/ . Embryol. exp. Morph. Vol. 59, pp. 341-353, 1980
Printed in Great Britain © Company of Biologists Limited 1980
341
Scanning electron microscopy of wound healing in
Xenopus and chicken embryos
By MARTIN STANISSTREET, 1 JENNIFER WAKELY 2 AND
MARJORIE A.ENGLAND 2
From the Departments of Zoology, University of Liverpool,
and Anatomy, Medical School, University of Leicester
SUMMARY
Wound closure in the ectoderm of Xenopus early neurulae and chick primitive-streak
embryos has been studied by scanning electron microscopy (SEM). Initial gaping of the
wound and a cobble-stone appearance of cells peripheral to the wound in both Xenopus and
chick confirm that the ectoderm is under lateral tension at these stages. Healing is rapid: in
Xenopus embryos wound closure has started within 5 min of wounding; in chick healing is
almost complete within 30 min in some cases. The SEM observations suggest that in Xenopus
embryos changes in cell shape are the major mechanism for wound closure. In chick embryos
wound healing is also accompanied by changes in the shape of the marginal cells, but evidence
is presented that in this system cell proliferation is important. The mechanisms of wound
healing in Xenopus and chick embryonic ectoderm are compared with those of wound healing
in other tissues.
INTRODUCTION
One of the reasons why amphibian and chick embryos have been used so
extensively in experimental embryology is that they show a remarkable ability
to heal following manipulation. However, although the wound-healing ability
of early embryos is well known, there has been little systematic study of the
phenomenon in multicellular embryos, although studies of wound healing in
amphibian uncleaved eggs have been made (Luckenbill, 1971; Bluemink, 1972).
Observations on wound healing in amphibian early embryos have been made in
passing during unrelated experiments. For example Jacobson & Gordon (1976),
in an extensive analysis of the mechanism of neurulation in amphibian embryos,
used wounding to show that the ectoderm of neurulae is under tension, and
so cannot 'push' the neural folds together. Similarly, Nakatsuji (1979) noted
that healing of the wound following microinjection of amphibian early gastrulae
was inhibited by cytochalasin B.
In contrast, the cellular activities involved in wound healing in the chick have
1
Author's address: Department of Zoology, University of Liverpool, P.O. Box 147,
Liverpool L69 3BX, U.K.
2
Authors' address: Department of Anatomy, Medical School, University of Leicester,
University Road, Leicester LEI 7RH, U.K.
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M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
received a few detailed examinations. England & Cowper (1977) and Mareel &
Vakaet (1977), in a scanning electron microscopy (SEM) study of the closure of
endodermal wounds in early chick embryos, found that endoderm cells on
opposite sides of the wound made contact by processes extending across the
wound, which healed as a result of active endodermal cell movement. However,
healing in chick ectoderm has not been studied.
The SEM has proved a valuable technique to describe morphogenesis at
cellular level, showing differences between cells of different germ layers (Smith,
Osborn & Stanisstreet, 1976; Stanisstreet & Smith, 1978), changes in cell shape
during morphogenesis (Tarin, 1971; Monroy, Baccetti & Denis-Donini, 1976;
England & Wakely, 1977; Bancroft & Bellairs, 1975), and changes in embryonic
cells when cultured in vitro (England & Wakely, 1978; Stanisstreet & Smith,
1978). We have therefore used the SEM to study and compare ectodermal wound
healing in embryos of the South African clawed toad, Xenopus laevis, and the
chick, Gallus domesticus.
