J. Embryo!, exp. Morph. Vol. 56, pp. 225-238, 1980
Printed in Great Britain © Company of Biologists Limited 1980
225
Analysis of glycosaminoglycans during
chondrogenesis of normal and brachypod
mouse limb mesenchyme
By JANET SHAMBAUGH 1 AND WJLLIAM A.ELMER 2
From the Biology Department, Emory University, Atlanta, Georgia
SUMMARY
Studies have been carried out to characterize radioactive incorporation rates and steadystate levels of hyaluronic acid (HA) and chondroitin sulfate (CS) as well as hyaluronidase
activity in the hind limbs of normal and brachypod mouse embryos between 11-14 days of
gestation. The results of the analysis show that changes in the synthetic and degradative rates
of HA and CS occur at about the 12-5-day stage in normal hind limbs. These changes include
an increased rate of CS synthesis, a decreased rate of HA synthesis, and a correspondingly
sharp, transitory rise in hyaluronidase activity. Similar changes also occur in brachypod hind
limbs but appear to be delayed in onset by approximately one-half day. In addition, the
mutant hind limb exhibits a slower loss of HA at a time when turnover would be expected
to be on the increase. These changes are concomitant with cell surface alterations and abnormal mesenchymal condensation formation which have been previously shown to occur
in this mutant.
INTRODUCTION
Changing physiological levels of the cartilage glycosaminoglycans (GAGs),
hyaluronic acid (HA) and chondroitin sulfate (CS), are thought to be important
to chondrogenesis, and therefore, to normal skeleton formation. Thus, a correlation has been shown between HA and morphogenesis in several developing
systems (Polansky, Toole & Gross, 1973; Toole, 1973 a; Pratt, Larson & Johnston, 1975; Solursh, 1976; Greenberg & Pratt, 1977; Solursh & Morriss, 1977;
Derby, 1978, Orkin & Toole, 1978), and removal of HA has been correlated with
subsequent cytodifferentiation of chondrocytes (Toole, 1972; Corsin, 1975;
Smith, Toole &~ Gross, 1975). While most studies of chondrogenesis have
focused on CS or CS proteoglycan in the chick limb system (Goetinck, Pennypacker & Royal, 1974; Hascall, Oegema, Brown & Caplan, 1976; DeLuca et al.
1977; Holtzer, Okayama, Biehl & Holtzer, 1978) and in the mouse limb system
(Royal & Goetinck, 1977), changing levels of both GAGs have been described
using only radioactive labeling in chick limb development (Toole, 1972) and
newt limb regeneration (Toole & Gross, 1971; Smith et al. 1975). A few studies
have measured actual levels of HA and CS - one in chick axial skeleton (Kvist
& Finnegan, 1970) and another in neurulating chick (Derby, 1978).
1
Author's address: Biology Department, Johns Hopkins University, Baltimore, Maryland
21218 U.S.A.
2
Author's address: Biology Department, Emory University, Atlanta, Georgia 30322, U.S.A.
226
J. SHAMBAUGH AND W. A. ELMER
The present study characterizes radioactive incorporation rates and steadystate levels of HA and CS, as well as hyaluronidase (HAdase) activity, during
the early stages of hind-limb development in normal ( + / + ) mice and mice
which carry the brachypod (bp^/bp3) mutation. This mutation, which affects
the differentiation and development of the appendicular skeleton, appears to
act by causing a delay and reduction in the formation of the mesenchymal
condensations which subsequently develop into the cartilaginous anlagen of the
skeletal elements (Griinebarg & Lee, 1973; Elmer, 1976). The defect in the condensation process has been correlated with alterations in the surface properties
of the prechondrogenic mesenchyme (Duke & Elmer, 1977, 1978, 1979; Hewitt
& Elmer, 1978). It has not been reported, however, whether biochemical differences of extracellular matrix components are being displayed concomitantly
during this developmental time period. The data presented in this paper indicate
that an alteration in the levels of the glycosaminoglycans is also occurring at this
time period.
MATERIALS AND METHODS
Source of tissue
Normal and mutant mice were obtained from Jackson Laboratory, Bar Harbor, Maine. Homozygous bp3 /bp3 mice were bred onto the same hybrid background as the phenotypically normal mice ^CAFi/J). Embryos of a particular
gestational age were generated using timed matings with the appearance of
the vaginal plug called day zero. Embryos were removed from the uterus in
warm, sterile Tyrode's solution and staged as described previously (Elmer &
Selleck, 1975). The hind-limb buds were excised using iridsctomy scissors and
either placed into organ culture (Kochhar & Aydelotte, 1974) or treated further
for cell culture (Elmer & Selleck, 1975). For culturing the whole limb bud, the
medium consisted of 75 % BGJ chemically defined medium (Difco Laboratories),
25 % fetal calf serum (GIBCO), 150 /tg/ml ascorbate (Nutritional Biochemicals
Corp.), and 50 /tg/ml gentamicin sulfate (Garamycin, Schering Pharmaceutical
Corp.). For cell culture, limb mesenchyme was obtained by removing the ectoderm with microneedles after a 5 min incubation at room temperature in 2-25 %
trypsin (GIBCO) plus 0-75 % pancreatin. The resulting mesodermal core was
placed in calcium-magnesium-free Tyrode's solution for 30 min at 25 °C to
dissociate the cells. The cells were plated in 35 mm plastic tissue culture dishes
(Falcon) at a density of 1 x 106 cells/ml in 3 ml Eagle's Basal Medium with
Hank's salts supplemented with glutamine (GIBCO), 10% horse serum
(GIBCO), and 50 ^g/ml gentamicin sulfate. Both cell and organ cultures were
incubated in a humidified atmosphere of 5 % CO2, 95 % air, at 37 °C.
