/. Embryol. exp. Morph. Vol. 72, pp. 1-18, 1982
Printed in Great Britain © Company of Biologists Limited 1982
\
Cell contact and surface coat alterations
of limb-bud mesenchymal cells during
differentiation
By BERND ZIMMERMANN 1 , EVAMARIA SCHARLACH
AND ROMAN KAATZ 2
From the Institut fur Toxikologie und Embryonal-Pharmakologie,
Freie Universitdt, Berlin
SUMMARY
Measurements of the cell perimeter, cell-cell contact number and length and of the thickness of the surface coat were performed in limb buds of mouse embryos during the process of
cell condensation of the chondrogenic cell mass. This study starts with measurements of
'non-limb' mesenchyme.of day-9 embryos and ends with the young cartilage of day 13. It is
shown that until day 12 all cells in the limb bud are interconnected by cell contacts of the
gap-junction type. Contact number and contact length increase during development, but
increase about twice as much between central, prechondric cells as between peripheral or
distal cells. Later in development, chondroblasts lose their contacts almost completely,
whereas between peripheral cells, the amount of cell contacts drops to the initial value of
mesenchymal cells. The cell size decreases during chondrogenesis. A decrease in the
thickness of the surface coat of the cells during the whole differentiation period is shown.
It may be assumed that this 'wave of cell contacts' is the last step for the initiation of the
chondrogenic differentiation process.
INTRODUCTION
The development of the limb skeleton starts with the formation of condensations of mesenchymal cells in the prospective chondrogenic areas (Fell,
1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). This process
of blastema formation is not yet fully understood. A local increase in cell
proliferation can be excluded (Hornbruch & Wolpert, 1970; Janners & Searls,
1970; Cairns, 1977), but there are some indications concerning the participation
of cell movements and changes of cell adhesion and intercellular matrix (Toole,
Jackson & Gross, 1972; Ede & Flint, 1975; Ede, Flint, Wilby & Colquhoun,
1977; Holmes & Trelstad, 1980).
Polarization of mesenchymal cells towards the centre of the condensation
were shown by Trelstad (1977) and Ede & Wilby (1981). The importance of cell
1
Author's address: Institut fur Toxikologie und Embryonal-Pharmakologie, Freie Universitat, Berlin, Garystr. 9, D-1000 Berlin 33, FRG.
2
Author's address: Anatomisches Insititut, Freie Universitat Berlin, Garystr. 9, D-1000
Berlin 33, FRG.
2
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
adhesion and slight cell movements was indicated by comparison of chondrogenesis in normal and talpid3 chick embryos (Ede & Agerback, 1968; Ede,
Flint & Teague, 1975; Ede et al. 1977). A decrease in intercellular spaces by
action of cellular hyaluronidase may be also involved in the condensation
process (Toole, 1972, 1973; Zimmermann, 1981).
The morphological changes during blastema formation are well known in
the chick embryo. In prospective chondrogenic regions, the loose mesenchyme
condenses, the width of the intercellular spaces decreases and an enhanced
formation of cell-cell contacts occurs (Searls, Hilfer & Mirow, 1972; Summerbell, Lewis & Wolpert, 1973; Thorogood & Hinchliffe, 1975; Kelly & Fallon,
1978).
Similar events are described in the development of the skeletal blastema in
mammals (Jurand, 1965; Neubert, Merker & Tapken, 1974; Borck, 1977).
Participation of cell movements (Holmes & Trelstad, 1977, 1980) and cell
adhesion (Duke & Elmer, 1978, 1979) was shown.
Although the importance of the formation of cell contacts during blastema
formation in the limb (Searls et al. 1972; Kelly & Fallon, 1978) as well as in
other organs (Trelstad, Hay & Revel, 1967; De Haan & Sachs, 1972; Bunge
et al. 1979; Gilula, 1980; Loewenstein, 1980) has been pointed out, no quantitative estimations are available. Regarding cell adhesion, quantitative measurements of the cell surface coat in the condensing mesenchymal cells are also
lacking.
In this study, cell size, quantity of cell contacts and the thickness of the cell
surface coat in limb skeletal development of mouse embryos were measured.
The whole developmental period was covered starting with 'non-limb' mesenchyme at day 9 of development until early chondrogenesis at day 13.
MATERIALS AND METHODS
Upper limb buds of mouse embryos of days 10, 11, 12 and 13 (mouse strain
NMRI, day 0 = day of conception) were removed and fixed immediately.
