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/. Embryol. exp. Morph. Vol. 31, 3, pp. 761-785, 1974
Printed in Great Britain
761
A scanning electron microscope study
of the early limb-bud in normal and talpid3 mutant
chick embryos
By D. A. EDE, 1 R. BELLAIRS 2 AND M. BANCROFT 2
From the Department of Zoology, University of Glasgow and the
Department of Anatomy and Embryology, University College London
SUMMARY
Scanning electron microscope studies, supported by transmission electron microscope and
light microscope observations, have been made of the wing-bud apex in the region of the apical
ectodermal ridge (AER) in both normal and talpid3 mutant embryos.
In normal embryos: the ectoderm consists of two layers, the periderm and the basal layer,
resting upon a basal lamina. The external surface of the periderm cells bears numerous villi,
especially numerous at the cell boundaries. In the AER the cells of the basal layer are compacted into a fan-shaped form, remaining as a single layer, though displacement of their
nuclei gives the appearance of a stratified epithelium; the periderm cells tend to round up and
many are necrotic. Microfilaments and microtubules are present in greater numbers in the
AER than in other ectoderm cells and are orientated along the long axis in the very narrow
elongated cells which comprise the middle of the ridge. The mesenchymal cells of the mesoderm tend to be flattened, with extensive flattened surface areas separated from each other by
long edges of lamellar cytoplasm, from which many longfilopodialextensions arise. Often the
cells are elongated, with the filopodia arising predominantly at the anterior and posterior ends.
In talpid*embryos: when these were compared with normals no differences were detected in
the general ectoderm or in the AER, but in the mesoderm a statistical analysis revealed
significant differences in the occurrence of sections through very fine cytoplasmic processes in
transmission electron microscope micrographs. Taken in conjunction with visual examination
of scanning electron microscope pictures the differences suggested that talpid3 cells have
filopodia distributed more extensively around the cell, but that these extensions do not extend
so far through the intercellular spaces or produce such fine terminal arborizations as in
normal mesenchyme.
At the ectodermal/mesodermal boundary there is a web of extracellular material under
the basal lamina, separated from the underlying mesoderm by a very narrow gap in normal
embryos. Fine cytoplasmic extensions from the mesenchyme cells extend across the gap towards
the basal lamina and many are in contact with it. The gap is wider in talpid3 wing-buds and a
statistical analysis confirmed that far fewer filopodia approach closely to the basal lamina
or make contact with it.
The significance of these findings in relation to problems of AER formation and activity
and motility of mesenchymal cells in the wing-bud is discussed.
1
Author's address: Department of Zoology, University of Glasgow, Developmental
Biology Building, 124 Observatory Road, Glasgow G12 9LU, U.K.
2
Authors' Address: Department of Anatomy and Embryology, University College London,
Gower Street, London WC1E 6BT, U.K.
762
D. A. EDE AND OTHERS
INTRODUCTION
Classical studies on limb morphogenesis were mainly concerned with the
relations between tissues, in particular with the interaction between mesoderm
and ectoderm (reviewed by Zwilling, 1961), but more recently the emphasis has
shifted, towards concentration upon the activity of individual cells and to the
relations between them (e.g. Ede, 1971, 1972). Hitherto, our knowledge of the
structure and arrangements of these cells in the limb-bud has been based largely
upon the two-dimensional views furnished by light and transmission electron
microscopy (e.g. Jurand, 1965; Gould, Day & Wolpert, 1972) and no threedimensional study using the scanning electron microscope has been published.
The activity and appearance of living mesenchyme cells derived from primary
explants of limb-bud fragments in vitro have been investigated by Ede & Flint
(1974tf), who noted differences between the structure and behaviour of cells
from normal chick embryos and those from talpid3, a mutant in which the pattern of development of the limb-bud is strikingly abnormal (Ede & Kelly, 1964;
Hinchliffe & Ede, 1967; Hinchliffe & Thorogood, 1974). In order to decide
whether these differences are reflected in the cells of whole embryos and whether
they are related to the differences in morphogenesis between normal and mutant
limb-buds, it is necessary to compare the normal and talpid3 cells both in vivo
and in vitro.
In this paper we report a scanning electron microscopic study of the wing-buds
of normal and talpid3 embryos examined after fixation /'// vivo. This is supplemented by studies using both transmission electron microscopy and light
microscopy, and also by a statistical analysis of the electron micrographs.
A report on the structure of normal and talpid3 cells cultured in vitro is in
preparation.
