/. Embryol. exp. Morph. Vol. 20, 1, pp. 81-100, August 1968
With 3 plates
Printed in Great Britain
81
Cell adhesion
and movement in relation to the developing limb
pattern in normal and talpid3 mutant chick
embryos
By D. A. EDE 1 & G. S. AGERB AK2
From the Agricultural Research Council Poultry Research Centre, Edinburgh
Descriptive studies of the talpid3 chick embryonic lethal mutant (ta3/ta3) have
suggested that the multiple effects produced by this gene are of mesodermal origin,
and that they arise from defective mesenchymal cell movement and condensation (Ede & Kelly, 1964 a, b). It may be argued that condensation in vivo is
comparable to cell reaggregation of dissociated cells in vitro, and that defects in
the former are likely to be reflected in the latter. In this case it should be possible
to obtain experimental verification of this effect of the gene at the cellular level,
using the quantitative methods for assessing aggregation developed by Moscona
(1961 a, b) and Curtis & Greaves (1965). The experiments reported here show
a clear genetic effect upon cell adhesion in the wing-bud mesenchyme of the
talpid3 mutant.
The wing-bud was chosen because it was hoped to establish a connexion
between the effect of the gene at the cellular level and its dramatic effect on limb
morphogenesis. A theoretical model of its development is proposed, which
demonstrates how changes in cell adhesiveness and motility may account for the
distorted shape of the mutant wing-bud, and also the role that these factors may
play in the production of the normal limb-bud pattern.
MATERIALS AND METHODS
Four- and five-day embryos (stages 24 and 26) were obtained from matings
between known talpid3 heterozygotes, giving approximately one talpid3: three
normal phenotypes. Mutant and normal embryos were easily distinguishable
at these ages (Plate 1), and any talpids which were not clearly healthy (as judged
1
Author's address: Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh, 9, U.K.
2
Author's address: Microbial Genetics Research Unit, Hammersmith Hospital, London
W. 12, U.K.
6
.1 E E M 2O
82
D. A. EDE & G. S. AGERBAK
by size, vigorous heart-beat and absence of capillary haemorrhages) were
discarded.
The methods used for obtaining single cell suspensions of wing-bud mesoderm cells and for producing rotation-mediated reaggregation follow those of
Moscona (1961 a) with slight variations. Wing-buds were dissected offin Tyrode's
solution, removed to 15 ml centrifuge tubes, rinsed in Ca2+- and Mg2+-free
Tyrode's solution (CMF) and incubated for 10 min at pH 8-0. Incubation for
this and all other operations was at 37-5 °C. They were then rinsed and incubated
for 10 min in trypsin solution (1 % Difco 1:250 in CMF at pH 8-0), then transferred into CMF in cavity slides where the ectoderm was carefully removed and
the mesoderm divided into small fragments by means of tungsten needles. The
fragments were placed in centrifuge tubes with fresh trypsin solution and incubated for 20 min, then rinsed in CMF and the CMF finally replaced by 1 ml of
culture medium. A cell suspension was obtained from the fragments by flushing
them 15-20 times through the tip of a fine Pasteur pipette (0-75 mm tip diameter),
transferred to a 25 ml conical flask and made up to 2 ml with culture medium.
Samples of this stock suspension were taken for cell counting in a haemocytometer, for checking for complete dispersion of cells and for viability testing.
Suspensions obtained in this way contained not more than 3-4 % of clusters (two
or more adhering cells) among cells counted, and viability, tested by eosin
staining, was between 94 and 95 % for both talpid3 and normal cells.
(a) Aggregation over 3-day periods
The culture medium consisted of Eagle's basal medium based on Earle's BSS
(Flow Laboratories) with 0-02 % glutamine, 10 % horse serum (Oxoid), 2 %
embryo extract (50:50 in Tyrode's solution; 9-day chicks embryos) and penicillinstreptomycin at a concentration of 100 units/ml, added. Media were sterilized
by Millipore filtration.
The stock suspension was diluted to give 5 x 105 cells in 3 ml culture medium
in 25 ml conical flasks. The flasks were gassed with 5 % CO2-95 % air mixture
jto give pH 7-2, tightly stoppered and placed on a gyratory shaker inside a waterac keted incubator. The shaker (to be described in a separate publication) was
constructed in the laboratory work-shop and was designed to give a very smooth
swirling action. For these experiments it was run with a fin. radius rotary
motion, at a speed of 50 rev/min. In each experiment about six flasks each of
talpid3 and normal cell suspensions were prepared from embryos of the same
age, either 4- or 5-day; eight experiments were done with 4-day and six with
5-day embryos.
At 7, 25, and 70 h after beginning rotation each flask was removed from the
shaker, its stopper replaced by a glass cover, and the aggregates photographed;
then it was gassed again, re-stoppered and put back on the shaker. Measurements and counts were made on the photographs.
After photographing at 70 h the aggregates were fixed in Bouin's fluid,
/. Embryol. exp. Morph., Vol. 20, Part 1
PLATE 1
Figs. A and B. Normal embryos, 4- and 5-day.