MATERIALS AND METHODS
Xenopus embryos
Embryos were obtained by injecting pairs of adult Xenopus laevis with
chorionic gonadotrophin ('Chorulon', Intervet Ltd.) and were staged according
to Nieuwkoop & Faber (1956). The jelly coats were removed chemically by a
modification of the method of Dawid (1965). Embryos were placed in 2 %
cysteine hydrochloride in 10 % Steinberg saline (Steinberg, 1957) brought to
pH 7-8 with 2 M NaOH. The embryos were subsequently washed, cultured and
wounded in 10 % Steinberg saline, pH 7-3. When the embryos had reached the
late gastrula stage (stage 13) the vitelline membranes were removed using
watchmakers' forceps. The embryos were then handled over 1 % purified agar
in 10 % Steiner saline, pH 7-3. The embryos were wounded at the early neurula
stage (stage 14-15) using an electrolytically sharpened tungsten needle. A
longitudinal incision approximately 0-45 mm (about 25 cells) long was made in
the lateral ectoderm and the embryos were fixed for electron microscopy at 0,
5, 15, 30 and 45 min after wounding. Some intact embryos were fixed to serve
as controls.
Embryos were fixed overnight in 2-5 % glutaraldehyde plus 2 % paraformaldehyde with 2-5 mM calcium chloride in 0-1 M cacodylate buffer, pH 7-2 (modified from Karnovsky, 1965). They were washed in changes of cacodylate buffer
containing 2-5 mM calcium chloride, and then dehydrated in a graded acetone
series. The absolute acetone was replaced with liquid CO2, and the embryos
were dried using the critical-point method. The embryos were fixed to stubs with
'Durofix' (Rawlplug Ltd.), coated with gold-palladium, and observed and
photographed using a Cambridge 'Stereoscan' scanning electron microscope.
SEM of wound healing in Xenopus and chick
343
Chick embryos
Fertile White Leghorn hens' (Callus domesticus) eggs were incubated at 37 °C
until the embryos had reached the primitive-streak stage (stages 3-5, Hamburger
& Hamilton, 1951). The embryos were then removed from the eggs and mounted
as for New Culture (New, 1955). Ectodermal wounds were made in two ways.
In 10 embryos the wound was made from the ectoderm side. The anterior edge
of the blastoderm was detached from the vitelline membrane and folded back
on itself to expose the ectoderm as far posteriorly as Hensen's node. A single
straight cut was made with a tungsten needle lateral to the node and on the same
level as the node. The embryo was then unfolded and laid back on the vitelline
membrane. In the alternative method applied to 12 embryos the embryo was
left attached to the vitelline membrane, and the wound was made from the
endoderm side so as to penetrate through the germ layers on a level with
Hensen's node.
Control embryos were fixed without wounding. Wounded embryos were either
fixed immediately or reincubated at 37 °C for 30 min, 1 h and 2 h before fixation.
Specimens were fixed in Karnovsky's fixative (Karnovsky, 1965) overnight.
They were washed in 0-2 M sodium cacodylate buffer (Plumel, 1948), post-fixed
in cacodylate-buffered osmium tetroxide and dehydrated in ascending concentrations of ethanol/water. From 100 % ethanol they were transferred to 100 %
acetone. They were critical-point dried in a Polaron apparatus after replacing
the acetone with liquid CO2. Dried embryos were mounted ectoderm side
uppermost on aluminium stubs using colloidal silver adhesive and coated with
40 nm of gold in an Edwards High Vacuum S-150 coating unit. They were
examined in an International Scientific Instruments I.S.I. 60 scanning electron
microscope.
RESULTS
Xenopus embryos
Preliminary microscopic observations on Xenopus embryos showed that the
wound healed rapidly: immediately after incision the wound gaped, after 5 min
the wound had contracted and after 45 min it appeared almost healed. One
problem was that the wound became filled with loose cells and cell debris, which
obscured the process of healing. The early neurula was chosen for these experiments because the ectoderm is not yet ciliated at this stage. Observations on
control embryos confirmed our previous results (Smith, Osborn & Stanisstreet,
1976). The margins of the cells were slightly sunken and at higher magnification
it could be seen that the ectoderm cells had discrete borders with some marginal
pits.
Immediately after the incision, the wound gaped and the cells around the
wound, especially around the sides, showed a pronounced cobble-stoned
appearance, suggesting that the ectoderm was originally under a lateral tension
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M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
SEM of wound healing in Xenopus and chick
345
which was released by the wound. At the edges of the wound there were many
yolk platelets released from broken cells, and at the ends of the wounds the
peripheral cells were smoother, suggesting that they were being stretched
(Fig. 1).
