Download PDF

/ . Embryo!, exp. Morph. Vol. 56, pp. 59-70, 1980
Printed in Great Britain © Company of Biologists Limited 1980
59
Changing patterns of proteoglycan synthesis during
chondrogenic differentiation
By MICHAEL OVADIA 1 , CHARLES H. PARKER 2
AND JAMES W. LASH2
From the Department of Anatomy, School of Medicine, University
of Pennsylvania
SUMMARY
The pattern of proteoglycan synthesis was examined in the cartilages of the anterior and the
posterior limbs, the vertebra and the sterna of the developing chick embryo, during chondrogenic differentiation. At stage 18, the limb primordia synthesize small monomeric proteoglycans. In all nine cartilages examined, there was a shift during differentiation from small
to larger intermediate forms followed by a transition toward the aggregate forms. As
development proceeds, the proportion of aggregates increases and the small proteoglycans
almost disappear. Chondrogenic differentiation is thus marked by a increase in the size of
the proteoglycan molecules, and an increase in the proportion of the large proteoglycan
aggregates.
INTRODUCTION
The primary assays for chondrogenic differentiation have been either morphological observations or the quantitation of sulfated glycosaminoglycans
after proteolytic treatment of these tissues. Although quantitation of glycosaminoglycan synthesis has been useful in studying chondrogenesis (Kosher,
Lash & Minor, 1973; Lash, et al. 1973; Kosher & Lash, 1975) or corneal stroma
production (Meier & Hay, 1973; Hay & Meier, 1974; Hay, 1977), moremeaningful analyses can now be performed on the proteoglycans, a unique constituent
of the cartilage extracellular matrix. Dissociative solvents such as guanidine
hydrochloride, calcium chloride, or magnesium chloride show that cartilages
from a wide variety of vertebrates contain proteoglycans that exhibit great
heterogeneity and exist as four discrete forms: aggregated (PGA), intermediate
size, aggregating (PGM) and non-aggregating (PGN) monomers (Hardingham,
Fitton-Jackson & Muir, 1972; Hardingham & Muir, 1972, 1973; Bjelle, Gardell
& Heinegard, 1974; Hascall & Heinegard, 1974; Pita, Muller & Howell, 1975;
Shipp & Bowness, 1975; Hascall, Oegema, Brown & Caplan, 1976; Vasan
& Lash, 1977,1978,1979; Lash & Vasan, 1977, 1978; Muir, 1977; Lash, Ovadia
1
Author's Address: Department of Zoology, Tel-Aviv University, Tel-Aviv, Israel.
Author's Address: (For reprints) Department of Anatomy, School of Medicine G3,
University of Pennsylvania, Philadelphia, Pennsylvania 19104 U.S.A.
2
60
M. OVADIA, C. H. PARKER AND J. W. LASH
& Vasan, 1978). The relatively small monomeric proteoglycans may or may not
be capable of associating with hyaluronic acid to form very large proteoglycan
aggregates (Hardingham, Ewins & Muir, 1976).
It is now becoming clear that chondrogenesis can also be characterized by
biochemical changes in proteoglycans. These may be an increase in total sulfate
incorporation, coupled with a preferential increase of the aggregated forms.
Some of these changes have been demonstrated in the differentiation of chick
somites (Lash, 1968; Kosher et al. 1973; Kosher & Lash, 1975; Lash & Vasan,
1978) and in the differentiation of dissociated mesenchymal cells derived from
mouse limb buds (DeLuca et al. 1977; Royal & Goetinck, 1977).
During chondrogenesis, the growing zone of the epiphyseal cartilage is both
avascular and non-calcified. Shortly before it transforms into bone, it calcifies
and becomes invaded by capillary sprouts (Anderson & Parker, 1966; Schenk,
Spiro & Weiner, 1967). Recent studies by Kuettner et al. (1975) indicate that
lysosymes play a role in the mechanism of cartilage calcification by inducing a
shift from large proteoglycan aggregates (PGA) of the epiphyseal growing zone
to small aggregates and non-aggregating monomeric forms (PGN) in the hypertrophic zone. This shift forms a new microenvironment which presumably
enables the accretion of calcium phosphate. A high proportion of aggregates
inhibits calcifications (Kuettner et al. 1975). This result led to the working hypothesis that epiphyseal cartilage should accumulate a higher proportion of
aggregated proteoglycans during its development, in order to prevent calcification in the growing zone of the cartilage. The present work examines this
hypothesis by investigating the changes of proteoglycans during the development of the anterior and the posterior limbs, sterna and vertebra.