Figs. C and D. talpid3 embryos, 4- and 5-day.
D. A. EDE & G. S. AGERBAK
facing p. 82
J. Embryol. exp. Morph., Vol. 20, Part 1
PLATE 2
K
Aggregates from dissociated wing-bud mesenchyme cells from 4- and 5-day embryos at
different stages of rotation culture.
Fig. E. 4-day normal aggregate, 7 h.
Fig. F. 4-day talpid3 aggregate, 7 h.
Figs. G-I. 5-day normal aggregates; the same culture at 7, 25 and 50 h.
Figs. J-L. 5-day talpid3 aggregates; the same culture at 7, 25 and 50 h.
D. A. EDE & G. S. AGERBAK
Cell adhesion and development
83
sectioned at 5 fi and stained variously with haematoxylin and eosin, iron haematoxylin, Masson's saffron and alcian blue with chlorantine fast red.
In order to estimate changes in cell number some flask contents, obtained
from 5-day embryos, were disaggregated again after 50 h of reaggregation, and
cell counts made with a haemocytometer.
(b) Aggregation over 5 h periods
Conditions for these experiments differed from the above in the following
respects: horse serum and embryo extract were omitted from the culture medium;
each flask contained 2 x 106 cells in 3 ml of medium; rotation was at 70 rev/min.
In each experiment one flask each of talpid3 and normal cell suspensions was
prepared, gassed and stoppered and placed on the shaker; thereafter samples
were taken for haemocytometer counts of single cells remaining non-aggregated,
following the method of Curtis & Greaves (1965), at hourly intervals up to 5 h.
Cell suspensions from 4- and 5-day embryos were used for four experiments in
each case.
RESULTS
3
Normal and talpid wing-buds dissected from living embryos contained
approximately equal numbers of cells at 4 days (Table 1), but at 5 days there
were over \\ times as many in talpid3. However, there was no indication of more
Table 1. Average number of mesoderm cells in 4- and 5-day wing-buds, and in
aggregates from 5 x 105 5-day cells after 50 h rotation culture. Total number of
wing-buds of flasks in parentheses
Wing-bud, 4-day
Wing-bud, 5-day
Aggregates
Normal
talpid3
311000(84)
943000(110)
160000 (9)
326000(42)
1598000(44)
183000(10)
ta3/N
10
1-7
1-1
rapid multiplication of talpid3 cells in culture when cells from 5-day embryos
were reaggregated and then redissociated for counting after 50 h rotation at
50 rev/min. Only about one-third of the original cell number was recovered in
each case, indicating that any increase by cell multiplication had been offset by
cell loss during the processes of reaggregation and redissociation.
During the 3-day period reaggregation proceeded in the way described by
Moscona (1961a), the large number of very small aggregates appearing after
a few hours reducing to smaller numbers of larger aggregates as rotation continued. Direct observation suggested a difference between normal and talpid3 in
the pattern of reaggregation as regards size and number of aggregates, and also
in their form, stabilizing at about 50 h (Plate 2, figs. G-L). The data regarding
size and number in cultures from 4-day embryos are summarized in the histo6-2
84
D. A. EDE & G. S. AGERBAK
gram (Text-fig. 1), which omits aggregates of 0 1 mm or less since these are
difficult to count and measure accurately. It shows little difference between
normal and talpid3 at 7 h, but thereafter normal aggregates are generally larger
and less numerous than talpicP aggregates, this tendency increasing up to 50 h,
when the pattern becomes stabilized.
Changes in total aggregate number (not including aggregates of 0 1 mm and
under) and aggregate size are shown in Table 2 and in Text-fig. 2. In order that
the significance of the larger aggregates should not be obscured by measurements
Hours
10
Normal
talpid3
0-5-
10
25
50
a;
70
0-5
0-1
0-5
1-0
1-5 0-1
0-5
10
Size of aggregates (maximum diameter, mm)
1-5
Text-fig. 1. Number and size distribution of 4-day normal and talpid*
aggregates > 0-1 mm at 7, 25, 50 and 70 h. semilog.
Cell adhesion and development
85
of the smaller ones (which would include at the extreme limit any group of two
or more cells) size is expressed as an index d, where d = 2J 4 /2d 3 , d is the maximum diameter of an aggregate and summation is over aggregates. In this way,
the aggregate to which a given cell belongs is effectively averaged over cells rather
Table 2. Average size {all aggregates, 100 log d index) and average number (per
flask, aggregates > 0-1 mm) at 7, 25, 50 and 70 h, with analysis of variance
(*= p < 005, ** = P < 001, n.s. = not significant)
25 h
7h
Normal
talpid3
5-day Normal
talpid3
Normal v. talpid3
4-day v. 5-day
4-day
50 h
70 h
Size
Number
Size
Number
Size
Number
Size
Number
37-0
249
407
34-5
*
23-5
12-6
24-2
13-2
*
101-5
610
88-2
77-0
**
133-6
80-4
110-3
86-2
**
4-7
19 6
16-2
25-1
**
127-4
88-6
113-8
86-2
**
4-5
19-9
13-7
27-4
**
n.s.
n.s.
n.s.
