/. Embryo exp. Morph. Vol. 48, pp. 225-237, 1978
Printed in Great Britain © Company of Biologists Limited 1978
225
Cell contacts and sorting out in vivo:
the behaviour of some embryonic tissues implanted
into the developing chick wing
By C. TICKLE, 1 M. GOODMAN 1 AND L. WOLPERT 1
From the Department of Biology as Applied to Medicine,
The Middlesex Hospital Medical School, London
SUMMARY
The interaction of cells from embryonic liver, neural retina and mesonephros with cells
from limb-bud mesenchyme has been investigated in vivo by grafting these tissues into the
developing chick wing-bud. The implanted cells were in all cases from quail tissue which
can be recognized histologically. As embryonic liver and neural tube are tissues that sort
externally to limb-bud mesenchyme in mixed aggregates, it would be expected, from a differential adhesiveness hypothesis, that heterotypic adhesions along the borders of graft and
host would be favoured over cell-cell adhesions in the graft. No morphological signs of this
were evident: rather the grafted cells maximized like-like contacts. The cells of the grafts,
including those from control mesenchyme, did not invade into the wing. The results were
the same irrespective of whether the graft was a fragment of tissue or a pellet of reaggregated cells. This supports the idea that cells within tissues are not actively moving around
and also provides controls for assaying the invasiveness of other cell types, such as malignant cells into the wing.
INTRODUCTION
If suspensions of two types of cell are allowed to form mixed aggregates, the
cells often segregate according to type (Moscona, 1962; Steinberg, 1964, 1970).
Not only do like cells tend to stick together but the cells also take up a defined
position relative to the inside and outside of the aggregate. This segregation and
positioning of cells in aggregates is known as sorting out.
Steinberg (1964) has proposed, that differential adhesiveness results in sorting
out as soon as there is a choice of adhesions and that weaker adhesions are
exchanged for stronger until an equilibrium is reached: often this is when
one cell type surrounds the other. The internally segregating cell type is the
more adhesive, the externally segregating the less adhesive. The strength of
heterotypic adhesions would be intermediate between those of the homotypic
adhesions. This prediction is peculiar to the differential adhesiveness hypothesis
and accounts for the sorting out of cells into a concentric layer surrounding
1
Authors' address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, Cleveland Street, London, W1P 6DB, U.K.
226
C. TICKLE, M. GOODMAN AND L. WOLPERT
an inner core of cells of the other type. In addition it also provides an explanation
for the behaviour of fused fragments of different cell types where one fragment
spreads to enclose the other. The positioning of cell types in this case mimics
that obtained by sorting out in aggregates of mixtures of the same cell types.
Embryonic chick limb-bud cells have been widely used in sorting out experiments. Cells from the precartilage regions have ended up the internal core of
aggregates formed with a variety of cell types (Steinberg, 1970). The positioning
of the limb-bud cells suggests that they are the most adhesive cell type in
these combinations in aggregates. However, a rather different arrangement
has been found in two-dimensional cultures (Steinberg & Garrod, 1975) with
limb-bud cells taking up an external position. It would thus be of interest to
test the behaviour of different tissues in vivo as sorting out has often been
supposed to involve mechanisms similar to those of morphogenesis. It is easy
to graft tissues into the developing chick wing and we have been able to test
the behaviour of cells from different embryonic tissues with limb-bud cells
in vivo.
The mesenchyme at early stages of limb development is a loose meshwork
with only small areas of the cells being in contact (Gould, Day & Wolpert,
1972). We might therefore expect that cells from tissues which segregate
externally in aggregates with chick limb mesenchyme cells, would interdigitate
along the extensive free surfaces of the limb-bud cells at the borders of graft
and host. This would occur because grafted cells should be more adhesive to
the host cells than to the other cells in the graft. This point about the heterotypic adhesions have also been made by Armstrong (1970, 1971). We might
even expect, in our case, cells from the graft to migrate into the limb. This
might occur without breaking the strong adhesions between the limb-bud cells
because there are many spaces between them.
