J. Embryol. exp. Morph. 73, 263-274, 1983
Printed in Great Britain © The Company of Biologists Limited 1982
263
Analysis of perinotochordal materials. 2. Studies on
the influence of proteoglycans in somite
chondrogenesis*
By NAGASWAMISRI VASAN 1
From the Department of Anatomy, Cell Biology Division, New Jersey Medical
School
SUMMARY
Notochordal proteoglycans influence somite chondrogenesis. In earlier work we learned
that notochord cultured under improved conditions for 8 days contains three different classes
of proteoglycans (Vasan, 1981). The question was whether these three different classes of
perinotochordal proteoglycan molecules, are necessary for inducing somite chondrogenesis.
When the notochords were exposed to substances causing metabolic alterations (BUdR,
excess vitamin A, and hyaluronic acid), only small proteoglycans accumulated in the
perinotochordal sheath. Such treated notochords or the proteoglycans extracted from them
failed to support somite chondrogenesis, suggesting that the large aggregate in the
perinotochordal matrix may be necessary for somite induction. Adding a crude proteoglycan
prepared from embryonic sternal cartilage increased the induction of chondrogenesis in
somites, but papain treatment of the proteoglycan diminished its inductive capacity. This
result further suggests that large aggregate proteoglycan may be necessary for the tissue
interactions that promote chondrogenesis.
INTRODUCTION
Extracellular matrix materials involved in tissue interaction and resulting morphogenesis (Grobstein, 1956; 1957), have been found to stimulate somite chondrogenesis in the notochord (Strudel, 1971; Kosher & Lash, 1975; Lash & Vasan,
1978; Vasan, 1980,1981; Belsky, Vasan & Lash, 1980; Cheney & Lash, 1981),
to differentiate corneal epithelium (Meier & Hay, 1975), and to affect salivary
gland morphogenesis (Bernfield, Banerjee & Cohn, 1972), and tooth germ
development (Galbraith & Kollar, 1976). Ventral spinal cord and notochord
influence somite differentiation in vivo (Holtzer & Detweiler, 1953; Watterson,
Lowler & Fowler, 1954; Grobstein & Holtzer, 1955). These findings led to a
search for the component of extracellular matrix which induces somite chondrogenesis. While this inducer has not been identified yet, recently developed
* Presented in part at The American Society for Cell Biology 20th Annual meeting. Cincinnati, Ohio, November 14-18, 1980.
1
Author's address: Department of Anatomy, Cell Biology Division, New Jersey Medical
School, 100 Bergen Street, Newark, New Jersey 07103, U.S.A.
264
N. VASAN
methods for (1) culturing tissues in vitro, (2) obtaining various extracellular
matrix components in pure fractions, and (3) analysing micro-quantities of
macromolecules have led to progress. Perinotochordal matrix material which
includes collagen (Linsenmayer, Trelstad & Gross, 1973; Von der Mark & Gay,
1976), glycosaminoglycans (Kvist & Finnegan, 1970; Kosher & Lash, 1975), and
proteoglycans (Vasan, 1979-81a,6) induces somite chondrogenesis. Hence, an
in vitro system with embryonic notochord-somite has been used to study extracellular matrix material in tissue interaction and differentiation.
The size of the proteoglycan accumulated in the perinotochordal sheath of in
vitro chick notochord increases with culture time (Vasan, 1981a). Molecularsieve chromotography resolves three different proteoglycan classes from 8-day
cultures. Cartilage proteoglycans are large heteropolysaccharide complexes
made of several different substituent molecules. Sulphated glycosaminoglycans
(chondroitin sulphate and keratan sulphate) are covalently linked to a protein
core to form proteoglycan monomers. Many such monomers interact with
hyaluronic acid to form large aggregates, and such interactions are stabilized by
glycoprotein link factors. The protein core of the monomers is known to have
chondroitin sulphate-rich regions, keratan sulphate-rich regions, and hyaluronic
acid-binding regions (Hascall & Heinegard, 1979).
