/. Embryo!, exp. Morph. Vol. 55, pp. 195-209, 1980
Printed in Great Britain © Company of Biologists Limited 1980
195
Sulfated proteoglycan accumulation
during development of the embryonic chick limb
bud studied by electron microscopic
autoradiography
By MOIRA CIOFFI, 1 ROBERT L. SEARLS 1
AND S. ROBERT HILFER 1
From the Department of Biology, Temple University,
Philadelphia, U.S.A.
SUMMARY
Cartilage cells are characterized by a high concentration of extracellular sulfated proteoglycan. Electron microscopic autoradiography was used to compare the incorporation of
sulfate into proteoglycan by limb-bud chondrogenic and myogenic cells. From stage 19 to
stage 21 there was no significant difference between the cartilage- and muscle-forming regions
in the number of silver grains over either the extracellular space or the intracellular space.
From stage 22 to 25 the number of extracellular silver grains was significantly greater
in the chondrogenic region than in the myogenic region, but the number of intracellular
silver grains was the same. Since most of the silver grains were intracellular, no significant
difference in the total number of grains was found between the two tissues. Stage-26 and
-27 embryos showed a significantly greater number of silver grains over both the cells and
the extracellular space in the cartilage region than in the muscle region. Thus, the first
step of cartilage differentiation involves a decrease in the extracellular deposition of sulfated
proteoglycan in the myogenic region rather than an increase in deposition in the chondrogenic region between stage 22 and 25. After stage 25 there is an increase in sulfated proteoglycan synthesis in the chondrogenic region relative to the myogenic region.
INTRODUCTION
During limb development, an apparently homogeneous population of cells
is converted into specialized cell types including fibroblasts, muscle and
chondrocytes. Mature chondrocytes are characterized by an extracellular matrix
rich in collagen and sulfated proteoglycans. The synthesis of sulfated proteoglycans by limb-bud cells has been investigated qualitatively by light microscopic
autoradiography after administration of [35S]sulfate (Searls, 1965a; Hinchliffe
&Thorogood, 1974); the radioactivity in the limb after histological processing
was shown to be due to proteoglycans (Searls, 1965 b). From the beginning of
limb outgrowth to the middle of stage 22 (Hamburger & Hamilton, 1951),
1
Authors' address: Department of Biology, Temple University, Philadelphia, PA 19122,
U.S.A.
196
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
sulfate uptake was uniform throughout the limb. After stage 22 a distinct pattern of uptake emerged, with labeling higher in the central proximal region,
intermediate in the distal subridge region and lower in the dorsal and ventral
proximal regions. At stage 27, essentially all labeling was over the central
proximal region, where the cells were surrounded by a matrix that stained
metachromatically with toluidine blue. Thus, differentiation seemed to involve
a restriction to the chondrocytes of a process that was initially ubiquitous.
The technique of light microscopic autoradiography does not permit a
quantitative determination to be made of the rate of proteoglycan synthesis
within the chondrogenic region as compared to the myogenic regions. Repeated
attempts have been made in this laboratory to develop such a procedure. Biochemical assays after growth of the two regions in organ culture are subject
to question because cells from the myogenic region tend to form cartilage in
organ culture (Zwilling, 1966). Assay of surgically isolated myogenic and
chondrogenic regions is subject to the criticism that lack of cross contamination
of one cell type with the other cannot be demonstrated. We finally chose to
measure uptake of [35S]sulfate in ovo by means of quantitative electron microscopic autoradiography, the procedures for which are well established (Salpeter
& Bachman, 1964, 1972; Farquhar, Reid & Daniell, 1978). Chondrogenic and
myogenic cells could be recognized by their position in the limb and by their
ultrastructural appearance. All regions of the limb bud receive the same
concentration of isotope so myogenic and chondrogenic regions can be compared
in the same section. The relative amount of sulfate uptake could be measured
by counting the number of silver grains in each region.