We chose to look at the behaviour of embryonic neural tube, liver and
mesonephros. Neural tube and liver have been shown to segregate externally
to limb-bud precartilage cells in mixed aggregates (Steinberg, 1970). The
behaviour of mesonephros with limb precartilage has not, to our knowledge,
been tested. However on the basis of observations by Bresch (1955), Steinberg
(1970) suggested that mesonephros was more adhesive than liver, which was
the only tissue that was looked at by both of them. The interaction of mesonephric cells with chick limb cells therefore cannot be predicted. The reason
for using mesonephros was that it has been claimed that fibroblasts move
around within this tissue (Armstrong & Armstrong, 1973). In other tissues
such as heart and liver no evidence for cell movement in intact tissues has been
obtained (Weston & Abercrombie, 1967). We therefore wanted to test whether
cells from these embryonic tissues would migrate into the limb. These studies
have provided us with a base-line for our investigations on the invasiveness
of a variety of malignant cells in the chick limb-bud (Tickle, Crawley &
Goodman, 1978 a, b).
Cell contacts and sorting out in vivo in chick wing
227
METHODS
Chicken embryos, stage 20-21 (Hamilton-Hamburger stages), were used as
hosts for grafts. The embryonic tissues tested in the wing were from quail
embryos. Quail tissue was used because the cells can be distinguished from
chick cells by the staining properties of the nuclei both in light and electron
microscope sections (Le Douarin, 1973).
The general procedure was as follows (see also Tickle et al. 1978 a). The
wings of chick embryos were prepared to accept a graft by cutting a square
piece of tissue (mesenchyme and overlying ectoderm) out of the dorsal surface
of the right wing with sharpened tungsten needles. The piece of tissue removed
was between a third and half the depth of the limb at this stage.
The following tissues were implanted in the holes cut into the wings; quail
limb mesenchyme (control), mesonephros, neural tube and liver. These implants
were either fragments cut from the tissue or part of a pellet of reaggregated
cells.
Quail limb mesenchyme was prepared from wings (stage 22) by soaking
them in 2 % trypsin (Difco 1:250) in calcium- and magnesium-free Hanks'
solution for 1 h at 0 °C (Szabo, 1955). After this treatment the ectoderm can
be peeled from the mesenchyme and pieces of mesenchyme can be cut out and
grafted or alternatively the mesenchyme can be disaggregated into a cell suspension by mechanically flushing the tissue through a Pasteur pipette. A pellet
of cells was prepared by centrifuging at 2000 g for 10 min. The pellet of cells
was then consolidated at 38 °C for 1 h, then removed from the centrifuge tube
and cut into pieces and grafted into the chick wing.
Mesonephros was dissected from quail embryos (stage 24) and treated with
2 % trypsin for 30 min at 0 °C. After this time the Wolffian duct could be
freed from the mesonephros. The mesonephros was then treated in the same
way as the wing mesenchyme; fragments of mesonephros or pieces of reaggregated mesonephros were grafted into the chick wings. The liver of quail
embryos (stage 24) was cut into fragments for grafting or was disaggregated
into single cells after treatment with 0-001 M EDTA for 10 min (Elton & Tickle,
1971). Pieces of the pellet of reaggregated cells were then grafted. The neural
tube of quail embryos (stage 22) was cleaned of adjacent tissue in the same
way as the mesonephros and grafted as fragments. The grafts were held in
position with pins made from platinum wire (25 /an in diameter). These were
removed 4 h or more after grafting, if electron microscopy was to be carried
out, otherwise they were left in place.
After the grafts had been made the chick hosts were allowed to develop for
a further day or 2 days. At these times, the wings bearing grafts were cut off
and fixed in half-strength Karnovsky fixative (Karnovsky, 1965) at 4 °C overnight. The tissue was then rinsed in 0-1 M cacodylate buffer. Wings from
duplicate experiments were processed in the following way. One set of wings
228
C. TICKLE, M. GOODMAN AND L. WOLPERT
was stained with 0-05 % Alcian green in acid alcohol (70 % alcohol with
1 % concentrated HC1) and then dehydrated in alcohols, cleared and
embedded in araldite: 2/mi sections were cut and arranged in alternate
groups of four on parallel slides. One set of sections was stained with 0-1 %
toluidine blue in borax and the other set was treated with a modified Feulgen
technique to show the distribution of quail cells. The other set of wings were
stained with 1 % osmium tetroxide for 1 h at 4 °C, then dehydrated, cleared
and embedded in araldite for electron microscopy. Thin sections showing
graft/host border were collected on grids, stained with lead citrate (Reynolds,
1963) and examined in a Philips EM 300 microscope.