Although perinotochordal proteoglycan occurs as three sizes, it is not clear
which size induces somite chondrogenesis. We undertook an investigation to
answer this question. However, it is unrealistic to attempt to separate various
sizes from the notochord because the quantity of proteoglycan material is
limited. To obtain material to study, we inhibited proteoglycan synthesis and
thus indirectly reduced the large aggregate in the perinotochordal sheath. We did
this with substances that alter metabolic alteration. Perinotochordal
proteoglycans obtained from such notochord were used to investigate somite
chondrogenesis. The result confirms findings of our earlier indirect study (Lash
& Vasan, 1978), that the large proteoglycan aggregates are probably important
in somite chondrogenesis, even though they form only 30-35 % of the
perinotochordal sheath proteoglycan (Vasan, 1981a).
MATERIALS AND METHODS
Culture techniques
Trunks were dissected from White Leghorn chick embryos previously incubated at 38 ° for 72 h (Stage 19 series of Hamburger & Hamilton, 1951). Under
methods described previously (Vasan, 1981a), trunks were suspended in a
solution of 1-25 trypsin (GIBCO, NY, USA) for 10-15 min in a humidified
CO 2 /air (5 %/95 %) incubator. Somites and notochords were separated from
the rest of the tissues by first flushing back and forth in a Pasteur pipet and
subsequently by microdissection. The isolated notochords were pooled, washed
several times with 10 % foetal calf serum, and cultured on Millipore filters (Type
Perinotochordal proteoglycans and somite chondrogenesis 265
HA, 0-45 /im pore size) resting on Nitex screens (656/im mesh size, Tetko, Inc.,
Elmsford, NY, USA). The whole set up was placed in a 35 mm Petri dish containing 1-5 ml of feeding medium. The liquid nutrient consisted of Simms balanced
salt solution (SBSS), foetal calf serum, and nutrient supplement F12X in 2:2:1
proportion, 50/ig/ml of ascorbic acid, and 100/iCi/ml of radioactive sulphate
(Na2 35 SO 4 - carrier free, New England Nuclear, Boston, MA, USA) (Vasan,
1981a). All cultures were maintained at 38°C in a humidified incubator. After
8 days of culture proteoglycans were extracted from the notochordal tissues
along with the Millipore filter.
Somites were isolated by the modified method of Lash (1967); i.e., the lateral
mesenchyme, appendages, mesonephros and endoderm were dissected and
removed from the trunks of stage-19 embryos. The remaining tissues, consisting
of paired somites, spinal cord, notochord and epidermis, were trypsinized in
1-25% trypsin and incubated at room temperature for 5min. Somites and
notochords were isolated and cultured on Millipore filters (Type HA, 0-45 /im
pore size) as described earlier.
When somites were cultured with notochords, the notochordal tissue was
surrounded by clusters of somites all resting on Millipore filters. When somites
were cultured in the presence of proteoglycans extracted from the notochords,
the proteoglycan was dissolved in the feeding medium (composition described
earlier) and somites rested on the Millipore filters. The cultures were grown for
6 days, and then proteoglycans were extracted from the tissues and Millipore
filter.
To determine the effect of metabolic inhibitors on notochordal proteoglycan
synthesis, the nutrient feeding medium was supplemented with (1) 150/ig/ml of
BUdR; (2) lOi.u./ml of Vitamin A; (3) lOOjUg/ml of rooster comb high
molecular weight hyaluronic acid (generous gift from Dr Balazs, Columbia
University). Lower concentrations of these inhibitors failed to produce regular
change in proteoglycan synthesis, while higher concentrations were cytotoxic.
The control cultures in all the experiments were supplied with normal medium
containing no added substances. The cultures were grown for 8 days and then the
proteoglycan was extracted from the tissues and the Millipore filter.
Extraction and fractionation of proteoglycans
Proteoglycans were extracted for 36 h in buffered 4-OM-guanidinium chloride
in 0-05M-sodium acetate, pH5-8, containing proteolytic inhibitors (Oegema,
Hascall & Dziewiatkowski, 1975) at 4°C on a rotary shaker. The extract was
dialysed against 0-04M-sodium sulphate (when proteoglycan was labelled with
35
S sulphate) for 12 h, followed by extensive dialysis in cold distilled water for
48h. The extract was cleared by centrifugation for 30min at 17300g, and the
supernatant lyophilized.