MATERIALS AND METHODS
Ten normal ( + / + or ta3l + ) control and ten talpid3 (ta3/ta3) embryos were
used in this investigation, obtained either from the flock maintained in Glasgow
by D. A.E. or from the related flock kept by Dr J. R. Hinchliffe at Aberystwyth.
The control embryos were the phenotypically normal siblings of the talpid3
mutants. Eggs were incubated for 5 days, by which time they had reached stages
24-26 (Hamburger & Hamilton, 1951) when the mutant embryos could be
easily distinguished by the expanded fan shape of the wing-buds (Ede & Kelly,
1964).
Wing-buds of both normal and talpid3 embryos were fixed for scanning electron
microscopy (SEM), for transmission electron microscopy (TEM) and for thinsection light microscopy.
Electron microscopy of chick limb-bud
763
Preparation for SEM
Two methods were used:
(i) (Seven normal and seven talpid3 embryos.) Wing-buds were fixed for 1 h
in Karnovsky's fixative (Karnovsky, 1965), washed for 15min in cacodylate
buffer and treated with 1 % osmium tetroxide. They were rinsed in 70 % ethanol
and stored in 70 % ethanol. Each limb was cut into two parts vertically along the
proximo-distal axis with a razor blade or a new scalpel blade and dehydrated
through an ethanol series followed by an ethanol-freon series. Embryos were
dried in a critical point 'bomb' from liquid CO2 before being mounted on
aluminium stubs with UHU glue. Specimens were coated with carbon and gold
and then examined in a Cambridge 'Stereoscan' MKI operated at 10 kV.
(ii) (Three normal and three talpid3 embryos.) Wing-buds were fixed in 3 %
glutaraldehyde in 0-15 M cacodylate buffer at 37 °C for 2 h, then allowed to cool
at room temperature and continue fixing overnight, giving a total fixation time
of about 24 h. They were washed for 30 min in distilled water and then dehydrated and treated as in the first series.
Preparation for TEM and light microscopy
After each egg was opened the embryo was removed quickly, the wing-buds
snipped rapidly from the body and plunged into the fixative at pH 7-2-7-4 at
room temperature. Specimens were usually fixed in Karnovsky's fixative with
either phosphate or cacodylate buffers but three normal and three talpid3 limbbuds were fixed instead by the technique used by Burnside (1971). Similar
results were produced by the two fixatives, except that the microtubules appeared
to be preserved more readily by the Burnside fixative. In either case, fixation was
for 1 h, followed by a number of washes in the buffer used previously as a component of the fixative. After immersion in 1 % osmium tetroxide at pH 7-2-7-4
specimens were given several brief washes in maleic acid buffer at pH 515 before
treatment for I h in 1 % uranyl acetate, made up in 6% maleic acid. The specimens were then dehydrated with graded ethanols and embedded in either
Araldite or Spurr epoxy resin. Sections were cut on a Porter-Blum Mark II
ultramicrotome and examined in a Siemens Elmiskop 1 electron microscope.
Sections about 1 /cm thick were also cut and, after staining with a 1 % solution
of toluidine blue, were examined by light microscopy.
Statistical investigation
This was carried out entirely on material fixed with Karnovsky's fixative.
Large montages at magnifications of about 6000-9000 x, each usually consisting of 10-15 consecutive photographs, were prepared of areas of ectoderm and
mesoderm adjacent or nearly adjacent to, but not including, the apical ectodermal ridge (AER). This region was selected as being readily located so that valid
comparisons could be made between areas from normal and talpid3 embryos.
764
D. A. EDE AND OTHERS
Fig. 1. Diagram to illustrate the sampling technique used in the analysis of the
electron micrographs. A polythene sheet, marked with a grid of intersecting lines,
is placed over the photograph. A count is made of the number of intersections falling
respectively on intercellular spaces, on nuclei, and on cytoplasm. The cytoplasm is
itself treated as being of three types: the mitochondria, processes less than 0-5 /<m
in diameter, and the remainder of the cytoplasm.
These montages were used in making a statistical analysis, applying the sampling technique described by Bellairs (1959). In this technique, large transparent
sheets of polythene were divided by fine intersecting ink lines into \ in squares.
