/ . Embryol. exp. Morph. Vol. 46, pp. 207-213, 1978
Printed in Great Britain © Company of Biologists Limited 1978
207
Cell adhesiveness and embryonic differentiation
By RUTH BELLAIRS 1 , A. S. G. CURTIS 2 AND
E. J. SANDERS 3
From the Department of Anatomy & Embryology,
University College, London
SUMMARY
The aim of the investigation was to decide whether changes in cell to cell adhesiveness
took place during embryonic differentiation. The technique of Curtis (1969) was used to
measure the adhesive behaviour of several types of ectodermal, neural and mesodermal cells
of the chick embryo at stages 7 and 12 of differentiation. Cells dissected from segmented
mesoderm were found to be more adhesive than cells from unsegmented mesoderm. Cells
from the ectoderm were more adhesive than those from the neural tissue, at both stages 7
and 12. Cells from both ectoderm and neural tissue became more adhesive between stages
7 and 12. It is concluded that an increase in adhesiveness may play a role in somite segmentation, but not in neural tube formation.
INTRODUCTION
It has often been suggested that changes in the relative adhesiveness of cells
may play an important role in embryonic differentiation (e.g. Townes &
Holtfreter, 1955; Curtis 1967; Ede & Agerbak, 1968; Steinberg, 1970; Johnson,
1970). There have however been curiously few attempts to investigate whether
or not adhesive changes take place in the cells of a particular tissue as it differentiates. Several authors have proposed that changes of this type are a major
factor in the segmentation of somites (e.g. Waddington, 1956; Zeeman, 1971),
or during the formation of the neural plate (e.g. Brown, Hamburger & Schmidt,
1941; Gustafson & Wolpert, 1967), but to the best of our knowledge these ideas
have never been tested. The present experiments were designed to answer the
question: 'Does the adhesiveness of one cell to another change during differentiation?'.
We have used the technique of Curtis (1969) to measure the adhesive behaviour of several types of ectodermal, neural and mesodermal cells at two
stages of differentiation.
1
Author's address: Department of Anatomy and Embryology, University College London,
U.K.
2
Author's address: Department of Cell Biology, The University of Glasgow, U.K.
3
Author's address: Department of Physiology, The University of Alberta, Edmonton,
Alberta, Canada.
208
R. BELLAIRS, A. S. G. CURTIS AND E. J. SANDERS
MATERIAL AND METHODS
Experiments were carried out on embryos which had been incubated for
about 30 h and were at about stage 7 of Hamburger & Hamilton (1951), and
on embryos which had been incubated for about 40 h and were at about stage
12. In each experiment, batches of 60 embryos were removed from the yolk
and treated with 0-1% trypsin in Ca2+- and Mg2+-free Tyrode's solution at
37 °C until it was possible to dissect the tissues cleanly from one another.
The dissected tissues were collected in normal Tyrode's.
The experiments on the ectodermal and neural tissues were carried out on
embryos of stage 7 and stage 12; the tissues were taken solely from the trunk
region, the head levels being discarded (Fig. 1 A, B). The experiments on the
mesodermal cells were carried out on embryos of stage 12 only; the posterior
somites and the unsegmented presumptive somite mesoderm were collected
separately (Fig. 1C), care being taken to avoid the most anterior somites which
were already beginning to differentiate into dermo-myotomes and sclerotomes.
Similarly, the part-formed somites which lie in the transitional zone between
the segmented and unsegmented mesoderm were discarded.
Each collection of tissue was dissociated into individual cells using 0-1 %
EDTA in Ca2+- and Mg2+-free Tyrode's for about 20 min. The dissociation
was aided mechanically by drawing the tissues up and down a pipette. More
difficulty was experienced in obtaining good dissociation with ectodermal
and neural tissues than with the mesodermal ones. In particular, the ectodermal
tissues from the stage-12 embryos were especially difficult to dissociate. In
each case, the degree of separation was checked with a phase contrast microscope.
The adhesiveness of the cells to one another was investigated using a Couette
viscometer.
The technique and its theoretical basis were described in detail by Curtis
(1969). The suspension of cells is placed in the narrow gap between two concentric cylinders of known diameter. As one of the cylinders rotates a laminar
shear flow is established and the shear rate can be calculated. Under appropriate conditions the shear promotes reaggregation of the cells.
