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Environmental enhancement of in vitro chondrogenesis
DEVELOPMENTAL
BIOLOGY
36, 210-220 (1973)
Environmental
Enhancement
IV. Stimulation
of ln vitro Chondrogenesis
of Somite Chondrogenesis
by Exogenous
Chondromucoprotein’ F~OBERT A. KOSHER, JAMES Department
of
Anatomy, University
of
W. LASH, AND RONALD R. MINOR
Pennsylvania School
of
Medicine, Philadelphia, Pennsylvania 19174
Accepted June 22, 1973 Proteoglycan complex extracted from embryonic cartilage (chondromucoprotein) with 4.0 M guanidinium chloride greatly stimulates in vitro somite chondrogenesis. In the presence of exogenous chondromucoprotein (CMP) which consists predominantly of proteochondroitin sulfate, there is a large increase in the amount of differentiating cartilage which can be detected visually in somite explants. There is a 2-3-fold increase in the amount of sulfated glycosaminoglycans (including chondroitin 4- and 6-sulfate) accumulated by somite explants supplied with exogenous CMP complex. These results are of potential significance, since during the period of interaction between the notochord or sninal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan. INTRODUCTION
Embryonic notochord and spinal cord have been shown by many workers to promote, or “induce” chondrogenesis of somitic mesoderm (see Lash, 1968a, for review). It has also been established, however, that embryonic chick somites can synthesize chondroitin sulfate and can undergo chondrogenesis in vitro in the absence of spinal cord or notochord (Lash et al., 1962; Lash, 1967, 1968a-c; Mar&lo and Lash, 1967; Ellison et al., 1969; Ellison and Lash, 1971). The evocation of this chondrogenic expression was brought about by improving the culture environment (Ellison and Lash, 1971; Minor et al., 1973), by the addition of extractable tissue components (Lash et al., 1962), or by minor alterations in the potassium concentration of the balanced salt solutions in the media (Lash, 1972; Lash et al., 1973). Nevertheless, the inducing tissues (notochord and spinal cord) do greatly enhance the rate and the amount of matrix accumulation in vitro (Lash, 1967; Ellison and Lash, 1971; Gordon and Lash, 1974). In ‘Supported by grant HD-00380 from the U.S. Public Health Service (JWL) and a U.S.P.H.S. postdoctoral fellowship (RAK) .
addition, earlier in vivo studies demonstrated the enhancing role of the spinal cord and notochord in somite chondrogenesis (Holtzer and Detwiler, 1953; Strudel, 1953; Watterson et al., 1954). Thus on the above-mentioned considerations, it is firmly established that the role of the spinal cord and notochord is primary to the enhancement or stabilization of a preexisting chondrogenic bias of somites. The mechanism by which the spinal cord and notochord evoke an enhancement of the chondrogenic bias of somites is unknown. Cohen and Hay (1971) have demonstrated that during the time of interaction between spinal cord and somitic mesoderm (2-4 days of development), the chick spinal cord synthesizes and secretes collagen (see also Trelstad et al., 1973). Although these authors stressed the importance of collagen in the tissue interaction, they also observed that other extracellular matrix materials were being secreted, (presumably proteoglycan granules), as seen with electron microscopy. Minor (1973) has demonstrated that the spinal cord and notochord in uiuo and in vitro synthesize and secrete not only thin unbanded collagen fibrils, but also 200-800 A proteoglycan granules, and his results also suggested an
210 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
KOSHER,
LASH, AND MINOR
Exogenous Chondromucoprotein
association of the induction of somite chondrogenesis with the synthesis and secretion of these matrix components by the inducer tissues. In addition, Ruggeri (1972) has observed that glycosaminoglycans and collagen fibrils secreted by the notochord ih uiuo become distributed amongst the adjacent sclerotomal cells. This author suggested that slcerotomal cells begin to overtly differentiate into cartilage at the time they interact with this extracellular material. In addition to these electron microscopic, histochemical, and autoradiographic analyses, there are many reports presenting biochemical evidence that the embryonic spinal cord and notochord synthesize sulfated glycosaminoglycans, including chondroitin sulfate (FrancoBrowder et al., 1963; Lash et al., 1964; Marzullo and Lash, 1967; Lash, 1968a; Kvist and Finnegan, 1970a, b; Manasek, 1970; Strudel, 1971). In addition, it is of interest that the only other tissue (i.e., hypertrophying embryonic cartilage) which has been demonstrated to stimulate somite chondrogenesis irz vitro is actively synthesizing and secreting glycosaminoglycans (Cooper, 1965). The above studies, coupled with Nevo and Dorfman’s (1972) observation that exogenous proteoglycan stimulates proteoglycan production by chondrocytes in shortterm suspension culture, have prompted us to investigate the possibility that proteoglycan synthesized and secreted by the notochord and spinal cord is involved in the enhancement of somite chondrogenesis. In the experiments to be reported here, we have found that proteoglycan complex (chondromucoprotein) consisting predominantly of proteochondroitin sulfate greatly stimulates in vitro somite chondrogenesis. MATERIALS
AND
METHODS
Preparation and assay of cultures. Somites were dissected from stage 17 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks as previously described (Ellison and Lash, 1971). Explants
and Chondrogenesis
211
consisting of 8-10 somites were cultured on nutrient agar containing Simms’ balanced salt solution (SBSS), fetal calf serum (FCS), and the nutrient supplement F12X in the proportions 2 : 2 : 1 (see Gordon and Lash, 1974; Minor, 1973). Somites were derived from the region of the embryo trunk between the anterior border of the fore limb bud, and the posterior border of the hind limb bud. The explants of randomized somites were fed with liquid nutrient medium containing SBSS, FCS, and F12X (2 : 2 : 1) For labeling the chondroitin sulfate synthesized, the nutrient agar and liquid nutrient feeding medium contained 5.0 &i/ml of carrier free NaZs5S0, (Amersham/Searle). To determine the effect of upon chonexogenous proteoglycan drogenesis, the liquid feeding medium was supplemented with approximately 200 rg/ml of chondromucoprotein (see below). Cultures were examined daily for visual signs of chondrogenesis. Preparation of chondromucoprotein complex. Chondromucoprotein complex
(i.e., proteoglycan, or protein polysaccharide complex) was extracted from lo-day embryonic chick vertebral cartilage and 13-l&day embryonic chick sternal cartilage with 4.0 M guanidinium chloride, 0.05 M Tris.HCl (pH 7.5), as described by Sadjera and Hascall (1969). The extract was clarified by centrifugation and dialyzed for 48 hr at 4°C against distilled water. The dialyzate was further clarified by centrifugation and filtration, and then lyophilized (see Sadjera and Hascall, 1969). Characterization of the chondromucoprotein complex. The uranic acid content
of the chondromucoprotein preparations was determined by the Bitter and Muir (1962) modification of the carbazole method of Dische (1947) using sodium glucuronate as standard. The amount of chondroitin 4-sulfate and chondroitin 6-sulfate present in the extracts was determined by the following procedures. Aliquots of the chondromuco-
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DEVELOPMENTALBIOLOGY
protein extracts were incubated at 37°C with 1 unit of chondroitinase ABC (Miles Laboratories, Inc.) in the presence of enriched Tris buffer, pH 8.0 (prepared as described by Saito et al., 1969). After 3 hr, an additional 1 unit of enzyme was added, and the reaction mixture was incubated an additional 16 hr at 37°C. The amount of dissaccharides unsaturated 4-sulfated (ADi-4s; 2-acetamido-2-deoxy-3-0-(/3-ngluco-4-enepyranosyluronic acid)-4-sulfon-galactose) and 6-sulfated disaccharides (ADi-6s; 2-acetamido-2-deoxy-3-O-@-0gluco-4-enepyranosyluronic acid)S-sulfon-galactosel released by the enzyme was determined by the thiobarbituric acid procedure described by Hascall et al. (1972) using ADi-4S (Miles Laboratories, Inc.) as standard. The release of ADi-6S was specifically monitored by the Reissig et aZ. (1955) modification of the Morgan and Elson (1934) procedure for N-acetylhexosamine using ADi-6S as standard. Disaccharides sulfated in position 4 are unreactive in the hexosamine assay (see Hascall et al., 1972). The hydroxyproline, and hence collagen, content of the chondromucoprotein extracts was determined on hydrolyzates of the extracts by the Switzer and Summer (1971) modification of the procedures of Juva and Prockop (1966) and Kivirikko et al. (1967). The DNA content of the extracts was determined by the Santoianni and Ayala (1965) modification of the procedure of Kissane and Robins (1958), except that fluorescence was determined in 1 N HCl instead of 0.6 N PCA (Hinegardner, 1971). RNA was determined by the orcinol procedures of Schneider (1955) and Dische (1955). Analysis of sulfated glycosaminoglycan synthesis. The amount of sulfated glycosaminoglycan (GAG) accumulated by somite explants has been shown to be a sensitive index of their chondrogenic expression (Ellison and Lash, 1971; Lash et al., 1973).