Quantification of glycosaminoglycans
The radioactive precursor D- [6-3H]glucosamine-HCl (sp. act. 20-30 Ci/
mmole, Amersham-Searle) used in the synthesis of HA, CS and glycoproteins
Glycosaminoglycan levels in normal and brachypod limbs
227
(GPs) was added at t0 to the medium at a concentration of 5 /tCi/ml for the
organ cultures and 1 /tCi/ml for the cell cultures. Since the incorporation rate
was observed to be linear for at least 12 h for both genotypes, a 6 h labeling
period was used throughout all of the experiments. Cell and organ cultures were
also labeled concurrently with 5 /*Ci/ml of Na235SO4 (carrier free, AmershamSearle) as an additional indicator of sulfated GAGs. The GAGs were extracted
from either one cell-culture plate (cell layer plus medium) or from 10 limb buds
combined with 20 unlabeled limb buds by shaking in 0-5 M-NaOH and 1-0 M
sodium borohydride overnight at 25 °C. After dialysis against running water
overnight, an aliquot was removed for DNA determination (Giles & Myers,
1965). For the limb-bud retentate, the GAGs were precipitated with three volumes of cold ethanol plus 5 % potassium acetate, and chromatographed on a
column of DEAE-52 cellulose (Whatman) with a linear gradient of 0-1-2 MLiCl in 0-05 M tris buffer, pH 7-2 (Pratt, Goggins, Wilk & King, 1973). Fractions
were collected (3 ml) and a 0-5 ml aliquot of each fraction was assayed for
radioactivity in 10 ml of ACS (Amersham-Searle) liquid scintillant using a
Beckman LS 100 counter to determine the elution profile. The fractions within
each peak (fractions 14-20 for HA and 22-37 for CS) were pooled, dialyzed,
concentrated to 1-0 ml using a Molecular Separator Kit (Millipore) and assayed
for uronic acid content (Bitter & Muir, 1962) and radioactivity. The identity
of the peaks was confirmed by digesting the concentrated effluent with enzymes
of narrow specificity: l-0mg/ml leech head hyaluronidase (Biotrics, Inc.,
Arlington, Mass.), 5 units/ml chondroitinase AC or ABC (Sigma) in enriched
tris buffer (Saito, Yamagata & Suzuki, 1968), and l-0mg/ml heparanase
(kindly supplied by A. Linker, V. A. Hospital, Salt Lake City, Utah) in 0 1 M
sodium acetate buffer, pH 7-0 (Linker & Hovingh, 1972).
HA levels in limb-bud extracts were also quantified using the Reissig test
(Reissig, Strominger & Leloir, 1955) after digestion with Streptomyces HAdase
(Calbiochem, Grade B) as described by Hatae & Makita (1975). For this assay
the NaOH extract was first deproteinated by precipitation with 10% trichloroacetic acid (TCA). The TCA was extracted with three changes of an equal
volume of ethyl ether, and the GAGs were precipitated from the aqueous phase
with 3 vol. of cold ethanol containing 5 % potassium acetate. The GAGs
were dissolved in 0-2 ml of 0-05 M sodium acetate buffer, pH 5-0, and two
Turbidity Reducing Units (TRU) of Streptomyces HAdase were added. After
incubation overnight at 55 °C, the pH was adjusted to 9-2 before measuring free
hexosamine reducing ends.
Cell cultures were extracted in a similar manner. As described above, excess
protein was first removed from the dialysis retentate using TCA. Separation of
GAGs was then accomplished by sequential collection on a filter of pore size
0-45 /tm (Millipore) according to methods of Saarni & Tammi (1977). The
scintillant used was ACS (Amersham-Searle).
228
J. SHAMBAUGH AND W. A. ELMER
Loss of glycosaminoglycans in culture
Approximation of GAG turnover in limb-bud organ cultures was measured
by pulse labelling for 6 h followed by a 4-day incubation in medium minus
the radioactive precursor and containing a 50-fold excess (10 fiu) of unlabeled
glucosamine. Cultures were fed by changing 1 ml of medium each day. Cultures
were terminated at 1-day intervals by extracting four limb buds as described
above. The extract was dialyzed, and DNA content and radioactivity determined.