After 5 min the wound had already started to close and was no longer gaping,
and the pronounced cobble-stoned effect was no longer observed (Fig. 2),
suggesting that the tension in the ectoderm had been re-established. The cells
around the edge of the wound, and especially at the ends, were elongated and
tapered towards the wound (Fig. 3). Connexions, such as filopodia and lamellopodia, linking the marginal ectoderm with the exposed underlying cells, and
which might have been responsible for the closure of the wound, were not
observed.
At 15 min the wound appeared similar except that the edges were more
closely apposed. The peripheral ectoderm cells were smooth, and the marginal
ectoderm cells at the end of the wound were tapered. In the centre of the wound,
the marginal ectoderm appeared to be curling under (Fig. 4), and occasional
connexions with the underlying tissue could be seen.
After 30 min the wound was considerably smaller than previously, and in
some cases had become almost circular (Fig. 5), and the cells around the margin
of the wound were elongated radially. The ectoderm still appeared to curl under
at the edge of the wound. At 45 min the majority of wounds were almost completely healed. The centre of the wound appeared smooth, and cell margins
could not readily be observed (Fig. 6), although artifactual cracks in some
specimens may have indicated lines of weakness at the junctions of recently
joined cells. At both 30 and 45 min after wounding the ectoderm peripheral to
the wound appeared similar to that in the controls.
FIGURES
1-6
Fig. 1. End of wound in ectoderm of Xenopus neurula immediately after incision
(0 min) showing stretching of cells, x 310.
Fig. 2. Ectoderm of Xenopus neurula 5 min after wounding. Wound is starting to
close and peripheral ectoderm is smoother, x 140.
Fig. 3. End of wound in ectoderm of Xenopus neurula 5 min after incision, showing
cells tapering towards the wound, x 620.
Fig. 4. Margin of wound in ectoderm of Xenopus neurula 15 min after incision,
showing edges of ectoderm curling under, x 1040.
Fig. 5. Ectoderm of Xenopus neurula 30 min after wounding. Wound has become
smaller, and cells around the wound are elongated radially, x 230.
Fig. 6. Ectoderm of Xenopus neurula 45 min after wounding. Wound is almost
closed. x320.
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M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
Fig. 7. Freshly made incision in chick ectoderm. The wound (*) is surrounded by
damaged cells and debris (d), and gapes widely, x 500.
Fig. 8. Healing wound in chick ectoderm 30 min after surgery. The incision is filled
with debris (d). The marginal cells are convex, elongated, and aligned towards the
wound margins. The edges of the wound are almost straight and parallel, x 1000.
Fig. 9. End of a healing wound in chick ectoderm 30 min after surgery. The incision
line is obscured by debris (d) remaining from the wounding procedure. Ectoderm
cells (ec) converge on the end of the wound and are fiat and wedge shaped, x 1900.
Fig. 10. Healed wound in chick ectoderm 2 h after surgery. The incision is closed
and is marked by a double epithelial fold (arrowed). The wound edges are straight,
x 1000.
SEM of wound healing in Xenopus and chick
347
Chick embryos
No differences were seen in the process of wound healing whether the wounds
included ectoderm only or ectoderm and endoderm. In the chick freshly made
wounds gaped widely forming a circular hole (Fig. 7). Regardless of whether the
wound was made from the ectodermal or endodermal aspect of the embryo the
edges of the wound rolled inwards towards the endoderm. The chosen wound
site was anterior to the furthest extent forwards of the mesoderm so that mesoderm cells were not seen in the wound. The wound margins were rimmed with
cell debris. This remained attached to the wound throughout the healing process
until the wound was completely healed when the debris was extruded on to the
ectoderm surface. The ectodermal cells immediately behind this line of debris
became convex, giving a cobble-stoned appearance similar to that seen in the
wounded ectoderm of Xenopus but less pronounced (Fig. 8).