Low-salt-extracted proteoglycan (non-aggregates) was obtained by subjecting
the sternal cartilage to extraction in a solvent of 0-15M-sodium acetate pH6-0
266
N. VASAN
containing proteolytic inhibitors for 16 h at 4°C on a gentle rotary shaker (Hardingham & Muir, 1974). Proteoglycan extract was subjected to guanidinium
chloride/CsCl density gradient centrifugation to obtain pure proteoglycan
aggregate (Al) and monomer fractions (Al-Dl) according to the method of
Heinegard (1972).
In experiments where notochordal proteoglycan was included in the feeding
medium, guanidinium chloride or low-salt-extracted proteoglycan solution was
dialysed extensively against distilled water and lyophilized. The lyophilized
material was dissolved in minimum volume of 4-0 M-guanidinium chloride buffer
and dialysed extensively against Simms balanced salt solution. Proteoglycan
containing SBSS buffer, foetal calf serum and F12X was added to this sample in
the same proportion as described earlier. The proteoglycan aggregate (Al) used
in this study contained greater than 90 % large aggregate and 10 % intermediatesize molecule. The proteoglycan monomer (Al-Dl) however, did not contain
any aggregate molecules.
The low-salt-extracted molecules used in this report did not bind with large
hyaluronic acid and hence is considered pure non-aggregates. For this testing,
the cartilage pieces were exposed to radioactive sulphate for 12 h prior to extraction with 0-15M-sodium acetate buffer.
Controlled pore glass-bead (CPG-10-2500) column chromatography
The lyophilized proteoglycan was dissolved in a small volume of
4-0 M-guanidinium chloride and dialysed against 0-5 M-NaCl. The retentate was
then resolved on the column and eluted with 0-5 M-NaCl (Vasan & Lash, 1978).
The gravity-fed flow rate was 0-4ml/min. The eluate was collected in 1-0 ml
fractions, and the radioactivity was determined in a liquid scintillation counter.
Proteoglycan isolated from 16-day-old chick embryonic sternum was digested
with papain to obtain glycosaminoglycan side chains (De Luca et al. 1977). The
digested material was resolved on a Sephadex G-200 column (0-9 cm x 80 cm)
and the fractions containing polysaccharide chain were pooled, dialysed against
water, and lyophilized. The lyophilized material was dissolved in small volumes
of normal saline, and dialysed against Simms balanced salt solution. The retentate containing polysaccharide chains in Simms solution was used in making the
feeding medium. Somite cultures were provided with medium having 50, 100,
150 and 200/ig of uronic acid material per millilitre, while the control received
normal medium, as already discussed.
Biochemical analysis
DNA determinations: Radioactive glycosaminoglycans were related to the
amount of DNA measured by the method of Santoianni & Ayala (1965), as
described by Daniel, Kosher, Lash & Hertz, (1973).
Glycosaminoglycans: After 6 days of culture, radioactive sulphate incorporated in the glycosaminoglycans were determined (Kosher, Lash & Minor,
Perinotochordal proteoglycans and somite chondrogenesis 267
1973) as an index of the amount of glycosaminoglycans synthesized. Uronifc acid
was determined according to the method of Bitter & Muir (1962/, using
glucuronolactone as standard.
RESULTS
Improved feeding medium with a higher proportion of foetal calf serum and
ascorbate helped successfully culture notochords for long periods of time. Such
notochords were viable and actively incorporated radioactive sulphate into large
proteoglycan molecules with increase in culture time (Vasan, 1981a).
Notochords cultured for 8 days in vitro accumulated proteoglycans which resolved on chromatography into three classes of molecules (Fig. 1). This pattern,
resembles that of proteoglycans synthesized by other differentiated cartilage,
such as 16-day embryonic sternum or 11-day vertebral cartilage, but differs in
that the larger aggregates are only 30-35 % of the total, less than half that in the
matured cartilage (Fig. 1, dotted lines). The intermediate-size molecule, with a
kav 0-3-0-38, predominated and constituted 65 % of the total proteoglycans.