MATERIALS AND METHODS
Tissue culture
Limb buds, clean epiphyseal cartilages of femur, tibia, humerus, the whole
cartilage of fibula, radius and ulna, sterna and the centrum of sacral vertebra
were dissected from White Leghorn chick embryos at various stages of development. Embryonic stages were determined according to the staging series of
Hamburger & Hamilton (1951). The tissues were cultured for 24 h upon Nuclepore filters (0-8/*m pore size, Nuclepore, Inc., Pleasanton, Calif., U.S.A.)
which were cut into 5 mm x 5 mm squares, and placed upon a Nitex screen, to
support the filters at the liquid medium interface. The liquid nutrient medium
consisted of Simms' balanced salt solution (SBSS), fetal calf serum (FCS), and
the nutrient supplement F12X in the proportions 2:2:1 (Minor, 1973). Radioactive sulfate (Na235SO4, New England Nuclear, Boston, Mass., U.S.A.) was
added to the nutrient medium at a concentration of 30 /tCi/ml. All cultures
were maintained at 37 °C in a humidified atmosphere of 95 % air, 5 % CO2.
Proteoglycan synthesis during chondragenic differentiation
61
Proteoglycan extraction
The labelled proteoglycans were extracted with 4-0 Mguanidinium hydrochloride, GuHCl (Ultra Pure grade, Schwarz/Mann, Orangeburg, N.Y., U.S.A.),
buffered at pH 5-8 with 0-05 M sodium acetate (Hascall & Heinegard, 1975),
which contained 0-01 M EDTA, disodium salt (Sigma Chemical Co., St Louis,
Mo., U.S.A.), 0-1 M 6-aminohexanoic acid (Aldrich Chemical Co., Milwaukee,
Wise, U.S.A.) and 0-005 M benzamidine hydrochloride (Eastman Kodak,
Rochester, N.Y., U.S.A.) to inhibit proteolysis (Oegema, Hascall & Dziewiatkowski, 1975). The tissues were extracted in the GuHCl solvent by agitation
on a rotary shaker for 48 h at 4 °C. Under these conditions, the proteoglycan
aggregates in the cartilage matrix are dissociated and go into solution as
monomeric proteoglycans. The extract was dialyzed against ten changes of
deionized water for 5 days. This procedure causes the proteoglycans in the 4-0
M-GuHCl extract to re-aggregate. The extracts were centrifuged at 20000 # for
30min. The supernatants were then lyophilized and kept in a freezer until
used.
Molecular size of proteoglycans
Molecular sieve chromatography was performed according to the methods
of Lever & Goetinck (1976), using controlled-pore glass beads (Electro-Nucleonics, Fairfield, N.J., U.S.A.) as modified by Lash & Vasan (1978). The
size bead used was CPG-10-2500B (mean pore diameter of 257-3 nm). The
dimensions of the column packed with CPG beads was 1-5 x 85 cm. The lyophilized proteoglycans were dissolved in 0-5 ml of 4-0 M-GuHCl by shaking
for 2-4 h and dialyzed overnight against 0-5 M-NaCl containing 0-02 % sodium
azide. The retentate was then added to the column and eluted at 4 °C with the
same solution. The flow rate was 6 ml/cm 2 h and the eluate was collected in
1-0 ml fractions.
The void volume (Vo) of the column was determined with polyvinyltoluene
latex microspheres (2-02 /im in diameter, Duke Scientific, Palo Alto, Calif.
U.S.A.). The elution of the spheres was examined in an optical microscope.
The total volume was determined with radioactive sulfate. The aggregated
proteoglycans (PGA) were found in the void volume (Vo), followed by smaller
molecules of intermediate size. Monomers were eluted just before the total
volume (Vt) of the column.
Radioactivity determinations.
Radioactivity was determined by scintillation fluid containing 6-0 gm/liter
PPO, 3-0 gm/liter POPOP (Packard Instruments, Downers Grove, 111., U.S.A.)
and 10% Bio-Solv. (Beckman, Fullerton, Calif, U.S.A. Fomular BBS-3).