The time taken for wound closure in the chick was much more variable
between individual embryos than in the amphibians. Thirty minutes after surgery
some wounds showed the initial signs of healing, while others were almost
closed.
As wounds healed the initial round opening narrowed to an oval and then to a
slit with occasional points of contact between the two sides before finally
closing, without overall shortening. As the wound edges straightened and
approximated to each other the cells immediately behind the line of debris
became flattened and elongated towards the edges. Cells converging on the end
points of the wound became wedge shaped (Fig. 9).
Changes were seen on the ectoderm surrounding the wound for up to 20 cells
distance from the wound site and three different types of processes were seen.
Some cells showed long cytoplasmic threads which crossed the ectodermal
surface connecting the cells together, sometimes across one or two intervening
cells. Many cells displayed similar processes, but with a cytoplasmic bead at
some point along their length (Fig. 9). Threads of this type were also seen on
unwounded ectoderm elsewhere in the embryo, in smaller numbers, but numbers
near the wound appeared to increase during the early stages of approximation
of the wound margins, and to decline as healing was completed. The third type
of process, seen during the early stages of wound closure, was a broad lamellipodium extending from an ectodermal cell across the upper surface of a
neighbouring cell for a short distance. These were uncommon, and their
direction was not related to the position of the cell relative to the wound.
Two hours after surgery all wounds were healed. In the majority of embryos
the position of the wound was recognisable by the double straight line of dead
cells representing the original edges of the wound. Where it was possible to
examine the wound margins through gaps in the debris they were seen to meet
along a straight line with no overlap of cells. Particularly in embryos operated
from the ectoderm side the line of closure was flanked by a straight ridge of
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M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
ectoderm on either side (Fig. 10). In the most completely healed specimens this
double ridge flattened out leaving an almost continuous epithelium.
Beyond this stage the wound site could no longer be identified.
DISCUSSION
The results confirm that wound healing in Xenopus and chick embryos is
rapid, and show that there are similarities between the responses to ectodermal
wounding in Xenopus and chick embryos. In both there is an initial gaping of
the wound with bulging of the peripheral cells giving a cobble-stoned appearance,
followed by a narrowing of the wound to a straight slit, which then progressively
closes.
The initial widening of the wound can be attributed to the ectoderm being
under lateral tension in both amphibian (Jacobson & Gordon, 1976) and chick
embryos. The same phenomenon is seen in freshly wounded avian endoderm
(England & Cowper, 1977) and indeed the whole blastoderm is in a stretched
condition due to its expansion over the vitelline membrane as development
proceeds (New, 1959; Bellairs, Bromham & Wylie, 1967). Additional tension in
the opposite direction exists in the area pellucida ectoderm because of the
morphogenetic movement of cells from this layer towards the primitive streak
(Rosenquist, 1966; Nicolet, 1971). When the ectoderm is cut these tensions are
released locally at the wound site but continue to act on the surrounding cells,
hence pulling the wound edges apart and causing the bulging of marginal cells
to give a cobble-stoned appearance.
The healing of Xenopus and chick embryonic ectoderm shows some similarities. In both, wound closure occurs in two phases. First, the cells at the ends of
the wound become wedge shaped as the wound narrows with little obvious
shortening. Thus it is possible to imagine an initial closure of the wound
resulting from an active change in the shapes of the marginal cells, of the type
that contribute towards gastrulation in amphibians and effect morphogenesis
of the neural tube (Schroeder, 1970; Handel & Roth, 1971, amongst others).
Similar changes in cell shape are also seen in wound healing in chick endoderm
(England & Cowper, 1977). Subsequent closure proceeds in part in 'zipper-like'
fashion from the ends of the wound so that in Xenopus the wound at 30 min is
considerably shorter than original.
In one respect, however, the repair process in Xenopus and chick ectoderm
differs. In the chick embryo mitosis increases around the wound during the
first half hour after surgery. Beaded threads, identified by Bellairs & Bancroft
(1975) as mitotic mid-bodies connecting pairs of cells formed from a single
mitosis, are a normal feature of chick ectoderm (Bancroft & Bellairs, 1975).