VOLUME 35,1973
The incorporation of *%-sulfate into sulfated GAG is a sensitive assay of sulfated GAG synthesis, since sulfation occurs concomitant with chain elongation during the synthesis of the polysaccharide chains of chondroitin sulfate (Telser et al., 1966; Meezan and Davidson, 1967; DeLuca and Silbert, 1968; Derge and Davidson, 1972). Therefore, the incorporation of YS-sulfate into sulfated GAG by explants in the presence or absence of exogenous chondromucoprotein complex was determined by three different procedures. Virtually identical results were obtained using each procedure. In the first procedure, explants which had been continuously exposed to Nal%O, for the time interval under study, were washed three times with 100 volumes of SBSS, sonicated in 0.3 ml of distilled water, and aliquots (50 ~1) were removed for DNA determinations. The sonicates were placed in boiling water for 3 min, cooled, and then digested for 24 hr at 55°C in 0.2 A4 Tris.HCl (pH 8.0) containing 400 &ml of Pronase (Calbiochem). The digests were made 10% with respect to trichloroacetic acid (TCA) at 2”C, and then the precipitates compacted by centrifugation in an International clinical centrifuge. The supernatants were dialyzed overnight against 0.05 M NaSO,, and then for 24 hr against distilled water. The dialyzates were lyophilized, dissolved in distilled water, and aliquots were removed for determination of radioactive GAG in a toluene based scintillation fluid containing 5% BioSolv (Beckman). The incorporation of %-sulfate into sulfated GAG was also determined by the method of Fratantoni et al. (1968) and by a modification of the method of Overman and Beaudoin (1971). The latter procedure was modified as follows. After treatment with saturated Na&SO,, the explants were sonicated, and aliquots of the sonicates were removed for DNA determinations. The remainder was solubilized in NCS (Amersham/Searle), supplied with scintil-
KOSHER, LASH, AND MINOR
Exogenous Chnndromucoprotein and Chondrogenesis
lation fluid (PPO and POPOP in toluene), and the amount of radioactivity was determined with an Intertechnic liquid scintillation spectrometer. This procedure proved to be adequate for the determination of 36S-sulfate-labeled GAG in small numbers (5-10) of explants, since it was determined that virtually all of the radioactivity extracted by the saturated NalSOI was dialyzable. More than 97% of the labeled material remaining in the tissue was protease resistant and nondialyzable (i.e., sulfated GAG). Identification of sulfated glycosaminoglycans. The relative amounts of the iso-
merit chondroitin sulfates synthesized by the somite explants were determined by the enzymatic method of Saito et al. (1968) using chondroitinase ABC and AC as previously described (Kosher and Searls, 1972). RESULTS
Characterization of the Chondromucoprotein Complexes
The dialyzed chondromucoprotein complex extracted from 13-X-day embryonic chick sternal cartilage (sternal CMP) with 4.0 M guanidinium chloride contained 2.01 pmoles/ml of uranic acid and the lo-day vertebral CMP extract contained 1.65 Ccmoles/ml of uranic acid. Following chondroitinase ABC treatment, 1.95 pmoles/ml (889 &ml) of unsaturated disaccharide residues could be detected in the sternal CMP complex by the thiobarbituric acid (TBA) procedure of Hascall et al. (1972), and 1.53 hmoles/ml of unsaturated disaccharide residues could be detected in the vertebral CMP extract. Therefore, the number of pmoles of unsaturated disaccharides released from each complex by chondroitinase ABC treatment is about equal to the number *of micromoles of uranic acid in the extracts. This indicates that nearly all of the uranic acid containing polysaccharides of the extracts are de-
213
graded to disaccharide residues after chondroitinase ABC treatment, and are therefore chondroitin sulfate. In addition, following chondroitinase treatment, 0.68 pmoles/ml and 0.77 pmoles/ml of MorganElson reactive N-acetylhexosamine were detected in the sternal and vertebral CMP preparations, respectively. Since 4-sulfated disaccharides are unreactive in the hexosamine assay, the amount of MorganElson reactive N-acetylhexosamine present after chondroitinase digestion is a measure of the amount of unsaturated 6-sulfated disaccharides released by the enzyme, i.e., the amount of degraded chondroitin g-sulfate. Comparison of the total uranic acid content of the extracts with the total amount of unsaturated disaccharides released by chondroitinase (TBA procedure) and the amount of g-sulfated disaccharides released by the enzyme (MorganElson reaction) indicates that the uranic acid containing polysaccharides of the sternal CMP complex consist of approximately 65% chondroitin 4-sulfate and 35% chondroitin g-sulfate, while the vertebral CMP complex consists of approximately 50% chondroitin 4-sulfate and 50% chondroitin g-sulfate. That the polysaccharide chains of the molecules are attached to protein is indicated by the fact that the uranic acid is rendered TCA-soluble after Pronase digestion. The collagen content of the CMP extracts was estimated from the amount of hydroxyproline in hydrolyzates of the extracts. The hydroxyproline content of the sternal CMP extract was 0.003 pmole/ml, and the hydroxyproline content of the vertebral CMP extract was 0.0023 pmolel ml. Therefore, the amount of hydroxyproline in both the sternal and vertebral CMP extracts is less than 0.5% of the amount of uranic acid. DNA was not detectable in as much as 3 ml of either the sternal or vertebral CMP extract using an extremely sensitive fluorometric procedure (Santoianni and Ayala,
214
DEVELOPMENTALBIOLOGY
1965) which is able to detect approximately 5 ng of DNA. Similarly, RNA was not detectable in either extract.
Accumulation
Although both control somite explants and those supplied with exogenous chondromucoprotein (CMP explants) possessed numerous translucent areas after 18-24 hr, the number of these areas was greater in the CMP explants. These translucent areas have been shown to contain the same structured matrix components as differentiated cartilage, although in lesser amounts (Minor, 1973). The presence of these translucent areas is consistently associated with chondrogenic differentiation in cultured somites (Gordon and Lash, 1973; Minor, 1973; Minor et al., 1973). After 3 days of culture, the incidence of chondrogenesis was virtually 160% in both control and CMP explants. The amount of cartilaginous tissue in the CMP explants, however, was considerably greater than the amount in the control explants. Since it is difficult to accurately quantitate the amount of this tissue in explants by visual observations, the major criterion used was the incorporation of *%-sulfate into sulfated glycosaminoglycans. (A1
of Glycosaminoglycans
The accumulation of 85S-sulfate-labeled glycosaminoglycans (GAG) by control and CMP explants is shown in Fig. 1. There was little difference in the amount of sulfated GAG accumulated by control, vertebral-CMP, or sternal-CMP explants during the first 46 hr. By 72 hr, however, the amount of GAG accumulated by the CMP explants was 2-3-fold greater than the amount accumulated by control explants (Fig. 1). During day 4 of culture, the amount of GAG accumulation by CMPexplants continued to increase, whereas there was little additional increase in the amount of GAG accumulation by control explants (Fig. 1). The pattern of accumulation of sulfated GAG by vertebral- and sternal-CMP explants was similar. The only difference observed was that vertebral-CMP explants began to accumulate GAG above control levels earlier than sternal-CMP explants (Fig. 1). The data shown in Fig. 1 are the results of typical experiments. In several similar experiments, an increase in the accumulation of s5S-sulfate-labeled GAG by CMP explants was always evident by the second or third day of culture, and the amount of GAG
Visual Appearance of Somite Explants
,
VOLUME 35.1373
,
1 IBI
FIG. 1. Accumulation of 86S-sulfate-labeled glycosaminoglycans by control explants (C) and by explants supplied with exogenous chondromucoprotein. (A) Exogenous chondromucoprotein derived from sternal cartilage (S). (B) Exogenous chondromucoprotein derived from vertebral cartilage (V). Amount of isotope incorporation determined as dpm x lo-‘/pg DNA.