MATERIALS AND METHODS
Labeling of the embryos
Fertilized White Leghorn eggs were incubated at 37 °C and windowed on
the third day of incubation. The activity of the carrier-free [35S]sulfate was
calculated from the half life of [35S]- and the [35S]sulfate was diluted with
Hanks saline to give 5 mCi per ml. 0-1 ml of this solution (500 juCi) was dropped
directly on top of the embryo after tearing the chorion and amnion with
forceps, and the egg returned to the incubator. Embryo stages 19-21 were
sacrificed 4 h after application of the sulfate. For each stage from 22 to 27,
one embryo was sacrificed at -|, 1, 2, 3 and 4 h after application of the sulfate.
The number of silver grains observed over sections by means of autoradiography
can vary with the specific activity of the isotope, the thickness of the section
or the emulsion, the exposure time and the photographic processing. Every
attempt was made to keep the experimental conditions uniform throughout
the whole series of experiments. However, it is difficult to make quantitative
comparisons between tissues processed for autoradiography at different times,
because it is impossible to be sure that small changes in technique do not
Sulfated proteoglycans during chick development
197
occur from month to month. Since the sections from embryos of the same
stage were to be compared with each other, embryos from one stage were
administered [35S]sulfate and processed through to development of the
emulsion as a group. The stage-19 to -21 embryos were processed together
and the embryos of stages 22 and 24 were also processed together in order to
obtain some comparison of labeling in closely related stages. The only exception
to this general rule was the stage-24 embryo that had been labeled for 1 h; this
embryo was processed with the embryos of stages-19-21 embryos. The time
from administration of the [35S]sulfate to development of the emulsion was
about the same for all of the groups.
Electron microscopic autoradiography
The wing buds were cut off and fixed in half-strength Karnovsky fixative
(Karnovsky, 1965) at pH 7-3 for 1 h, post-fixed for 1 h in 1 % osmium in
0-1 M sodium cacodylate buffer and embedded in Araldite. The right wing
buds were cross-sectioned using an LKB Huxley ultramicrotome. Starting
from the proximal end of the limb, sectioning was continued until ectoderm
was present on both the dorsal and ventral sides of the wing, indicating that a
position about half way along the prospective humerus region of the wing
had been reached (Stark & Searls, 1973). Thin sections of the whole crosssection of the wing were taken for EM autoradiography. Gold sections were
selected, and 20-40 sections were taken from each wing. The grids were
covered with Ilford L4 emulsion (Caro & Tubergen, 1962; Caro, 1969). The
emulsion was melted at 45°, diluted 1:1 with distilled water, and cooled until
it became quite viscous. A wire loop was dipped into the emulsion and withdrawn slowly so that a thin film formed in the loop. This film gelled almost
immediately and could be placed over the grids. The thickness of the emulsion
was checked periodically by placing the film on a formvar-coated grid, exposing
it immediately and examining the emulsion by electron microscopy. Since a
quantitative study was being attempted it was essential for the emulsion to
consist of a tightly packed monolayer of silver halide crystals, and this method
was found to provide such monolayers reproducibly. The grids were stored in
the presence of Drierite for 3 months at 0-4 °C.
With 35S, each silver halide crystal has to be hit by a number of electrons
before a latent image is produced, so there could be a loss of quantitative
accuracy when comparing regions of very high and very low activity. The
process of gold latensification (Salpeter & Bachman, 1964) partly negated this
objection and produced a three- to four-fold increase in sensitivity by decreasing
the number of times a silver halide crystal has to be hit by an electron before
a developed silver grain can be obtained, while leaving the background unchanged. A solution of gold thiocyanate was prepared using the method
described by Salpeter & Bachman (1964), and diluted 1:20 with distilled water.
Before developing, the grids were dipped in distilled water for 10 sec and then
198
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
in the dilute solution of gold thiocyanate for 30 sec. They were washed for
1 min in distilled water, developed in Kodak D-19 developer for 2 min and
fixed in Kodak Rapid Fix for 5 min (Caro, Tubergen & Kolb, 1962). The grids
were washed for 5 min in several changes of distilled water, blotted dry and the
emulsion cleared by soaking the grids in 0-05 M-NaOH for 4 min (Revel &
Hay, 1963). Each grid was then stained for 1|- min in a saturated solution of
uranyl acetate followed by 3 min in lead citrate (Venable & Coggeshall, 1965).