Their number increases dramatically near wounds undergoing the initial stages
of closure, and falls to a normal level after apposition of the wound edges,
within about 2 h of wounding. Thus it appears that beaded threads have a
SEM of wound healing in Xenopus and chick
349
limited life span. Their presence on an epithelium denotes cell division very
shortly before the time of fixation. Thus there seems to be a burst of mitosis
closely related to the healing process. Some rearrangement of ectoderm cells can
also be seen in the chick, and the occasional lamellipodia observed on cells a
short distance from healing wounds may assist the cells in making such small
changes of position. In addition the cells immediately behind the wound margins
flatten and elongate thus increasing their surface area to help in approximating
the wound edges. In both chick and Xenopus ectoderm, mitosis and local internal
cellular rearrangement are normal features of epithelial growth (Bancroft &
Bellairs, 1975; Keller, 1975). The need for a greater than normal rate of increase
in epithelial area due to wound repair seems simply to temporarily exaggerate
this normal behaviour in the chick.
In Xenopus, no such marker for mitosis is seen under the scanning electron
microscope, and so it is not possible to observe as directly whether wound
healing is accompanied by a local increase in mitosis. However, Xenopus
embryos which have been treated with colchicine to block mitosis and disrupt
microtubules still show the ability to heal wounds (Stanisstreet & Panayi,
1980). Thus repair of embryonic ectoderm in the chick appears to involve
three mechanisms; cell proliferation and adjustments of cell shape and position.
In Xenopus embryonic ectoderm, eel) shape changes are the important factor
and increased mitosis does not appear to play an essential role in wound
healing.
The present results may also be compared with observations on wound
healing in other amphibian and avian embryonic and adult systems. Wound
closure in tadpole epidermis is accomplished by active cell migration, the
migrating cells forming lamellipodia which spread over the underlying cells
(Radice, 1977, 1978). The present results suggest that the mechanism of wound
healing in earlier embryos is rather different.
Although some similarities between healing in chick ectoderm and endoderm
are seen (above), there is a major difference between the repair process in chick
ectoderm described here, and that seen in chick endoderm and a variety of adult
systems such as skin and cornea, which are ectodermal derivatives (Croft &
Tarin, 1970; Krawzcyk, 1971; Pfister, 1975; Trinkaus, 1976; Anderson, 1978;
Pang, Daniels & Buck, 1978; Gabbiani, Chapponnier & Huttner, 1978). In
chick endoderm and in these mammalian systems the predominant cellular
activity is spreading of epithelial cells from the wound edges into the wound,
frequently using the connective tissue or basement membrane exposed by
epithelial wounding as a substrate. Cell migration may also be accompanied by
proliferation of epithelial cells surrounding the wound (Selden & Schwartz,
1979). Active ingrowth of marginal cells is also seen in the repair of the cornea
of late chicken embryos (Takeuchi, 1976) and in the epidermis of amphibian
tadpoles (Derby, 1978) and adult newts (Repesh & Oberpriller, 1978). Thus the
fundamental difference between chick and Xenopus ectodermal repair and the
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M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
adult and embryonic systems above is that in the present study no evidence was
seen of active spreading of cells from the wound margins, across the wound
opening. Epithelia thus appear to be divisible into two groups: firstly the chick
embryo endoderm and the majority of adult stratified epithelia which repair by
a process which includes active cell spreading at the wound margins, and secondly
chicken and Xenopus embryonic ectoderm which does not show migratory
activity. This difference may be related to the organization and the structure of
the epithelial cells. Wounded chick endoderm at stages 3-5 rests on a substrate
formed by the ectodermal basement membrane, as do adult stratified epithelia.
