vitreous. Organ cultures of 5-6 day embryonic cornea1 epithelia, devoid of mesenchymal cells, synthesize a collagenous matrix which, in the electron microscope, appears similar to the primary cornea1 stroma as seen in vitro (see Hay et al., 1979). When incubated with H-proline, these cultures synthesize two major types of labelled collagens which by a number of criteria, are indistinguishable (K)l~-1835/82/030371+09 $01.00/O
0 1982 Academic Press Inc. (London) Limited
372
T. F. I~IESEh’MAYEK.
ET AL
from types 1 and 11 (Linsenmayer, Smith and Hay, 1977). Likewise. using biochemic*al criteria, in vitro cultures of 6-7 day whole embryonic neural retinas (Smith. cultures of neural retina cells Linsenmayer and Newsome, 1976) or monolayer (Linsenmayer and Little, 1978), synthesize type II as their predominant collagen type. In the present investigation we have used immunoradiochemical criteria to further analyse the synthesis of collagens within the eyes of embryos labelled in ovo. The results confirm the presence of a type II collagen molecule in both the early embryonic cornea and vitreous. Also in this study, since the embryos were labelled in ova, these results show that the data previously obtained by in vitro labelling of isolated tissues do indeed reflect the situation in the developing embryo.
2. Materials
and Methods
Labelling of embryos and extraction of collqens Groups of 60 embryos were labelled in ovo during +6 or 5-a days of development. Eggs were injected with 50 ,uCi of 3H-proline (New England Nuclear, NET-323) plus 300 pg of P-aminopropionitrile (BAPN) in a total volume of 100 pl. The /3-aminopropionitrile prevents crosslinking, thus rendering the collagen soluble. The eggs were returned to the incubator and about l$ days later embryos were removed and the corneas and vitreous were cleanly dissected into physiological saline. In some cases, to produce collagens with a higher specific activity, B-day corneas were labelled in vitro for 24 hr. The labelling medium consisted of Dulbecco Modified Eagle’s Medium supplemented with 50,~g/ml BAPN, lOO,~g/ml ascorbic acid and 50,&i/ml 3H-proline. All tissues were extracted for 24 hr in @4 ionic strength potassium phosphate buffer, pH 7.6 at 4%. From previous experiments we know this extraction removes the majority of the collagen from these two tissues. The extracts were clarified by centrifugation and the supernatants dialysed exhaustively against the same buffer to remove unincorporated counts. In most experiments to furt,her remove non-collagenous material, the extracts were dialysed into 0~5 M-HAc and treated with pepsin (2OO,~g/ml at 4% for 24 hr). The pepsin was inactivated by neutralizing the solution and any proteolytically released material was removed by dialysis against potassium phosphate buffer. Preparation of antibodies Highly purified collagens were used both as immunogens and to test the purity of the antibodies. The type I collagen was a neutral salt soluble fraction extracted from 3-week-old lathyritic chicken skins. The type II was extracted from adult sternal cartilages and was extracted by limited pepsin digestion at 4’C (Miller, 1972). All collagens were purified by numerous salt precipitation steps from both neutral and acid solutions and low ionic strength precipitations at neutral pH. In addition, ethanol precipitations (14 %) were performed and the type II collagen was separated from type I by fractional salt precipitations (Trelstad, Kang, Toole and Gross, 1972). Antibodies against these two types of collagens were produced in rabbits according to von der Mark, von der Mark and Gay (1976). Five milligram quantities of antigens were administered subcutaneously with the initial injection emulsified in complete Freunds adjuvant and subsequent boosters in incomplete adjuvant. Serum was collected and the IgG fraction isolated by precipitation with 45 “/b ammonium sulfate followed by DEAE-cellulose fractionation. Finally, the antibodies were purified by affinity chromatography using immunoabsorbants of the collagens bound to CNBr-activated sepharose 4B. Cross reactive anticollagen antibodies were removed by passing the IgG over an affinity column of the heterologous collagen type until, by passive hemagglutination. (Beil, Furthmayer and Timpl, 1972), all detectable antitype I activity was removed from the type II preparation and vice versa. Then any non-collagen antibodies present were removed by subsequent passage of the preparations over affinity columns of the homologous collagen type. In the latter purification