Body segments of day-9 embryos in the prospective limb-forming region
(somites 8-11) were excised and fixed. For this study, embryos of three to four
different litters were used, and two to three embryos per litter were examined.
Fixation and embedding procedure
Fixation was performed in 2 % glutaraldehyde (Ferak, Berlin) plus 1 %
tannic acid (E. Merck, Darmstadt) in 0.1 M-phosphate buffer, pH 7-2, for 1 h
at room temperature. After rinsing, post-fixation was done in 1 % OsO4 in
0-1 M-phosphate buffer, pH 7-2, for about 1 h at +4 °C. After rinsing thoroughly,
the explants were dehydrated in ethanol and embedded in Epon or Mikropal
(Ferak, Berlin).
Cell contact and surface coat alterations
Fig. 1. Regions measured for cell circumference, contact number and contact
length, and surface coat thickness. (1) Distal; (2) central; (3) peripheral. Example
showing a day-11 limb.
Electron microscopy
Different parts of the limbs were prepared for electron microscopy after
orientation on semithin sections. From day-9 embryos, the lateral body wall
between somatopleura and the outer epithelium was used. From day-10
embryos, the whole limbs could be sectioned horizontally in the medial plane.
The apical ectodermal ridge served as reference point. Electron microscopic
pictures were taken in distal regions under the apical ectodermal ridge, in
central regions and the periphery. The limbs of day-11 and -12 embryos were
divided into a distal and a proximal part. From the distal parts, pictures were
taken under the apical ectodermal ridge and around the marginal sinus; from
the proximal part, in the centre in the area of the prospective humerus, and
from the periphery, under the lateral basement membrane (Fig. 1). In day-13
limbs, only the proximal/central part (humerus) and the peripheral regions were
examined.
Electron microscopic pictures were taken at primary magnifications of 2000
and 40000. Measurements of the cell perimeter and of contact number and
contact length were done on enlargements of 6000, measurements of the surface
coat on enlargements of 120000.
Measurements of the cell perimeter and the contact length
Cell perimeter, contact length and the number of contacts of each measured
cell were performed with an Interactive Image Analysing System IBAS 1
(Kontron, Echingen, Miinchen). In this system, lines of the measured structure
are covered by a contact pen manually. The position data of x and y are computed and processed in an integrated microprocessor. Histograms, single data,
mean and standard deviations were printed out. One cell contact length was
defined from one intercellular space to the next.
Measurements of the cell surface coat
The thickness of the surface coat was estimated on pictures of a magnification of 120000 by using a measuring magnifier. In each litter series, 40 positions
4
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
were measured in every region. Only exact cross sections through the cell
membrane with well-recognizable bilayer structure were used.
Statistical analysis
For statistical analysis, a PDP-11/34A computer (digital equipment, Minnesota) was used with a RSX-11M operating system. Mean and standard deviations
of the surface coat thickness were calculated for each litter series separately to
show the variance in the different series. The /-test was used for testing the
significance of differences between the data of different stages and regions.
Mean and standard deviations of cell perimeter were printed by the IB AS.
The Mest was performed with a PDP-11/34A computer. Since the data of the
contact length are not normally distributed, the t test could not be used for
statistical analysis of the data. Therefore, the single data printed by the IBAS
were fed into the PDP-11/34A. The Mann-Whitney-Wilcoxon test for the
difference between two populations was performed using the statistical program 'minitab', release 81-1 (Copyright Penn. State University 1981,
University of Toledo).
RESULTS
Between the outer epithelium and the somatopleura in the presumptive
limb-forming region of day-9 mouse embryos, the mesenchymal cells are
almost completely separated by wide intercellular spaces. The cells exhibit an
irregular cell surface, and many cell processes cross the intercellular space.
Only a few focal cell-cell contacts are visible (Fig. 2 a). Most of the contacts
can be considered as gap junctions (Fig. 2b). The mean perimeter of a cell
amounts to 30 /im, a single contact exhibits a mean length of about 1.0 /*m,
and about 2-3 contacts per cell are present (Table 1). The histogram of the
contact length shows a relatively high amount of very short cell contacts
(Fig. 7).
The surface coat of these cells has a thickness of about 8 nm (Fig. 2c, Table 2).
Very often, larger structures of up to 80 nm extend beyond the dense surface
layer.