16-3
32-5
27-8
36-7
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
2
40 r
100
A
20
8
50
0 L—I0 7
50
25
Hours
70
0
7
25
50
70
Hours
Text-fig. 2. Aggregates from 4-day normal (O, solid line) and talpid* ( • , dotted line)
at 7, 25, 50 and 70 h. A, Average size (100 log index 3) of all aggregates. B, Average
number of aggregates > 0 1 mm per flask.
than over aggregates. Put in another way, the contribution which any aggregate
makes to the average is weighted in proportion to its approximate volume (d3).
For statistical analysis it was found to be more convenient to use log d.
The graphs indicate that there is indeed only a small difference in aggregate
86
D. A. EDE & G. S. AGERBAK
size at 7 h, but that this difference increases up to 50 h. Aggregate number is
greater in normal cultures at 7 h, but from 25 h it is smaller than in talpid*.
The same picture emerges from the experiments using cells from 5-day
embryos. Table 2 suggests that there is also a difference between cultures from
4- and 5-day embryos, showing a consistently higher average number of aggregates in the latter, but an analysis of variance shows that this apparent difference
is not significant at the 5 % level. On the other hand the differences between
normal and talpid* are significant in all cases, except for the difference in aggregate number at 25 h, where an inexplicably wide divergence from the general
pattern in one out of the six experiments reduced the statistical significance of
the result.
Table 3. Number of single cells (% of original suspension of 2 x 106 cells) at 0-5 h>
with analysis of variance of steepness of decline between 0-1, 2-3 and 4-5 h
(* = P < 005, n.s. = not significant)
Hours
4-day Normal
talpid3
5-day
Normal
talpid3
Normal v. talpid3
4-day iv. 5-day
0
1
2
3
4
5
1000
1000
801
54-7
46-3
51-7
38-9
37-2
34-9
33-7
17-4
22-9
24-8
20-5
11-7
16-7
20-6
11-5
64-3
78-8
1000
1000
691
5-3
*
n.s.
n.s.
n.s.
*
*
In both 4-day and 5-day experiments normal aggregates are generally not only
larger but also irregular in shape, whereas talpid* aggregates are nearly spherical.
This is very marked at 7 h (Plate 2, figs. E, F, G and J), when normal aggregates
are extremely irregular, with cells adhering loosely at their surfaces, and tangling
with neighbouring aggregates. Talpid* aggregates at this stage are rounded, have
few loose cells at their surfaces, and aggregates are clearly separated from their
neighbours.
This difference in the form of normal and talpid* aggregates is evident in
histological sections made from aggregates fixed after 3 or 4 days or rotation
(Plate 3, figs. M-P). Cells at the boundary of normal aggregates usually remain
rounded and project irregularly to give a rough surface, whereas those at the
boundary of talpid* aggregates are usually flattened and closely packed together,
forming a virtual epithelium around each aggregate and giving it a smooth
surface.
The cells of the chondrogenic interior of the aggregates at the end of the
culture period are stellate in normal aggregates, and are in contact with each
other chiefly at the tips of their cytoplasmic projections, whereas in talpid
/. Embryol. exp. Morph., Vol. 20, Part 1
PLATE 3
O
Figs. M and N. Section of a 4-day normal aggregate,fixedat 70 h. Stained haematoxylin and
eosin.
Figs. O and P. Section of a 4-day talpid3 aggregate, fixed at 70 h. Stained Masson's saffron.
Fig. Q. Section of the chondrogenic interior of a 5-day normal aggregate, fixed at 70 h.
Stained Masson's saffron.
Fig. R. Section of the chondrogenic interior of a 5-day talpid3 aggregate, fixed at 70 h.
Stained Masson's saffron.
Fig. S. Longitudinal section of the wing of a 6-day normal embryo showing chondroblast
orientation in the ulna region. Stained haematoxylin and eosin.
Fig. T. Longitudinal section of the wing of a 6-day talpid3 embryo showing failure of
chondroblast orientation in the ulna region. Stained haematoxylin and eosin.
D. A. EDE & G. S. AGERBAK
facing p. 86
Cell adhesion and development
87
aggregates these cells are more rounded, and adhere to each other along more
extensive regions of the cell boundaries (Plate 3, figs. Q, R).
The results of the experiments in which cells were rotated at 70 rev/min over
a 5 h period in order to observe the decline in number of single (non-aggregated)
cells are given in Text-fig. 3, and in Table 3 together with the results of an analysis
of variance of the differences in steepness of decline at the beginning, middle and
end of this period. They show that talpid3 declines more steeply than normal
during the first hour, after which there is no significant difference between them
in this respect. They also show that 5-day cultures decline more steeply than
4-day cultures; the difference in this case is not significant during the first hour
but becomes significant during the middle and end periods.