KOSHER, LASH, AND MINOR
TABLE
Exogenous Chondromucoprotein and Chondrogenesis
1
AMOUNT OF T%~JLFATE-LABELED GLYCOSAMIETOCLYCANS ACCUMULATED BY CONTROLAND STERNAL-CMP EXPLANTS AFTER 72 I-k IN CULTURES
DPM/pg DNA CMP
Control
1587 1440 1401 1384 1230
802 780 704 695 610
0 One hundred control and sternal-CMP explants were prepared on the same dav. Bach value represents the amount of 9-sulfate-labeled GAG accumulated by 20 explants.
accumulated was always 2-3-fold greater than control explants. In one series of experiments, 200 control and 200 sternal CMP explants were exposed to *%-sulfate for 72 hr, and the amount of asS-sulfate-labeled GAG accumulated by each of 5 groups of 20 explants of each type was determined. Although there was some variability in the total amount of GAG accumulation, the amount accumulated by the CMP explants was always at least 2-fold greater than the control explants (Table 1).
215
48 hr after transfer, the amount of *%-sulfate-labeled GAG present in the explants was determined. No differences could be detected in the rate of degradation of previously synthesized sulfated GAG in either the control or CMP explants (Fig. 2). Therefore, the increased accumulation of sulfated GAG by the CMP explants is the result of increased synthesis. Viability
of Control and CMP Explants
Exogenous CMP does not increase the viability of somite explants. There is little difference in the DNA content of CMP- or control explants during any period of culture (Table 2). In both types of explants, the initial period of culture is marked by a decline in total DNA content which is indicative of cell death (see Ellison and Lash, 1971; Minor, 1973; Gordon and Lash, 1973; Minor et al., 1973). Identification of Sulfated Glycosaminoglycans
The types and relative amounts of the various sulfated GAG accumulated by the explants was determined after 2, 3, and 4 days of culture by analysis with the en-
Degradation of Accumulated Glycosaminoglycans
Since the difference in accumulation of sulfated GAG in control and CMP explants could be due to a decreased rate of degradation in the CMP-explants rather than an increased synthesis, the degradation of previously synthesized GAG in each type of culture was determined. Control and CMP explants were cultured for 48 hr in the presence of YS-sulfate, then removed from the nutrient agar, washed with 100 volumes of SBSS, and transferred to fresh nutrient agar lacking the isotope. Both types of explants were then fed with nonradioactive medium. The nutrient medium fed to the CMP explants was supplemented with CMP. At zero time, 24 hr, and
Pm. 2. Percentage of previously labeled glycosaminoglycan remaining in control (C) and stemalCMP explants after chase in unlabeled medium for 2 days. Control and CMP explants were cultured for 48 hr in the presence of “S-sulfate, washed, and transferred to nonradioactive CMP and control medium. At zero time, 24 hr, and 48 hr after transfer, the amount of *%-sulfate-labeled GAG present in the explants was determined.
216
DEVELOPMENTALBIOLOGY
zymes chondroitinase ABC and AC (see Kosher and Searls, 1973). Explants of each type (control and sternal- and vertebralCMP treated) accumulated chondroitin 6sulfate, chondroitin 4-sulfate, and chondroitinase-resistant material in the same relative amounts (Table 3). In all explants chondroitin 6-sulfate synthesis predominated. Thus, the increased accumulation of sulfated GAG by the CMP explants is not the result of a preferential synthesis of a particular type of chondroitin sulfate. TABLE
2
TOTAL DNA CONTENT OF CONTROL, STERNAL-CMP, AND VERTEBRAL-CMP SOMITE EXPLANTS AT VAIUOUS DAYS OF CULTURE Percentage of original DNA” Days Stemal-
Vertebral-
CMP
CMP
47 42 40 51
45 46 46 50
Control 1 2 3 4
46 44 44 50
“Data are expressed as percentage of original DNA, since the DNA content of freshly explanted somites varied from experiment to experiment because of slight differences in the age of the embryos or size of the explants. Zero time values ranged from approximately 4.0 pg DNA to 5.0 pg DNA/20 explants. Results are the mean of 4 determinations. Standard deviation was *2-3% in each case. TABLE
VOLUME 35,1973 DISCUSSION
The results of this investigation indicate that exogenous proteoglycan complex (chondromucoprotein) consisting predominantly of proteochondroitin sulfate stimulates in vitro somite chondrogenesis. Stage 17 embryonic chick somites, although synthesizing low levels of chondroitin sulfates, are not cartilaginous at the time of their explantation to the culture environment. The somites undergo chondrogenic differentiation in uitro. Although a small amount of cartilaginous tissue is visible in control somite explants by the third day of culture, the amount of cartilaginous tissue which forms on subsequent days, and the amount of glycosaminoglycan which accumulates, is greatly increased in the presence of exogenous chondromucoprotein. The control and the CMP explants accumulate *?S-sulfate labeled glycosaminoglycans at similar rates for the first 2 days of culture. Beginning on the third day of culture, the control explants show less *YS-sulfate accumulation and have fewer visible cartilaginous areas. One effect of the exogenous proteoglycan complex seems to be to maintain the initial rate of synthesis which, in the control culture environment, becomes seriously impaired after the first 2 days of culture. It is not known whether this failure in the maintenance of 3
RELATIVE AMOUNTS OF THE VARIOUS SULFATED GLYCOSAMINOGLYCANS(GAG) ACCUMULATED BY CONTROL AND CMP-EXPLANTS AVER 3, 4, AND 5 DAYS IN CULTURE AS DETERMINED BY CHONDRO~TINASEANALYSIS
Chondroitin g-sulfate” Chondroitin 4-sulfate* Chondroitinase-resistantc Chondroitin 6-sulfate Chondroitin 4-sulfate
5 Days
4 Days
3 Days GAG
Control (So)
CMP @)
Control (%)
CMP (OJO)
%?l0
CMP (S)
53 16 31
51 16 33
51 16 33
51 18 31
50 20 30
50 22 28
77123
76124
76123
74126
71/26
69/31
o Determined as the relative amount of radioactivity associated with g-sulfated disaccharide residues after chondroitinase ABC and AC treatment. b Relative amount of radioactivity associated with I-sulfated disaccharide residues after chondroitinase ABC and AC treatment. c S5-Sulfate-labeled GAG which is not degraded by chondroitinase ABC or AC, likely heparan sulfate or keratan sulfate.
KOSHER, LASH, AND MINOR
Exogenous Chondromucoprotein
synthetic activity involves an inhibitory condition in the control cultures. Although the lower *‘S-sulfate accumulation in the control explants may be partly due to undersulfation of the chondroitin sulfate molecules, this cannot be the major reason for the differences in ‘%-sulfate accumulation. The CMP explants invariably possess considerably more cartilaginous tissue than do the control explants, as well as accumulate 2-3 times as much “S-sulfated glycosaminoglycans. The differentiation of chondroblastic mesenchyme (somite sclerotome) into cartilage is enhanced by exogenous proteoglycan complex. These results are of potential significance in view of the following published observations. (1) Embryonic notochord and spinal cord influence somite chondrogenesis in vitro (Lash, 1963, 1967; Ellison and Lash, 1971) and in uivo (Holtzer and Detwiler, 1953; Strudel, 1953; Watterson et al., 1954). (2) Extracts from the embryonic spinal cord and notochord have been reported to stimulate somite chondrogenesis (Lash et al., 1962; Lash, 1963). These experiments were hindered by a lack of a suitably sensitive assay and the difficulty in obtaining adequate amounts of extracted material. These experiments are being reinitiated to compare the effect of the previously extracted material (Lash et al., 1962) with our currently available chondromucoprotein. (3) During the period of interaction between the notochord or spinal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan (France-Browder et al., 1963; Marzullo and Lash, 1967; Lash, 1968a-c; Kvist and Finnegan, 1970a,b; Manasek, 1970; Strudel, 1971; Cohen and Hay, 1971; Ruggeri, 1972; Minor, 1973), which becomes distributed among adjacent sclerotomal cells. Furthermore, it has been suggested that this extracellular matrix may be a causal factor in the condrogenic differentiation of the sclerotome cells (Strudel, 1971; Ruggeri, 1972; Minor, 1973).