Radioactivity, expressed as DPM, was determined using a Model 4200 Intertechnique Scintillation Counter which was programmed to present the data in
5
EMB 56
62
M. OVADIA, C. H. PARKER AND J. W. LASH
bar graphs form (IN/US Service Corp., Fairfield, N.J., U.S.A.). The proteoglycan profiles presented in this report were transcribed from the computerprinted data sheets.
RESULTS
Molecular size proteoglycans
Three major populations of proteoglycans have been reported in extracts of
dissociated embryonic chick chondrocytes which had been cultured for 13
days (Lever & Goetinck, 1976; McKeown & Goetinck, 1979). To determine
whether proteoglycans exhibit the same heterogeneity when whole tissues are
cultured, precartilaginous and cartilaginous embryonic chick limbs were
cultured for 24 h and the proteoglycans were extracted and analyzed. Figure 1
shows that during chondrogenesis at least three major populations can be
distinguished. Since the terminology of these populations is unsettled (cf.
McKeown & Goetinck, 1979), we designate them as PGA (large proteoglycan
aggregates in the void volume), Intermediates (a heterogeneous population
between the void and total volumes), and PG (small proteoglycans, characteristic of early embryonic tissues).
Proteoglycans at a precartilaginous stage of limb development
The presence of at least three predominant forms of proteoglycans in embryonic cartilages provokes the question of whether these forms can be detected
in the mesenchymes of precartilaginous limb buds. Therefore, anterior and
posterior limb buds were dissected from 40 embryos at stage 18 and cultured
for 24 h. The labeled proteoglycans of these tissues are seen in the top profiles
in Figs. 1-3, and show a predominant small molecular form (PG). Most of these
molecules are found in a peak near the total volume of the column, which indicates that the mesenchymes of the limb primordia synthesize small proteoglycans.
Proteoglycans of limb cartilages at various stage of development
Our working hypothesis was that epiphyseal cartilage should accumulate
aggregated proteoglycans during development. In order to examine this hypo-
Fig. 1. Molecular sieve chomatography of proteoglycans synthesized by femur and
fibula cartilages at stages 34,38 and 39. Posterior limb primordia (PL) synthesize proglycans (PG) of predominately small molecular size. In Fig 1-5 proteoglycan aggregates (PGA) are found in the void volume (Vo) and small monomeric proteoglycans
(PG) are found near the total volume (Vt). The heterogeneous proteoglycans in
between are designated as 'Intermediates.'
Fig. 2. Molecular sieve chromatography of proteoglycans synthesized by tibia
(stage 34 and 38) and phalanges (stages 34 and 39). Even at stage 39 the phalanges
synthesize primarily intermediate size proteoglycans.
Proteoglycan synthesis during chondrogenic differentiation
PGA
intermediates
60
i
63
PG
80
100
Fraction number
120
A
60
80
100
Fraction number
120
.
V,
Fig. 1
Fig. 2
5-2
64
M. OVADIA, C. H. PARKER AND J. W. LASH
100
50
I
100
I
I
Humerus
st. 34
50
100 i-
st. 38
50 -
100 |-
100
Fig. 3. Molecular^sieve chromatography of proteoglycans synthesized by various
stages of humerus, radius and ulna. The characteristic shift towards larger proteoglycans is apparent. At comparable stages of development these cartilages make
smaller proteoglycans than do more proximal cartilages (c/. stage-38 humerus and
stage-38 radius and ulna). AL, anterior limb primordia.
thesis, the growing zone was dissected from cartilages of the femur, tibia, phalanges, humerus, fibula, radius and ulna at various stages of differentiation (chick
embryos at stages 32-43; days 7-17). The cartilaginous tissues were cultured
separately for 24 h. and the proteoglycans extracted and chromatographed.
In all the cartilages examined, there was a shift in the proteoglycans which
Proteoglycan synthesis during chondrogenic differentiation
65
were synthesized at different stages of the development, from monomeric to
intermediate forms followed by a transition towards the aggregate forms (Figs.