2x10* «-
2x10*
1x104 -
0
1
2
3
4
5
Hours
Text-fig. 3. Decline in number of single cells over the first 5 hours of reaggregation.
A, Cells from 4-day normal (O, solid line) and talpid3 ( • , broken line) embryos.
B, Cells from 5-day normal (A, solid line) and talpid3 (A, broken line) embryos.
DISCUSSION
1. Aggregation patterns in normal and talpid3, and their
relation to cell adhesion
From these experiments it is clear that a difference in aggregation pattern
exists between normal and talpid3 wing-bud mesenchyme cells, visible at 7 h and
stabilizing at about 50h in rotation-mediated cultures; in general, normal
aggregates are larger and irregular in shape while talpid3 aggregates are smaller
and more nearly spherical. The number of cells in pooled aggregates after 50 h
is approximately the same in each, so that the difference between them is a matter
of cell distribution, uncomplicated by differences of cell number.
Moscona (1961a) showed that, under conditions similar to these, different
cell types (e.g. neural retina, liver, mesonephros) produced aggregates of characteristic size and shape, and, on the basis of experiments in which speed of rotation
and temperature were varied, concluded that there was a direct relation between
size and cell cohesiveness: the stronger the cohesiveness, the larger the aggregates
produced from a given initial cell suspension. If this rule is applicable to our case
88
D. A. EDE & G. S. AGERBAK
it would indicate that talpid3 cells are less adhesive to each other than normal
cells, since they form smaller aggregates.
However, estimates of the decline in number of single cells, which gives a
direct measure of cell adhesiveness during the first stage of aggregation, show
a steeper fall in talpid3 than in normal, indicating that on the contrary talpicP
cells are the more adhesive. The histological appearance of the aggregates is
consistent with this conclusion and at the same time suggests why it is that
normal aggregates should, nevertheless, be the larger. The appearance in sections
of the cells in the chondrogenic interior of aggregates at the end of the culture
period strongly suggests that talpidz chondroblasts are more closely adherent to
each other than normal chondroblasts. More important for our present argument is the contrast between the rather rough surface, produced by loosely
adhering cells, of normal aggregates and the smooth quasi-epithelial surface of
talpid3 aggregates, which is evident as early as 7 h.
These observations may all be resolved if in this instance aggregate size is
indirectly related to cell adhesiveness, not directly as Moscona's rule requires
—'The size distribution of aggregates thus formed reflects an equilibrium
between cell cohesiveness under the given conditions, and the shearing forces of
the system: the lesser the capacity of cells to cohere (or the fewer the number of
cohesive cells, or the stronger the shearing forces), the smaller the resulting
aggregates.' (Moscona, 1961 Z>). It is therefore important to see whether this
generalization, which was proposed for its heuristic value, does not require
modification.
The production of aggregates in this system is due chiefly, after the first hour
or so, not to collisions of single cells but of smaller aggregates, which may
unite to give larger aggregates, and if 'aggregate' were substituted throughout
for 'cell' the rule would be indisputable. However, it is not permissible to
equate aggregate cohesiveness with cell adhesiveness; that is to say, the mutual
adhesiveness of individual cells may not be reflected in the mutual adhesiveness
of the aggregates they make up, since whether two colliding bodies stick together
or not depends not only upon the adhesiveness of their surfaces but also upon
how much of these surfaces come into mutual contact, i.e. upon their shape and
deformability.
The characteristic shapes of aggregates are not accounted for by Moscona,
except as depending on a process of sorting out within aggregates of cells of
different degrees of adhesiveness. Thus, liver cells produce one or a few large
irregular aggregates under standard conditions while limb-bud cells produce
many small subspherical ones. According to Moscona (1965), 'One assumes
that in 24 h the aggregates acquire a surface layer of cells which are relatively
non-adhesive at the side exposed to the medium. Hence a cell population comprising many such cells may be expected to yield an aggregation pattern
consisting of many small aggregates, and vice versa. Cells from dissociated
whole limb-buds produce aggregates which, perhaps because they are rapidly
Cell adhesion and development
89
covered by epidermal cells, become non-adhesive and remain quite small; liver
cells, on the other hand, cohere into large masses'. However, in earlier work,
Moscona (19616) obtained similar results using only the central core (skeletomyogenic mesoblast) of 4-day chick limb rudiments; in fact the character of the
limb-bud aggregates does not depend upon the presence of epidermal cells, and
there is no reason to believe that at this stage the central mesoblast cells are
heterogeneous with respect to adhesiveness. In our experiments differences in
aggregate shape are apparent at 7 h, when no sorting will have occurred, and we
suppose them to be due not to any heterogeneity within the cell populations, but
to differences in the way in which the cells become packed together.
A single cell in rotating medium will assume a spherical shape, which will be
deformed if it collides with another cell and adheres to it. The degree of adhesion
will be related to the degree of deformation, and will be strongest when the cells
are deformed into hemispheres (i.e. adhere over a maximum area), producing
a spherical aggregate (i.e. one with minimal surface) conjointly. Looser adhesions
will occur when there is less deformation of each cell, producing a dumb-bell
shaped aggregate, with a smaller area of adherence and a larger surface. Other
cells colliding with these aggregates and adhering to them will do so with a
maximum of contact and produce nearly spherical aggregates if they are very
adhesive, and with less contact and produce irregular aggregates if they are
less so.