RESULTS
General
The grafts ended up in the proximal part of the wing. After 1 day the chick
mesenchyme surrounding the graft was relatively undifferentiated, but after
a further day cartilage and muscle had formed adjacent to the grafts. The cells
in all the grafts looked healthy and mitoses were often observed.
Wing mesenchyme (control)
When a fragment of quail limb mesenchyme was grafted, so that we replaced
a piece of chick wing tissue with an homologous piece of quail wing tissue, the
quail cells remained together (Figs. 1-3). No quail cells had migrated from the
graft into the chick mesenchyme. At the border between graft and host there
was not a clear demarcation between individual cells of the two types (see
Fig. 3). However in these 2/«n thick araldite sections every cell that lacked
a Feulgen-positive nucleolus may not be a chick cell. Even if some of the
FIGURES
1-5
Fig. 1. Border between quail wing mesenchyme (G) and chick wing mesenchyme
(H). The boundary runs from approximately the bottom left-hand corner up to
the top right-hand corner. 2/*m section stained with toluidine blue. Scale bar is
25/*m.
Fig. 2. Low power view of region from which Fig. 1 was taken. Same orientation
as Fig. 1. Scale bar is 50 ju,m.
Fig. 3. Adjacent section to that shown in Figs. 1 and 2, stained by Feulgen technique to show quail cells. Note the rather indistinct border between graft and
host but that no quail cells appear to have invaded into the host chick tissue. Scale
bar is 25 /*m.
Fig. 4. Border between a graft (G) of reaggregated quail mesenchyme and host
chick mesenchyme (H). Section stained by Feulgen technique to show quail cells.
Note the graft is compact and no quail cells have moved into host mesenchyme.
Scale bar is 50 jam.
Fig. 5. Ultrastructure of the border between quail mesenchyme graft and host
chick mesenchyme. Note quail cell (arrowed) with characteristic nucleolus and
other cells which can be identified as quail (Q) and chick (H). Scale bar is
Cell contacts and sorting out in vivo in chick wing
229
230
C. TICKLE, M. GOODMAN AND L. WOLPERT
6
Sr«a8:.>T's;:v>^:-
IJ^^^MrMt'^rt
m£'"*
/§*
Cell contacts and sorting out in vivo in chick wing
231
quail cells along the border were mistaken for chick, there did not seem to be
an absolute dividing line between host and graft cells; rather cells from graft
and cells from host seemed to mesh together.
Essentially the same result was obtained when quail wing cells were disaggregated and pieces of the reaggregate implanted into the chick wing.
Figure 4 shows the border of such a graft with the host wing. No quail cells
were found to have moved from the graft into the adjacent mesenchyme. The
exact border between the graft and host was not easily identified even at the
ultrastructural level (Fig. 5).
Mesonephros
Fragments of mesonephros remained discrete (Figs. 6, 7). Loose connective
tissue between the tubules was also part of the grafts. This could be identified
as quail in sections stained with Feulgen technique (Fig. 8). These loose connective tissue cells remained with the tubules in the graft and did not migrate
into the adjacent limb mesenchyme. The tubules formed a smooth border with
the mesenchyme and a basal lamina was present. Two types of tubule were
found; in one type the cells had microvilli on the apical surfaces, in the other
there were no microvilli.