1-5). As development progresses, the proportion of aggregates becomes higher
and the small monomers almost disappear. This is the rule for all the limb
cartilages. This transition towards the intermediate and the aggregate form
occurs earlier in proximal cartilages of the limb. The molecular size of the intermediates and of the aggregates becomes more homogeneous in later stages of
development. The population of proteoglycans between the intermediates and
the large aggregates almost disappear; presumably they are converted into
large aggregates.
Proteoglycans of vertebrae and sternae at various stages of development
The cartilages of vertebrae and sternae were chosen to examine the proteoglycan profiles in other developing cartilages. The centrum of the vertebrae
and whole sternae were dissected from chick embryos at various stages of
development, cut into small pieces and treated as above. Figs 4 and 5 show that
the pattern of the proteoglycans synthesis in the developing sterna and vertebra
resembles that of limb cartilages. The transition, however, towards the intermediate and aggregate forms occurs at different stages of development. This is
probably due to the fact that different cartilages start their development at
different embryonic stages.
DISCUSSION
Proteoglycans are one of the two classes of molecules which characterize
cartilage tissue. Collagen Type II is the other class, but will not be considered
in this discussion. Biochemical techniques used in this work have made it
possible to isolate and characterize the proteoglycans during the differentiation
of the cartilage. The profiles obtained with molecular sieve chromatography,
using controlled pore glass beads, indicates that there are major size classes in the
heterogeneous population of the proteoglycans. The large proteoglycan aggregates pass through the column with the void volume, intermediate size molecules
pass through the column demonstrating their heterogeneity, and the small
proteoglycans pass through in the region of the total volume mark.
When proteoglycans were extracted from limb primordia of stage-18 chick
embryos and chromatographed on columns of CPG-2500, all the radioactive
macromolecules were found in a peak near the total volume of the column.
This indicates that the mesenchymes of the limb primordia synthesize small
proteoglycans of monomeric types. In all the cartilages examined there was a
shift in the amount of the proteoglycans from the monomeric and intermediate
forms towards the aggregate forms with progressive stages of development. As
development proceeds, the proportion of the aggregates becomes higher and the
66
M. OVADIA, C. H. PARKER AND J. W. LASH
100
Sternum
st. 36
100 r
100 r
100 i-
60
80
100
Fraction number
120
80
100
Fraction number
v,
Fig. 4
Fig. 5
Fig. 4. Molecular sieve chromatography of proteoglycans synthesized by embryonic
vertebral cartilages at various stages of development. The earlier stages of vertebral
chondrogenesis have been published in Lash & Vasan (1978), and are consistent
with the transitions seen in Fig. 1-3.
Fig. 5. Molecular sieve chromatography of proteoglycans synthesized by various
stages of embryonic sterna. The sternum begins chondrogenesis at a late stage of
embryonic development, and the transitional stages toward large proteoglycans
occurs at relatively late stages. It can be noted that at no stage of embryonic
development are proteoglycan aggregates found without the intermediate size
molecules.
monomers almost disappear. The molecular size of the intermediates and of the
aggregates becomes more homogeneous in later stages of development.
The shifts in the patterns of proteoglycan populations during the development is readily apparent in all nine cartilages examined. If, as we suggest, an
increased synthesis of large monomers and of aggregates is indicative of differentiation, then another aspect of limb cartilage differentiation can be seen in
these profiles. It has long been known that chick embryo limbs develop in a
proximal-distal sequence (Saunders, 1948). With this in mind, it can be seen
that the more proximal femur has more 'mature' cartilage than the more
Proteoglycan synthesis during chondrogenic differentiation
67
distal fibula, and that the most distal phalanges contain the smallest and most
heterogeneous population of proteoglycans, and are hence the least differentiated of the three cartilages. It is thus apparent that the molecular size distribution of proteoglycans can be a useful index of chondrogenic differentiation
in developing chick limbs.
These results support the working hypothesis that the growing zone of
older embryonic cartilage synthesizes a higher proportion of large aggregated
proteoglycans as development proceeds. These results are also in agreement
with those of Royal & Goetinck (1977), and DeLuca et ah (1977), who cultured
isolated mesenchymal cells in a petri dish for different periods of time. During
chondrogenesis, they found a shift of the predominant molecular species from
the small monomeric proteoglycans at 24 h to large aggregates at 5 days.