After some time aggregation by collision of single cells with other single cells
and with small aggregates will be superseded by collision of aggregates with
aggregates. Spherical aggregates will come into contact over an area which
becomes relatively smaller as their bulk becomes greater, and though they will
still tend to stick together more frequently because their cells are more adhesive
there will be an increasing opposing tendency, eventually dominant, for them
to bounce off each other. Irregular aggregates, on the other hand, will make
contact at a number of points and will tend increasingly to tangle with each
other, so that although their cells are less adhesive the chances of fusion will be
greater. More adhesive cells will, in fact, tend to produce numerous small
spherical aggregates, less adhesive cells a few large irregular ones.
How this may be related to our data, on the hypothesis that talpid3 cells are
more adhesive than normal cells, may be explained in conjunction with the
accompanying diagram (Text-fig. 4), in which aggregation is divided into four
phases, as follows.
Phase 1. The initial single cell suspension, before aggregation has begun.
Phase 2. Single cells are colliding together, and since they adhere more frequently the number of single cells declines more steeply and the number of small
aggregates is larger in talpid3 (Text-fig. 3 A, B, 0-1 h).
Phase 3. Single cells are colliding with small aggregates, which are also
colliding with each other; the irregular normal aggregates are already fusing
more often than the subspherical talpid3 aggregates, and consequently becoming
90
D. A. EDE & G. S. AGERBAK
larger and less numerous (Text-fig. 2 A, 7 h; Plate 2, figs. G, J). In the number
graph (Text-fig. 2B, 7 h) normal aggregates appear as more numerous, since
aggregates of 01 mm and less are not included, and a greater proportion of
talpid3 aggregates fall into this category.
Normal
falprd'
Phase 1
a0 °
Phase 2
&<?
o oo
0,o J
Phase 3
Phase 4
Text-fig. 4. Diagram illustrating proposed relationship between cell adhesiveness and
aggregate size and number in normal and talpid3. The number of cells is 150 in each
case and remains constant through all phases. The top left half of each frame represents single cells and aggregates < 0-1 mm, the bottom right half aggregates
> 01 mm.
Cell adhesion and development
91
Phase 4. The tendency for irregular aggregates to fuse more readily than
spherical ones continues; all aggregates have reached countable size and they
are clearly fewer and larger in normal than in talpid3 cultures (Text-fig. 2 A, B,
25 and 50 h; Plate 2, figs. H, I, K, L).
If this interpretation is accepted, all data indicate that the mutual adhesiveness of wing-bud mesenchyme cells is greater in talpid3 mutant than in normal
embryos. Whether the talpid3 gene produces this effect by altering some property
of the cell membrane or by affecting the production of some extracellular
substance will be a matter of further investigation.
2. Adhesion and motility in talpid3 cells
The adhesiveness of cells may play an important part in determining their
motility; sarcoma cells, fibroblasts and epithelial cells appear to lie on a scale of
increasing adhesiveness and decreasing motility. If cells are very adhesive their
motility might be much reduced, and the following evidence suggests that on this
scale talpid3 cells are towards the epithelial end:
1. The appearance of cells at the surface is more epithelioid in talpid3 than in
normal aggregates (Plate 3, figs. N, P).
2. The cells of the chondrogenic interior appear to be united by more extensive regions of cytoplasm in talpid3 than in normal aggregates (Plate 3,figs.Q, R).
3. Hinchliffe & Ede (1968) found that whereas in normal development the
chondroblasts of the shoulder girdle elements become orientated at right angles
to the long axis of the rudiments, no such orientation occurs in talpid3. This
absence of chondroblast orientation is also found in the cartilage rudiments of
the limb in 6-day talpid3 embryos, when it is clearly established in normals (Plate 3,
figs. S, T). The emergence of this order within a rudiment in which the chondroblasts are at first arranged randomly must entail some movement of these cells
upon one another, and inhibition of this movement because of their abnormally
high adhesiveness would account for its absence in talpid3.
We conclude that talpid3 cells are not only more adhesive to each other than
normal cells, but that as a consequence their motility is reduced.
3. Defective mesenchymal condensation andpolydactyly in talpid3 mutants
Defective segregation of mesenchyme cells in talpid3 was first described in the
head (Ede & Kelly, 1964a), where the distortions of the face—e.g. eyes drawn
together in the mid-line, absence of upper beak and fusion of the halves of the
lower beak—were attributed to the prechordal mesoderm failing to separate to
form the lateral maxillary rudiments, remaining instead as a mesial block of
mesenchyme. Widespread failure of separate precartilage condensations to
appear during skeletal development was also found (Ede & Kelly, 19646), and
in particular extensive fusion between the elements of the limb skeleton. These
fusions are well shown in histochemical studies on the development of the latter,
especially as regards mucopolysaccharide distribution (Hinchliffe & Ede, 1967).