At day 10, upper limb buds of about 0.5 mm length have formed, whereas
the lower limb buds become just recognizable. The mesenchyme in the upper
Fig. 2(a). Loose mesenchyme between the somatopleura and the epithelium of the
body wall in the prospective limb-forming region (between somites 8 and 11)
of day-9 mouse embryos. Irregularly shaped cells show many cell processes and
some cell-cell contacts (-*•). x6000; bar = 1 /im. (b) High magnification of a
cell-cell contact of the loose mesenchymal cells reveals the typical morphology of a
gap junction, x 120000; bar = 100 nm. (c) The surface coat of the mesenchymal
cells in the prospective limb-forming region of day-9 mouse embryos exhibits a
thickness of about 8 nm (-*•). Some filamentous structures extend to up to 80 nm
into the extracellular space ( • ) . x 120000; bar = 100 nm,
Cell contact and surface coat alterations
2(b)
>*f?w&SF**
5
6
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
Table 1. Measurements of cell perimeter and cell contact number and length on
cells in the limb anlage of different stages
Cell perimeter
Stage/
region1
Length of cell contacts
>
S.D.
N
M5
X2
S.D.3
N*
(X
10 d
10 c
10 p
30
28
30
28
9-8
7-8
8-3
6-6
40
45
26
25
1-09
1-09
1-43
0-80
10
1-0
1-0
0-55
93
110
53
79
0-72
0-72
1-04
0-58
lid
lie
Up
30
29
25
7-5
7-6
6-0
51
33
50
2-15
3-10
1-60
1-47
2-05
1-15
266
195
256
12 d
23
26
24
4-3
5-8
6-0
48
39
103
1-15
2-60
1-50
158
182
327
23
4-3
43
1-40
2-61
1-70
1-40
105
21
28
4-0
7-1
38
34
0-58
0-92
0-45
0-78
9
12c
12 p
12 dp8
13c
13p
Contacts per cell
Length 6
%7
2-32
2-44
2-04
3-15
2-56
2-66
2-92
2-52
8-5
9-5
9-8
90
1-78
2-51
1-34
5-22
5-89
5-15
11-20
18-30
8-15
35-4
630
32-6
3-30
4-68
3-18
310
4-61
12-20
5-40
4-34
200
47-0
22-5
133
0-92
1-92
1-10
1-22
32
75
0-43
0-65
0-85
2-20
0-49
2-02
2-3
7-2
No.
190
1
Stage: day of embryonic development; region: d = distal, c = central, p = peripheral.
x = mean (jim)
3
S.D. = standard deviations.
4
Number of cells or contacts resp. measured.
5
M = median (/«n); since the values of contact length do not show a normal distribution
(see histograms Fig. 7), the median is necessary for statistical analyses (see 'statistics' in
Materials and Methods).
6
Total contact length per cell is a calculated value of the number of cell contacts multiplied by the mean contact length.
7
% of contacts describes the percentage of cell membranes of one cell involved in cell
contacts.
8
dp = distal-peripheral, measurements in a region between distal and peripheral, serving
as a control of reproducibility, reveals the correspondence to the values of 12 d and 12 p.
2
limb anlage is very uniform. The cells are interconnected by small cell contacts,
irregularly shaped and separated by large intercellular spaces, such as the
mesenchymal cells at day 9. Measurements of the cell perimeter in different
areas of the limb bud (distal, central, peripheral) show no differences. The
portion of the cell membrane involved in cell contacts is about 9 % and quite
similar in all regions (Table 1).
The lengths of the cell contacts show significant differences. In the central
part of the limb, the cell contacts exhibit a length of about 1-4 fim (mean =
1-4/an, median = 1-0 fim), which is significantly longer than in the distal and
peripheral areas (Table 1). On the other hand, the number of cell contacts is
only about 2 per cell in the central parts, 2-4 in the distal and 3-2 in the peripheral parts. Therefore, the percentage of cell contacts is not different in the
measured areas. A tendency of increasing contact length is also demonstrated
in the histograms (Fig. 7).
Cell contact and surface coat alterations
Table 2. Thickness of the cell surface coat on cells in the limb buds of mouse
embryos of different stages
Stage/region
Distal
Central
Peripheral
—
9-8 + 1-9
7-1 ±1-6
8-3±l-8
6-4+1-4
6-2±H
—
Day 9
Day 12
5-6±l-0
6-2±M
5-4 + 0-8
6-3 ±1-3
5-6 + 0-9
7-0±l-8
3-4 ±0-7
4-9±l-0
4-2 ±0-8
Day 13
—
Day 10
Day 11
6-5 ±1-6
5-4 ±0-9
6-1 ±1-1
4-0 ±0-9
3-3 ±0-7
3-7 + 0-7
4-1 ±1-0
1-8 + 0-4
2-1 ±0-6
1-5 + 0-5
5-2±l-0
6-7 ±1-4
6-0 ±1-0
5-5 ±0-8
6-2 ±0-9
4-9+1-1
5-5+1-1
3-9 ±0-8
5-1 + 1-4
3-7±l-0
5-4+1-2
Data are given in nm.