HAdase activity in limb-bud homogenates
HAdase activity was measured in a crude homogenate of 50-100 limb buds
by the Reissig test (Reissig et al. 1955) using 100 fig of HA as substrate (Sigma,
Umbilical Cord) for a 20 h incubation at 37 °C (Polansky, Toole & Gross,
1973). Alternatively, [3H]HA isolated from limb-bud cultures by DEAE chromatography was used as substrate. A unit of activity is denned as fig HA
digested/10 limb buds/24 h.
RESULTS
Glycosaminoglycans were quantified over developmental time in two ways
in order to obtain a more complete description of the changes occurring in
the limb bud. In one assay, a colorimetric analysis of the GAG levels was
carried out to determine the quantity of GAGs at different stages of early
development. The other assay involved the use of the radioactive precursors,
[3H]glucosamine and Na35SO4, to determine the amount of HA, CS and GPs
Table 1. Deoxyribonucleic acid levels in normal and brachy pod limb buds*
Day of gestation
bpu/bpK
No. of determinations
11-5
12-5
13-5
14-5
9-l±2-4f
9-2 ±1-3
9
17-2±3-2
17-4 ±3-8
12
23-6±0-9
23-2 ±0-8
5
31-3±41
30-0+1-7
4
* DNA was quantified by the diphenylamide reaction in dialyzed sodium hydroxide extracts of limb buds.
t Mean±s.D.
produced during a particular 6 h period. However, since these values are determined by synthesis minus degradation, the loss of radioactivity was also
measured to determine the importance of degradation to rate. The data are
expressed on either a per limb bud or per fig of DNA basis since the DNA
content was not significantly different between the two genotypes in limb buds
of the same age (Table 1).
To perform the colorimetric and radioactive assays, HA, CS and GP were
Glycosaminoglycan levels in normal and brachypod limbs
229
first separated by anion exchange chromatography as described under Methods.
A sample chromatograph is shown in Fig. 1, indicating how the fractions were
pooled to give three groups. Peak one co-elutes with HA standard, whereas,
peak two which contains both 3 H and 35S co-elutes with CS standard. The exact
nature of the components present in the two major peaks was confirmed using
enzymes with narrow specificities. These results are summarized in Table 2.
The first major peak is 90% digestible by leech hyaluronidase. This enzyme
has absolute specificity for HA. The second, broader peak is about 40%
chondroitin sulfate A and C, 30% chondroitin sulfate B, and 20% heparan
sulfate. The remaining 10% could be keratin sulfate since this GAG is not
degraded by any of the enzymes used. Since the first major peak contains mainly
HA and the second peak contains mainly CS, HA and CS were routinely quantified by summing the fractions (14-20 for HA and 22-37 for CS) within their
respective peaks. All fractions collected before the HA peak were also pooled
to quantitate the GPs (Pratt et al. 1973). The percent composition of the peaks
did not differ significantly between the two genotypes (Table 2), therefore HA
and CS may be compared between genotypes by measuring the material collected
within the two peaks.
Table 2. Identification of components in the DEAE column
effluent using specific enzyme digestions
Peak 1 *
% digested by
leech HAdase
Peak 2
% digested by
CSase ABC
CSase AC
Heparanase
Leech HAdase
Day 11-5
Day 12-5
Day 13 5
Day 14-5
72 (74)f
88 (92)
90
94 (86)
58 (49)
65 (68)
75
12 (61)
—
—
0(0)
35 (35)
17 (16)
—
40
19
—
—
—
0(0)
* Peak 1 (fractions 14-20) represents HA, peak 2 (fractions 22-37) is CS.
f Values in parentheses are results from digestion of pb^/bp™. All other values are from
+ / + limb mesenchyme extracts.
Chondroitin sulfate measurements
The radioactive incorporation of [3H]glucosamine into CS (Fig. 2 A) displays
a constant rate until 12-5 days of gestation, when the rate increased linearly
(r2 = 0-97 for linear regression of line described by last four points, n = 2). The
timing of this abrupt change corresponds to the onset of visible areas of cell
condensation and cartilage matrix deposition in the normal limb. In the mutant,
condensations are also visible, however, they are smaller in size (Griineberg &
Lee, 1973) and contain a lesser amount of matrix material (Duke & Elmer,
230
J. SHAMBAUGH AND W. A. ELMER
Glycoproteins
'Peak one
Peak two
45
x
£
30
15
12
18
24
Fraction number
30
36
Fig. 1. A representative DEAE-cellulose chromatogram of glycosaminoglycans
from 13-day + / + mouse hind limbs. [3H]Glucosamine and Na235SO4, each at
5 /iCi/ml, were added at t0 to the organ culture medium for a period of 6 h. Ten limb
buds were extracted together with 20 unlabeled buds, and the GAGs precipitated with
ethanol before being applied to the column. Fractions were tested for radioactivity,
and the material within each peak was pooled as indicated by the vertical lines. The
concentrated peaks were tested for uronic acid content and enzyme susceptibility.
Peak one represents HA and peak two is CS. Solid line, PH] counts; broken line,
[35S] counts.