Proteoglycans from 8-day notochord culture, when dissolved in the feeding
medium and provided to somite culture, resulted in a 65 % increase in radioactive sulphate incorporation into sulphated glycosaminoglycans. However,
proteoglycans from 3-day notochord culture failed to elicit such induction (Table
1). We reported earlier (Vasan, 1981a) that in proteoglycans from 3-day
notochord culture small molecules predominate.
Subsequent experiments determined whether the large, intermediate, and
2-51-
VT
Fig. 1. Molecular sieve chromatogram (CPG-10-2500) of proteoglycans extracted
from notochord cultured in vitro for 8 days (solid line). The broken line showing the
elution pattern of proteoglycan extracted from 16-day-old chick embryonic sternum
or 11-day vertebral cartilage compares material from differentiated cartilage.
268
N. VASAN
Table 1. Sulphated glycosaminoglycan synthesis
DPM/nG of DNA ± S.D.
Somites
Somites + proteoglycan obtained from 8 days
notochord culture
Somites + proteoglycan obtained from 3 days
notochord culture
19-4 ±4-1
33-1 ±6-3
21-6 ±5-7
The data are from five experiments. Every attempt was made to maintain each culture to
same tissue size.
small proteoglycans in the perinotochordal sheath are necessary for inductive
interaction. We could not isolate enough of these three classes of proteoglycans
from the notochord and conduct experiments, since not enough material was
available. We used chemical compounds dissolved in the feeding medium for 6
days to alter the type of perinotochordal proteoglycans synthesized. Notochords
treated with 5-bromodeoxyuridine, excessive vitamin A, and hyaluronic acid
accumulated smaller size proteoglycans (Fig. 2), whereas the control culture
accumulated 30-35 % larger proteoglycans. Differentiated chondrocyte exposed
to BUdR (Palmoski & Goetinck, 1972; Levitt & Dorfman, 1973; Daniel et al.
1973), excessive vitamin A (Solursh & Meier, 1973; Vasan & Lash, 1975; Vasan,
19816), and hyaluronic acid (Toole, Jackson & Gross, 1972; Solursh, Vaerwyck
S 2
Pu
U
0
10
20 t
30
40
I Fraction number
Vo
50
601
70
|
V-,
Fig. 2. Molecular sieve chromatographic (CPG-10-2500) profile of proteoglycan
extracted from notochord after 8 days in culture exposed to normal mediumcontrol (broken line), BUdR (dashed line), and hyaluronic acid (solid line). Note the
loss of large molecular weight proteoglycans in the treated tissues. Brief exposure of
BUdR also caused irreversible alteration in the synthesis of large molecular weight
proteoglycan.
Perinotochordal proteoglycans and somite chondrogenesis 269
& Reiter, 1974), lost chondrogenic expression and accumulated small
proteoglycan molecules. The absence of larger molecular weight proteoglycan
in the treated notochords made it possible to evaluate the role of this molecule
in tissue induction. When somites were cocultured with such treated notochords,
or were treated in the presence of proteoglycan extracted from these notochords,
notochords failed to induce synthesis of large proteoglycan molecules (Fig. 3).
The somite culture fed on a medium containing proteoglycans from normal
notochords increased the synthesis of proteoglycans excluded from the column.
The experimental culture synthesized molecules that are smaller and eluted at
the total volume of the column. Notochord was also exposed to BUdR for 24 h
and cultured (after washing in BUdR-free medium) for 6 days in normal
medium. Proteoglycans extracted from such notochords also failed to induce
somite chondrogenesis (figure not shown).
Crude proteoglycan extract obtained from the 16-day embryonic sternum
markedly stimulated radioactive sulphate incorporation (47-1 dpm/ng DNA)
into sulphated chondromucoprotein synthesized by the somites. However,
purified proteoglycan aggregate (Al) and monomer preparation (Al-Dl)
elicited different stimulation in sulphated chondromucoprotein synthesis
(36-3 dpm/ng DNA and 29-8 dpm/ng DNA respectively). Interestingly, treating
the crude proteoglycan preparation with papain resulted in considerable loss of
the inductive capacity (Table 2). Low-salt-extracted proteoglycan, which is
mostly smaller size molecules (also known as non-aggregating proteoglycans,
o
X
5p.