TYPE
II COLLAGEN
373
step the antibodies which bound to the column were eluted with 3 M-KSCN and were immediately dialyzed against 605 M-Tris pH 7.5.0.15 M-NaCl. The final antibody concentrations were adjusted to 64 mg/ml. Immunoprecipitation of labelled collagens Immunoprecipitations of radiolabelled collagens were performed using killed S. aureu.3 (Staph A) as a solid phase immunoprecipitating agent (Kessler, 1975). Lyophilized Staph A was obtained from the Enzyme Center (Tufts University, Boston, MA) and was precpcled through 95 y0 Triton X-100 according to their directions. Antibody reactions were performed in buffer A which consisted of 605 M-T+ pH 55. 915 M-NaCl. 0005 M-EDTA and O.l”/” Triton X-100. Each incubation consisted of 100 yl of labelled antigen, a volume of antibody (IgG content 94 mg/ml) which varied from 5 to 506 ~1, an amount of buffer A to bring the volume to 6OOg1, plus 6yl of bovine serum albumin (100 mg/ml) and sodium azide to a final concentration of 601~~. The tubes were incubated overnight at 4OC and then 100 ,el of a 25 “/o suspension of Staph A in buffer A was added to each t,ube and the tubes were placed on a rocking table at room temperature for 1 hr. The Staph A antibody complex was collected by centrifugation, washed once with buffer A and the supernatants pooled. The pellet was extracted with 2 94 SDS in 606 M-Tris pH 6.8 at 1OOY’ for 10 min; the Staph A was removed by centrifugation, washed once with the 2 o/oSDS and the supernatants pooled. The supernatants were counted in 10 ml of Hydroflour (National Diagnostics) in a Beckman Scintillation Counter. Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions using 6 y0 gels (Laemmeli, 1970) and processed for fluorography using X-ray film (Royal X-OMAT) that had been prefogged (Bonner and Laskey. 1974; Laskry and Mills. 1975). They were exposed at -9O’C. OH-proline determination8 To determine the relative proportions of proline to 4-hydroxyproline, some fractions were hydrolized in 6 N-HCl for 24 hr and the two amino acids were separated on a Jelco automated amino acid analyser with a portion of the effluent being diverted by stream splitting. The effluent fractions were counted in 10 ml of hydrofluor in a Beckman scintillation counter.
3. Results Antibody
specificity
in immunoprecipitation
The purity of the antibodies was monitored at each step of their purification by passive hemagglutination. The final titers for type I antibody preparations, expressed as twofold serial dilutions, ranged from 8 to 10 ( -logz); and those of type II preparations ranged from 11 to 13 (-log,). No preparations had detectible reactivity to the heterologous collagen type as judged by this assay procedure. The antibodies were also tested for specificity and ability to interact with purified 3H-proline labelled collagen types I and II in the solid phase immunoprecipitation assay described in Methods. The standard collagens were extracted from in vitro labelled l7-day embryonic calvaria and from 12-day embryonic sterna. Calvarium, being a membrane bone, is almost entirely type I and contains no type II, whereas the sternum is predominately type II but may contain a small amount of type I cont,ributed by the perichondrium (see Miller, 1976). When increasing amounts of antitype II antibody were added to the sternal collagen preparation, increasing radioactivity was precipitated [Fig. l(a)]. In different experiments the maximum concentration of antibody (250 yl) precipitated 55-62 “/o
374
---
\ 0 s ‘Z ” .a !
--+I
-- ___-------I I
4 I
3co(b) 250 200-
_&----+--
_----
- -----4
1%
Y-1 I loo ; 50:
0
o:z
: 100
200
xx,
400
, 500
Antlbody odded (pl)
FIG. 1. Lmmunoprecipitation calvaria
(b) using affinity
curves of radiolabelled collagens from chick sternal cartilage (a) and chick purified antibodies against collagen types I (--O- --) and II (-a-).