The sections were examined using a Philips EM 300 electron microscope.
Analysis of the sections
All of the photographs were taken at 1600 x . This magnification was high
enough to allow identification of silver grains, and low enough to include an
average of 25 nuclei at stage 19, although by stage 27 this value had increased
two- to three-fold. From stage 19 to 21 there is no observable different between
cells in the chondrogenic and myogenic regions. The chondrogenic region was
located by its central position in the wing along both the dorsal-ventral and
anterior-posterior axes. The myogenic regions of the wing were assumed to
occupy the area dorsal and ventral to the chondrogenic region and about
five cell diameters from the dorsal and ventral ectoderm. After stage 22, the
chondrogenic and myogenic regions were easily recognized by their ultrastructural appearance (Searls, Hilfer & Mirow, 1972; Hilfer, Searls & Fonte,
1973). Photographs of the two regions were taken in two to six sections of
each wing.
A portion of each section not containing tissue was examined, and the
background found to be between 5 and 15 silver grains per 3000 ju,m2 (the
approximate size of the area photographed). After 1-4 h of [35S]sulfate uptake,
when counts were as high as 900 silver grains per photograph,the background
could be considered as negligible. Silver grains over each of the following cell
compartments were counted: the nucleus, the nuclear membrane, the cytoplasm,
the cell membrane and the extracellular space. The results were expressed as
silver grains per nucleus for each compartment, and were compared by the
test for significance of differences between two means for independent samples
(Bliss, 1967).
RESULTS
Examples of cells from the chondrogenic and myogenic regions of the wing
are shown for stage 19 (Figs. 1 and 2) and for stage 27 (Figs. 3 and 4). Careful
examination of the sections revealed no subpopulations of cells in either region
with different rates of [35S]sulfate uptake, and even cells undergoing mitosis
had about the same number of silver grains as other cells. The silver grains
over the cytoplasm were not localized to any particular organelle, although
the Golgi region was often more heavily labeled. The counts obtained for
each time of sulfate incorporation at each stage of development were averaged,
Sulfated proteoglycans during chick development
Fig. 1. An electron microscopic autoradiograph showing cells from the pre-cartilage
region of a stage-19 wing bud. Approximately 70% of the silver grains are over
the cells, x 3700. Bar = 10/tm.
199
200
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
Fig. 2. An electron microscopic autoradiograph showing a section through the premuscle region of a stage-19 wing bud. The distribution and number of silver grains
over the section are not significantly different from the pre-cartilage region
(Fig. 1). x3700. Bar = 10 /un.
Sulfated proteoglycans during chick development
Fig. 3. An electron microscopic autoradiograph of a section through the cartilage
region of a stage-27 wing bud. Approximately 70% of the silver grains are over
the cells, x 3700. Bar = 10 /*m.
201
202
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
Fig. 4. An electron microscopic autoradiograph of a section through the muscle
region of a stage-27 wing bud. There is very little extracellular space, and
approximately 95 % of the silver grains are over the cells. The total number of
silver grains over the section is two to three times less than in the cartilage region
(Fig. 3). x3700. Bar = 10/*m.