<J
O
C/5
0
10
20 t
30
40
| Fraction number
VO
50
60 t
70
|
VT
Fig. 3. Molecular sieve chromatogram (CPG-10-2500) of proteoglycan extracted
from 6-day-old somite cultures under normal and experimental conditions. The
somites were exposed to proteoglycan extracted from notochords grown in normal
medium for control (broken line), hyaluronic acid-containing medium (solid line)
and BUdR-containing medium (dashed line). The proteoglycan used in these experiments are just 4-OM-guanidinium chloride extract not subjected to any purification.
A similar effect occurred when treated notochords were cultured (after several
washings with SBSS buffer) with somites.
EMB73
270
N. VASAN
Table 2. Sulphated glycosaminoglycan synthesis
DPM/nGofDNA±s.D.
Untreated Somites
18-2±3-l
Somites + crude proteoglycan preparation
47-116-2
Somites + purified proteoglycan aggregate (Al)
36-3 ±3-1
Somites + purified proteoglycan monomer (Al-Dl)
29-8 ±2-8
Somites + papain-treated crude proteoglycan preparation
25-4 ±4-3
Somites + low-salt-extracted proteoglycan
19-7 ±2-6
The data are from five different experimental analyses. Tissue volume was maintained
almost the same in all the experiments. Various proteoglycan preparations were substituted
in the medium at 150 ^g of uronic acid/ml.
(Hardingham & Muir, 1974), did not show any stimulatory effect when included
in the feeding medium of somites (Table 2). The concentration dependent
stimulatory effect of crude proteoglycans on somite chondrogenesis was studied
by using various levels (50-200 /ig/ml) in the feeding medium. Maximum stimulation was seen at 150/ig/ml, while higher concentration (in a few experiments) did not result in any appreciable increase (Fig. 4).
X
S
v 1
O
C/5
50
100
150
200
250
uronic acid/ml
Fig. 4. Radioactive sulphate incorporated into sulphated glycosaminoglycans of
6-day-old somite culture. Tissue volume was maintained at the same level in every
culture. Proteoglycan was dissolved in the feeding medium and provided to the
somite culture (open circle), while the control culture (closed circle) received regular
feeding medium. The results are the average of three experiments. 250 ^g/ml (values
not shown) did not increase the incorporation level compared to 200jUg/ml.
Perinotochordal proteoglycans and somite chondrogenesis 271
DISCUSSION
Recent advances in embryonic tissue culture technology and procedures for
microanalysis of matrix enabled us to investigate the inducer components involved in tissue interaction. Improved in vitro culture of notochord for long times
is possible (Vasan, 1981a). Molecular sieve chromatography resolves three sizes
of proteoglycans in guanialium chloride extract of 8-day notochord culture (Fig.
1). In differentiated cartilage which has also been shown to synthesize three
proteoglycan populations (Lash, Ovadia & Vasan, 1978; Vasan, 1981a) large
aggregate (PG-A) forms up to 75 % of the total; intermediates (PG-I) form
15-20%, and small monomers the remainder (Fig. 1). These percentages
contrast with those from 8-day notochord culture where the aggregates form less
than half the percentages found in a typical differentiated cartilage. If cultured
for longer than 8 days, the notochord may or may not accumulate greater proportions of large aggregates, but such culture has not been possible. Notochords may
degenerate after 8 days, in culture, as in vivo because they have a programmed
fate, (Vasan, 1981a).
Subsequent experiments were directed to understanding the importance of the
large perinotochordal proteoglycan aggregates in somite chondrogenesis. Earlier in vitro studies showed that the notochord accumulates mostly smaller (PGM) molecules on the third day, and that on the eighth day the size of the
proteoglycans increases (Vasan, 1981a). Extracts from 3-day cultures stimulated
somite chondrogenesis very little compared to 8-day materials (Table 1). The
large proteoglycan molecules in the perinotochordal sheath seemed to induce
somite chondrogenesis more effectively than the smaller monomers.
Subsequent studies were conducted to establish this view. Notochords exposed to substances causing metabolic alterations lost large proteoglycans in the
perinotochordal sheath (Fig. 2). Normal chondrogenic stimulation was not observed in somites cultured in the presence of treated notochords or proteoglycans
extracted from them (Fig. 3). This effect was not due to traces of the inhibitor
in the tissues or attached to the proteoglycans since chick sternal proteoglycan
aggregate included with the treated material stimulated normal chondrogenesis.