92
D. A. EDE & G. S. AGERBAK
We believe that the failure of these mesenchymal cells to segregate properly to
form normal clearly separated condensations is due to their abnormal adhesiveness and reduced motility.
Within the limb-bud, development of the pentadactyl precartilage pattern
in the mesoderm entails two processes whose mutual relationships are still
obscure:
1. Condensation, i.e. its division into a central core of closely packed and a
peripheral region of loosely packed mesenchyme cells, followed by subdivision
of the core into condensed regions representing the skeletal elements.
2. Differentiation, proceeding in parallel with condensation, in which the cells
of the core become transformed into chondroblasts, with characteristic intercellular matrix, and separated by sheaths of spindle-shaped perichondrial cells
from the myogenic and other tissues developing in the peripheral mesenchyme.
The aspect of differentiation will be dealt with in a further study; for the present
condensation only will be considered, and it will be supposed that it is produced
by aggregative cell movements. Of the two other mechanisms suggested by Trinkaus
(1965), i.e. (1) local increase in cell division and (2) contraction of the whole
cellular mass, there is no evidence for the first in this instance, and the experiments
of Hampe (1960) suggest that individual cell movement is involved rather than
the second. The condensations appear at definite locations within the developing
limb-bud, and their foci are presumably the centres of production of some
morphogenetic substance (morphogen) leading to aggregation in their neighbourhood. The process appears to be similar to the appearance of aggregation
centres in dispersed populations of slime mould amoebae (Bonner, 1963), though
whether the morphogen of the limb bud produces its effect chemotactically as
the hormone acrasin does in slime moulds, or by some sort of trapping by cell
immobilization, is an open question.
Whatever the mechanism, the tendency to aggregate will be greatest at the
focal point and diminish with increasing distance from it, so that in a twodimensional diagram regions of decreasing aggregation potential may be represented by concentric lines around it (Text-fig. 5 A). Neighbouring regions overlap, and the degree of overlapping is related to their position on the proximodistal axis of the limb bud; those at the distal end overlap least because, being
at the border of the expanded portion of the bud, they are the most widely
spaced. Where lines overlap, the regions they enclose become fused.
According to this scheme, cells move away from the periphery towards the
centre, and normally condense into the black regions in the diagram, forming
(omitting the carpals for the sake of simplicity) four bands of elements in the
wing: (1) humerus, (2) radius and ulna, (3) four metacarpals and (4) four sets of
phalanges. Talpid3 cells, because of their abnormal adhesiveness and reduced
motility, manage to condense only into the outer rings, so that radius and ulna
elements and metacarpal elements become fused, only the phalanges appearing
as separate condensations. Expressing this in another way, we may say that as it
Cell adhesion and development
93
grows out from the trunk the limb bud generates a sequence of individuation
fields (Waddington, 1967) of chondrogenic potential, and that each of these
fields (except the most proximal) normally becomes divided into subfields, but
that in talpid3 this subdivision manifests itself only in the most distal field.
However, talpid3 is characterized not only by fusion of normally separate
elements, but also by polydactyly in the formation of up to eight phalangeal
elements in the wing, and this is clearly related to the distinctive fan shape of the
limb-bud. In the normal embryo the number as well as the spacing of elements
in each field is related to its position on the proximo-distal axis: 1 (humerus)
proximally, where the limb is narrowest, 2 (radius and ulna) next distally,
4 (metacarpals) and 4 (phalanges) most distally. This in itself suggests that the
number of condensations is related to the breadth of the field, and this would
account for the increased number of phalanges in talpid3, where the distal margin
Normal
Ulpid
Text-fig. 5. Diagram showing precartilage condensation fields
in normal and talpid wing-buds.
is extremely extended. The fan-shaped distortion of the limb-bud will also increase the number of metacarpal foci, but since condensations extend to the
outer ring these elements remain fused and indistinguishable. If the effect of the
gene on cell adhesion were less strong, so that condensation proceeded up to the
middle ring, the increased number of metacarpals would be revealed (Textfig. 5B). This is precisely what is shown in photographs of the cartilage limb
skeleton of the talpid2 chick embryo (Goetinck & Abbott, 1964), a lethal mutant
which shows the same general effects as talpid3, but which survives longer and
whose genetic effects on cell properties are therefore probably less intense.
The same sort of relationship was found by Coulombre, Coulombre & Mehta
(1962) to exist between the diameter of the eye and the number of scleral ossicles
produced in the conjunctival mesenchyme. Both are clear examples of the type
of development characterized (Smith, 1960) by the production of an integral
94
D. A. EDE & G. S. AGERBAK
number of structures from a homogeneous field of an extent which can vary
continuously from individual to individual, with a preferred spacing between
structures, the actual number formed being a compromise between the preferred
spacing and the requirement that the number be integral. Whether in this case
the field is strictly homogeneous, requiring explanation of the origin of subfields
by reference to a Turing-type system, or whether gradients and competitive
interactions alone would be sufficient mechanisms, must remain open questions
for the present.