of the labelled hydroxyproline. The precipitate contained about 42 y0 of the total labelled imino acid as 4-hydroxyproline. In the same assay, the antitype I antibody precipitated less than 3 y0 of the counts, thus attesting to its lack of cross-reactivity with the type II collagen. Conversely, when the two antibody preparations were tested on the collagen extracted from calvaria [Fig. l(b)], in various experiments the curves obtained with the antitype I antibody plateaued when 68-80 y0 of the hydroxyproline counts were precipitated, whereas the antitype II antibody removed less than 1 ‘x of the counts. In the type I precipitates, 4-hydroxyproline accounted for 4244 T/bof the total proline. Antibody
precipitation
of in ovo labelled cornea and vitreous
Neutral salt soluble, 3H-proline labelled material was extracted from corneas and vitreous bodies that had been labelled in ovo during 4+6 or 5-g days of embryonic development (five dozen embryos per experiment). The material was also subjected to limited pepsin digestion t,o remove most noncollagenous proteins and t,hen assayed by immunoprecipitation. Labelled cornea1 material was precipitate by both the antitype I and the antitype II collagen antibodies [Fig. 2(b) and Table I]. In four different experiments, the labelled collagen precipitated by the two antibodies, when added together, amounted to 46-75 y0 of the total CPM in the samples. Of the collagen precipitated, 3M6 V0 was accounted for by the antitype II antibodies. Radiolabelled material from the vitreous bodies reacted only with the antitype II collagen antibody [Fig. 2(a) and Table I]. in these preparations, the antitype I and type II antibodies together precipitated 41-42 y0 of the total counts and greater than 90% was due to the type II antibody.
TYPE
0
loo
IT (‘OLI ,.l(:ES
200
MO
400
500
Antibody added (pl)
FIG. 1. Immunoprecipitation c,hivk (‘ornea (h) using affinity
curves of in ova-labelled collagens from chick embryonic vitreous (a) and purified antibodies against’cnllagen types I (- -0 --- ) and II (-0 -- -).
TAHI,F: 1
Prwipitntions Tisnur ROWW (lahelling periods of individual rxperiments)
Total c/min sample
SIM- PAGE analysis
with antibodies c/min Precipitated by antitype I (% of total c/min)
of antibody
agtimt
rollayen
c/min Precipitated by antitgpe II ( “‘. of total r/min)
types I md
OOPrecipitated counts in type II
I1
oo Total sample c/min precipitated by both antibodies
precipitates
Due to the small amounts of labelled collagen obtainable from in ovo precipit,ations. we were unable to further analyse the antibody precipitates and supernatants. Thus. to generate collagen with enough radioactivity for subsequent analysis by SDS-PACE, we used in vitro labelled &day corneas as the source of labelled material. In this case. after precipitation with antibodies, the labelled material remaining in the supernatants and the material stripped from the precipitates with boiling SDS were analyzed by SDS-PAGE fluorography. As can be seen in Fig. 3, the cornea1 material precipitated by t.hc antitype 1 antibody
376 (bl
(0) Type I! P
Front -
_“\
s
TYPO ’ P
S
-
-
FIG. 3. Fluorogram of immunoprecipitates (P) and supernatents (S) of radiolabelled collagens after SDS-PAGE. Collagens obtained from in vitro-labelled 5-day embryonic corneas. (b) results obtained with antitype I collagen antibody. (a) results obtained with antitype II collagen antibody. (type I, P) contained bands corresponding to al and a2 chains in the 2 : 1 ratio expected for a type I collagen. The material remaining in the supernatant (type I, S) had a strong al-chain band and a much lighter one corresponding to a2 chain. This result would be expected if the type I antibody specifically precipitated most of the type I collagen leaving the type II plus a small amount of type I in the supernatant. Analysis of antitype II antibody precipitate (type II, P) by SDS-PAGE revealed a single band which correspond to an al chain. The antitype II supernatant (type II, S) contained both al and a2 chain bands. It can also be seen in Fig. 3, that the antitype II supernatant (type II, S) contains several extremely light bands migrating slower than the al chain. Some of these bands. at times could also be detected in antibody precipitates. Their identities are unknown, however they may represent P-chain dimers and y-chain trimers of both collagen types and possibily grace amounts of some other collagens.