1979). Although the synthetic rates coincide closely, the change at 12-5 days
does not appear to be quite as sharp in the mutant limb bud as compared to the
normal control.
The increase in micrograms of CS over developmental time is shown in
Fig. 2D. The quantitative levels in the limb buds at the different ages are consistent with the integral of the rate function, as expected. In other words, the
linear increase in micrograms (Fig. 2D) corresponds to the constant segment
in the rate (Fig. 2A) while a change to quadratic increments (Fig. 2D) after
12-5 days of development corresponds to the linear portion of the rate function
in Fig. 2A.* When this regression line is used to calculate x values (time in
days) for the actual y values for + / + (micrograms of CS), the accumulation
of CS in the brachypod limbs is observed to lag significantly (P < 0-01) behind
their normal counterparts for each point past day 12 (paired t test for last four
time points in Fig. 2D). The lag amounts to nearly one-half day by day 12-5
through 14-5 (mean ± S.D. = 10 h ± 0-5 h).
* A regression line can be fitted to these data by weighting the time points that correspond
to the quadratic increase by coefficients which give a linear transformation. Thus, when the
time points of days 1.1-5 through 14-5 are weighted by the following coefficients: — 2, —1,0,
.1, 4, 9, 16, the resulting linear regression line produces a correlation coefficient, r2 = 0-99
for + / + points, n = 8.
Glycosaminoglycan levels in normal and brachypod limbs
231
250
200
100 r
150
en
50
1 ioo
25
XI'
6 50
40
o
x
60
30
2P 40
2G
20
15
20
15
10
10
12
13
14
12
13
14
15
Gestational age (days)
Fig. 2. Incorporation of [3H]glucosamine and uronic acid concentration of macromolecules from different-aged mouse limb buds. (A, D) Chondroitin sulfate; (B,
E) hyaluronic acid; (C) glycoproteins; (F) units of hyaluronidase activity from
different-aged mouse limb buds. Vertical bars in (E) and (F) represent 95% confidence limit of the mean {x±t 0-975 times S.E.). In (E) 4-8 determinations were
carried out for each point by the uronic acid assay and 25 determinations were
made with the Reissig test after Streptomyces HAdase digestion. In (F) three
determinations were made for each point. Assay variances for the incorporation of
[3H]glucosamine, uronic acid assay, and the Reissig test were all within 10%
of the mean. Recovery of known quantities of HA and CS by the methods described
was 90 % with a variance of 3 % within the mean, x — x, + / + ; O—O,bpH/bpyt.
Although the synthetic rates in the organ culture system coincide closely
(Fig. 2A), differences were observed in the incorporation of [3H]glucosamine
by 12-5-day limb mesenchyme grown in cell cultures (Table 3). Under these
culture conditions the normal cells had a significantly higher incorporation rate
than the mutant cells (alpha less than 0-05, two-sided t test, n = 4). The clearer
232
J. SHAMBAUGH AND W. A. ELMER
Table 3. Incorporation of [*H]glucosamine by 12-5-day normal and
brachypod limb mesenchyme in culture (sp. act. cpm/fig DNA)*
Genotype
bpH/bpH
Product
+/+
Glycoproteins
Hyaluronic acid
Chondroitin sulfate
14-01 ±2-16f
42-16± 11-38$
28-96 ±4-39§
17-46±6-69
29-48 + 7-39
20-85 ±2-98
* /^gDNA: + / + , 20-23±3-78; bpH/bpK, 23 08±600.
t Mean ± S.D.
X Statistically significant at a. < 010.
§ Statistically significant at a < 005.
Table 4 . Incorporation of ^S]sulfate by normal and brachypod
limb buds in organ culture
Genotype
Gestational age (days)
+ /+
bpH/bpK
11-5-12-0
12-25-12-75
13-0-140
31212±7204*
23 868 ±6472
80383 ±15379
27094±1485
24145 ±3012
7O33O±29363
* Mean ± S.D., n = 3. Data are expressed as cpm/30 limb buds.
Table 5. Incorporation of [35S]sulfate by 12-5-day normal and
brachypod limb mesenchyme in culture
Genotype
+ /+
bp^/bp*
cpm/mg DNA
4990 ±830*
3380 ±370
* Mean±S.D., n = 4. Difference in means is significant at P < 005, 2-sided t test.
expression of a difference at 12-5 days could be related to the greater availability
of the radioactive precursor to cells in a monolayer as opposed to an organ
culture.
The radioactive precursor, [35S]sulfate, was also used to determine sulfate
incorporation into CS for normal and brachypod limb buds. It is apparent from
the data in Table 4 that the variance in the incorporation of [35S]sulfate in organ
culture is too large to determine if any significant difference exists. However, as
observed with the incorporation of [3H]glucosamine (Table 3), the incorporation of [35S]sulfate by 12-5-day limb mesenchyme grown in cell culture was
significantly lower in the mutant as compared to controls (Table 5). The lower
Glycosaminoglycan levels in normal and brachypod limbs
3
233
35
incorporation of both [ H]glucosamine and [ S]sulfate into CS by brachypod
cell cultures suggests that a decrease in synthesis is occurring rather than a
decrease in sulfation of individual CS molecules.