We conclude that the fusion of cartilage elements in the talpid3 wing skeleton
is accounted for by the increased adhesiveness and reduced motility of the
mesenchyme cells, and that this also accounts, in conjunction with the distorted
shape of the limb-bud, for the characteristic phalangeal polydactyly.
4. Cell motility in relation to limb-bud shape
It may be asked whether the characteristic distortion of the talpid3 limb bud
is produced by an intrinsically abnormal rate of cell division. There are about
70 % more cells in the mutant than in normal wing buds at 5 days, but there is
no difference in numbers of dissociated and reaggregated cells after 2 days in
culture (Table 1); although more evidence is required on this point, we suspect
that the cell multiplication rate is potentially equal, but that because most
proliferation occurs just behind the apical ectodermal ridge (see below), and
because this ridge is up to 65 % more extensive in the mutant, more cells are in
fact dividing in it than in the normal. The increased cell number is a result of the
abnormal geometry of the limb-bud rather than vice versa.
In its outward growth the limb-bud becomes flattened and the main features
of its growth can be seen in lateral view, as in Text-fig. 6, where tracings from
stage 22 to stage 29 have been superimposed. On the right of the diagram the
pictures have been idealized by smoothing the outlines, eliminating the elbow
joint in the normal and making the pre-axial and post-axial portions symmetrical
in order to emphasize the essential difference between the two. It becomes clear
that this lies in the fact that whereas the normal limb bud grows outwards
without much increase in breadth for some time, forming a stem, and only later
begins to fan out to form a distal paddle, in talpid3 there is no stem because the
limb bud begins to increase in breadth immediately.
The mechanisms of limb growth have not been fully worked out even at the
descriptive level. Saunders (1948) established that growth and differentiation
of new prospective regions in the mesoderm occurs at the distal end of the bud,
while the more proximal regions established previously continue to develop.
Mitosis is most intense just behind the apical ectodermal ridge, but it is not
certain whether there is a distinct zone of proliferation here as Saunders believed,
and for which Searls (1965) provides some evidence, or a gradient of mitotic
activity with its high point distally as postulated by Camosso, Jacobelli &
Pappalettera (Amprino, 1965).
Cell adhesion and development
95
In order to account for elongation of the stem region of the normal limb bud
by cell division alone it would be necessary for the mitotic spindles to be orientated along the proximo-distal axis, and this has not been observed. The simplest
alternative is to suppose that there is a tendency to cell movement in a distal
direction; if this were necessary for normal development, inhibition of movement in talpid3 cells would account for the absence of a stem region and the
consequent distortion of the mutant limb-bud. In order to test this hypothesis,
programmes are currently being designed for the generation of models simulating relevant aspects of limb-bud growth on a digital computer (Ede & Law,
Normal
Talpid3
Text-fig. 6. A. Superimposed outlines of wing-bud stages 22-29, normal and talpid3.
B. Idealized diagrams of the same.
unpublished). Pending completion of this work, when the detailed design of the
programmes will be published, their basic principles have been used to produce
simpler models on graph paper, examples of which are shown in Text-figure 7.
The diagrams show a series of models representing increasing additions of
simple rules controlling cell reproduction and the position of the cells when they
are produced. In spite of the simplicity of the rules it is clear that distinct
patterns of growth are generated, that some of these patterns approximate to
patterns found in developing limb-buds in normal embryos, and that slight
changes in the rules produce dramatic changes in shape which are comparable
to those found in the mutant embryos. The models suggest that in addition to
cell multiplication with a higher rate of proliferation distally, cell movement
towards the distal end is also a necessary factor in normal growth of the limb
D. A. EDE & G. S. AGERBAK
96
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5a
Text-fig. 7. For legend see opposite page.
Cell adhesion and development
97
bud. Where this movement is restricted there is a distortion of its shape such as is
found in talpid?. Models 4 and 5 incorporate a change in motility, suggested by
the experimental evidence that in both normal and talpid3 cells from 5-day
embryos are more adhesive than cells from 4-day embryos. Their resemblance
to the outlines of 5-day or 6-day normal and talpid3 wing-buds respectively is
very striking, suggesting that changes in motility of the mesenchyme cells with
time may play a part in determining the basic pattern of the limb-bud shape.
No such movement of limb-bud mesenchyme cells has been observed, but as
demonstrated in the models it need be only of a very small order, amounting to
slight displacements of the cells on each other in an orientated way, and it is
difficult to see how such small movements could be demonstrated directly.
However, Thornton (1960) showed that in regenerating limbs of Amblystoma
the epidermal cap exercised an orientating influence on the aggregation and
outgrowth of mesodermal cells to form the new limb, and pointed out, as has
Faber (1965), that the apical cap in amphibia is essentially similar to the apical
ectodermal ridge in higher vertebrates. It may be that one of the functions of
the apical ectodermal ridge is to initiate and direct the outward cell movement
postulated here. More generally, this recalls the demonstration by Clarkson &
Wolpert (1967) that bud elongation in hydra is caused not so much by cell
division, as previously thought, but rather by orientated movements of cells
immigrating from surrounding tissues, directed by the tip of the bud.