Cells spreading from the wound margins thus may move on a fairly normal
substrate of extracellular materials. Neither chick nor Xenopus ectoderm have a
coherent endodermal basement membrane as a substrate for locomotion (Low,
1967). The absence of a scaffolding of extracellular material in these situations
may be unfavourable to cell migration so that proliferation and changes in cell
shape and position are more in evidence than cell movements. Additionally
ectoderm and endoderm in the chick differ in the way in which neighbouring
cells are attached to each other. Chick endoderm cells show only small junctions
whereas ectoderm cells have more highly developed junctional complexes
(Trelstad, Hay & Revel, 1967). Thus endoderm cells may be more free to spread
and change their shape than the more tightly connected ectoderm. Similarly in
stratified epithelia, the basal cells are the ones seen to migrate into wounded
areas. They may have an inherent ability to readily loosen their junctions with
neighbouring cells to enable them to move into the upper epithelial strata
during normal epithelial renewal, which is diverted towards outward migration
during repair.
The situation with which the repair of chick ectoderm appears to have most in
common is the healing of small wounds in the epithelium of the mouse lens
(Rafferty & Smith, 1976). In this tissue wound healing occurs with no morphological evidence of cells moving out from the wound margins into the wound
area and proliferation is the main method of repair. Cells immediately around
the wound are stimulated to divide and pass through one cell cycle after wounding. The resulting increase in cell numbers, together with some expansion and
minor 're-shuffling' of the cells to restore the normal orderly epithelial arrangement closes the wound. It is possible that in the expanding blastoderm of the
chick, as in the lens epithelium from which peripheral cells are continually lost
by differentiation into lens fibres, the radial forces pulling the wound apart are
too great to be overcome by changes in individual cells. Therefore some compensatory expansion of the entire epithelium towards the wound margins is
necessary to ensure that the edges appose.
Characteristically, closed wounds showed some buckling of the epithelial
surface on either side of the line of closure (Trinkaus, 1976). Similar buckling
was seen in healed wounds of chick, but not Xenopus, ectoderm. This phenomenon occurs fairly widely in wound repair (Trinkaus, 1976), as though the
SEM of wound healing in Xenopus and chick
351
marginal cells from each side of the wound meet, make contact and stop moving
but that movement does not immediately cease in the non-marginal cells, thus
causing cells to pile up along the wound, suggesting that movement does not
immediately cease when cells make contact. Possibly there is a delay between the
restoration of morphological continuity in the ectoderm and the establishment
of intercellular communication (Sheridan, 1968) between its cells after the
inevitable disruption of cell junctions as the cells rearrange. A similar phenomenon is seen in normal development during the fusion of digits in mammals
(Maconnachie, 1979), closure of the neural tube (Waterman, 1976), and the
development of the eyelids and pinna (Maconnachie, 1979). The extrusion of
dead cells and cellular debris on to the epithelial surface is also seen in normal
developmental processes involving the fusion of epithelial edges, such as the
formation of sensory placodes (Meier, 1978; Wakely, 1976) and the closure of
the neural tube.
M. S. wishes to thank Mr C. Veltkamp and Mr B. Lewis for their expert help with the
electron microscopy and photography, and Mrs J. Clumpus for technical assistance. M. A.E.
and J.W. are grateful for the excellent technical assistance of Miss A. Cole and Mrs S.
Bulman. Mr G. L. C. McTurk provided expert assistance with the scanning electron microscopy. Miss Dorothy Smith typed the manuscript.
REFERENCES
ANDERSEN, L. (1978). Quantitative analysis of epithelial changes during wound healing in
palatal mucosa of guinea pigs. Cell Tiss. Res. 193, 231-246.
BANCROFT, M., & BELLAIRS, R. (1975). The onset of differentiation in the epiblast of the chick
blastoderm (SEM and TEM). Cell Tiss. Res. 155, 399-418.
BELLAIRS, R., BROMHAM, D. R. & WYLIE, C. C. (1967). The influence of the area opaca on the
development of the young chick embryo. J. Embryol. exp. Morph. 17, 195-212.
BELLAIRS, R. & BANCROFT, M. (1975). Midbodies and beaded threads. Am. J. Anat. 143,
393-398.