We will use the term particle to mean either one cell or one aggregate. The
population density of the suspended particles is measured with a haemocytometer, using a Quantimet 720 image analysing computer to count cells and
aggregates just before the shear is applied and at short intervals during the
experiment. As reaggregation occurs, the number of particles drops and the
rate of formation of aggregates can be calculated. If the shear rate, the concentration of cells and the sizes of the cells are all known, the collision rate can be
calculated. The actual rate of formation of aggregates is now compared with
the collision rate to obtain a measure of the collision efficiency. The collision
efficiency is a reliable indicator of the adhesiveness of the cells.
Cell adhesiveness and embryonic differentiation
209
t ^ j = lateral ectoderm
|ff§| = neural tissue
= segmented mesoderm
= segmental plate mesoderm
(unsegmented)
Fig. 1. Diagram to show the regions from which tissues were dissected.
(A) Stage 7 to show the source of the lateral ectoderm and the neural plate.
(B) Stage .12 to show the source of the lateral ectoderm and the neural tube.
(C) Stage 12 to show the source of the segmented mesoderm and the segmental
plate (unsegmented mesoderm). Note that neither the anterior somites nor the
part-formed somites were used.
RESULTS
Individual cells from all the tissues became rounded after dissociation.
The diameters were as follows:
At stage 7, the neural cells measured about 12 /im whereas the ectoderm
cells measured about 10 /tin.
At stage 12, the neural cells measured about 10 fim whilst the ectoderm
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210
R. BELLAIRS, A. S. G. CURTIS AND E. J. SANDERS
Table 1. Collision efficiencies of dissociated cells at two stages of
development
Stage 12
Stage 7
Diameters
of cells
Ectoderm
Neural
Unsegmented
Segmented
N*
Means
S.D.
(jim)
15
15
15-66
7-75
—
—
6-42
5-82
—
—
10
12
—
—
Diameters
of cells
TV*
Means
S.D.
8
7
34-4
18-28
8-93
171
7-64
6-65
4-77
7-26
12
12
7
10
12
12
* N = number of embryos.
cells measured about 7 jam. The mesoderm cells measured about 12 jam in
diameter.
The viability of cells after dissociation was tested by explanting them in
tissue culture (see Bellairs, Sanders & Portch, 1978#). Most of the cells from
each tissue survived and many of them spread out on the surface of the Petri
dish.
The population density at the start of each dissociation experiment was
about 106 cell/ml. Reassociation took place at 37 °C and measurements were
taken every 7 min over a period of half an hour. The collision efficiencies are
shown in Table 1. The significance of the results was examined by means of
the modified /-test of Cochran & Cox (see Snedecor & Cochran, 1967). The
following relationships were obtained:
(a) The collision efficiency of the cells from the segmented mesoderm was
about twice that of the cells from the unsegmented mesoderm. The difference
was statistically significant at the 4 % level.
(b) The collision efficiency of cells from the ectoderm was about twice that
of cells from the neural tissue. This was so whether the cells were both taken
from embryos at stage 7, or from embryos at stage 12. At each stage, the
difference between ectodermal and neural cells was statistically significant at
the 4 % level.
(c) The collision efficiencies of both the ectodermal and neural cells more
than doubled between stages 7 and 12, and these differences were statistically
significant at the 4 % level.
DISCUSSION
These results indicate (1) that at stage 12 cells derived from the segmented
mesoderm are more adhesive to one another than are cells derived from unsegmented mesoderm; (2) that at both stages 7 and 12 cells derived from the
ectoderm are more adhesive to one another than are cells from the neural
Cell adhesiveness and embryonic differentiation
211
plate; and (3) that the cells of all three tissues (ectoderm, neural plate and
somites) become more adhesive with differentiation.
The first point for discussion is that the relative adhesiveness of the cells to
one another parallels the relative adhesiveness of these types of cells to glass
and/or plastic. In a recent investigation, Bellairs & Portch (1977) found striking
behavioural differences between segmented and unsegmented somite mesoderm
when the two tissues were dissected from the embryo and explanted in vitro.