Sulfated proteoglycans during chick development
203
Table 1. Silver grains per nucleus at stages 19, 20 and 21 after 4 h of [35S]sulfate
uptake presented plus and minus one standard deviation
Intracellular
Extracellular
Total
25-8 ±4-1
27-4±4-8
9-2 ±2-4
12-4±2-5
38-9±7-5
430±70
17-9±l-5
170 ± 3 0
4-7 ±1-9
6-9 ± 1 0
24-9 ±3-8
26-3 ±2-9
101 ±2-1
11-9 ±2-9
30 ±1-1
5-3 ±0-9
15-8±l-9
18-8±3-9
Stage 19
Myogenic
Chondrogenic
Stage 20
Myogenic
Chondrogenic
Stage 21
Myogenic
Chondrogenic
Table 2. Silver grains per nucleus at stage 22 after \-4 h of [35S]sulfate uptake
presented plus and minus one standard deviation
Myogenic
Time Tntracellular Extracellular
•h
1
2
3
4
0-6 ±0-3
3-2 ±0-5
31 ±0-6
8-3± 1-7
7-7 ±0-8
Chondrogenic
Total
0-2 ± 0 1
0-9 ±0-6
0-4 ±0-2
4 0 ±0-6
0-6 ±0-4
41 ±10
11 ±0-3 10-7±20
0-9 ± 0 1
9-6 ± 1 0
Intracellular Extracellular
1-1 ±0-5
31 ±0-7
29 ±0-4
81 ±2-6
60 ±0-7
Total
0-5 ± 0 1
1-7 ±0-7
1-2 ±02* 4-7 ±0-8
1-2 ±0-4
4-6 ± 1 0
3-2±0-7* 129±36
2-8 ±0-4* 9-8 ±1-2
* Chondrogenic significantly greater (P < 001) than myogenic.
and the results are shown in Tables 1-6. The intracellular counts include silver
grains observed over the nucleus and nuclear membrane, which constituted
about 25 % of the intracellular count. The total count does not add up to the
sum of the intracellular and extracellular counts, since a small percentage of
the grains was over the cell membrane.
From stage 19 to 21, when the physical characteristics of the muscle- and
cartilage-forming cells in the limb bud are essentially the same, there was no
significant difference in the number of extracellular or intracellular silver
grains, or in the total number of grains per nucleus (Table 1). From stages 22
to 25 the pre-muscle and pre-cartilage regions of the limb become progressively
more distinguishable by morphological criteria, although the extracellular
matrix in the chondrogenic region does not stain metachromatically until
stage 25, and muscle straps are not yet evident. In wing buds of stages 22-25
no significant difference in the number of intracellular silver grains was observed
between the two regions, although the chondrogenic region had two to three
times as many extracellular silver grains as the myogenic region (Tables 2-4).
This difference was not reflected in the total silver grain counts per nucleus
204
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
Table 3. Silver grains per nucleus at stage 24 after \-4 h of [Z5S]sulfate uptake
presented plus and minus one standard deviation
Myogenic
Time Intracellular Extracellular
10±01
3-3 ±0-2
4-4 ±0-7
5-5±0-8
5-6±l-3
01
0-4±01
0-3 ± 0 1
O-6±O-3
0-2 + 0-2
Chondrogenic
Total
1-2
4-6±01
5-2 ±0-9
6-9 ± 1 0
70±l-3
Intracellular Extracellular
l-2±0-l
4-8 ±0-7
3-3 ± 0 1
51 ± 1-4
5-5 ±0-7
0-3
1-6 ±0-3*
10 ±0-2*
l-3±O-3
20 ± 0 1 *
Total
1-6
7-6±l-2
4-7 ±0-2
7-2 ±1-8
8-3 ±0-9
* Chondrogenic significantly greater (P < 001) than myogenic.
Table 4. Silver grains per nucleus at stage 25 after |—5 h of [z5S]sulfate uptake
presented plus and minus one standard deviation
Myogenic
Time Intracellular Extracellular
0-9 ±0-3
2-2 ±0-4
3-4 ±0-9
2-2 ±0-4
0-2 ± 0 1
0-6±0-l
0-6 ±0-4
Chondrogenic
Total
10 ±0-4
2-7±0-5
4-4±l-l
3-3±O-9
Intracellular Extracellular
0-4 ±0-2
0-8±0-2*
1-4 ±0-4*
1-5 ±0-6
11 ±0-4
2-4 ±0-6
40 ±0-7
2-9 ±0-9
Total
1-6 ±0-7
3-5±0-6
6-2 ±1-3
5-4 ±1-8
Chondrogenic is significantly greater (P < 001) than myogenic.