The synthesis of small proteoglycans by the precartilaginous limb buds (anterior and posterior) at stage 18 suggests that the tissue is already programmed
for the production of proteoglycans at very early stages of development.
Whether these molecules are indeed homogeneous, or whether they have anything at all to do with chondrogenesis is unclear. From the profiles we present, it
is obvious that, even at early stages, there are small amounts of intermediates
(cf. PL and AL in Figs 1-3). It is of interest that the proportion of aggregates and
intermediates is also affected by the amount of lapsed time between dialysis and
placing the extract on the column. All of the experiments in this report were
performed by placing the extract directly on the column after dialysis. The longer the time between dialysis and column chromatography, the greater the
proportion of aggregating molecules. If extracts from stage-18 limb buds are
kept for a week before submitting to column chromatography, small amounts of
aggregates can be detected (Stephens, Vasan & Lash, submitted for publication).
It is apparent from these results, and former reports, that the exact nature of
these small proteoglycans is unsettled (Lash, 1976; Lash, Belsky & Vasan, 1977;
Lash & Vasan, 1978 - i n somites, and Kleine, 1973; 1974; Levitt & Dorfman,
1974; Goetinck, Pennypacker & Royal, 1974; Royal & Goetinck, 1977; McKeown & Goetinck, 1979; Vasan & Lash, 1979 - i n limb buds).
The later transition from a predominance of small proteoglycans to a predominance of aggregate forms (PGA) raises the question of the induction of this
process. Is the aggregation mediated by a new factor(s) synthesized later in
development (e.g. the link glycoprotein), or is aggregation induced by the
proteoglycans and collagens already present in the tissue? It may be of significance that none of the cartilage extracts examined consist only of aggregates.
Intermediate size proteoglycans are always present, possibly at very early stages
of development. Whether this is an artifact of the extraction procedures, or a
true reflexion of the molecular constitution in vivo has yet to be determined. In
addition, the intermediate size proteoglycans have yet to be fully characterized.
Lever & Goetinck (1976), DeLuca et al. (1977) and McKeown & Goetinck
(1979) have reported from the actively synthesizing chondrocytes an inter-
68
M. OVADIA, C. H. PARKER AND J. W. LASH
mediate size of molecule that is capable of aggregating (type PGS-la of McKeown & Goetinck, 1979). This may be the eventual fate of the intermediate
molecules we observe in differentiating cartilage. Not all of the intermediates
are capable of aggregating (Cheney & Lash, in preparation), and the heterogeneity of the intermediates decreases with differentiation, as shown in this
report.
A fact that has not been addressed satisfactorily in past reports on proteoglycan assays is the exact nature of what is being assayed. The proteoglycans
that are presented in graph form as 'aggregates' are actually the molecules
that have the ability to re-aggregate, since they were dissociated during the
extraction procedures. Thus the inability of some intermediates to aggregate
could be a true reflection of how they exist in vivo, or it could be due to changes
in the molecule effected by the extraction procedures. Indeed, evidence has been
obtained that the hyaluronic acid-binding region is labile in the early stages of
limb chondrogenesis (Vasan & Lash, 1979).
In spite of the fact that the intermediate size molecules in extracts from differentiating cartilages have not yet been fully characterized, there is enough consistency between the results reported here and those of Lever & Goetinck
(1976), DeLuca et al. (1977) and Vasan & Lash (1979) to indicate that the transitory populations of proteoglycans during differentiation are real, and of
significance with regard to the regulation of chondrogenesis.
We are grateful for the assistance of Gladys Treon in dissecting embryos, and N. S. Chandrasekar for help in performing the biochemical analyses. We also thank Dr Clarissa Cheney
for thoughtful and helpful discussions. The work was supported by NIH research grant
HD-O0380to J.W.L.
REFERENCES
C. E. & PARKER, J. (1966). Invasion and resorption of endochondral ossification.
/. Bone Jt Surg. 48-/1 (5), 899-914.
BJELLE, A., GARDELL, S., & HEINEGARD, D. (1974). Proteoglycans of articular cartilage from
bovine lower femoral epiphysis. Connect. Tissue Res. 2, 111-116.