and Chondrogenesis
217
In addition, it can be stated that the chondrogenic stimulation does not qualitatively alter the chondroitin sulfates synthesized. The indictment by Abbott et al. (1972) of our previous report that chondroitin 6-sulfate is the predominant glycosaminoglycan synthesized by embryonic somites (Marzullo and Lash, 1967) cannot be substantiated. Embryonic cartilage was chosen as the source of proteoglycan in the present study not only because of the ease of obtaining an adequate amount of material, but also because Minor (1973) has shown that the structured matrix components synthesized by the embryonic spinal cord and notochord are similar to those seen in lo-day vertebral cartilage. Experiments are currently in progress to determine whether the proteoglycan complex extracted from embryonic notochord and spinal cord, which would be of greater developmental significance, will also promote somite chondrogenesis. Preliminary results indicate that such a stimulation is achieved. By far the major components of the chondromucoprotein complexes used in the present study to stimulate somite chondrogenesis are proteochondroitin 4- and g-sulfate. The extracts do not contain DNA or RNA, and contain only trace amounts of collagen. The amount of hydroxyproline is less than 0.5% of the amount of uranic acid. It has however, been demonstrated by Hascall and Sadjera (1969) that the proteoglycan complex extracted from bovine nasal cartilage with 4.0 M guanidinium chloride contains minor glycoprotein components, which constitute less than 5% by weight of the complex. Although we feel it is unlikely that the trace amount of collagen or minor glycoprotein components of our chondromucoprotein extracts are responsible for the observed stimulation of in vitro somite chondrogenesis (simply because they are present in such small amounts compared to the large amount of proteoglycan), this possibility cannot at this time be conclusively eliminated. Since the proteoglycan molecules can be sepa-
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DEVELOPMENTAL
BIOLOGY
rated from the minor glycoprotein components and trace amount of soluble collagen by cesium chloride density gradient centrifugation, it will be important to determine the effect of purified proteoglycan subunit molecules on somite chondrogenesis. That polyanionic proteoglycans can stimulate chondrogenesis is certainly not an untenable view, since in another system it has been demonstrated that chondromucoprotein and other polyanions stimulate proteoglycan synthesis (Nevo and Dorfman, 1972). Even if the trace amount of collagen present in our extracts is involved in the stimulation along with the major proteoglycan components, it is significant that extracellular materials influence differentiation. At this point, it is admittedly speculative to discuss the mechanism whereby extracellular proteoglycan may stimulate the expression of the chondrogenic genotype. A likely site of action, however, is the cell membrane. It is conceivable, as suggested by Nevo and Dorfman (1972), that an interaction of proteoglycan with components of the cell surface can result in an activation or stimulation of already existing metabolic patterns. It is also tempting to relate the stimulation of chondrogenesis by extracellular proteoglycan to recent experiments which indicate that the extracellular cationic environment can have profound effects upon chondrogenic expression (Lash, 1972; Lash et al., 1973). A high molecular weight polyanion such as the proteoglycan complex would have the potential to bind, sequester, and possibly regulate the external ionic microenvironment of the chondrogenic cells. Attempts to correlate the results presented here with the effects of extractable tissue components (Lash et al., 1962; Lash, 1963) and of the ionic environment (Lash, 1972; Lash et al., 1973) will have to await further experimentation. REFERENCES ABaol~, J., MAYNE, R., and HOLTZER, H. (1972). Inhibition of cartilage development in organ cul-
VOLUME35,197s
tures of chick somites by the thymidine analog, 5-bromo-2-deoxyuridine. Develop. Biol. 26. 430-442. Brrrxx, T., and Mum, H. M. (1962). A modified uranic acid carbazole reaction. Anal. B&hem. 4, 339334. COHEN,A. M., and HAY, E. D. (1971). Secretion of collagen by embryonic neuroepithelium at the time of spinal cord-somite interaction. Develop. Biol. 26, 578-805. COOPER,G. W. (1965). Induction of somite chondrogenesis by cartilage and notochord: A correlation between inductive activity and specific stages of cytodifferentiation. Develop. Biol. 12, 185-212. DELUCA,S., and SILBERT,J. E. (1968). Biosynthesis of chondroitin sulfate. II. Incorporation of sulfate-*% into microsomal chondroitin sulfate. J. Biol. Chem. 243, 27252729. DERGE,J. G., and DAVIDSON,E. A. (1972). Proteinpolysaccharide biosynthesis. Membrane-bound saccharides. Biochem. J. 126, 217-223. DISCHE, Z. (1947). A new specific color reaction of hexuronic acids. J. Biol. Chem. 167, 189-198. DISCHE, Z. (1955). Color reactions of nucleic acid components. In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, pp. 285-305. Academic Press, New York. EUIBON, M. L., and LASH,J. W. (1971). Environmental enhancement of in vitro chondrogenesis. Develop. Biol. 26, 466-496. ELLISON,M. L., AMBROSE,E. J., and EASTY, G. C. (1969). Chondrogenesis in chick embryo somites in vitro. J. Embryol. Exp. Morphol. 21, 331-340. FRANCO-BROWDER, S., DERYDT, J., and DORPIIIAN,A. (1983). The identification of a sulfated mucopolysaccharide in chick embryos, stages 11-23. Proc. Nat. Acad. Sci. U.S. 49, 643-647. FRATANTONI, J. C., HALL, C. W., and NEUFELD,E. F. (1988). The defect in Hurler’s and Hunter’s syndromes: Faulty degradation of mucopolysaccharide. Rot. Nat. Acad. Sci. U.S. 60, 699-706. GORDON,J. S., and LASH, J. W. (1974). In vitro chondrogenesis and differential cell viability. Develop. Biol. In press. HAMBURGER, V., and HAMILTON,H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 33, 49-92. HUCALL, V. C., and SADJERA,S. W. (1969). Proteinpolysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. J. Biol. Chem. 244, 2364-2396. HASCALL,V. C., Rro~o, R. L., HAYWMD, J., JR., and REYNOLDS,C. C. (1972). Treatment of bovine nasal cartilage proteoglycan with chondroitinases from Flavobacterium heparinum andProteus vulgaris. J. Biol. Chem. 247, 4521-4526. HINEGARDNER, R. T. (1971). An improved fluorometric assay for DNA. Anal. Biochem. 39, 197-201. HOLTZER,H., and I%TWILER,S. R. (1953). An experi-
KOSHER,LASH, ANDMINOR
Exogenous Chondromucoprotein
mental analysis of the development of the spinal column. III. Induction of skeletogenous cells. J. Exp. 2001. 123, 335-366. JUVA,K., and PROCKOP,D. J. (1966). Modified procedure for the assay of Ha- or C”-labeled hydroxyproline. Anal. Biochem. 15, 17-83. KISSANE,J. M., and R.CJRINS, E. (1958). The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Ckem. 233, 184-188. KIYIRIKKO, K. I., LAITINEN, O., and PROCKOP,D. J. (1967). Modifications of a specific assay for hydroxyproline in urine. Anal. B&hem. 19: 249-255. KOSHER,R. A., and SEARLS,R. L. (1973). Sulfated mucopolysaccharide synthesis during the development of Rana pipiens. Develop. Biol. 32, 50-68. K~BT, T. N., and FINNEGAN,C. V. (1970a). The distribution of glycosaminoglycans in the axial region of the developing chick embryo. I. Histochemical analysis. J. Exp. 2001. 175, 221-240. KVIST, T. N., and FINNEGAN,C. V. (1970b). The distribution of glycosaminoglycans in the axial region of the developing chick embryo. II. Biochemical analysis. J. Exp. Zool. 175, 241-258. LASH, J. W. (1963). Tissue interaction and specific metabolic responses: Chondrogenic induction and differentiation. In “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), pp. 235-260. Academic Press, New York. LAs.H,J. W. (1967). Differential behavior of anterior and posterior embryonic chick somites in vitro. J. Exp. Zool. l&47-56. LASH, J. W. (1968a). Chondrogenesis: Genotypic and phenotypic expression. J. Cell Physiol. 72, Suppl. 1, 35-46. LASH, J. W. (196813). Phenotypic expression and differentiation: In vitro chondrogenesis. In “The Stability of the Differentiated State” (H. &sprung, ed.), pp. 17-24. Springer-Verlag, Berlin and New York. LASH, J. W. (1966c). Semitic mesenchyme and its response to cartilage induction. In “EpithelialMesenchymal Interactions” (R. F. Fleischmajer and R. E. Billingham, eds.), pp. 165-172. Williams & Wilkins Co., Baltimore, Maryland. LASH, J. W. (1972). In “The Comparative Molecular Biology of Extracellular Matrices” (H. Slavkin, ed.). Academic Press, New York. LASH, J. W., HOMMES,F. A., and ZILLIKEN,F. (1962). Induction of cell differentiation. The in vitro induction of vertebral cartilage with a low molecular weight tissue component. Biochim. Biopkys. Acta 56, 313-319. LASH,J. W., GLICK,M. C., and MADDEN,J. W. (1964). Cartilage induction in vitro and sulfate-activating enzymes. Nat. Cancer Inst. Monagr. 13, 39-49. LASH,J. W., ROSENE,K., MINOR, R. R., DANIEL,J. C., and KOSHER,R. A. (1973). Environmental enhancement of in uitro chondrogenesis. III. The influence
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of external potassium and chondrogenic differentiation. Develop. Biol. In press. MANASW, F. J. (1970). Sulfated extracellular matrix production in the embryonic heart and adjacent tissues. J. Exp. Zool. 174, 415-440. MARZIJLLO,G., and LASH,J. W. (1967). Acquisition of the chondrocytic phenotype. In “Experimental Biology and Medicine” (E. Hagen, W. Wechsler, and F. Zilliken, eds.), Vol. I, pp. 213-219. Karger, Basel. MEEZAN,E., and DAVIDSON,E. A. (1967). Mucopolysaccharide sulfation in chick embryo cartilage. II. Characterization of the endogenous acceptor. J. Biol. Chem. 242,49564962.