Table 5. Silver grains per nucleus at stage 26 after \-4 h of [ZbS]sulfate uptake
presented plus and minus one standard deviation
Myogenic
Time Intracellular Extracellular
29±0-5
5-8 ±0-2
6-4 ±0-8
7-3 ±0-3
0-2 ±01
0-3±01
0-3
1-0 + 0-1
Chondrogenic
Total
3-3±0-6
6-5±0-3
7-2 ±0-8
90±0-4
Intracellular Extracellular
3-8±O-8
8O±O-6
12-3±l-4*
8-8±0-9
Total
2-3 ±0-4* 6-4± 1-3*
2-8±0-l* ll-9±0-8*
4-4±l-l* 18-6±2-2*
5-4±0-4* 15-5±l-2*
* Chondrogenic is significantly greater (P < 001) than myogenic.
for each region, since the extracellular counts represented only 10-30% of
the total. A significant difference in the intracellular and total number of
silver grains between the two regions of the limb was first observed at stage 26
(Table 5). At stages 26 and 27 the cartilage region contains a well developed
extracellular matrix and the muscle region contains multinucleate straps. By
stage 27 the cartilage region had 25 times as many extracellular and two times
as many intracellular silver grains as the muscle region. In the cartilage region
Sulfatedproteoglycans
during chick development
205
Table 6. Silver grains per nucleus at stage 27 after \-4 h of [3bS]sulfate uptake
presented plus and minus and one standard deviation
Myogenic
Chondrogenic
Time Intracellular Extracellular
i
1
2
3
4
3-3±O-5
2-9±0-2
4-3±01
4-8±0-3
6-7±l-0
0
01
0-4±01
0-2
0-4±0-2
Total
Intracellular Extracellular
3-5±0-5
31 ±0-2
5-4±0-2
5-3±0-2
80±l-2
12O±3-8*
3-2±0-7
10-2±2-4*
9-2±2-l*
14-5±3-6*
3-4±0-l*
0-9±0-l*
5-3±0-l*
6-1 ±10*
8-8±2-0*
Total
16-6±4-l*
4-3±0-9
17-6±2-3*
16-7±3-4*
27-0±6-3*
* Chondrogenic is significantly greater (P < 001) than myogenic.
Table 7. Fraction of the total silver grains over the extracellular space
Hours
Stage 19 myogenic
Stage 19 chondrogenic
Stage 20 myogenic
Stage 20 chondrogenic
Stage 21 myogenic
Stage 21 chondrogenic
Stage 22 myogenic
Stage 22 chondrogenic
Stage 24 myogenic
Stage 24 chondrogenic
Stage 25 myogenic
Stage 25 chondrogenic
Stage 26 myogenic
Stage 26 chondrogenic
Stage 27 myogenic
Stage 27 chondrogenic
*
1
2
3
4
—
—
—
—
—
—
—
—
—
—
—
—
010
—
—
—
—
—
—
015
0-26
0-26
—
—
—
—
—
—
010
0-25
009
018
018
0-28
0-24
0-29
019
0-26
019
0-28
009
0-29
010
0-24
0-22
0-29
008
005
—
009
006
0-21
0-25
0-23
006
005
0-36
0-27
—
003
0-21
0-21
014
0-23
004
0-24
007
0-30
0-20
007
—
0-28
004
0-37
—
—
011
0-35
005
0-33
70-80 % of the grains were intracellular as compared to 90-95 % in the muscle
region, so that the difference in the total number of grains between the two
regions was only about threefold (Table 6).