Enzymatic removal of perinotochordal matrix material caused the notochords
to lose the capacity to induce somite chondrogenesis. But in subsequent culturing, the treated notochords accumulated perinotochordal materials and induced
somite chondrogenesis (Kosher & Lash, 1975). In vivo treatment of chick embryos with substances like L-azetidine-2-carboxylic acid or 6-diazo-5-oxo-Lnorleucine which cause abnormal development also resulted in reduced accumulation of perinotochordal materials and loss of somite differentiation
(Strudel, 1975; Vasan, unpublished observation). This again establishes the
effectiveness of proteoglycans in somite chondrogenesis.
Both crude proteoglycan extract of embryonic sternal cartilage (Kosher &
Lash, 1975; Lash & Vasan, 1978), and purified proteoglycan aggregates (Lash,
272
N. VASAN
Belsky & Vasan, 1977; Belsky, Vasan & Lash, 1980) induced somite chondrogenesis more effectively than purified monomer fractions (Lash & Vasan,
1978). When low-salt-extracted non-aggregate proteoglycan (Hardingham &
Muir, 1974) was included in the feeding medium of the somite cultures, the
characteristic induction of somite chondrogenesis was not observed (Table 2).
Crude chick embryo sternum proteoglycan was digested with papain and resolved on a Sephadex G-200 column to obtain glycosaminoglycan chains. The large
included fraction which contains these chains incorporated very little radioactive
sulphate into somites, while the untreated specimen incorporated much more
(Table 2). The chondroitin sulphate and keratan sulphate chains of proteoglycan
are poor inducers of somite chondrogenesis. This result agrees with the earlier
report of Lash and his co-workers (Lash & Vasan, 1978). Urea and guanidium
chloride were also used in solubilizing fibronectin from the tissues (Hynes,
Destree & Mautner, 1976; Yamada, Schlesinger, Kennedy & Pastan, 1977). It
is possible that the 4-OM-guanidium chloride used to extract proteoglycan from
the sternal cartilage also removed some fibronectin contaminant in the preparation. Crude proteoglycan may exert its stimulating effect because of the fibronectin. SDS polyacrylamide gel electrophoresis of the sternal proteoglycans extracted with 4-OM-guanidinium chloride showed a protein migrating with catalase
(232 x 10~3 relative molecular mass), suggesting the presence of fibronectin. We
are preparing fibronectin-free proteoglycans in order to investigate the role of
fibronectin in embryonic induction.
The present study supports the hypothesis of Grobstein (1956) that extracellular matrix materials play a vital role in embryonic development. It also
shows that the microenvironment of perinotochordal proteoglycans modulates
the somite chondrogenesis. The reason for a specific need to have large
proteoglycan molecules in the perinotochordal sheath for maximum somite induction is not clear. At this point, we can only speculate on the mechanism.
Perhaps, as Nevo & Dorfman (1972) suggest, an interaction of proteoglycan with
components of the cell surface activates or stimulates existing metabolic patterns. Such interactions might also influence the transport of essential substances
to and from the cells. Because of its negatively charged sulphate and carboxylate
groups, a high molecular weight polyanion, such as proteoglycan aggregate, will
influence the diffusion of ions. This effect may have important consequences.
For example, it may attract calcium ions whose concentration affects a wide
range of physiological processes. This view has been supported by Shulman &
Opler (1974), who have reported the stimulatory effect of calcium on the
synthesis of cartilage proteoglycan. However, attempts to correlate our results
with the effects of extractable tissue components and of the ionic microenvironment will have to await further experimentation.
This investigation was supported in part by a grant from NIH 5 S07 RR05393 and NICHHD1RO1HD16147. The author wishes to thank Marge Pascavage for typing the manuscript and
Dr Al Butrym for the editorial help.
Perinotochordal proteoglycans and somite chondrogenesis 273
REFERENCES
S. & LASH, J. W. (1980). Extracellular matrix components and somite
chondrogenesis: A microscopic analysis, Devi Biol. 79, 159-180.