Measurements were done on micrographs of a magnification of 120000.
Mean and standard deviations of different series are shown (N of each series = 40).
Measurements were done only at exact cross sections of the cell membrane.
In all measured regions, the surface coat of the cells is quite similar and the
thickness comes to 6 nm (Fig. 3 a, Table 2). Some structures are present at the
cell surface, which extend by more than 50 nm into the extracellular space.
Sometimes cell contacts which are about to form are detectable. These structures
have a regular cell-cell distance of about 20 nm; the space is filled by electrondense material. At one end of such wide contacts, the membranes of the adjacent cells come together to form a gap junction (Fig. 3b).
In the limb buds of day-11 mouse embryos, the first formation of cell condensation occurs in the region of the prospective humerus. Measurements of
the cell contacts in the three different regions reveal a high increase in both the
number of contacts per cell as well as in the mean (or median) length of a
contact. This results in a very high percentage of cell membranes involved in
cell contacts.
Cells in the peripheral and the distal areas show a similar behaviour. The cells
are close together, but considerable intercellular spaces are present (Fig. 4a).
The number of cell processes seems to be diminished; this is indicated in the
peripheral cells by the decrease of the cell perimeter (P < 001, significant
against the mesenchymal cells of day 9, day 10 and central or distal regions of
day 11) (Table 1).
8
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
In the central parts, the percentage of contacts has increased to more than
60%, the cells are very close together and only a small intercellular space is
visible (Fig. 4 b). All the contacts between the cells are typical gap junctions
with an electron-dense space of about 2 nm between the two outer lamellae
of the adjacent cells (Fig. 4c). In the contacts, small distensions cut the junction
into regular sections of 100-150 nm. This dimension is probably the 'real
length' of a gap junction, but in the measurements done here, one cell contact
is defined from one intercellular space to the next.
No clear-cut changes in the surface coat are recognizable. The cells in all
estimated areas exhibit a surface coat of about 6 nm thickness (Fig. 4d, Table 2).
In the limb buds of day-12 mouse embryos, no distinct differences were
detected between the distal and the peripheral regions (Table 1). Again the
organization of the tissue rather resembles that of the mesenchyme. On the
other hand, about 47 % of the membranes of the cells in the central part (humerus) are involved in contacts. The cells are still close together, but a dilatation
of the intercellular space is visible (Fig. 5 a). In the intercellular space as well
as in the cells, sdme myelin-like membrane structures are detectable (Fig. 5 a).
At higher magnification, these structures look like coiled gap junctions (Fig. 5 b).
This possibly reflects the, dis-assembly of cell contacts.
The thickness of the surface coat of the cells in all three areas is now diminished (Fig. 5d). Measurements reveal a thickness of about 4 nm (Table 2).
In the central region (humerus) of the limb of day-13 mouse embryo, chondrogenesis has started. The cells show the typical morphology of young chondroblasts (Fig. 6 a). The perimeter of a cell has further decreased significantly
(P > 0-01) to a mean of 21 /im. Only very few cell contacts are detectable:
0-85 contacts per cell are counted. The intercellular space has widened and is
filled with the typical chondrogenic matrix. The surface coat of these cells is
only very thin; the thickness has further decreased to about 2nm (Fig. 6b,
Table 2).
The cells in the distal region are difficult to define. Distally to the measured
central area the anlagen of forearm, wrist and hand are localized. They are now
advanced in development, so is the central part at day 12. Cells lying directly
under the distal epithelium resemble peripheral cells. Since the apical ectodermal ridge is now absent, no clear-cut position finding was possible, so that
measurements in this area were renounced.
Perimeter, contact length and contact number as well as the morphology of
the peripheral cells resemble those of young mesenchymal cells at day 9 or 10
(Table 1). Also the thickness of the surface coat comes to 5 /tm, a value similar
to that of the cells at day 10 (Table 2).