Grafts of pellets of reaggregated mesonephros were almost indistinguishable
from those of fragments of tissue, and consist of tubules and loose connective
tissue. Again, the implanted cells remained together within the graft and no
quail cells were found away in the mesenchyme. The cells in the graft became
reorganized into tubules (Fig. 9) with a basal lamina and looked very similar
to tubules in the intact tissue fragments (Fig. 10). In one case, where we
FIGURES
6-11
Fig. 6. General view of a wing-bud with graft of mesonephros, after 24 h. Note
section of pin (arrowed) which was used to hold graft in place. Mesenchyme of
the limb is still undifferentiated. Apical ectodermal ridge at *. Scale bar is 100 /tm.
Fig. 7. High power of graft from Fig. 6, showing tubules and loose connective
tissue. Scale bar is 20/tm.
Fig. 8. Adjacent section to that in Fig. 7, treated by Feulgen technique shows
that tubules and connective tissue between them are quail tissue. No quail cells
seen in chick mesenchyme (H). Scale bar is 20 /am.
Fig. 9. Tubule, formed in graft of reaggregated cells from mesonephros, abutting
mesenchyme cells (H). Note microvilli on apical surfaces of cells in the tubule (at *)
and also basal lamina around the bases of the cells (arrowed). Scale bar is 5 fim.
Fig. 10. Border between cells in a mesonephric tubule (M) and mesenchyme cells
(H) showing basal lamina (arrowed). Scale bar is 2/tm.
Fig. 11. Border of graft of a mixture of quail mesonephros and wing mesenchyme
cells, made into a chick wing. Section stained by Feulgen technique to show
distribution of quail cells, with none in adjacent mesenchyme. Note two tubules
(arrowed) in graft. Scale bar is 25 /tin.
232
C. TICKLE, M. GOODMAN AND L. WOLPERT
obtained a thin section through the lumen of a reorganized tubule, we found
that the cells making up the tubule were not all of one type; one cell had
microvilli on the apical surface, while the other 11 cells round the lumen had
bare surfaces.
It appears that the cells from the mesonephric tubules readily form tubules
again rather than remain in contact with the chick mesenchyme cells. To give
more opportunity for interaction between limb mesenchyme cells and cells
from the mesonephros we made mixed pellets of mesonephros and quail wing
mesenchyme cells and grafted pieces into the chick wing. Even in these cases
mesonephric tubules were formed and no quail cells were found to have
migrated into the wing mesenchyme (Fig. 11).
Liver
Liver fragments formed a smooth border with the chick wing mesenchyme
(Figs. 12-14), and no quail cells were found away from the graft. In the cases
where grafts were left in place 2 days, the chick wing mesenchyme cells had
started to differentiate. Often the graft came to lie adjacent to developing
cartilage; in these cases no perichondrium was formed (Fig. 12).
When the liver was disaggregated and the cells were grafted as a reaggregated
pellet, the result was almost indistinguishable from a graft of an intact liver
fragment (Figs. 15, 16). There was no basal lamina round the liver cells either
in the intact fragments (Fig. 14) or in reaggregated pellets (Fig. 16) and the
liver cells came into close contact with the mesenchyme cells (Fig. 16). In the
reaggregated pellets the cells formed well-organized epithelia around lumina
(Fig. 15), and were polarized as in intact liver; apical junctions were present,
the nuclei tended to take up a basal position within the cell and the endoplasmic
reticulum was aligned parallel to the lateral borders of the cells.
FIGURES
12-16
Fig. 12. Quail liver graft in chick wing after 2 days. Along lower edge graft abuts
developing cartilage (C) and no perichondrium is present. Note graft is well
vascularized. Sections of pin at P. Scale bar is 25/tm.
Fig. 13. Fragments of quail liver (L) abutting mesenchyme (M): graft left 2 days.
Scale bar is 10/*m.
Fig. 14. High power of Fig. 13 showing cell with distinctive quail nucleolar marker
in liver. Note there is no basal lamina between liver and mesenchyme cells. Scale
bar is 2 [im.
Fig. 15. Graft of reaggregated quail liver cells in chick wing after 2 days. Liver
cells are reorganized with well defined lumina at *. Chick mesenchyme adjacent
to graft is differentiating into cartilage. Scale bar is 10 /*m.
Fig. 16. High power of Fig. 15, showing region where liver cells (L) abut developing
cartilage. There is no basal lamina and liver cells come close to developing chondrocytes. Note developing extracellular cartilage matrix. Scale bar is 2/tm.