From Tables 2-6 it is evident that the main difference in [35S]sulfate accumulation between the myogenic and chondrogenic regions of the limb is in the
number of silver grains present over the extracellular space. This value was
calculated as a fraction of the total number of silver grains for each time of
[35S]sulfate incubation and stage of development, and the results are shown
in Table 7. In general, the fraction of the total silver grains over the extracellular space did not vary over the £-4 h allowed for incorporation of [35S]sulfate. In the chondrogenic region of the wing, the fraction of silver grains
over the extracellular space was between 0-2 and 0-3, and changed very little
14
EMB 55
206
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
from stage 19 to stage 27. In the myogenic region the fraction of silver grains
over the extracellular space was similar to that in the chondrogenic region
until stage 22, but then decreased to reach a value of 0-05 by stage 27.
DISCUSSION
It has previously been shown (Searls, 19656) that if limb buds labeled with
[ S]sulfate are processed for light microscopy (fixed in Bouin's fluid, dehydrated
through alcohols to xylene, etc.), all of the radioactivity remaining in the limbs
is bound to proteoglycan, predominantly chondroitin-4- and chondroitin-6sulfates. The only significant difference in processing for electron microscopy
is that the tissue is post-fixed in osmium tetroxide, preserving lipids that would
have been lost in Bouin's fixed tissue. However, sulfated lipids have only been
found in myelin (Rouser & Yamamoto, 1969), so that the sulfate uptake
observed in the limb cells must be into sulfated proteoglycans. Therefore by
using electron microscopic autoradiography to measure uptake of [35S]sulfate,
the present study has provided quantitative data on the synthesis of sulfated
proteoglycan by developing muscle and cartilage cells in the chick wing bud.
The first difference observed between the two tissues involved a decrease in
extracellular sulfated proteoglycan in the soft tissue region from stage 22 to
25, rather than an increase in the chondrogenic region. However the intracellular sulfate uptake was similar between the two tissues, and constituted
such a large proportion of the total that it masked the difference in the extracellular proteoglycan as far as total uptake was concerned. At stages 26 and
27 there was a further decrease in extracellular proteoglycan in the muscle
region of the wing, coupled with a two- to three-fold increase in intracellular
proteoglycan in the chondrogenic region. However, even after 4 h of [35S]sulfate
incubation, only about 30 % of the proteoglycan was extracellular.
The reduction in the number of extracellular silver grains in the soft tissue
region of the wing from stage 22 to 27 is hardly a surprising result, since there
is very little extracellular space in the soft tissue regions at later stages (compare Figs. 3 and 4). Gould, Selwood, Day & Wolpert (1974) have suggested
that the orientation and shape of chondrogenic cells may be a mechanical
effect of matrix secretion. It was also mentioned earlier that the percentage
of extracellular silver grains in the chondrogenic region remained fairly constant
from stage 19 to 27, although matrix accumulation cannot be recognized
histologically until stage 25. The reason for this may be that at early stages
of development the mesenchyme cells are dividing too rapidly for sufficient
accumulation of sulfated proteoglycan to take place.
35
Unlike the present study, previous investigations using light microscopic
autoradiography found a clear difference in [35S]sulfate uptake between the
pre-muscle and pre-cartilage regions of wing buds of stages 22-25 (Searls,
1965a; Hinchliffe & Thorogood, 1974). In an earlier study (Searls, 1965a) the
sections were examined at a magnification of 60 x using dark-field optics. When
Sulfated proteoglycans during chick development
207
the sections were examined at 500 x the difference between the two areas was
not so striking, but it was assumed that the limited field of view and the
difficulty in comparing sections were responsible. It is possible that the stained
cells tended to hide the intracellular silver grains at low magnification in dark
field, so that only the extracellular grains were noticed, giving the impression
of greater labeling in the chondrogenic as compared to the myogenic region.
In agreement with the present study are some preliminary biochemical investigations carried out by Kosher and Searls (unpublished). Their results suggested
that at stages 26 and 27 sulfated proteoglycan accumulation in the chondrogenic
region is only about two times greater than in the myogenic region.