The contact length of the cells in different areas and at different stages are
demonstrated in the histograms of Fig. 7. It is shown that the distribution of the
contact length differs characteristically. Before cell condensation occurs (day
9 and 10), most of the cell contacts are very short, but during the condensation
Cell contact and surface coat alterations
Fig. 3(o). A surface coat of about 6nm thickness (-*•) is detectable on the cell
membrane of day-10 mesenchymal cells. Some structures extend to up to 50 nm
( • ) into the extracellular space, x 120000; bar = 100nm. (b) A developing gap
junction is shown in this micrograph. A regular intercellular space of about 20 nm,
filled with electron-dense material, and a forming gap junction at one end ( ^ ) are
visible, x 120000; bar = 100 nm.
10
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
Cell contact and surface coat alterations
11
process and at the onset of chondrogenesis (day 11 and 12), longer contacts are
detectable. These alterations in the distribution pattern are more pronounced
in the central and distal regions than in the periphery. During chondrogenesis
on day 13, the short contacts are present again in the central and in the peripheral areas.
The percentage of cell membranes involved in cell-cell contacts are plotted
against the stage in Fig. 8. It is shown that in the peripheral and distal regions
cell contact behaviour is identical. It starts from about 9 % at day 10, reaching
a maximum of about 35 % at day 11 and decreases to the 5 % level at day 13.
The cells in the central part also start at the 10 % level at day 10. Then, however, they reach a maximum at day 11 with more than 60% and decline to
less than 1 % at day 13, when the cells are differentiated. That 'wave of cell-cell
contact' therefore is highest in the cells of the central region, which results in
chondrogenic differentiation, and is lower in the cells of the distal and peripheral parts of the limb, where the cells do not undergo chondrogenic development.
DISCUSSION
Measurements of cell perimeter and cell contacts together with electron
microscopic findings on the cells in the limb bud at different stages of development allow a more detailed description of chondrogenic differentiation in the
limb. The results can be summarized as follows:
(1) From the earliest stage of development until day 12 all the cells in the
limb are more or less interconnected by cell-cell contacts of the gap-junction
type.
(2) During the cell condensation process, cell contacts increase in all regions
of the limb, but maximal formation of cell contacts occurs in the central parts,
which will further develop into cartilage.
(3) After completion of the condensation process, chondrogenesis starts in
the central parts, whereas the cells in the periphery revert to mesenchymal
characteristics. The chondroblasts lose their contacts almost completely.
Fig. 4(a). Mesenchymal cells in the distal region of day-11 mouse limb buds. Similar
morphology can be shown in peripheral parts. Cell density has increased and cells
are interconnected by gap junctions (-*•). x6000; bar = 1 ftm. (b). Cells in the
central region of day-11 mouse limb buds exhibit a maximal condensation density.
The intercellular space (X) has diminished. Cells are interconnected by long cell
contacts (-*•). x 6000; bar = 1/tm. (c). High magnification of the cell contacts
reveals the typical morphology of gap junctions, x 120000; bar = 100 nm. (d).
The surface coat of the cells in all measured regions exhibits a thickness of about
6 nm (-•). Only delicate structures extend into the extracellular space ( • ) . x 120000
bar = 100 nm.
12
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
Cell contact and surface coat alterations
Fig. 6 (a) Chondroblasts in the central part of day-13 mouse limb buds are separated
by wide intercellular spacesfilledwith collagenfilaments.Only very few cell contacts
are present (-*•). x 6000; bar = 1 /im. (b) The surface coat of such chondroblasts
is only very thin. Extracellular proteoglycan granules are present (-•) near the cell
membrane, x 120000; bar = 100 nm.
Fig. 5 (a). In the central region of day-12 mouse limb buds, the intercellular space
shows little enlargement. Most of the cells are yet interconnected by cell contacts
(•*•). Myelin-like figures are detectable in the cells as well as in the intercellular
space ( • ) . x6000; bar = 1 /tm. (b) The morphology of a myelin-like structure
shown at high magnification resembles that of coiled gap junctions, x 120000;
bar =100 nm. (c) The thickness of the surface coat of all cells in the central
region of day-12 limb buds is reduced to about 4nm. x 120000; bar = 100 nm.
13
14
B. ZIMMERMANN, E. SCHARLACH AND R. KAATZ
40-
20
Day 9
40
20Day 10
8
40-
20
Day 11
40-
20
Day 12
40'
20
Day 13
Distal
Central
Peripheral
Fig. 7. Histogram of contact length in different regions and at different stages
of mouse limb buds. Each section on the abscissa represents a contact length of
0-5 /tm. Note the relative preponderance of short contacts between the cells in
limb buds of days 9, 10 and day 13. During the cell condensation process at days
11 and 12 the number of longer cell contacts increases, but more in the central and
distal parts than in the periphery.