Cell contacts and sorting out in vivo in chick wing
233
234
'17
C. TICKLE, M. GOODMAN AND L. WOLPERT
18
Fig. 17. Neural tube implanted into chick wing after 1 day. Scale bar is 100/tm.
Fig. 18. High power of adjacent section to that shown in Fig. 17, stained by Feulgen
technique. Shows quail cells in neural tube and none in adjacent mesenchyme.
Lumen of neural tube (arrowed). Scale bar is 20 /im.
Neural tube
When fragments of neural tube were implanted into the wing there was a
clear demarcation between graft and host and no quail cells were found away
from the graft after 1 or 2 days (Figs. 17, 18). Pellets of disaggregated neural
tube were not tested.
DISCUSSION
Both embryonic liver and neural tube, which have been shown to segregate
externally to limb-bud cells, formed discrete grafts in the chick wing. There
was no sign that heterotypic adhesions were favoured over homotypic ones.
The adhesions between like cells, for example in liver, were much more extensive
than between graft and host cells and also showed specialized contacts. Ironically,
these compact grafts excluding limb-bud cells, seem to resemble more closely
the arrangement of these cell types in two-dimensional cultures (Steinberg &
Garrod, 1975) than that in aggregates.
It may not seem surprising that cells from intact tissue fragments did not
form strong adhesions with the limb mesenchyme or migrate away from the
graft. However, the same result was obtained when pieces of a pellet of reaggregated liver cells were used. In fact the appearance of the graft was more
or less identical to that of an intact liver fragment. So even after contacts
between cells had been disrupted, associations of cells within the graft were
favoured over those of host and graft. There may be, however, rapid reorganization of cells within the pellet. Specialized contacts have been observed
between neural retina cells shortly after aggregation although reorganization
of cells and elaboration of junctions was not seen until 2 h later (Sheffield &
Moscona, 1969; Sheffield, 1970). Similarly junctions have been found between
cultured fibroblasts after aggregation for a few minutes (Lloyd, Rees, Smith &
Cell contacts and sorting out in vivo in chick wing
235
Judge, 1976). So although some contacts between the cells in the grafts may have
formed, it seems unlikely that reorganization of cells would have taken place
within the graft prior to grafting.
Mesonephros fragments or pellets behaved in the same way as liver and
neural tube: the grafted cells formed discrete regions and did not penetrate
into adjacent chick tissue. However, in grafts of mesonephros, unlike liver,
there was a basal lamina preventing contact between cells in tubules and host
mesenchyme. We were particularly interested, however, to find that the
mesenchymal component of the graft did not penetrate into the adjacent wing
mesenchyme. This is contrary to the claim that cells of loose connective
tissue of mesonephros are able to migrate long distances in fused fragments.
However, it has been suggested that this movement may be reduced if mesonephric tubules are present (Armstrong & Armstrong, 1973) although a
mechanism which would restrict cell movement only when tubules are present
is obscure. Armstrong & Armstrong (1973) suggest that a similar association
between heterogeneous types of cells may explain why Weston & Abercrombie
(1967) found little evidence for cell movements in heart or liver. We have
found that in neither case did cells from liver or heart (Tickle et al. 1978 a)
move into the chick wing. However, if we consider other confrontations
between mesenchyme and mesenchyme, our results, here, and those of Searls
(1967) and Stark & Searls (1973) show that the mesenchyme cells of the limb
move around little in vivo. Overall, it would seem therefore that most findings
support the conclusions of Weston & Abercrombie (1967) that there is little
gross displacement of cells relative to each other in intact tissues.
We should add here an aside about our use of quail tissue as grafts. There
seems to be ample evidence that quail tissue can be used in combination
with chick tissue, in experiments of this type: the fact that the tissues come
from different avian species does not affect the results. This has been shown
by Armstrong & Armstrong (1973) who used both quail tissue and tritiated
thymidine as markers and is implicit in experiments where quail cells are
used to trace the origin of cells during development (for example, Chevalier,
Kieny, Mauger & Sengel, 1977) and their migration (for example, Teillet &
Le Douarin, 1970). In addition we used quail mesenchyme grafts whereas
Searls (1967) and Stark & Searls (1973) used tritiated thymidine as a cell
marker with the same results.