MINOR, R. R. (1973). Somite chondrogenesis: A structural analysis. J. Cell Biol. 56, 27-50. MINOR,R. R., LASH,J. W., and KOSHER,R. A. (1973). Environmental enhancement of in vitro chondrogenesis. II. Factors affecting early chondrogenic differentiation in cultured somites (In preparat.ion). MORGAN, W. T. J., and EISON, L. A. (1934). CXXXVIU. A calorimetric method for the determination of N-acetylglucosamine and N-acetylchondrosamine. Biochem. J. 28, 999995. Nxvo, Z., and DORPMAN,A. (1972). Stimulation of chondromucoprotein synthesis in chondrocytes by extracellular chondromucoprotein. Proc. Nat. Acad. Sci. U.S. 69, 2069-2072. OVERMAN, D. O., and BEAUDOIN,A. R. (1971). Early
biochemical changes in the embryonic rat heart after teratogenic treatment. Teratology 4,183-190. Rxas~c, J. L., STROMINGER, J. L., and LELOIR,L. F. (1955). A modified calorimetric method for the estimation of N-acetylamino sugan. J. Biol. Chem. 217,959-966. RUGGERI,A. (1972). Ultrastructural, histochemical, and autoradiographic studies on the developing chick notochord. Z. Anat. Entwicklungsgesch. 138, 20-33. SADJERA,S. W., and HASCALL,V. C. (1969). Proteinpolysaccharide complex from bovine nasal cartilage. Comparison of low and high shear extraction procedures. J. Biol. Chem. 244, 77-97. SAITO, H., YAMAGATA, T., and SUZUKI, S. (1966). Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 15361542. SANTOIANNI,P., and AYALA, M. (1965). Fluorometric ultramicroanalysis of deoxyribonucleic acid in human skin. J. Invest. Dermatol. 45, 99-103. SCHNEIDER,W. C. (1955). Determination of nucleic acids in tissues by pentose analysis. Methods Ensymol. 3, 690-694.
STRUDEL,G. (1953). L’influence morphogene du tube nerveux sur differentiation de la colonne vertebrale. C. R. Sot. Biol. 147, 132-133. STRUDEL,G. (1971). Material extracellulaire et chondrogenese vertebrale. C. R. Acad. Sci. Ser. D 272, 473-476. SWITZER,B. R., and SUMMER,G. K. (1971). Improved
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DEVELOPMENTAL
BIOLOGY
method for hydroxyproline analysis in tissue hydrolyzates. Anal. Biochem. 39, 437-491. TEU~R, A., RORINSON, H. C., and DORPMAN, A. (1966). The biosynthesis of chondroitin sulfate. Arch. Biothem. Biophys. 116, 459-465. TIWSTAD, R. L., KANG, A. H., COHEN, A. M., and HAY, E. D. (1973). Collagen synthesis in vitro by
VOLUME
35,1973
embryonic spinal cord epithelium. Science 179, 295-297. WATTERSON, R. L., FOWLIZR,I., and FOWLFS,B. J. (1954). The role of the neural tube and notochord in development of the axial skeleton of the chick. Amer. J. Anat. 95,337-399.
BIOLOGY
36, 210-220 (1973)
Environmental
Enhancement
IV. Stimulation
of ln vitro Chondrogenesis
of Somite Chondrogenesis
by Exogenous
Chondromucoprotein’ F~OBERT A. KOSHER, JAMES Department
of
Anatomy, University
of
W. LASH, AND RONALD R. MINOR
Pennsylvania School
of
Medicine, Philadelphia, Pennsylvania 19174
Accepted June 22, 1973 Proteoglycan complex extracted from embryonic cartilage (chondromucoprotein) with 4.0 M guanidinium chloride greatly stimulates in vitro somite chondrogenesis. In the presence of exogenous chondromucoprotein (CMP) which consists predominantly of proteochondroitin sulfate, there is a large increase in the amount of differentiating cartilage which can be detected visually in somite explants. There is a 2-3-fold increase in the amount of sulfated glycosaminoglycans (including chondroitin 4- and 6-sulfate) accumulated by somite explants supplied with exogenous CMP complex. These results are of potential significance, since during the period of interaction between the notochord or sninal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan. INTRODUCTION
Embryonic notochord and spinal cord have been shown by many workers to promote, or “induce” chondrogenesis of somitic mesoderm (see Lash, 1968a, for review). It has also been established, however, that embryonic chick somites can synthesize chondroitin sulfate and can undergo chondrogenesis in vitro in the absence of spinal cord or notochord (Lash et al., 1962; Lash, 1967, 1968a-c; Mar&lo and Lash, 1967; Ellison et al., 1969; Ellison and Lash, 1971). The evocation of this chondrogenic expression was brought about by improving the culture environment (Ellison and Lash, 1971; Minor et al., 1973), by the addition of extractable tissue components (Lash et al., 1962), or by minor alterations in the potassium concentration of the balanced salt solutions in the media (Lash, 1972; Lash et al., 1973). Nevertheless, the inducing tissues (notochord and spinal cord) do greatly enhance the rate and the amount of matrix accumulation in vitro (Lash, 1967; Ellison and Lash, 1971; Gordon and Lash, 1974). In ‘Supported by grant HD-00380 from the U.S. Public Health Service (JWL) and a U.S.P.H.S. postdoctoral fellowship (RAK) .