DELUCA, S., HEINEGARD, D., HASCALL, V., KIMURA, J. H. & CAPLAN, A. I. (1977). Chemical
and physical changes in proteoglycans during development of chick limb bud chondrocytes
grown in vitro. J. biol. Chem. 252, 6600-6608.
GOETINCK, P. F., PENNYPACKER, J. P. & ROYAL, P. D. (1974). Proteochondroitin sulfate
synthesis and chondrogenic expression. Expl Cell Res. 87, 244-248.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. / . Morph. 88, 48-92.
ANDERSON,
HARDINGHAM, T. E. EWINS, R. J. F. & MUIR, H. (1976). Cartilage proteoglycans. Structure
and heterogeneity of the protein core and the effects of specific protein modifications on the
binding to hyaluronate. Biochem. J. 157, 127-143.
HARDINGHAM, T. E., FITTON-JACKSON, S. & MUIR, H. (1972). Replacement of proteoglycans
in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase.
Biochem. J. 129, 101-112.
HARDINGHAM, T. E. & MUIR, H. (1972). Biosynthesis of proteoglycans in cartilage slices.
Biochem. J. 126, 791-803.
HARDINGHAM, T. E. & MUIR, H. (1973). Binding of oligosaccharides of hyaluronic acid to
proteoglycans. Biochem. J. 136, 905-908.
Proteoglycan synthesis during chondrogenic differentiation
69
HASCALL, V.
C. & HEINEGARD, D. (1974). Aggregation of cartilage proteoglycans. III. Characteristics of ths protein isolated from trypsin digests of aggregates. / . biol. Chem. 249,
4232-4241.
HASCALL, V. C. & HEINEGARD, D. (1975). The structure of cartilage proteoglycans. In Extracellular Matrix Influences on Gene Expression (ed. H. C. Slavkin & R. C. Greulich), pp.
423-434. New York: Academic Press.
HASCALL, V. C , OEGEMA, T. R., BROWN, M. & CAPLAN, A. I. (1976). Isolation and characterization of proteoglycans from chick limb bud chondrocytes grown in vitro J. biol.
Chem. 251, 3511-3519.
HAY, E. D. (1977). Interactions between cell surface and extracellular matrix in corneal
development. In Cell and Tissue Interactions (ed. J. W. Lash & M. M. Burger), pp. 115138. New York: Raven Press.
HAY, E. D. & MEIER, S. (1974). Glycosaminoglycan synthesis by embryonic inductors: neural
tube, notochord and lens. / . Cell Biol. 62, 889-898.
KLEINE, T. O. (1973). Biosynthesis of chondroitin sulfate proteins: Isolation of four pools
of chondroitin sulfate proteins differing in their solubility and labeling rates with radio
sulfate calf rib cartilage. FEBS Lett. 31, 170-174.
KLEINE, T. O. (1974). Biosynthesis of chondroitin sulfate proteins. Pulse labeling experiments
with radio sulfate of four pools of chondroitin sulfate proteins in calf rib cartilage. FEBS
Lett. 39, 255-258.
KOSHER, R. A. & LASH, J. W. (1975). Notochordal stimulation of in vitro somite chondrogenesis before and after enzymatic removal of perinotochordal materials. Devi Biol. 362378.
KOSHER, R. A., LASH, J. W. & MINOR, R. R. (1973). Environmental enhancement of in vitro
chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein. Devi Biol. 35, 210-220.
KUETTNER, K. E., PITA, J. C , HOWELL, D. S., SORGENTE, N. & EINSTEIN, R. (1975). Regulation of epiphyseal cartilage maturation. In Extracellular Matrix Influences on Gene Expression (ed. H. C. Slavkin & R. C. Greulich), pp. 435-440. New York: Academic Press.
LASH, J. W. (1968). Somitic mesenchyme and its response to cartilage induction. In Epithelial
Mesenchymal Interactions ed. R. Fleischmajer & R. Billingham), pp. 165-172. Baltimore:
Williams and Wilkins.
LASH, J. W. (1976). Extracellular matrix products and differentiation: Somite chondrogenesis.
In vitro Tests of Teratogenicity (ed J. D. Ebert & M. Marois), pp. 367-374. Amsterdam:
North Holland.