4. Discussion The present investigation both confirms and extends the results obtained in our previous biochemical characterizations of the collagens synthesized by in vitro organ
TYPE
II
COLLAGES
377
cultures of cornea1 epithelium (Linsenmayer et.al., 1977 j, and of organ and cell cultures of the neural retina (Smith et al., 1976; Linsenmayer and Little, 1978). In the present invest,igation we deemed it desirable to use in ovo labelled material, since it has been shown that under certain in vitro conditions, some connective tissue cells. including those of the cornea1 stroma, can change their biosynthetic patterns (Conrad, Dessau and von der Mark, 1980). Compared to in vitro labelled tissue, however, the amount of proline labelled collagen obtained from in ovo labelled tissue is much less. The antibodies showed the required specificity when tested in the immunoprecipit’ation assay using labelled sternal and calvaria collagen standards. although as stated they did not precipitate all of the homologous labelled collagen leaving 20-40 ?b in the supernatant fractions. Thus the data presented here, are only semiquantitative. It is not known why the antibody preparations did not precipitate all of the labelled collagens. However, different antibody preparations tended to behave similarly, both with respect to the shape of the precipitation curves obtained with increasing amounts of antibody and the total percentages of collagen precipitated. One possibility is that certain antigenic sites within collagen molecules can be destroyed by various proteolytic cleavages during extraction and other artifactual antigenic sights can be created, thus rendering a percentage of the molecules antigenically different. Alternatively, what is considered to be a single, homogenous collagen type may represent more than one gene product. Also, various post-translational modifications may be incomplete (Balian, Click, Hermodson and Bornstein, 1972), changing the antigenicity of a portion of the molecules. Giving credence to these latter two possibilities is the observation that the monoclonal antibodies produced against collagen types I (Linsenmayer, Hendrix and Little, 1979) and II (Linsenmayer and Hendrix, 1980), which have the narrowest specificity, precipitate a considerably smaller proportion of the respective collagen types than do the conventional antibodies*. The in ovo-labelled primary cornea1 stroma had 3(r47 “I0 of its antibody-precipitable collagen as type II. This fits well with our previous chromatographic analyses of the collagen+ synthesized in culture by isolated cornea1 epithelia (Linsenmayer et al.. 1977). In that study, the al :a2 ratios of chains obtained by carboxymethyl-cellulose chromatography suggested a type II content of 33-42 y/o. That the antibody precipitations showed the vitreous at this stage to be predominantly type II is also consistent with previous biochemical investigations. Newsome et al. (1976) showed that the collagen within the vitreous at this stage contained only al type chains, and also showed that this collagen was probably synthesized by the neural retina which then secreted it into the vitreous space. Subsequently, Smith et al. (1976) showed that the major CNBr peptides of this in vitro synthesized retina collagen are homologous t,o those of type II. The only other collagen type that has been detected in neural retina cell cultures is a series of procollagen forms which are probably related to type V (AB) (Linsenmayer and Little, 1978). These type V precursors account for less than 10 96 of the collagen synthesized in vitro, and in ovo they may not even be secreted along with the type II into the vitreous space. Instead they may remain associated with the neural retina cell layer. It is now apparent that the type II collagen of the cornea and vitreous are similar if not’ identical to the type II of hyaline cartilage by a number of criteria including the immunological cross-reactivity shown here and in the immunohistochemical study of von der Mark et al. (1977). * Since the monoclonal antibodies of labelled collagens. we used affinity
against collagen types purified. conventional
I and II precipitate only a small percentage antibodies in the present investigation.
37x
7’. F. I~Ih’SESMAl’EK
ET AI,
(in press) have In addition, Hendrix, Hay. von der ,Mark and Linsenmagrr performed an immunohistochemical study of t’he developing cornea at’ hot11 the fluorescence and electron microscopical levels. using monoclonal antibodies aga,inst collagens type 1 (Linsenmayer et al.. 1979) and type II (Linsenmayrr and Hendrix. 1980). In that study we observed by immunofluorescence that in the primary st,roma of the early embryonic !-day chick, equivalent to those used here. the type I and 11 are uniformly distributed throughout’ the stroma. lmmunoferritin labelling showed both collagens occur within the striated fibrils. After fihroblast invasion and during subsequent development of the secondary, mature stroma t,he immunofluorescence staining for type II collagen disappears from all but t’he anterior (epithelial) and posterior (endothelial) regions. At all stages the stromas retain uniform staining for type I with either staining procedure. Eventually, the type I1 staining disappears from the anterior portion of the adult stromas, but remains within t*hr posterior region forming part of the chick Descemet’s membrane. The fate, of the type II in the vitreous is different. Here the type I1 remains as the only detectable collagen, even into the adult (Swann. Constable and Harper. 1972). During development, however, the t,issue responsible for its synthesis changes from the neural retina which is responsible for early synthesis, to a cell population within the vitreous itself (possibly hyalocytes) which is responsible for later synthesis (Newsome et’ al., 1976). The function of type II collagen in the avian stroma is still not known, which is not surprising since it is not yet possible to correlate molecular properties of any of the collagen types with a specific function. However, the most striking features of this extracellular matrix is the precise orthogonal arrangement of the collagen layers and the uniform diameters of the fibrils which compose these layers (see Hay et al.. 1979). Possibly the type I and II molecules interact to determine fibril diameter, orthogonality or both. We are currently examining the possibility that fibril diameter is controlled by the two types of molecules acting in consort.