addition, earlier in vivo studies demonstrated the enhancing role of the spinal cord and notochord in somite chondrogenesis (Holtzer and Detwiler, 1953; Strudel, 1953; Watterson et al., 1954). Thus on the above-mentioned considerations, it is firmly established that the role of the spinal cord and notochord is primary to the enhancement or stabilization of a preexisting chondrogenic bias of somites. The mechanism by which the spinal cord and notochord evoke an enhancement of the chondrogenic bias of somites is unknown. Cohen and Hay (1971) have demonstrated that during the time of interaction between spinal cord and somitic mesoderm (2-4 days of development), the chick spinal cord synthesizes and secretes collagen (see also Trelstad et al., 1973). Although these authors stressed the importance of collagen in the tissue interaction, they also observed that other extracellular matrix materials were being secreted, (presumably proteoglycan granules), as seen with electron microscopy. Minor (1973) has demonstrated that the spinal cord and notochord in uiuo and in vitro synthesize and secrete not only thin unbanded collagen fibrils, but also 200-800 A proteoglycan granules, and his results also suggested an
210 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
KOSHER,
LASH, AND MINOR
Exogenous Chondromucoprotein
association of the induction of somite chondrogenesis with the synthesis and secretion of these matrix components by the inducer tissues. In addition, Ruggeri (1972) has observed that glycosaminoglycans and collagen fibrils secreted by the notochord ih uiuo become distributed amongst the adjacent sclerotomal cells. This author suggested that slcerotomal cells begin to overtly differentiate into cartilage at the time they interact with this extracellular material. In addition to these electron microscopic, histochemical, and autoradiographic analyses, there are many reports presenting biochemical evidence that the embryonic spinal cord and notochord synthesize sulfated glycosaminoglycans, including chondroitin sulfate (FrancoBrowder et al., 1963; Lash et al., 1964; Marzullo and Lash, 1967; Lash, 1968a; Kvist and Finnegan, 1970a, b; Manasek, 1970; Strudel, 1971). In addition, it is of interest that the only other tissue (i.e., hypertrophying embryonic cartilage) which has been demonstrated to stimulate somite chondrogenesis irz vitro is actively synthesizing and secreting glycosaminoglycans (Cooper, 1965). The above studies, coupled with Nevo and Dorfman’s (1972) observation that exogenous proteoglycan stimulates proteoglycan production by chondrocytes in shortterm suspension culture, have prompted us to investigate the possibility that proteoglycan synthesized and secreted by the notochord and spinal cord is involved in the enhancement of somite chondrogenesis. In the experiments to be reported here, we have found that proteoglycan complex (chondromucoprotein) consisting predominantly of proteochondroitin sulfate greatly stimulates in vitro somite chondrogenesis. MATERIALS
AND
METHODS
Preparation and assay of cultures. Somites were dissected from stage 17 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks as previously described (Ellison and Lash, 1971). Explants
and Chondrogenesis
211
consisting of 8-10 somites were cultured on nutrient agar containing Simms’ balanced salt solution (SBSS), fetal calf serum (FCS), and the nutrient supplement F12X in the proportions 2 : 2 : 1 (see Gordon and Lash, 1974; Minor, 1973). Somites were derived from the region of the embryo trunk between the anterior border of the fore limb bud, and the posterior border of the hind limb bud. The explants of randomized somites were fed with liquid nutrient medium containing SBSS, FCS, and F12X (2 : 2 : 1) For labeling the chondroitin sulfate synthesized, the nutrient agar and liquid nutrient feeding medium contained 5.0 &i/ml of carrier free NaZs5S0, (Amersham/Searle). To determine the effect of upon chonexogenous proteoglycan drogenesis, the liquid feeding medium was supplemented with approximately 200 rg/ml of chondromucoprotein (see below). Cultures were examined daily for visual signs of chondrogenesis. Preparation of chondromucoprotein complex. Chondromucoprotein complex
(i.e., proteoglycan, or protein polysaccharide complex) was extracted from lo-day embryonic chick vertebral cartilage and 13-l&day embryonic chick sternal cartilage with 4.0 M guanidinium chloride, 0.05 M Tris.HCl (pH 7.5), as described by Sadjera and Hascall (1969). The extract was clarified by centrifugation and dialyzed for 48 hr at 4°C against distilled water. The dialyzate was further clarified by centrifugation and filtration, and then lyophilized (see Sadjera and Hascall, 1969). Characterization of the chondromucoprotein complex. The uranic acid content
of the chondromucoprotein preparations was determined by the Bitter and Muir (1962) modification of the carbazole method of Dische (1947) using sodium glucuronate as standard. The amount of chondroitin 4-sulfate and chondroitin 6-sulfate present in the extracts was determined by the following procedures. Aliquots of the chondromuco-
212
DEVELOPMENTALBIOLOGY
protein extracts were incubated at 37°C with 1 unit of chondroitinase ABC (Miles Laboratories, Inc.) in the presence of enriched Tris buffer, pH 8.0 (prepared as described by Saito et al., 1969). After 3 hr, an additional 1 unit of enzyme was added, and the reaction mixture was incubated an additional 16 hr at 37°C. The amount of dissaccharides unsaturated 4-sulfated (ADi-4s; 2-acetamido-2-deoxy-3-0-(/3-ngluco-4-enepyranosyluronic acid)-4-sulfon-galactose) and 6-sulfated disaccharides (ADi-6s; 2-acetamido-2-deoxy-3-O-@-0gluco-4-enepyranosyluronic acid)S-sulfon-galactosel released by the enzyme was determined by the thiobarbituric acid procedure described by Hascall et al. (1972) using ADi-4S (Miles Laboratories, Inc.) as standard. The release of ADi-6S was specifically monitored by the Reissig et aZ. (1955) modification of the Morgan and Elson (1934) procedure for N-acetylhexosamine using ADi-6S as standard. Disaccharides sulfated in position 4 are unreactive in the hexosamine assay (see Hascall et al., 1972). The hydroxyproline, and hence collagen, content of the chondromucoprotein extracts was determined on hydrolyzates of the extracts by the Switzer and Summer (1971) modification of the procedures of Juva and Prockop (1966) and Kivirikko et al. (1967). The DNA content of the extracts was determined by the Santoianni and Ayala (1965) modification of the procedure of Kissane and Robins (1958), except that fluorescence was determined in 1 N HCl instead of 0.6 N PCA (Hinegardner, 1971). RNA was determined by the orcinol procedures of Schneider (1955) and Dische (1955). Analysis of sulfated glycosaminoglycan synthesis. The amount of sulfated glycosaminoglycan (GAG) accumulated by somite explants has been shown to be a sensitive index of their chondrogenic expression (Ellison and Lash, 1971; Lash et al., 1973).
VOLUME 35,1973
The incorporation of *%-sulfate into sulfated GAG is a sensitive assay of sulfated GAG synthesis, since sulfation occurs concomitant with chain elongation during the synthesis of the polysaccharide chains of chondroitin sulfate (Telser et al., 1966; Meezan and Davidson, 1967; DeLuca and Silbert, 1968; Derge and Davidson, 1972). Therefore, the incorporation of YS-sulfate into sulfated GAG by explants in the presence or absence of exogenous chondromucoprotein complex was determined by three different procedures. Virtually identical results were obtained using each procedure. In the first procedure, explants which had been continuously exposed to Nal%O, for the time interval under study, were washed three times with 100 volumes of SBSS, sonicated in 0.3 ml of distilled water, and aliquots (50 ~1) were removed for DNA determinations. The sonicates were placed in boiling water for 3 min, cooled, and then digested for 24 hr at 55°C in 0.2 A4 Tris.HCl (pH 8.0) containing 400 &ml of Pronase (Calbiochem). The digests were made 10% with respect to trichloroacetic acid (TCA) at 2”C, and then the precipitates compacted by centrifugation in an International clinical centrifuge. The supernatants were dialyzed overnight against 0.05 M NaSO,, and then for 24 hr against distilled water. The dialyzates were lyophilized, dissolved in distilled water, and aliquots were removed for determination of radioactive GAG in a toluene based scintillation fluid containing 5% BioSolv (Beckman). The incorporation of %-sulfate into sulfated GAG was also determined by the method of Fratantoni et al. (1968) and by a modification of the method of Overman and Beaudoin (1971). The latter procedure was modified as follows. After treatment with saturated Na&SO,, the explants were sonicated, and aliquots of the sonicates were removed for DNA determinations. The remainder was solubilized in NCS (Amersham/Searle), supplied with scintil-
KOSHER, LASH, AND MINOR
Exogenous Chnndromucoprotein and Chondrogenesis