Hyaluronic acid measurements
The incorporation for HA increases until day 12-5 of gestation and then
declines (Fig. 2B). After day 12-5 the radioactivity is significantly higher in the
+ / + limb bud as compared to bpn/bpa (alpha less than 0-01, two-sided paired
t test for last four time points; mean±s.D. of + / + minus bp^/bp11 = 5592±
114 cpm, n = 6). On the other hand, the micrograms of HA at and before 12-5
days is greater in the bp11 but returns to normal levels after day 12-5 (Fig. 2E;
alpha less than 0-05, two-sided paired / test for time points 12 and 12-5; mean ±
S.D. of + / + minus bp^fbp^ = -9-75 ±6-07, n = 4). Thus the levels of HA
in the mutant limb bud are greater than expected from the incorporation
rate over the entire time period tested and in fact are actually in excess before
day 12-5. In addition, the levels of HA in both genotypes do not reflect the
expected integral of the rate function as was true for CS. Instead, a pronounced
dampening of HA levels occurs during days 12-13 of gestation (Fig. 2E).
It has been reported that prior to the onset of overt chondrogenesis in the
chick limb there is a decreased incorporation of labeled precursor into HA
concomitant with an increase in hyaluronidase (HAdase) activity (Toole,
1972). In view of these observations, experiments were carried out to determine
whether similar changes in enzyme activity were also occurring in the mouse
limb system. As seen in Fig. 2F there is a sharp increase in HAdase activity
between days 12 and 13. The magnitude of the increase is the same for both
genotypes, however, the two curves are not exactly coincident. The delay in the
onset of HAdase activity in the bp^/bp11 limb buds could contribute to the
higher amounts of HA observed before 12-5 days of gestation, even though
no statistical difference was demonstrated.
A series of experiments was carried out, therefore, to determine the turnover time of HA in whole limb buds which were first cultured in the presence
of [3H]glucosamine for 6 h, then washed and further cultured in non-radioactive
medium containing an excess of unlabeled glucosamine (Fig. 3). The rate of
degradation of HA in the normal limb bud shows only a slight change up to 2
days of incubation, but increases sharply after the second day. When the slopes
for the entire lines of both normal and brachypod are calculated, the slope for
bp^/bp3- is less negative (alpha less than 0-001) than for + / + , indicating a
slower loss of radioactive GAGs. The total turnover time was determined to be
be 12 days for + / + and 13 days for bp
Equivalency of glycoprotein measurements
An explanation other than turnover for the differences seen between genotypes
in HA levels and incorporation rates is that the precursor pool of UDP-iV-
234
J. SHAMBAUGH AND W. A. ELMER
2
Days
Fig. 3. Turnover of glycosaminoglycans in organ culture. Twelve-day limb buds were
labeled at t0 with 5 /iCi/ml [3H]glucosamine for 6 h, followed by 4 days of incubation in the absence of tritiated precursor with a 50-fold excess of glucosamine.
Vertical bars represent 95% confidence limit of the mean (x±t 0-975 times S.E.),
n = four determinations for each point from three separate experiments. Slopes are
significantly different P < 0-001, 2-sided / test for regression in two populations.
x — x , + / + ; O - O , bpv/bp*.
acetylglucosamine is larger in the mutant and thus contains relatively less radioactive glucosamine than the control. Thus, the incorporation rates when compared to + / + would appear less than expected based on the actual levels of
HA at all ages tested. An excessively large precursor pool might also be related
to the occurrence of excess HA in the mutant. However, arguing against this
possibility is the incorporation of [3H]glucosamine into GPs (Fig. 2C). Although
GPs utilize the same precursor as HA, the radioactivity from mutant and normal
are not different from day 12-5 on (mean±s.D. of + / + minus bp^/bp11 =
62 cpm, paired t test, n = 9; a at 0-05). In addition, there is no correlation over
developmental time between either [3H]HA and [3H]GP (r2 = 0-30) or [3H]CS
and [3H]GP (r2 = 0-02, n = 9).
DISCUSSION
The low level of CS synthesis at day 11 of gestation corresponds to the time
during which the non-specific CS proteoglycan is found in the mouse limb, while
the sharp increase in synthetic rate at day 12-5 corresponds to the first appear-
Glycosaminoglycan levels in normal and brachypod limbs
235
ance of the cartilage-specfic molecule (Royal & Goetinck, 1977). The delay in
accumulation of CS in the mutant supports previous observations made at the
ultrastructural level (Duke & Elmer, 1979). The decreased CS might be related
to the excess HA found before day 12-5 of gestation. It has been shown that
HA is inhibitory to CS synthesis (Wiebkin & Muir, 1975, 1977; Handley &
Lowther, 1976). Thus, the greater level of HA found in the mutant should act
to suppress chondrogenesis. Alternatively, excess HA may block cell surface
interactions thought to be important to the initiation of chondrogenesis (Toole,
1972; Levitt & Dorfman, 1974). Such a physical barrier between mutant cells
may account for the observed delay in rearrangement of surface molecules that
correlates with differentiation (Hewitt & Elmer, 1978). A third possible action
of excess HA in the mutant involves the observation that cell adhesion is greater
among the mutant cells (Duke & Elmer, 1977). This aberrant adhesion may
account for the abnormal mesenchymal cell condensations observed both in
vivo (Griineberg & Lee, 1973) and in vitro (Elmer & Selleck, 1975) that occur
just prior to chondrification. Since it has recently been shown that HA is an
important factor in SV 3T3 cell adhesion (Underhill & Dorfman, 1978), excess
HA in the mutant might be responsible for the increased adhesion of bpli cells.