BLUEMINK, J. G. (1972). Control wound healing in the amphibian egg: an electron microscopical study. /. Ultrastruct. Res. 41, 95-114.
CROFT, C. B. & TARIN, D. (1970). Ultrastructural studies of wound healing in mouse skin.
I. Epithelial behaviour. / . Anat. 106, 66-77.
DAWID, I. B. (1965). Deoxyribonucleic acid in amphibian eggs. /. molec. Biol. 12, 581-599.
DERBY, A. (1978). Wound healing in tadpole tailfin pieces in vitro, J. exp. Zool. 205, 277-284.
ENGLAND, M. A. & COWPER, S. V. (1977). Wound healing in the early chick embryo studied
by scanning electron microscopy. Anat. Embryol. 152, 1-14.
ENGLAND, M. A. & WAKELY, J. (1977). Scanning electron microscopy of the development of
the mesoderm layer in chick embryos. Anat. Embryol. 150, 291-300.
ENGLAND, M. A. & WAKELY, J. (1978). Evidence for changes in cell shape from a 2-dimensional
to a 3-dimensional substrate. Experentia 35, 664-665.
GABBIANI, G., CHAPONNIER, C. HUTTNER, I. (1978). Cytoplasmic filaments and gap junctions
in epithelial cells and myofibroblasts during wound healing. /. Cell Biol. 76, 561-568.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. / . Morph. 8, 49-92.
HANDEL, M. A. & ROTH, L. E. (1971). Cell shape and morphology of the neural tube:
implications for microtubule function. Devi Biol. 25, 78-95.
23-2
352
M. STANISSTREET, J. WAKELY AND M. A. ENGLAND
A. G. & GORDON, R. (1976). Changes in the shape of the developing vertebrate
nervous system analysed experimentally, mathematically and by computer simulation.
/. exp. Zool. 197, 191-246.
KARNOVSKY, M. J. (1965). A formaldehyde/glutaraldehyde fixative of high osmolality for use
in electron microscopy. /. Cell Biol. 27, 137-138A.
KELLER, R. E. (1975). Vital dye mapping of the gastrula and neurula of Xenopus laevis. I.
Prospective areas and morphogenetic movements of the superficial layer. Devi Biol. 42,
222-241.
KRAWCZYK, W. S. (1971). A pattern of epidermal cell migration during wound healing. /.
Cell Biol. 49, 247-263.
Low, F. N. (1967). Developing boundary (basement) membranes in the chick embryo. Anat.
Rec. 159, 231-238.
LUCKENBILL, L. M. (1971). Dense material associated with wound closure in the axolotl egg
(A. mexicanum). Expl Cell Res. 66, 263-267.
MACONNACHIE, E. (1979). A study of digit formation in the mouse embryo. /. Embryol. exp.
Morph. 49, 259-276.
MAREEL, M. & VAKAET, L. C. (1977). Wound healing in the primitive deep layer of the young
chick blastoderm. Virchows Arch. B. Cell Path. 26, 147-157.
MEIER, S. (1978). Development of the embryonic chick otic placode. II. Electron microscopic
analysis. Anat. Rec. 191, 459-478.
MONROY, A., BACCETTI, B. & DENIS-DONINI, S. (1976). Morphological changes in the surface
of the egg of Xenopus laevis in the course of development. III. Scanning electron microscopy
of gastrulation. Devi Biol. 49, 250-259.
NAKATSUJI, N. (1979). Effects of inserted inhibitors of microfilament and microtubule function on the gastrulation movement in Xenopus laevis. Devi Biol. 68, 140-150.
NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro.
J. Embryol. exp. Morph. 3, 326-337.
NEW, D. A. T. (1959). The adhesive properties and expansion of the chick blastoderm.
/. Embryol. exp. Morph. 7, 146-164.
NICOLET, G. (1971). Avian gastrulation. Advances in Morphogenesis 9, 231-262.
NIEUWKOOP, P. D. & FABER, J. (1956). A Normal Table o/Xenopus laevis Daud., Amsterdam:
North Holland Publishing Co. Ltd.