These differences included the fact that the proportion of explants which
successfully attached to the glass substrate was consistently higher with the
segmented than with the unsegmented mesoderm. A similar result was obtained
when the two tissues were dissociated and plated out as individual cells in
plastic culture dishes (Bellairs et al. 1978 b). These results implied that the
cells from the segmented tissue were strongly adhesive to glass or plastic whereas
those of the unsegmented mesoderm were not. Thus the cells from the segmented mesoderm were relatively more adhesive than those from the unsegmented mesoderm whether the assessment was made on a cell to cell or on a
cell to substrate basis.
In a similar series of experiments (Bellairs et al. 1978a) it was found that
the ectoderm adhered more readily to a glass or plastic substrate than did the
neural cells. This was so whether the explants were of small pieces of tissue or of
dissociated cells. It appears therefore that the cells of the ectoderm are more
adhesive than those of the neural tissue whether the assessment is made on a cell
to cell or on a cell to substrate basis.
The second point for discussion is the significance of these findings for
embryonic differentiation. Our results support the idea that an increase in cell
to cell adhesiveness of the mesoderm plays a role in the process of segmentation.
We have no direct evidence as to whether the adhesiveness of each cell is the
same over its entire surface. We suggest however that each cell may become
polarized at least temporarily with regions of high and low adhesiveness. Thus
the cells might be expected to aggregate together at regions of high adhesiveness
which would then become the centres of the individual somites; it may be
significant that desmosomes appear in the centre of each somite soon after
segmentation (Bellairs et al. 1978b). Conversely, the cells would be expected
to separate easily at the regions of low adhesiveness, which would thus enable
furrows to form between somites and would also separate the somites from
adjacent tissues.
Our results do not support the idea of Brown et al. (1941) that neurulation
is brought about by a higher level of adhesiveness in the neural than in the
ectodermal cells. On the contrary we have shown that the neural cells have a
lower level of adhesiveness. It is true that the adhesiveness of the neural cells
more than doubles between stages 7 and 12, and that this corresponds with the
period when the neural plate thickens, rolls up and undergoes elaboration.
But the adhesiveness of the ectoderm cells also doubles during this period, and
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212
R. BELLAIRS, A. S. G. CURTIS AND E. J. SANDERS
yet this tissue does not increase in thickness (Bellairs et al. 1978 a) and it does
not roll into a tube. Similarly, our results do not support the suggestion made on
theoretical grounds by Gustafson & Wolpert (1967) that the neural plate cells
undergo elongation because of an increased adhesion along their lateral faces.
Indeed, the region of greatest adhesion is probably located near the apical
border of each cell since large intercellular spaces may often be seen toward
the basal sides of the tissue.
The most widely held view on the mechanism of neurulation is that it results
from the co-ordinated activities of microtubules and micron*]aments. It is
supposed that the microtubules of the neural cells elongate in a dorso-ventral
direction and that this results in an elongation of the cells. The microfilaments
of the same cells now become orientated parallel to the apical surface and then
contract, so that the cells become constricted apically and the neural plate
rolls into a tube. (See Karfunkel, 1974, for a review of the literature.) Our
morphological studies also lend support to this theory (Bellairs et al. 1978a).
The question now arises as to why the ectodermal cells possess such a high
level of adhesiveness in comparison with the neural cells. It is known that the
ectoderm is under considerable tension as the chick blastoderm expands over
the surface of the yolk (New, 1959; Bellairs, Bromham & WyJie, 1967). Much
of this force is generated by the peripheral cells of the area opaca ectoderm
which migrate steadily in a centrifugal direction. It seems likely therefore that
strong cell to cell adhesiveness in the area pellucida ectoderm prevents the
tissue from becoming torn apart.
The neural tissue is not under such tension, and is apparently in no danger of
tearing. It is perhaps for this reason that it has a cell to cell adhesiveness at
stage 12 which is relatively low, being comparable to that of the segmented
mesoderm. We propose to carry out further experiments to determine whether
a pattern exists in the distribution of cell to cell adhesiveness in the embryo
which may be related to the level of differentiation in the various tissues.
We wish to thank Miss Rose McKinney for her generous and skilled assistance, and Mrs
J. Astafiev for Fig. 1. We are also grateful to the Wellcome Trust who made funds available
for the purchase of the Quantimet 720 image analysing computer; the M.R.C. of Canada
who supported the work of E.J.S. and the University of Kuwait who supported the work
ofR.B.
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{Received 10 February 1978, revised 3 April 1978)