BERNFIELD, M. R., BANERJEE, S. S. & COHN, R. H. (1972). Dependence of salivary epithelial
morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. /. Cell Biol. 52, 674-689.
BITTER, T. & Mum, H. (1962). A modified uronic acid carbozole reaction. Anal. Biochem. 4,
330-334.
CHENEY, C. M. & LASH, J. W. (1981). Diversification within embryonic chick somites: Differential response to notochord. Devi Biol. 81, 288-298.
DANIEL, J. C , KOSHER, R. A., LASH, J. W. & HERTZ, J. (1973). The synthesis of matrix
components by chondrocytes in vitro in the presence of 5-bromodeoxyuridine. Cell Different. 2, 285-298.
DELUCA, S., HEINEGARD, D. H., HASCALL, V. C , 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.
GALBRAITH, D. B. & KOLLAR, E. J. (1976). Procollagen enhancement of mouse tooth germ
development in vitro. Amer. Zool. 16, 183a.
GROBSTEN, C. & HOLTZER, H. (1955). In vitro studies of cartilage induction in mouse somite
mesoderm. /. exp. Zool. 128, 333-357.
GROBSTEIN, C. (1956). Inductive tissue interactions in development. Advan. Cancer Res. 4,
187-236.
GROBSTEIN, C. (1957). Some transmission characteristics of the tubule-inducing influence on
mouse metanephrogenic mesenchyme. Expl Cell Res. 13, 575-587.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HARDINGHAM, T. E. & Mum, H. (1974). Hyaluronic acid in cartilage and proteoglycan
aggregation. Biochem. J. 139, 565-581.
HASCALL, V. C. & HEINEGARD, D. (1979). Structure of cartilage proteoglycans. In Glycoconjugate Research, (ed. J. D. Gregory & R. W. Jeanloz), pp. 341-374. New York: Academic
Press.
HEINEGARD, D. (1972). Extraction, fractionation and characterization of proteoglycans from
bovine tracheal cartilage. Biochim. Biophys. Ada. 285, 181-192.
HOLTZER, H. & DETWEILER, S. R. (1953). An experimental analysis of the development of the
spinal column. III. Induction of skeletogenous cells. /. exp. Zool. 123, 335-366.
HYNES, R. D., DESTREE, A. T. & MAUTNER, V. (1976). In Membranes and Neoplasia: New
Approaches and Strategies, (ed. V. Marchesi), pp. 189-201. New York: Alan R. Liss, Inc.
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.
KOSHER, R. A. & LASH, J. W. (1975). Notochord stimulation of in vitro somite chondrogenesis
before and after enzymatic removal of perinotochordal materials. Devi Biol. 42, 362-378.
KOSHER, R. A. & CHURCH, R. L. (1975). Stimulation of in vitro somite chondrogenesis by
procollagen and collagen. Nature 258, 327-330.
KVIST, T. N. & FINNEGAN, C. V. (1970). The distribution of glycosaminoglycans in the axial
region of developing chick embryo. 2: Biochemical analysis. /. exp. Zool. 175, 241-257.
LASH, J. W. (1967). Differential behaviour of anterior and posterior embryonic chick somites
in vitro. J. exp. Zool. 165, 47-56.
LASH, J. W., BELSKY, E. & VASAN, N. S. (1977). Stimulation chondrogenic differentiation
with extracellular matrix components: An analysis using scanning electron microscopy. In
Cell Interaction in Differentiation (ed. M. Karkinen-Jaaskelainen, L. Saxen & L. Weiss).
New York: Academic Press.
LASH, J. W. & VASAN, N. S. (1978). Somite chondrogenesis in vitro: Stimulation by exogenous
extracellular matrix components. Devi Biol. 66, 151-171.
BELSKY, E., VASAN, N.
274
N. VASAN
LASH, J. W., OVADIA, M. & VASAN, N. S. (1978). Microheterogeneities, nonequivalance, and
embryonic induction. Med. Biol. 56, 333-338.
S. & DORFMAN, A. (1973). Control of chondrogenesis in limb-bud cell cultures by
bromodeoxyuridine. Proc. natn. Acad. Sci., U.S.A. 70, 2201-2205.