Thus, the exact manner in which HA is affecting chondrogenesis is unclear at
present and could be multi-fold.
The relationship of HA and HAdase activity to cytodifferentiation is still
uncertain. Some reports suggest that HA is inhibitory to chondrogenesis
(Toole, 19736; Solursh, Vaerewyck & Reiter, 1974), whereas, others indicate
that it has no effect (Finch, Parker & Walton, 1978). In the present study no
large differences were found between genotypes in the magnitude of HAdase
activity. There was, however, a delay in the appearance of activity, and a slower
turnover of labeled HA was observed for bp^/bp11. Excess GAG and decreased
turnover was described for this mutant in the neonate also (Rhodes & Elmer,
1975). The loss of radioactivity as measured under the described conditions is
not great enough to account for either the differences in the incorporation rates
between the two genotypes at 12-5 days of age or the dampening of HA levels
observed between days 12-13. One explanation for this is that degradation does
not occur in culture at the same rate as it does in vivo. It should also be noted
that the presence of at least two metabolic pools for GAGs that turn over at
different rates has been previously described (Gross, Matthews & Dorfman,
1960; Lohmander, 1977). Differential labeling of such pools may give an erroneous impression of the magnitude of the turnover rate. However, based on the
data presented decreased turnover is still the most probable explanation for
excess HA accumulation in the mutant.
The onset of decreasing HA synthesis has the same inflection point as does
increasing CS synthesis, again indicating the opposing roles of these two molecules. The pronounced dip in the HA synthetic rate at day 13 corresponds to the
peak of HAdase activity. It is noteworthy that at the same time, CS appears
236
J. SHAMBAUGH AND W. A. ELMER
totally unaffected by the increase in HAdase, even though other known mammalian HAdases attack both CS and HA (Saito, Yamagata & Suzuki, 1968;
Hayashi, 1978). A similar specificity was reported in regenerating newt limb
(Toole & Gross, 1971).
The regulatory mechanisms of HA levels that may be defective in brachypod
are unknown at present. The excess HA in the mutant is not due to undersulfated or unsulfated CS since (1) measurements of HA levels made with Streptomyces HAdase, which is specific for HA, also demonstrated excess HA in
the mutant, (2) the HA peak from DEAE chromatography was equally susceptible to leech-head HAdase for each genotype at the stage of excess HA in the
mutant, and (3) the 35S/3H ratio was similar for both genotypes. Thus, this
mutation is apparently different from brachymorph, which displays a similar
limb phenotype but contains undersulfated CS (Orkin, Pratt & Martin, 1976).
In addition, the observations that CS metabolism is nearly normal except for a
delay in the onset of accumulation while HA levels are high indicate that bp11
is unlike other cartilage mutants that display reduced skeletal elements. For
example, nanomelic chicks synthesize no cartilage-specific CS proteoglycan
(Mathews, 1967; Pennypacker & Goetinck, 1976), while the hydrocephalic
(ch+/ch+) mouse shows reduced synthesis of all GAGs (Breen, Richardson,
Bondareff & Weinstein, 1973). In the achondroplastic (en/en) mouse, matrix
materials are normal (Kleinman, Pennypacker & Brown, 1977), and thus the
anomolous cell arrangements may account for the defective cartilage. In the
chondrodysplasia mutation (cho/cho) of the mouse, abnormal arrangement of
cells is thought to be due to abnormal matrix (Seegmiller, Eraser & Sheldon
1971). Brachypod may be related to the mouse mutation, amputated (Flint,
1977) in which the entire skeleton is affected. The abnormal cartilage is thought
to arise from increased cell adhesions that lead to defective migration of cells
into pre-cartilage condensations. A similar conclusion could be drawn for
brachypod. The abnormal HA levels might increase cell adhesion and interfere
with cell interactions of migration, and thereby lead to the incorrect or deficient
partitioning of cells into the mesenchymal condensations. In addition, the residual HA may also suppress the chondrogenic program, which consequently
results in the formation of an abnormal limb.
REFERENCES
T. & MUIR, H. M. (1962). A modified uronic acid carbazole reaction. Analyt. Biochem. 4, 330-334.
BREEN, M., RICHARDSON, R., BONDAREFF, W. & WEINSTEIN, H. G. (1973). Acidic glycosaminoglycans in developing sterno-costal cartilage of the hydrocephalic (ch+/ch+) mouse.