(4) The changes in contact behaviour are accompanied by a reduction of the
cell perimeter. The central cells lose their processes resulting in almost round
chondroblasts. Peripheral cells also reduce their processes but they become
recognizable again once the condensation process has been completed.
(5) The condensation process and the beginning of chondrogenesis are
accompanied by a reduction of the cell surface coat. While the chondroblasts
lose their surface coat almost completely, it is retained by the peripheral
cells.
Cell contact and surface coat alterations
15
70 T
10-
13
Day of development
Fig. 8. Percentage of cell membranes involved in cell contacts of mouse limb buds
of different stages. A = Central region (humerus); • = distal region; • =
peripheral region. The increase in the percentage of contact-participating cell
membranes is highest during the process of cell condensation between days 11 and 12
of development. The increase is much higher in the central parts where chondrogenesis is to start than in the distal and peripheral regions which show the same course.
When at day 13 chondrogenesis has begun, the contact in these (central) parts
comes to less than 1 %, whereas in the peripheral parts, the initial values of days
9 and 10 are measurable.
The process of cartilage development starting from the undifferentiated
mesenchyme is therefore describable in terms of increased cell contacts, reduced cell circumference and diminished surface coat. At the time of cell
condensation, all the cells present in the limb anlage are involved in these
alterations, but the greatest changes occur in presumptive chondrogenic cells.
All the other cells (an exception are blood capillaries, which are not considered
in this study) show a concomitant reaction with the same characteristics, but
of a much lower degree.
Cell condensation is always demonstrable just before chondrogenesis (Fell,
1935; Searls & Janners, 1969; Summerbell, 1976; Cairns, 1977). Some cell
activities, such as cell movements and cell adhesion, are involved in the condensation process (Ede & Agerbak, 1968; Ede et al 1977; Duke & Elmer, 1979;
Holmes & Trelstad, 1980). Occurrence of cell contacts in such cell condensations
has been reported (Gould, Day & Wolpert, 1972; Searls et al 1972; Thorogood
16
B. ZIMMERMANN, E. SC&ARLACH AND R. KAATZ
& Hinchliffe, 1975; Borck, 1977). It is, however, not possible to differentiate
between 'active' and 'inactive' gap junctions electron microscopically. Some of
the junctions shown here may be open, others may be closed. Nothing is known
about the relevance of cell contacts in chondrogenesis. On the other hand,
in vitro studies have shown that a certain number of mesenchymal cells and
cell contacts is necessary for chondrogenesis (Merker, Zimmerman & Grundmann 1980). Isolated mesenchymal cells are able to undergo chondrogenesis
only under special culture conditions, permitting high cell densities (Kelly,
Barker, Crissman & Henderson 1973; Dienstman, Biehl, Holtzer & Holtzer
1974; Goel & Jurand, 1975; Solursh, Ahrens & Reifer, 1978). This may indicate
the necessity for a certain extent of cell-cell communication. In limb buds of
chick embryos stage 22-24, Kelly & Fallon (1978) using freeze-etch replicas,
found 8 to 12 cell contacts per 100 cells. Although the importance of cell-cell
contacts has been discussed by these authors, the number of contacts per cell
seems to be too little for coupling. In limb buds of day-10 mouse embryos
which are similar to stage 22-24, two to three contacts per cell were detectable
(Table 1).
Another aspect extensively studied by Toole and co-workers should be
mentioned. When, as shown here, the number of cell contacts increases in the
limb bud during cell condensation, the intercellular space has to decrease. The
resulting space around the cell condensation has to be filled by new cells. This
is possible because the mitotic index decreases only in the condensed cell mass,
where the cell density has reached its maximum, while cell proliferation proceeds in the distal and peripheral regions (Hornbruch & Wolpert, 1970; Janners
& Searls, 1970). Toole and co-workers have shown an increase in the activity
of hyaluronidase in the limb bud just before and during cell condensation
(Toole & Gross, 1971; Toole, 1972; 1973), leading to a digestion of the hyaluronic acid-rich matrix. Our studies on hyaluronidase have shown a complete
disappearance of the intercellular space followed by a very dense cell packing
in the whole limb bud after treatment with hyaluronidase (Zimmermann, 1981).