lation fluid (PPO and POPOP in toluene), and the amount of radioactivity was determined with an Intertechnic liquid scintillation spectrometer. This procedure proved to be adequate for the determination of 36S-sulfate-labeled GAG in small numbers (5-10) of explants, since it was determined that virtually all of the radioactivity extracted by the saturated NalSOI was dialyzable. More than 97% of the labeled material remaining in the tissue was protease resistant and nondialyzable (i.e., sulfated GAG). Identification of sulfated glycosaminoglycans. The relative amounts of the iso-
merit chondroitin sulfates synthesized by the somite explants were determined by the enzymatic method of Saito et al. (1968) using chondroitinase ABC and AC as previously described (Kosher and Searls, 1972). RESULTS
Characterization of the Chondromucoprotein Complexes
The dialyzed chondromucoprotein complex extracted from 13-X-day embryonic chick sternal cartilage (sternal CMP) with 4.0 M guanidinium chloride contained 2.01 pmoles/ml of uranic acid and the lo-day vertebral CMP extract contained 1.65 Ccmoles/ml of uranic acid. Following chondroitinase ABC treatment, 1.95 pmoles/ml (889 &ml) of unsaturated disaccharide residues could be detected in the sternal CMP complex by the thiobarbituric acid (TBA) procedure of Hascall et al. (1972), and 1.53 hmoles/ml of unsaturated disaccharide residues could be detected in the vertebral CMP extract. Therefore, the number of pmoles of unsaturated disaccharides released from each complex by chondroitinase ABC treatment is about equal to the number *of micromoles of uranic acid in the extracts. This indicates that nearly all of the uranic acid containing polysaccharides of the extracts are de-
213
graded to disaccharide residues after chondroitinase ABC treatment, and are therefore chondroitin sulfate. In addition, following chondroitinase treatment, 0.68 pmoles/ml and 0.77 pmoles/ml of MorganElson reactive N-acetylhexosamine were detected in the sternal and vertebral CMP preparations, respectively. Since 4-sulfated disaccharides are unreactive in the hexosamine assay, the amount of MorganElson reactive N-acetylhexosamine present after chondroitinase digestion is a measure of the amount of unsaturated 6-sulfated disaccharides released by the enzyme, i.e., the amount of degraded chondroitin g-sulfate. Comparison of the total uranic acid content of the extracts with the total amount of unsaturated disaccharides released by chondroitinase (TBA procedure) and the amount of g-sulfated disaccharides released by the enzyme (MorganElson reaction) indicates that the uranic acid containing polysaccharides of the sternal CMP complex consist of approximately 65% chondroitin 4-sulfate and 35% chondroitin g-sulfate, while the vertebral CMP complex consists of approximately 50% chondroitin 4-sulfate and 50% chondroitin g-sulfate. That the polysaccharide chains of the molecules are attached to protein is indicated by the fact that the uranic acid is rendered TCA-soluble after Pronase digestion. The collagen content of the CMP extracts was estimated from the amount of hydroxyproline in hydrolyzates of the extracts. The hydroxyproline content of the sternal CMP extract was 0.003 pmole/ml, and the hydroxyproline content of the vertebral CMP extract was 0.0023 pmolel ml. Therefore, the amount of hydroxyproline in both the sternal and vertebral CMP extracts is less than 0.5% of the amount of uranic acid. DNA was not detectable in as much as 3 ml of either the sternal or vertebral CMP extract using an extremely sensitive fluorometric procedure (Santoianni and Ayala,
214
DEVELOPMENTALBIOLOGY
1965) which is able to detect approximately 5 ng of DNA. Similarly, RNA was not detectable in either extract.
Accumulation
Although both control somite explants and those supplied with exogenous chondromucoprotein (CMP explants) possessed numerous translucent areas after 18-24 hr, the number of these areas was greater in the CMP explants. These translucent areas have been shown to contain the same structured matrix components as differentiated cartilage, although in lesser amounts (Minor, 1973). The presence of these translucent areas is consistently associated with chondrogenic differentiation in cultured somites (Gordon and Lash, 1973; Minor, 1973; Minor et al., 1973). After 3 days of culture, the incidence of chondrogenesis was virtually 160% in both control and CMP explants. The amount of cartilaginous tissue in the CMP explants, however, was considerably greater than the amount in the control explants. Since it is difficult to accurately quantitate the amount of this tissue in explants by visual observations, the major criterion used was the incorporation of *%-sulfate into sulfated glycosaminoglycans. (A1
of Glycosaminoglycans
The accumulation of 85S-sulfate-labeled glycosaminoglycans (GAG) by control and CMP explants is shown in Fig. 1. There was little difference in the amount of sulfated GAG accumulated by control, vertebral-CMP, or sternal-CMP explants during the first 46 hr. By 72 hr, however, the amount of GAG accumulated by the CMP explants was 2-3-fold greater than the amount accumulated by control explants (Fig. 1). During day 4 of culture, the amount of GAG accumulation by CMPexplants continued to increase, whereas there was little additional increase in the amount of GAG accumulation by control explants (Fig. 1). The pattern of accumulation of sulfated GAG by vertebral- and sternal-CMP explants was similar. The only difference observed was that vertebral-CMP explants began to accumulate GAG above control levels earlier than sternal-CMP explants (Fig. 1). The data shown in Fig. 1 are the results of typical experiments. In several similar experiments, an increase in the accumulation of s5S-sulfate-labeled GAG by CMP explants was always evident by the second or third day of culture, and the amount of GAG
Visual Appearance of Somite Explants
,
VOLUME 35.1373
,
1 IBI
FIG. 1. Accumulation of 86S-sulfate-labeled glycosaminoglycans by control explants (C) and by explants supplied with exogenous chondromucoprotein. (A) Exogenous chondromucoprotein derived from sternal cartilage (S). (B) Exogenous chondromucoprotein derived from vertebral cartilage (V). Amount of isotope incorporation determined as dpm x lo-‘/pg DNA.
KOSHER, LASH, AND MINOR
TABLE
Exogenous Chondromucoprotein and Chondrogenesis
1
AMOUNT OF T%~JLFATE-LABELED GLYCOSAMIETOCLYCANS ACCUMULATED BY CONTROLAND STERNAL-CMP EXPLANTS AFTER 72 I-k IN CULTURES
DPM/pg DNA CMP
Control
1587 1440 1401 1384 1230
802 780 704 695 610
0 One hundred control and sternal-CMP explants were prepared on the same dav. Bach value represents the amount of 9-sulfate-labeled GAG accumulated by 20 explants.
accumulated was always 2-3-fold greater than control explants. In one series of experiments, 200 control and 200 sternal CMP explants were exposed to *%-sulfate for 72 hr, and the amount of asS-sulfate-labeled GAG accumulated by each of 5 groups of 20 explants of each type was determined. Although there was some variability in the total amount of GAG accumulation, the amount accumulated by the CMP explants was always at least 2-fold greater than the control explants (Table 1).
215
48 hr after transfer, the amount of *%-sulfate-labeled GAG present in the explants was determined. No differences could be detected in the rate of degradation of previously synthesized sulfated GAG in either the control or CMP explants (Fig. 2). Therefore, the increased accumulation of sulfated GAG by the CMP explants is the result of increased synthesis. Viability
of Control and CMP Explants
Exogenous CMP does not increase the viability of somite explants. There is little difference in the DNA content of CMP- or control explants during any period of culture (Table 2). In both types of explants, the initial period of culture is marked by a decline in total DNA content which is indicative of cell death (see Ellison and Lash, 1971; Minor, 1973; Gordon and Lash, 1973; Minor et al., 1973). Identification of Sulfated Glycosaminoglycans
The types and relative amounts of the various sulfated GAG accumulated by the explants was determined after 2, 3, and 4 days of culture by analysis with the en-
Degradation of Accumulated Glycosaminoglycans
Since the difference in accumulation of sulfated GAG in control and CMP explants could be due to a decreased rate of degradation in the CMP-explants rather than an increased synthesis, the degradation of previously synthesized GAG in each type of culture was determined. Control and CMP explants were cultured for 48 hr in the presence of YS-sulfate, then removed from the nutrient agar, washed with 100 volumes of SBSS, and transferred to fresh nutrient agar lacking the isotope. Both types of explants were then fed with nonradioactive medium. The nutrient medium fed to the CMP explants was supplemented with CMP. At zero time, 24 hr, and
Pm. 2. Percentage of previously labeled glycosaminoglycan remaining in control (C) and stemalCMP explants after chase in unlabeled medium for 2 days. Control and CMP explants were cultured for 48 hr in the presence of “S-sulfate, washed, and transferred to nonradioactive CMP and control medium. At zero time, 24 hr, and 48 hr after transfer, the amount of *%-sulfate-labeled GAG present in the explants was determined.