Biochim. biophys. Acta 304, 828-836.
CORSIN, J. (1975). Influence du hyaluronate et de la hyaluronidase sur la chondrogenese
cephalique chez les amphibiens. Acta Embryologiae Experimentalis 1, 15-22.
DELUCA, S., HEINEGARD, D., 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.
BITTER,
Glycosaminoglycan levels in normal and brachypod limbs
237
M. A. (1978). Analysis of glycosaminoglycans within the extracellular environments
encountered by migrating neural crest cells. Devi Biol. 66, 321-336.
DUKE, J. & ELMER, W. A. (1977). Effect of the brachypod mutation on cell adhesion and
chondrogenesis in aggregates of mouse limb mesenchyme. /. Embryol. exp. Morph. 42,
209-217.
DUKE, J. & ELMER, W. A. (1978). Cell adhesion and chondrogenesis in brachypod mouse
limb mesenchyme: fragment fusion studies. /. Embryol. exp. Morph. 48,161-168.
DUKE, J. & ELMER, W. A. (1979). Effect of a brachypod mutation on early stages of chondrogenesis in mouse embryonic hind limbs: an ultrastructural analysis. Teratology 19, 367375.
ELMER, W. A. (1976). Morphological and biochemical modifications of cartilage differentiation in the brachypod and other micromelic mouse embryos. In Mechanisms de la Rudimentation des Organes chez les Embryons de Vertebres (ed. A. Raynaud), pp. 235-242.
Paris: Centre National de la Researche Scientifique.
ELMER, W. A. & SELLECK, D. K. (1975). In vitro chondrogenesis of limb mesoderm from normal and brachypod mouse embryos. /. Embryol. exp. Morph. 33, 371-386.
FINCH, R. A., PARKER, C. L. & WALTON, S. T. (1978). The lack of an inhibitory effect of
hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture. Cell
Differentiation 7, 283-293.
FLINT, O. P. (1977). Cell interactions in the developing axial skeleton in normal and mutant
mouse embryos. In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 465-484. Cambridge University Press.
GILES, K. & MYFRS, A. (1965). An improved diphenylamine method for the estimation of
deoxyribonucleic acid. Nature, Lond. 206, 93.
GOETINCK, P. R., PENNYPACKER, J. P. & ROYAL, P. D. (1974). Proteochondroitin sulfate
synthesis and chondrogenic expression. Exp I Cell Res. 87, 241-248.
GREENBERG, J. H. & PRATT, R. M. (1977). Glycosaminoglycan and glycoprotein synthesis
by cranial neural crest cells in vitro. Cell Differentiation 6, 119-132.
GROSS, J. I., MATTHEWS, M. B. & DORFMAN, A. (1960). Sodium chondroitin sulfate-protein
complexes of cartilage. /. biol. Chem. 235, 2889-2892.
GRUNEBERG, H. & LEE, A. J. (1973). The anatomy and development of brachypodism in the
mouse. /. Embryol. exp. Morph. 30, 119-141.
HANDLEY, C. J. & LOWTHER, D. A. (1976). Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture. Biochim. biophys. Acta 444, 69-74.
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.
HAT\E, Y. & MAKITA, A. (1975). Colorimetric determination of hyaluronate degraded by
Streptomyces hyaluronidase. Analyt Biochem. 64, 30-36.
HAYASHI, S. (1978). Study on the degradation of glycosaminoglvcans by canine liver lysosomal
enzymes. /. Biochem. 83, 149-157.
HEWITT, A. T. & ELMER, W. A. (1978). Developmental modulation of lectin-binding sites on
the surface membranes of normal and brachypod mouse limb mesenchyme cells. Differentiation 10, 31-38.
HOLTZER, H., OKAYAMA, M., BIEHL, J. & HOLTZER, S. (1978). Chondrogenesis in chick
limb buds and somites. Experientia 34, 281-284.
KLEINMAN, H. K., PENNYPACKER, J. P. & BROWN, K. S. (1977). Proteoglycan and collagen of
'achondroplastic' (cn/cn) neonatal mouse cartilage. Growth 41, 171-177.
KOCHHAR, D. M. & AYDELOTTE, M. B. (1974). Susceptible stages of abnormal morphogenesis
in the developing mouse limb analyzed in organ culture after transplacental exposure to
vitamin A. / . Embryol. exp. Morph. 31, 721-734.
KVJST, T. N. & FINNEGAN, C. V. (1970). The distribution of glycosaminoglycans in the axial
region of the developing chick embryo. II. Biochemical analysis. /. exp. Zool. 175, 241258.
LEVITT, D. & DORFMAN, A. (1974). Concepts and mechanisms of cartilage differentiation.
Current Topics in Developmental Biology (ed. A. Moscona & A. Monroy) 8, 103-149.
DERBY,
l6
EMB 56
238
J. SHAMBAUGH AND W. A. ELMER
A. & HOVINGH, P. (1972). Heparinase and heparitinase from Flavo-bacteria. Meth.
Enzymol. 28, 902-911.