LINSENMAYER, T. F., TRELSTAD, R. L. & GROSS, J. (1973). The collagen of chick embryonic
notochord. Biochem. Biophys. Res. Commun. 53, 39-45.
MEIER, S. & HAY, E. D. (1975). Stimulation of corneal differentiation by interaction between
cell surface and extracellular matrix. I. Morphometric analysis of transfilter 'induction'. /.
Cell Biol. 66, 275-291.
NEVO, Z. & DORFMAN, A. (1972). Stimulation of chondromucoprotein synthesis in chondrocytes by extracellular chondromucoprotein. Proc. natn. Acad. Sci., U.S.A. 69,
2069-2072.
OEGEMA, T. R., HASCALL, V. C. & DZIEWIATKOWSKI, D. D. (1975). Isolation and characterization of proteoglycans from the Swarm rat chondrosarcoma. J. biol. Chem. 250, 6151-6159.
PALMOSKI, M. J. & GOETINCK, P. F. (1972). Synthesis of proteochondroitin sulphate by normal, nanomelic, and 5-bromodeoxyuridine-treated chondrocytes in cell culture. Proc. natn.
Acad. Sci., U.S.A. 69, 3385-3388.
SANTOIANNI, P. & AYALA, M. (1965). Flurometric ultramicroanalysis of deoxyribo-nucleic
acid in human skin. J. Invest. Dermatol. 45, 99-103.
SHULMAN, H. J. & OPLER, A. (1974). The stimulatory effect of calcium on the synthesis of
cartilage proteoglycan. Biochem. Biophys. Res. Commun. 59, 914-919.
SOLURSH, M. & MEIER, S. (1973). The selective inhibition of mucopolysaccharide synthesis by
vitamin A treatment of cultured chick embryo chondrocytes. Calcif. Tiss. Res. 13,131-142.
SOLURSH, M., VAERWYCK, S. A. & REITER, R. S. (1974). Depression by hyaluronic acid of
glycosaminoglycan synthesis by cultured chick embryo chondrocytes. Devi Biol. 41,
233-244.
STRUDEL, G. (1971). Material extracellilaire et chondrogenese vertebrale. C.r. hebd. Seanc.
Acad. Sci. Paris. 272, 473-476.
STRUDEL, G. (1975). Effect of an L-proline analogue, the L-azetidine-2-carboxylic acid on the
phenotypic differentiation of the chick somite mesenchyme. C.r. hebd. Sianc. Acad. Sci.,
Paris 280, 1007-1010.
TOOLE, B. P., JACKSON, G. & GROSS, J. (1972). Hyaluronate in morphogenesis: Inhibition of
chondrogenesis in vitro. Proc. natn. Acad. Sci., U.S.A. 69, 1384-1386.
VASAN, N. S. & LASH, J. W. (1975). Chondrocytes metabolism as affected by vitamin A.
Calcif. Tiss. Res. 19, 99-107.
VASAN, N. S. (1979). Notochordal tissue in chondrogenic differentiation of somites. Xlth
International Congress of Biochemistry. 07-6-H108 ABST.
VASAN, N. S. (1980). Perinotochordal proteoglycans in chondrogenic induction of somites. /.
Cell Biol. 87, 124a.
VASAN, N. S. (1981a). Analysis of perinotochordal materials. I. Studies on proteoglycan
synthesis. /. exp. Zool. 215, 229-233.
VASAN, N. S. (19816). Proteoglycan synthesis by sternal chondrocytes perturbed with vitamin
A. /. Embryol. exp. Morph. 63, 181-191.
VON DER MARK, H. K. & GAY, S. (1976). Study of differential collagen synthesis during
development of the chick embryo by immunofluorescence. I. Preparation of collagen Type
I and Type II. Specific antibodies and their application to early stages of the chick embryo.
Devi Biol. 48, 237-249.
WATTERSON, R. L., LOWLER, I. & FOWLER, 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.
YAMADA, K. M., SCHLESINGER, D. H., KENNEDY, D. W. & PASTAN, I. (1977). Characterization
of a major fibroblast cell surface glycoprotein. Biochemistry 16, 5552-5559.
LEVITT,
(Accepted 12 September 1982)