The only previous study of [35S]sulfate uptake in which silver grains were
counted in electron microscopic autoradiographs was by Godman & Lane
(1964). In their experiments, well differentiated mouse cartilage was labeled
either in culture medium containing [35S]sulfate or by injection of [35S]sulfate
into the mouse. Autoradiographs were prepared after different times of incubation. They reported that 2 3 % of the silver grains were over the 'capsular
matrix' after 3 min of incubation in vitro and 20-8 % after 30 min of incubation
in vitro. When well differentiated mouse cartilage was labeled in vivo, 27-7 %
of the silver grains were extracellular after 30 min and 33-6% of the silver
grains were extracellular after 60 min. These results agree well with the 20-30 %
extracellular silver grains observed in the chondrogenic area of the chick wing
at stage 27. Only after 15 h was a majority (55-6%) of the silver grains over
the extracellular space in the mouse cartilage.
The production of intracellular proteoglycan was observed in both myogenic
and chondrogenic cells from embryos of stages 19-27. This material is not
being processed for export, since the percentage of extracellular silver grains
is constant from 30 min to 4 h after administration of the label. If the intracellular proteoglycan was being processed for export there should be very
little extracellular label at early time periods, increasing rapidly after the time
required for processing, while the amount of intracellular label should plateau.
The production of intracellular proteoglycan does not seem to depend on the
stage of differentiation of the cells, or on whether or not the cells are dividing,
since about 25 % of the cells in the chondrogenic region and about 75 % of
the cells in the myogenic region are dividing at stages 22-24 (Janners & Searls,
1970) but all the cells are accumulating intracellular proteoglycan at about
the same rate.
The intracellular proteoglycan must turn over quite rapidly compared to
the extracellular proteoglycan. Non-dividing cartilage cells show more rapid
uptake of label into intracellular than extracellular material, yet sulfated proteoglycans accumulate in the extracellular space to concentrations that can
be recognized histologically, while no such accumulation is observed in the
cells. A similar suggestion has been made concerning the sulfated proteoglycans
surrounding the yolk platelets of frog's eggs (Kosher & Searls, 1973). If the
14-2
208
M. CIOFFI, R. L. SEARLS AND S. R. HILFER
intracellular and extracellular proteoglycans are metabolically different, it is
possible that they are also chemically different. The existence of several kinds
of proteoglycans in the embryonic chick limb has been reported (Palmoski &
Goetinck, 1972; Vasan & Lash, 1977). Proteoglycans have also been reported
in the membranes of pituitary secretory granules (Giannatasio & Zanini, 1976),
pancreatic zymogen granules (Reggio & Palade, 1978; Berg, 1978), mitochondria (Dietrich, Sampaio & Toledo, 1976) and cell nuclei (Bhavanandan &
Davidson, 1975; Fromme, Buddecke, Figura & Kresse, 1976; Margolis,
Crockett, Kiang & Margolis, 1976).
The existence of a large pool of intracellular sulfated proteoglycan is important
for chemical studies on the synthesis of cartilage matrix. If the differentiation
of cartilage is assayed by permitting the cells to take up [35S]sulfate for a short
period of time, homogenizing the cells and determining the incorporation of
[35S]sulfate into proteoglycan, 70% of the observed uptake is likely to be
into intracellular proteoglycan and not cartilage matrix.
From our experiments we can make no statements concerning changes in
the absolute rate of sulfate incorporation into proteoglycan. In the muscleforming region of the wing, the total number of silver grains per nucleus
tended to decrease with increasing stage until stage 22 and then levelled off.
In the cartilage-forming region of the wing the silver grain counts showed
the same pattern until stage 25 but increased from stage 26 to 27. The simplest
explanation for these changes is that the amount of cold endogenous sulfate
available to the embryo changes during development. The very high incorporation
of [35S]sulfate observed in stage-19 wing buds probably means that they had
relatively little endogenous sulfate available to them, so that after equilibration
the administered [35S]sulfate was of high specific activity. We still do not know
whether the absolute amount of sulfate uptake during stages 26 and 27 increases
in the cartilage cells, decreases in the muscle cells, or both.
This work was supported by National Institutes of Health grant no. HDO4669.
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(Received 5 March 1979, revised 30 July 1979)