LOHMANDER, S. (1977). Turnover of proteoglycans in guinea pig costal cartilage. Archs
Biochem. Biophys. 180, 93-101.
MATTHEWS, M. B. (1967). Chondroitin sulfate and collagen in inherited skeletal defects of
chickens. Nature, Lond. 213, 1255-1256.
ORKIN, R. W., PRATT, R. M. & MARTIN, G. R. (1976). Undersulfated chondroitin sulfate in
the cartilage matrix of brachymorphic mice. Devi Biol. 50, 82-94.
ORKIN, R. W. & TOOLE, B. P. (1978). Hyaluronidase activity and hyaluronate content of the
developing chick embryo heart. Devi Biol. 66, 308-320.
PENNYPACKER, J. & GOETINCK, P. F. (1976). Biochemical and ultrastructural studies of collagen and proteochondroitin sulfate in normal and nanomelic cartilage. Devi Biol. 50,
35-47.
POLANSKY, J. R., TOOLE, B. P. & GROSS, J. (1973). Brain hyaluronidase: changes in activity
during chick development. Science 183, 862-864.
PRATT, R. M., GOGGINS, J. F., WILK, A. L. & KING, C. T. G. (1973). Acid mucopolysaccbaride synthesis in the secondary palate of the developing rat at the time of rotation and fusion.
Devi Biol. 32, 230-237.
PRATT, R. M., LARSEN, M. A. & JOHNSTON, M. S. (1975). Migration of cranial neural crest
cells in a cell-free hyaluronate-rich matrix. Devi Biol. 44, 298-305.
REISSIG, J. L., STROMINGER, J. L. & LELOIR, L. F. (1955). A modified colorimetric method for
estimation of N-acetylamino sugars. /. biol. Chem. Ill, 959-966.
RHODES, R. K. & ELMER, W. A. (1975). Aberrant metabolism of matrix components in neonatal fibular cartilage of brachypod (bpH) mice. Devi Biol. 46, 14-27.
ROYAL, P. D. & GOETINCK, P. F. (1977). In vitro chondrogenesis in mouse limb mesenchymal
cells: changes in ultrastructure and proteoglycan synthesis. /. Embrvol. exp. Morph. 39,
79-95.
SAARNI, H. & TAMMI, M. (1977). A rapid method for separation and assay of radiolabeled
mucopolysaccharides from cell culture medium. Analyt. Biochem. 81, 40-46.
SAITO, H., YAMAGATA, T. & SUZUKI, S. (1968). Enzymatic methods for the determination of
small quantities of isomeric chondroitin sulfates. /. biol. Chem. 243,1536-1542.
SEEGMILLER, R., FRASER, P. C, & SHELDON, H. (1971). A new chondrodystrophic mutant
in mice. Electron microscopy of normal and abnormal chondrogenesis. /. Cell Biol. 48,
580-593.
SMITH, G. N., JR., TOOLE, B. P. & GROSS, J. (1975). Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating
limbs with the regenerates. Devi Biol. 43, 221-232.
SOLURSH, M. (1976). Glycosaminoglycan synthesis in the chick gastrula. Devi Biol. 50,
525-530.
SOLURSH, M. & MORRISS, G. M. (1977). Glycosaminoglycan synthesis in rat embryos during
the formation of the primary mesenchyme and neural folds. Devi Biol. 57, 75-86.
SOLURSH, M., VAEREWYCK, S. A. & REITER, R. S. (1974). Depression by hyaluronic acid of
glycosaminoglycan synthesis by cultured chick embryo chondrocytes. Devi Biol. 41,
233-244.
TOOLE, B. P. (1972). Hyaluronate turnover during chondrogenesis in the developing chick
limb and axial skeleton. Devi Biol. 29, 321-329.
TOOLE, B. P. (1973a). Hyaluronate and hyaluronidase in morphogenesis and differentiation.
Am. Zool. 13, 1061-1065.
TOOLE, B. P. (19736). Hyaluronate inhibition of chondrogenesis: antagonsim of thyroxine,
growth hormone, and calcitonin. Science, N.Y. 180, 302-303.
TOOLE, B. P. & GROSS, J. (1971). The extracellular matrix of the regenerating newt limb:
synthesis and removal of hyaluronate prior to differentiation. Devi Biol. 25, 57-77.
UNDERHILL, C. & DORFMAN, A. (1978). The role of hyaluronic acid in intercellular adhesion
of cultured mouse cells. Expl Cell Res. Ill, 155-164.
WIEBKIN, O. W. & MUIR, H. (1975). Influence of the cells on the pericellular environment.
Phil. Trans. R. Soc. Lond. B 271, 283-291.
WIEBKIN, O. W. & MUIR, H. (1977). Synthesis of proteoglycans by suspension and monolayer
culture of adult chondrocytes and de novo cartilage nodules - the effect of hyaluronic acid.
/. CellSci. 27, 199-211.
LINKER,
(Received 18 July 1979, revised 2 October 1979)