5. Cell contact and cell death in talpid3
If the above is accepted, the talpid? gene affects three processes, sequential in
time but overlapping and to some extent superimposed:
(1) The drift of mesodermal cells in the direction of the apical ectodermal
ridge to produce the basic shape of the limb bud, (2) the aggregation of mesenLegend for text-fig. 7
Text-fig. 7. Models of limb-bud growth, with outlines of 6-day normal (4a) and
talpid3 (5 a) wing-buds.
Growth starts from a single row of 'cells', representing prospective limb region of
the flank, and proceeds by generation of a series of pictures, each produced by scanning the previous picture and transforming it by causing some 'cells' to reproduce
and positioning new 'cells' according to simple rules. The diagrams represent the
35th picture in each case.
Model 1.1 in 5 of' cells' in each row is reproduced, and each new cell placed in the
nearest available space.
Model 2. A ' gradient' of reproduction is obtained by limiting reproduction to cells
in the 10 outer rows, giving a first approximation to talpid3 wing-bud shape.
Model 3. The' gradient' is retained, but new cells are moved distalwards by a small
amount (three places) before being placed in the nearest available space, giving a
first approximation to normal wing-bud shape.
Model 4. As model 3, but after an arbitrary number of pictures the amount of
distal movement is reduced, giving a closer approximation to the normal shape.
Model 5. As model 4, but with more restricted distal movement, giving a closer
approximation to the talpid3 shape.
7
I EEM 2O
98
D. A. EDE & G. S. AGERBAK
chymal cells to form the precartilage condensations of the limb skeleton, and
(3) the alignment of chondroblasts within the developing cartilages. Each of
these, it has been argued, may result from a genetic effect on cell adhesion and
motility.
In addition, Hinchliffe & Ede (1967) showed that certain regions of normal
cell death, the anterior and posterior necrotic zones, were absent in talpid3, and
suggested that this might play a part in producing the abnormal shape of the
limb bud: these zones might act as 'end-stops' to the apical ectodermal ridge,
which in their absence would extend beyond its normal limits and, by stimulating mesodermal proliferation beneath it, cause much more expansive growth
at the distal margin. There is no evidence at present to determine whether the
findings reported in this paper render this hypothesis superfluous by showing
that factors acting within the mesoderm alone account for its fan-shaped
development, or whether both mechanisms are acting and reinforcing each
other, but on either view the absence of these zones of cell death remains. The
inhibition of cell death in highly circumscribed regions where it normally occurs
seems a much more mysterious and arbitrary genetic effect than a general
increase of cell adhesion within the limb bud, and it cannot be coincidental that
both occur in the same mutant. It seems most likely that the first is a particular
result of the second, and that programmed embryonic cell death (Saunders, 1966),
like other types of differentiation, is not a property of individual cells but depends
upon forms of contact between them in cell groups.
SUMMARY
1. Differences between aggregation patterns in reaggregating wing-bud
mesenchyme cells from normal and talpid3 embryos are interpreted as indicating
that the mutant cells are abnormally adhesive.
2. The role of cell adhesion and motility is discussed in relation to the formation of the normal pattern of skeletal condensations in the embryonic wing, and
to its distortion in talpid mutants.
3. A model of normal wing-bud growth is proposed in which slight movements of the mesodermal cells towards the apical ectodermal ridge play an
important part, accounting for the characteristic shape of the talpid3 wing-bud
by inhibition of these movements owing to the abnormal mutual adhesiveness
of the cells.
RESUME
Adhesion et mouvement cellulaire en relation avec le developpement du
membre chez les embryons de Poulet normaux et mutants ' talpid 3 '
1. Des differences observees dans le mode d'agregation des cellules du mesenchyme du bourgeon alaire d'embryons normaux et 'talpid3' indiqueraient une
adhesivite anormale des cellules mutantes.
Cell adhesion and development
99
2. La discussion porte sur le role de l'adhesion et de la motilite cellulaire en
rapport avec la formation d'un type normal de condensations squelettiques dans
l'aile embryonnaire et l'alteration de ce type chez les mutants ' talpid\
3. On propose un modele de croissance pour le bourgeon d'aile normal ou de
faibles mouvements de cellules mesodermiques vers la crete apicale ectodermique
joueraient un role important; il est valable pour le bourgeon ltalpid3i avec sa
forme caracteristique, ou ces mouvements seraient inhibes a cause de l'adhesivite
mutuelle anormale des cellules.
We wish to thank Mr Bernard Dugdale for constructing the gyratory shaker, Mr. R. Morley
Jones for statistical advice, Mr Hamish Law for discussions on mathematical aspects of
model construction, and Miss Irene Thomson and Miss Morag Sylvester for technical
assistance.
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(Manuscript received 6 December 1967)