ACKNOWLEDGMENTS We thank Dr Jerome Gross for helpful suggestions throughout the course of this work. This is publication number 871 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. This study was supported by NIH grants EY02261 and AM03564.
Thomas
F. Linsenmayer
Award, AMO0031. Charles D. Little
was recipient
of an NIH
Research
was supported by Training
Career
Development
Grant, HD07092.
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P. (1972). Structure
of rat skin
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IT (‘ol,LA(:ES
379
Hay, E.. Linsenmayer, T. F‘., Trelstad, R. L. and van der Mark, K. (1979). Origin and distribution of collagens in the developing avian cornea. In C’urrent Topics In Zhy Rasearch. Vol. 1 (Eds Zadunaisky. J. and Davson. H.). Pp. l-35. Academic Press. New \;ork. Hendrix. M. ,J. C’.. Hay, E. D., von der Mark. K. and Linsenmayer, T. F. (In press). lmmunohistochemical localization of collagen types I and II in the developing chick cornea by electron microscopy. Invest Ophthulmol. Kessler. S. W. (1975). Rapid isolation of antigens from cells with a staphylococcal protein .I-antibody ahsorhent.: parameters of the interaction of antihotly-ant)igen complexes with Protein 4. J. Zmmunol. 115. 1617-24. Laskry. R. A. and Mills, A. D. (1975). Q uantitative tilm detection of 3H and *Y’ in polyacrylamide gels by Auorography. Ew. .I. Hiorhrlrl. 56. X35-41. Laemmli, C’. K. (1970). Cleavage of struct,ural proteins during the assembly of the head of bacteriophage T4. Nu~ure 227. 680-5. Linsenmayer. T. F. and Hendrix, M. J. C’. (1980). M onoclonal antibodies to connective tissue macromolecules: type II collagen. Biochem. Biophys. Kes. C’ommun. 92, 440-6. Linsenmayer. T. F.. Hendrix. M. J. (‘. and Litt,l*b. C. I). (1979). Produt+on and characterization of monclonal antibody against chicken Type 1 c.ollagen. I’mc. Sut. .Icc~rl.Sri. f’.S.=l. 76, 3703-7. Linsenmayer. T. F. and Little. C. I). (1978). Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of Type II plus a new genetic type. I’roc. :Vrrl. Acad. Sri. I’.S.A. 75. 3235-9. Linsenmayer. T. F.. Smith. 0. N. and Hay, E. (1977). Svnthesis of two collagen types by rmhryonic: chick cornea1 epithelium in vitro. Proc. :$ut. .&ad. Sri. I~,S.A. 74, 3%43. Miller, E. J. (1972). Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11. 4903-n. Miller, E. J. (1976). Biochemical characteristics and biological signifit*ancr of the geneticallydistinct collagens. Mol. Cell Biochpm. 13, 165-92. Newsome. D. A.. Linsenmayer, T. F. and Trelstad. R. L. (1976). Vitreous body collagen : Evidence for a dual origin from the neural retina and hyaloctyes. J. Cell Biol. 71. 59-67. Smith, G. X.. *Jr. Linsrntnayer, T. F. and Nrwsome. 1). A. (1976). Synthesis of Type 11 collagen in vitro by emhryonir chick neural retina tissue. f’roc. Nut. Acnd. Sri. I’.h’.A. 73. 448&X Swann. D. A.. (!onstable. I. ,J. and Harper, E. (1972). Vitreous structure III. Oomposition of bovine vitreous collagen. Invest. Ophthulmol. 11. 73558. Trelstad, R. L.. Kang. A. H., Toole, B. P. and ($ross. J. (1972). Collagen heterogeneity. High resolution separation of native [a 1(I)], at? and (al (II)], and their component a chains. .J. Hiol.
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van der Mark. H.. von der Mark, K. and Gay, S. (1976). Study of differential collagen sivnthesistiuring development of the chick embryo by immunofluorescence. 1. preparation of collagen Type I and Type II specific antibodies and their application to early stages of t,he chick emhryo. Dev. Biol. 48, 237-49. van der Mark. Ii., vor, der Mark, H.. Timpl. R. and Trrlstad. R. L. (1977). Immunofluorescent localization of collagen Types I. II and III in the embryonic chick eye. I)rv. Biol. 59. -7~-85.