If, according to Toole's predictions, the presumptive chondrogenic cells
produce hyaluronidase to form a condensed cell mass, the activity of the
enzyme may not be strictly limited in the central core, but it should also act to a
lesser extent via diffusion in distal and peripheral regions. This may explain
the occurrence of a 'wave of cell-cell contacts' in the whole limb bud.
Muscle blastemata are also present in the limb bud. They are formed at
about day 12 in the proximal region of the limb, perhaps also during the 'wave
of cell-cell contacts'. Our measurements were very distinctly restricted to the
central region of the humerus anlage and distally to just under the apical ectodermal ridge, mostly around the marginal sinus. We are sure that in these
regions no muscle blastemata have been measured. Regarding the peripheral
areas, muscle blastemata are expectable by day 12 the earliest in the region
between the central cell condensation and the periphery. Measurements were
Cell contact and surface coat alterations
17
done very carefully in the tissue just under the basement membrane. Furthermore, a control measurement was performed in the distal/peripheral region,
where muscle blastemata are not yet present at day 12. Here, we found data
similar to those of both the distal and the peripheral region. Hence we are
certain that no significant amount of muscle blastema was measured.
This work was supported by grants of the Deutsche Forschungsgemejnschaft awarded to
Sonderforschungsbereich 29 - 'Embryonale Entwicklung und Differenzierung - EmbryonalPharmakologie'
Our thanks are due to Mrs Heidi Somogyi and Mrs Heidi Kriiger for their excellent assistance in doing the electron microscopic sections and to Prof H.-J. Merker for his helpful,
advice in preparation of this manuscript. The translation assistance of Mrs Barbara Steyn is
gratefully acknowledged.
REFERENCES
(1977). Elektronenmikroskopische Untersuchungen an Mauseembryonen iiber
die Differenzierung des Blastems in den Extremitaten zum embryonalen Vorknorpel.
Acta anat. 97, 423-434.
BORCK, CH.
BUNGE, R., GLASER, L., LIEBERMAN, M., RABEN, D., SALZER, J., WHITTENBERGER, B. &
WOOLSEY, T. (1979). Growth control by cell to cell contact. / . Supramol. Struct. 11,
175-187.
J. M. (1977). Growth of normal and talpid2 chick wing buds. An experimental
analysis. In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe &
M. Balls), pp. 123-137. Cambridge University Press.
DE HAAN, R. L. & SACHS, H. G. (1972). Cell coupling in developing systems. Curr. Top.
Devi Bio I. 7, 193-228.
DIENSTMAN, S. R., BIEHL, J., HOLTZER, S. & HOLTZER, H. (1974). Myogenic and chondrogenic lineages in developing limb buds grown in vitro. Devi Biol. 39, 83-95.
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. E. (1979). Effect of the brachypod mutation on early stages of chondrogenesis in mouse embryonic hind limb. Teratology 19 (3), 367-376.
EDE, D. A. & AGERBAK, G. S. (1968). Cell adhesion and movement in relation to the developing limb pattern in normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph. 20,
81-100.
EDE, D. A. & FLINT, O. P. (1975). Cell movement and adhesion in the developing chick wing
bud: studies on cultured mesenchyme cells from normal and talpid3 mutant embryos.
J.Cell Sci. 18, 301-313.
EDE, D. A., FLINT, O. P., & TEAGUE, P. (1975). Cell proliferation in the developing wing bud
of normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph. 34, 589-607.
EDE, D.A., FLINT, O. P., WILBY, O. K. & COLQUHOUN, P. (1977). The development of
precartilage condensations in limb bud mesenchyme in vivo and in vitro. In Vertebrate
Limb and Somite Morphogenesis {ed. D. A. Ede, J. R. Hinchliffe and M.Balis), pp. 161-179.
Cambridge University Press.
EDE, D. A. & WILBY, O. K. (1981). Golgi orientation and cell behaviour in the developing
pattern of chondrogenic condensations in chick limb bud mesenchyme. Histochem. J. 13/4,
615-630.
FELL, H. B. (1935). Experiments of the development in vitro of the avian knee-joint. Proc. R.
Soc.Lond. 5116, 316-351.
GILULA, N. B. (1980). Cell-to-cell communication and development. In The Cell Surface:
Mediator of Developmental Processes (ed. Subtelny & N. K. Wessels), pp. 23-41. New
York: Academic Press.
GOEL, S. C. & JURAND, A. (1975). Electron microscopic studies on chick limb cartilage
differentiation in tissue culture. / . Embryol. exp. Morph. 34, 327-337.