The cells from the grafts described here did not move into the chick
mesenchyme. This should be contrasted with the behaviour of isolated cells
seeded onto the outside of cell aggregates. In this case individual cells have
later been found in the interior of the aggregates (Wiseman & Steinberg, 1973;
Gershman & Drumm, 1975). This migration of cells into aggregates has been
found even when the isolated cells would be expected, from their positioning
in mixed aggregates, to be less adhesive than the cells in the aggregates (Wiseman, 1977 a). Infiltration by these cells may require that strong adhesions
236
C. TICKLE, M. GOODMAN AND L. WOLPERT
between like cells in the aggregate would have to be broken. However, the
significance of this penetration of individual cells has been thrown into question
by the finding that inert glass beads and other similar objects also apparently
'invade' aggregates to the same extent (Wiseman, 1911 b). It seems therefore
that the localization within an aggregate may not involve active cell locomotion.
In any case the behaviour of isolated cells may be of an aberrant nature.
Contacts between cells in tissues must play a major role in determining the
behaviour of individual cells. It is for this reason that it is perhaps not surprising
that cells did not emigrate out of these tissues which we implanted into the
wing. Since cells from normal embryonic tissues did not move into the mesenchyme, we have been able to use this site to assay the invasive behaviour of a
variety of cell types (Tickle et al. 1978 a, b). We have found that a number of
different types of cell are able to invade into the limb, as indeed are nerves
during normal development.
No broadenings of heterotypic adhesions at graft/host border were found.
In fact there was very little contact between grafted and host tissues. We
cannot explain this discrepancy between our observations and the prediction
of the differential adhesiveness hypothesis. The few data available however
support our findings. Armstrong (1970, 1971) could find no obvious differences
in contacts between heterotypic and homotypic cells in aggregates of heart and
pigmented retina and of pigmented and neural retina. It appears, in our experiments, that like associations are strongest which makes it even more difficult
to interpret these results in terms of the differential adhesiveness hypothesis.
It is possible that extracellular matrix might play a role. We know, however,
of no discussion of the role of the extracellular matrix in relation to the
differential adhesiveness hypothesis. If one argues that cells adhere more strongly
to the matrix, then one cannot explain the continued contacts between mesenchyme cells. These strong like associations could be taken to indicate some
specificity of adhesion (Roth, 1968). However such specificity cannot account
for the spreading of one fragment over a fragment of a different type.
This work was supported by the Cancer Research Campaign.
REFERENCES
P. B. (1970). A fine structural study of adhesive cell junctions in heterotypic
cell aggregates. /. Cell Biol. 47, 197-210.
ARMSTRONG, P. B. (1971). Light and electron microscope studies of cell sorting in combinations of chick embryo neural retina and retinal pigment epithelium. Wilhelm Roux
Arch. EntwMech. Org. 168, 125-141.
ARMSTRONG, P. B. & ARMSTRONG, M. T. (1973). Are cells in solid tissues immobile? Mesonephric mesenchyme studied in vitro. Devi Biol. 35, 187-209.
BRESCH, D. (1955). Recherches preliminaires sur des associations d'organes embryonnaires
de Poulet en culture in vitro. Bull. Biol. Fr. Belg. 89, 179-188.
CHEVALIER, A., KIENY, M., MAUGER, A. & SENGEL, P. (1977). Developmental fate of the
somitic mesoderm in the chick embryo. In Vertebrate Limb and Somite Morphogenesis
(ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 421-433. Cambridge: Cambridge University Press.
ARMSTRONG,
Cell contacts and sorting out in vivo in chick wing
237
R. A. & TICKLE, C. A. (1971). The analysis of spatial distributions in mixed cell
populations: a statistical method for detecting sorting out. /. Embryol. exp. Morph. 26,
.135-156.
GERSHMAN, H. & DRUMM, J. (1975). Mobility of normal and virus-transformed cells in
cellular aggregates. /. Celt Biol. 67, 419-435.