PANG, S. C , DANIELS, W. H. & BUCK, R. C. (1978). Epidermal migration during the healing
of suction blisters in rat skin: a scanning and transmission electron microscopic study.
Am. J. Anat. 153, 177-192.
PFISTER, R. W. (1975). The healing of corneal epithelial abrasions in the rabbit: a scanning
electron microscope study. Invest. Ophthal. 14, 648-661.
PLUMEL, M. (1948). Tampon au cacodylate de sodium. Bull. Soc. Chim. biol., Paris 30,
129-130.
RADICE, G. P. (1977). Active and passive cellular movements during wound closure in vivo.
J. Cell Biol. 15, AUK.
RADICE, G. P. (1978). Analysis of the migration of epithelial cells in vivo and in vitro. J. Cell
Biol. 79, 270 A.
RAFFERTY, N. S. & SMITH, R. (1976). Analysis of cell populations of normal and injured
mouse lens epithelium. Anat. Rec. 186, 105-114.
REPESH, L. A. & OBERPRILLER, J. C. (1978). Scanning electron microscopy of epidermal cell
migration during limb regeneration in the adult newt. Notophthalmus viridescens. Am. J.
Anat. 151, 539-556.
ROSENQUIST, G. C. (1966). A radioautographic study of labelled grafts in the chick blastoderm. Development from primitive streak stages to stage 12. Contr. Embryol. Carnegie
Instn. 38, 71-110.
SCHROEDER, T. E. (1970). Neurulation in Xenopus laevis. An analysis and model based upon
light and electron microscopy. /. Embryol. exp. Morph. 23, 427-62.
SELDEN, S. C. & SCHWARTZ, S. M. (1979). Cytochalasin-B inhibition of endothelial proliferation at wound edges in vitro. J. Cell Biol. 81, 348-354.
JACOBSON,
SEM of wound healing in Xenopus and chick
353
J. D. (1968). Electrophysiological evidence for low resistance intercellular junctions in the early chick embryo. /. Cell Biol. 37, 650-690.
SMITH, J. L., OSBORN, J. C. & STANISSTREET, M. (1976). Scanning electron microscopy of
lithium-induced exogastrulae of Xenopus laevis. J. Embryol. exp. Morph. 36, 513-522.
STANNISSTREET, M. & PAHAYr, M. (1980). Effects of colchicine cytochalasin-B and papaverine
on wound healing in Xenopus early embryos. Experientia (in press).
STANISSTREET, M. & SMITH, J. L. (1978). Scanning electron microscopy of cells isolated from
amphibian early embryos. /. Embryol. exp. Morph. 48, 215-223.
STEINBERG, M. (1957). Carnegie Inst. Wash. Yearbook 56, 347 (report by J. D. Ebert).
TAKEUCHI, S. (1976). Wound healing in the cornea of the chick embryo. III. The influence of
pore size of millipore filters on the migration of isolated epithelial sheets in culture. Devi
Biol. 51, 49-62.
TARIN, D. (1971). Scanning electron microscopical studies of the embryonic surface during
gastrulation in Xenopus laevis. J. Anat. 109, 535-548.
TRELSTAD, R. L., HAY, E. D. & REVEL, J. P. (1967). Cell contact during early morphogenesis
in the chick embryo. Devi Biol. 16, 78-106.
TRINKAUS, J. P. (1976). On the mechanism of metazoancell movements. In The Cell Surface
in Animal Embryogenesis and Development (ed. G. Poste & G. L. Nicolson), pp. 225-329.
Amsterdam: North-Holland Biomedical Press.
WAKELY, J. (1976). Scanning electron microscopy of lens placode invagination in the chick
embryo. Expl Eye Res. 22, 647-651.
WATERMAN, R. E. (1976). Topographical changes along the neural fold associated with
neurulation in the hamster and mouse. Am. J. Anat. 146, 151-172.
SHERIDAN,
{Received 4 December 1979, revised 21 April 1980)