CAIRNS,
18
B. ZIMMERMANN, E. SCHLARLACH AND R. KAATZ
R., DAY, A. & WOLPERT, L. (1972). Mesenchymal condensation and cell contact
in early morphogenesis of the chick limb. Expl Cell Res. 72, 325-336.
HOLMES, L. B. & TRELSTAD, R. L. (1977). Patterns of cell polarity in the developing mouse
limb. DevlBiol. 59, 164-173.
HOLMES, L. B. & TRELSTAD, R. L. (1980). Cell polarity in precartilage mouse limb mesenchyme cells. Devi Biol. 78, 511-520.
HORNBRUCH, A. & WOLPERT, L. (1970). Cell division in the early growth and morphogenesis
of the chick limb. Nature, Lond. 226, 746-766.
JANNERS, M. Y. & SEARLS, R. (1970). Changes in rate of cellular proliferation during the
differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing.
Devi Biol. 23, 136-165.
JURAND, A. (1965). Ultrastructural aspects of early development of the forelimb buds in the
chick and the mouse. Proc. R. Soc. Lond. B 162, 387-405.
KELLY, R. O., BAKER, T. J., CRISSMAN, H. A. & HENDERSON, C. A. (1973). Ultrastructure
and growth of human limb mesenchyme (HLM 15) in vitro. Anat. Rec. 175, 657-671.
KELLY, R. O. & FALLON, J. F. (1978). Identification and distribution of gap junctions in the
mesoderm of developing chick limb buds. /. Embryol. exp. Morph. 46, 99-110.
LOEWENSTEIN, W. R. (1980). Junctional cell-to-cell communication and growth control. In
Growth Regulation by Ion Fluxes. Ann. N.Y. Acad. Sci. 339, 39-45.
GOULD,
MERKER, H. J., ZIMMERMANN, B. & GRUNDMANN, K. (1980). Differentiation of isolated
blastemal cells from limb buds into chondroblasts. In Tissue Culture in Medical Research
vol. II. (ed. R. J. Richards & K. T. Rajan), pp. 31-39. Oxford and New York. Pergamon
Press.
NEUBERT, D., MERKER, H.-J. & TAPKEN, ST. (1974). Comparative studies on the prenatal
development of mouse extremities in vivo and in organ culture. Naunyn-Schmiedeberg's
Arch. Pharmacol. 286, 251-270.
SEARLS, R. L. & JANNERS, M. Y. (1969). The stabilization of cartilage properties in the cartilage-forming mesenchyme of the embryonic chick limb. / . exp. Zool. 170, 365-376.
SEARLS, R. L., HILFER, S. R. & MIROW, S. M. (1970). An ultrastructural study of early
chondrogenesis in the chick wing bud. Devi Biol 28, 123-137.
SOLURSH, M., AHRENS, P. B. & REITER, R. S. (1978). A tissue culture analysis of the steps
in limb chondrogenesis. In Vitro 14, 51-61.
SUMMERBELL, D., LEWIS, J. H. & WOLPERT, L. (1973). Positional information in chick limb
morphogenesis. Nature, Lond. 244, 492-495.
SUMMERBELL, D. (1976). A descriptive study of the rate of elongation and differentiation of
the skeleton of the developing chick wing. J. Embryol. exp. Morph. 35, 241-260.
THOROGOOD, P. V. & HINCHLIFFE, J. R. (1975). An analysis of the condensation process
during chondrogenesis in the embryonic chick hind limb. J. Embryol. exp. Morph. 33,
581-606.
TOOLE, B. P. (1972). Hyaluronate turnover during chondrogenesis in the developing chick
limb and axial skeleton. Devi Biol. 29, 321-329.
TOOLE, B. P. (1973). Hyaluronate and hyaluronidase in morphogenesis and differentiation.
Am. Zool. 13, 1061-1065.
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.
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.
TRELSTAD, R. L., HAY, E. D. & REVEL, J.-P. (1967). Cell contact during early morphogenesis
in the chick embryo. Devi Biol. 16, 78-106.
TRELSTAD, R. L. (1977). Mesenchyme cell polarity and morphogenesis of chick cartilage.
DevlBiol. 59, 153-163.
ZIMMERMANN, B. (1981). Effect of hyaluronidase on limb blastema formation in vitro.
Communication presented in First International Symposium on Hyaluronidase, Rotterdam.
(Received 24 March 1982, revised 4 June 1982)