GOULD, R. P., DAY, A. & WOLPERT, L. (1972). Mesenchymal condensation and cell contact
in early morphogenesis of the chick limb. Expl Cell Res. 72, 325-336.
KARNOVSKY, M. J. (1965). A formaldehyde glutaraldehyde fixative of high osmolarity for
use in electron microscopy. /. Cell Biol. 27, 137a (Abstr).
LE DOUARIN, N. (1973). A biological cell labelling technique and its use in experimental
embryology. Devi Biol. 30, 217-222.
LLOYD, C. W., REES, D. A., SMITH, C. G. & JUDGE, J. F. (1976). Mechanisms of cell
adhesion: early-forming junctions between aggregating fibroblasts. /. Cell Sci. 22, 671684.
MOSCONA, A. A. (1962). Analysis of cell recombinations in experimental synthesis of tissues
in vitro. J. cell comp. Physiol. 60 (Suppl. 1), 65-80.
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in
electron microscopy. / . Cell Biol. 17, 208-212.
ROTH, S. A. (1968). Studies on intercellular adhesive selectivity. Devi Biol. 18, 602-631.
SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick
limb bud. /. exp. Zool. 166, 39-50.
SHEFFIELD, J. B. (1970). Studies on aggregation of embryonic cells: initial cell adhesions
and the formation of intercellular junctions. /. Morph. 132, 245-265.
SHEFFIELD, J. B. & MOSCONA, A. A. (1969). Early stages in the reaggregation of embryonic
chick neural-retina cells. Expl Cell Res. 57, 462-466.
STARK, R. J. & SEARLS, R. L. (1973). A description of chick wing bud development and
a model of limb morphogenesis. Devi Biol. 33, 138-153.
STEINBERG, M. S. (1964). The problem of adhesive selectivity in cellular interactions. In
Cellular Membranes in Development (ed. M. Locke), pp. 321-366. New York: Academic
Press.
STEINBERG, M. S. (1970). Does differential adhesion govern self-assembly processes in
histogenesis? Equilibrium configurations and the emergence of a hierarchy among
populations of embryonic cells. /. exp. Zool. 173, 395-434.
STEINBERG, M. S. & GARROD, D. R. (1975). Observations on the sorting-out of embryonic
cells in monolayer culture. /. Cell Sci. 18, 385-403.
SZABO, G. (1955). A modification of the technique of 'skin splitting' with trypsin. 7. Path.
Bact. 70, 545.
TEILLET, M-A. & LE DOUARIN, N. (1970). La migration des cellules pigmentaires etudiee
par la methode des greffes heterospecifiques de tube nerveux chez l'embryou d'oiseau.
C.r. hebd. Seanc. Acad. Sci., Paris 270, 3095-3098.
TICKLE, C, CRAWLEY, A. & GOODMAN, M. (1978O). Cell movement and the mechanism of
invasiveness: a survey of the behaviour of some normal and malignant cells implanted
into the developing chick wing bud. /. Cell Sci. 31, 293-322.
TICKLE, C, CRAWLEY, A. & GOODMAN, M. (19786). Mechanisms of invasiveness of epithelial
tumours: ultrastructure of the interactions of carcinoma cells with embryonic mesenchyme
and epithelium. /. Cell Sci. 33, 133-156.
WESTON, J. A. & ABERCROMBIE, M. (1967). Cell mobility in fused homo- and heteronomic
tissue fragments. J. exp. Zool. 164, 317-324.
WISEMAN, L. L. (1977a). Can the differential adhesion hypothesis explain how an aggregate
of strongly cohesive cells can be penetrated by more weakly cohesive cells? Devi Biol.
58,204-211.
WISEMAN, L. L. (19776). Contact inhibition and the movement of metal, glass and plastic
beads within solid tissues. Experientia 33, 734-735.
WISEMAN, L. L. & STEINBERG, M. S. (1973). The movement of single cells within solid
tissue masses. Expl Cell Res. 79, 468-471.
ELTON,
{Received 9 June 1978)
l6
EMB 48