216
DEVELOPMENTALBIOLOGY
zymes chondroitinase ABC and AC (see Kosher and Searls, 1973). Explants of each type (control and sternal- and vertebralCMP treated) accumulated chondroitin 6sulfate, chondroitin 4-sulfate, and chondroitinase-resistant material in the same relative amounts (Table 3). In all explants chondroitin 6-sulfate synthesis predominated. Thus, the increased accumulation of sulfated GAG by the CMP explants is not the result of a preferential synthesis of a particular type of chondroitin sulfate. TABLE
2
TOTAL DNA CONTENT OF CONTROL, STERNAL-CMP, AND VERTEBRAL-CMP SOMITE EXPLANTS AT VAIUOUS DAYS OF CULTURE Percentage of original DNA” Days Stemal-
Vertebral-
CMP
CMP
47 42 40 51
45 46 46 50
Control 1 2 3 4
46 44 44 50
“Data are expressed as percentage of original DNA, since the DNA content of freshly explanted somites varied from experiment to experiment because of slight differences in the age of the embryos or size of the explants. Zero time values ranged from approximately 4.0 pg DNA to 5.0 pg DNA/20 explants. Results are the mean of 4 determinations. Standard deviation was *2-3% in each case. TABLE
VOLUME 35,1973 DISCUSSION
The results of this investigation indicate that exogenous proteoglycan complex (chondromucoprotein) consisting predominantly of proteochondroitin sulfate stimulates in vitro somite chondrogenesis. Stage 17 embryonic chick somites, although synthesizing low levels of chondroitin sulfates, are not cartilaginous at the time of their explantation to the culture environment. The somites undergo chondrogenic differentiation in uitro. Although a small amount of cartilaginous tissue is visible in control somite explants by the third day of culture, the amount of cartilaginous tissue which forms on subsequent days, and the amount of glycosaminoglycan which accumulates, is greatly increased in the presence of exogenous chondromucoprotein. The control and the CMP explants accumulate *?S-sulfate labeled glycosaminoglycans at similar rates for the first 2 days of culture. Beginning on the third day of culture, the control explants show less *YS-sulfate accumulation and have fewer visible cartilaginous areas. One effect of the exogenous proteoglycan complex seems to be to maintain the initial rate of synthesis which, in the control culture environment, becomes seriously impaired after the first 2 days of culture. It is not known whether this failure in the maintenance of 3
RELATIVE AMOUNTS OF THE VARIOUS SULFATED GLYCOSAMINOGLYCANS(GAG) ACCUMULATED BY CONTROL AND CMP-EXPLANTS AVER 3, 4, AND 5 DAYS IN CULTURE AS DETERMINED BY CHONDRO~TINASEANALYSIS
Chondroitin g-sulfate” Chondroitin 4-sulfate* Chondroitinase-resistantc Chondroitin 6-sulfate Chondroitin 4-sulfate
5 Days
4 Days
3 Days GAG
Control (So)
CMP @)
Control (%)
CMP (OJO)
%?l0
CMP (S)
53 16 31
51 16 33
51 16 33
51 18 31
50 20 30
50 22 28
77123
76124
76123
74126
71/26
69/31
o Determined as the relative amount of radioactivity associated with g-sulfated disaccharide residues after chondroitinase ABC and AC treatment. b Relative amount of radioactivity associated with I-sulfated disaccharide residues after chondroitinase ABC and AC treatment. c S5-Sulfate-labeled GAG which is not degraded by chondroitinase ABC or AC, likely heparan sulfate or keratan sulfate.
KOSHER, LASH, AND MINOR
Exogenous Chondromucoprotein
synthetic activity involves an inhibitory condition in the control cultures. Although the lower *‘S-sulfate accumulation in the control explants may be partly due to undersulfation of the chondroitin sulfate molecules, this cannot be the major reason for the differences in ‘%-sulfate accumulation. The CMP explants invariably possess considerably more cartilaginous tissue than do the control explants, as well as accumulate 2-3 times as much “S-sulfated glycosaminoglycans. The differentiation of chondroblastic mesenchyme (somite sclerotome) into cartilage is enhanced by exogenous proteoglycan complex. These results are of potential significance in view of the following published observations. (1) Embryonic notochord and spinal cord influence somite chondrogenesis in vitro (Lash, 1963, 1967; Ellison and Lash, 1971) and in uivo (Holtzer and Detwiler, 1953; Strudel, 1953; Watterson et al., 1954). (2) Extracts from the embryonic spinal cord and notochord have been reported to stimulate somite chondrogenesis (Lash et al., 1962; Lash, 1963). These experiments were hindered by a lack of a suitably sensitive assay and the difficulty in obtaining adequate amounts of extracted material. These experiments are being reinitiated to compare the effect of the previously extracted material (Lash et al., 1962) with our currently available chondromucoprotein. (3) During the period of interaction between the notochord or spinal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan (France-Browder et al., 1963; Marzullo and Lash, 1967; Lash, 1968a-c; Kvist and Finnegan, 1970a,b; Manasek, 1970; Strudel, 1971; Cohen and Hay, 1971; Ruggeri, 1972; Minor, 1973), which becomes distributed among adjacent sclerotomal cells. Furthermore, it has been suggested that this extracellular matrix may be a causal factor in the condrogenic differentiation of the sclerotome cells (Strudel, 1971; Ruggeri, 1972; Minor, 1973).
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In addition, it can be stated that the chondrogenic stimulation does not qualitatively alter the chondroitin sulfates synthesized. The indictment by Abbott et al. (1972) of our previous report that chondroitin 6-sulfate is the predominant glycosaminoglycan synthesized by embryonic somites (Marzullo and Lash, 1967) cannot be substantiated. Embryonic cartilage was chosen as the source of proteoglycan in the present study not only because of the ease of obtaining an adequate amount of material, but also because Minor (1973) has shown that the structured matrix components synthesized by the embryonic spinal cord and notochord are similar to those seen in lo-day vertebral cartilage. Experiments are currently in progress to determine whether the proteoglycan complex extracted from embryonic notochord and spinal cord, which would be of greater developmental significance, will also promote somite chondrogenesis. Preliminary results indicate that such a stimulation is achieved. By far the major components of the chondromucoprotein complexes used in the present study to stimulate somite chondrogenesis are proteochondroitin 4- and g-sulfate. The extracts do not contain DNA or RNA, and contain only trace amounts of collagen. The amount of hydroxyproline is less than 0.5% of the amount of uranic acid. It has however, been demonstrated by Hascall and Sadjera (1969) that the proteoglycan complex extracted from bovine nasal cartilage with 4.0 M guanidinium chloride contains minor glycoprotein components, which constitute less than 5% by weight of the complex. Although we feel it is unlikely that the trace amount of collagen or minor glycoprotein components of our chondromucoprotein extracts are responsible for the observed stimulation of in vitro somite chondrogenesis (simply because they are present in such small amounts compared to the large amount of proteoglycan), this possibility cannot at this time be conclusively eliminated. Since the proteoglycan molecules can be sepa-
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rated from the minor glycoprotein components and trace amount of soluble collagen by cesium chloride density gradient centrifugation, it will be important to determine the effect of purified proteoglycan subunit molecules on somite chondrogenesis. That polyanionic proteoglycans can stimulate chondrogenesis is certainly not an untenable view, since in another system it has been demonstrated that chondromucoprotein and other polyanions stimulate proteoglycan synthesis (Nevo and Dorfman, 1972). Even if the trace amount of collagen present in our extracts is involved in the stimulation along with the major proteoglycan components, it is significant that extracellular materials influence differentiation. At this point, it is admittedly speculative to discuss the mechanism whereby extracellular proteoglycan may stimulate the expression of the chondrogenic genotype. A likely site of action, however, is the cell membrane. It is conceivable, as suggested by Nevo and Dorfman (1972), that an interaction of proteoglycan with components of the cell surface can result in an activation or stimulation of already existing metabolic patterns. It is also tempting to relate the stimulation of chondrogenesis by extracellular proteoglycan to recent experiments which indicate that the extracellular cationic environment can have profound effects upon chondrogenic expression (Lash, 1972; Lash et al., 1973). A high molecular weight polyanion such as the proteoglycan complex would have the potential to bind, sequester, and possibly regulate the external ionic microenvironment of the chondrogenic cells. Attempts to correlate the results presented here with the effects of extractable tissue components (Lash et al., 1962; Lash, 1963) and of the ionic environment (Lash, 1972; Lash et al., 1973) will have to await further experimentation. REFERENCES ABaol~, J., MAYNE, R., and HOLTZER, H. (1972). Inhibition of cartilage development in organ cul-
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