LASH, J. W., BELSKY, E. & VASAN, N. S. (1977). Stimulation of chondrogenic differentiation
with extracellular matrix components: An analysis using scanning electron microscopy. In
Cell Interactions in Differentiation (ed. M. Karkinen-Jaaskelainen, L. Saxen & L. Weiss),
pp. 263-272. New York: Academic Press.
LASH, J. W., OVADIA, M. & VASAN, N. S. (1978). Microheterogeneities, non-equivalence, and
embryonic induction. Med. Biol. 56, 333-338.
LASH, J. W., ROSENE, K., MINOR, R. R., DANIEL, J. C. & KOSHER, R. A. (1973). Environ-
mental enhancement of in vitro chondrogenesis. III. The influence of external potassium
ions and chondrogenic differentiation. Devi Biol. 35, 370-375.
LASH, J. W. & VASAN, N. S. (1977). Tissue interactions and extracellular matrix components.
In Cell and Tissue Interactions, (ed. J. W. Lash & M. M. Burger), pp. 101-114. New York:
Raven Press.
LASH, J. W. & VASAN, N. S. (1978). Somite chondrogenesis in vitro; Stimulation by exogenous extracellular matrix components. Devi Biol. 66, 151-171.
LEVER, P. L. & GOETINCK:, P. F. (1976). Molecular sieve chromatography of proteoglycans:
a comparative analysis. Analyt. Biochem. 75, 67-76.
LEVITT, D. & DORFMAN, A. (1974). Concepts and mechanisms of cartilage differentiation.
Current Topics in Developmental Biology 8, 103-145.
MCKEOWN, P. & P. F. GOETINCK (1979). A comparison of the proteoglycans synthesized in
Meckel's and sternal cartilage from normal and nanomelic chick embryos. Devi Biol. 71,
203-215.
70
M. OVADIA, C. H. PARKER AND J. W. LASH
S. & HAY, E. D. (1973). Synthesis of sulfated glycosaminoglycans by embryonic
corneal epithelium. Devi Biol. 35, 318-331.
MINOR, R. R. (1973). Somite chondrogenesis. A structural analysis. /. Cell Biol. 56, 27-50.
MUIR, H. (1977). Structure and function of proteoglycans of cartilage and cell-matrix components. In Cell and Tissue Interactions, (ed. J. W. Lash & M. M. Burger), pp. 87-100.
New York: Raven Press.
OEGEMA, T. R., HASCALL, V. C. & DZIEWIATKOWSKI, D. D. (1975). Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma. /. biol. Chem. 250,
6151-6159.
PITA, J. C , MULLER, F & HOWELL, D. S. (1975). Disaggregation of proteoglycan aggregate
during endochondral calcifications: Physiological role of cartilage lysosyme. In Dynamics
of Connective Tissue Molecules, (ed. P. M. C. Burleigh & A. R. Poole), pp. 248-258.
Amsterdam: North Holland.
ROYAL, P. D. & GOETINCK, P. F. (1977). In vitro chondrogenesis in mouse limb mesenchymal
cells: Changes in ultrastructure and proteoglycan synthesis. /. Embryol. exp. Morph. 39,
79-95.
SAUNDERS, J. W. (1948). The proximo-distal sequence of origin of the parts of the chick
wing and the role of the ectoderm. /. exp. Zool. 108, 363-403.
SCHENK, R. K., SPIRO, D. & WEINER, J. (1967). Cartilage resorption in the tibial epiphyseal
plate of growing rats. / . Cell Biol. 34, 275-291.
SHIPP, D. W. & BOWNESS, J. M. (1975). Insoluble non-collagenous cartilage glycoproteins
with aggregating sub-units. Biochim. biophys. Acta 379, 282-294.
VASAN, N. S. & LASH, J. W. (1977). Heterogeneity of proteoglycans in developing chick limb
cartilage. Biochem. J. 164, 179-183.
VASAN, N. S. & LASH, J. W. (1978). Proteoglycan heterogeneity in embryonic chick articular
and epiphyseal cartilages. Conn. Tiss. Res. 6, 191-199.
VASAN, N. S. & LASH, J. W. (1979). Monomeric and aggregate proteoglycans in the chondrogenic differentiation of embryonic chick limb buds. /. Embryol. exp. Morph. 49, 47-59.
MEIER,
(Received 7 May 1979, revised 16 October 1979)