The polythene sheets were pinned over the montages and the structure which lay at
each intersection was noted (see Fig. 1). The aim was to compare the incidence of
a number of cellular structures or of intercellular spaces in the normal and talpid3
wing-buds. Each intersection was scored for one of the following categories:
Electron microscopy of chick limb-bud
765
(i) nuclei (including nucleoli), (ii) mitochondria, (iii) cytoplasmic processes less
than 0-5 /im in diameter, (tv) the remaining cytoplasmic components, and
(v) intercellular spaces. Separate scores were made for ectoderm (basal layer
only) and mesenchyme. A further examination of the montages was made in
order to investigate the relationship between the ectoderm and the underlying
mesenchyme in normal and talpid3 wing-buds in this region. A count was made
of the number of fine mesenchymal processes touching the ectodermal basal
lamina or lying within 5-6 /tm of it. The length of the strip of ectoderm in each
montage was obtained by means of a map measurer graded in mm.
The statistical analysis is set out in Tables 1 and 2. In Table 1 the data for the
grid counts is set out as a table of averages with frequency measured per 100
intersections of the grid. The F value is calculated and the probability (P) of that
value having occurred by chance is obtained.
RESULTS
General appearance
The characteristic shapes of the normal and talpid* wing-buds are shown in
Figs. 2, 3, 12 and 13. Only half of each limb is present; in both the normal and
the talpid3 the AER is a conspicuous feature extending around the perimeter of
the distal part of the bud.
Ectoderm
Figures 4 and 5 show surface views of the AER and of the ectoderm adjacent
to it. In both normal and talpid* the cells vary in size and shape. Individually, they
bear many more villi than would be expected from a casual inspection of transmission electron micrographs, and the junctions between cells are particularly
well defined by the large number of villi which appear at the cell margins
(Figs. 6, 7). Jurand (1965), who carried out a careful examination of the normal
limb-bud from transmission electron micrographs, produced a graphic reconstruction of the outer layer of ectoderm cells which these observations show to
be strikingly accurate.
Light microscope studies (Figs. 14, 15), SEM pictures (Figs. 8, 9) and TEM
micrographs (Figs. 10, 11) show that the general ectoderm (i.e. adjacent to the
ridge in our studies) consists of two layers - the outer periderm, whose appearance in surface view is described above, and the deeper basal layer, which rests
upon a basal lamina. The cells of the basal layer are columnar and arranged in
a loose epithelium with large intercellular spaces separating them, except at the
basal lamina where they are in contact with their neighbours all round; there
are some other points of contact randomly distributed over the main body of
each cell.
The much thinner periderm of the ectoderm is composed of a single layer of
flattened cells, with correspondingly flattened nuclei, orientated at right angles
to the cells of the basal layer and in close contact with them. The nuclei make
766
D. A. EDE AND OTHERS
FIGURES
2-5
Figs. 2, 3. Low magnification SEM views of forelimb-buds of normal and talpid'1
embryos, respectively. Only half of each limb-bud is shown, the cut surface passing
proximo-distally at right angles to the apical ectodermal ridge, aer, Apical ectodermal ridge; d, distal region of limb-bud;/?, proximal region of limb-bud, x 140.
Figs. 4, 5. SEM views of the surface of the ectoderm and apical ectodermal ridge
of normal and talpid3 embryos respectively. The individual cells can be seen as raised
areas, x 800.
Electron microscopy of chick limb-bud
161
Figs. 6, 7. Higher magnification SEM views of the ectoderm near the apical ridge of
normal and talpicP embryos, respectively. Villi can be seen as white spots on the
surface of the cells and at the junctions between the cells, x 4000.
Figs. 8, 9. SEM views of ectoderm and underlying mesenchyme taken from regions
proximal to the apical ridge of normal and talpicP embryos, respectively, x 16000.
bulges which are clearly visible in the SEM pictures. Each cell makes close
contact with its neighbouring periderm cells around the whole of its periphery
and the junction between periderm cells usually shows a number of interlocking
protuberances and indentations.
The specialized contacts between ectoderm cells have already been described
by Jurand (1965) and consist of tight junctions, intermediate junctions and
desmosomes. Microiilaments and microtubules are present in both basal and
48
EMB 31
768
D. A. EDE AND OTHERS
10
F I G U R E S 10 AND 11
TEM views of ectoderm and underlying mesenchyme just proximal to apical
ectodermal ridge in normal and talpicP limb-buds, respectively. Note that the gap
between the mesenchyme cells and the ectoderm is less in the normal than in the
talpid*. b, cell of basal layer; p, periderm; s, intercellular space, x 8000.
Electron microscopy of chick limb-bud
769
11
For legend see opposite.
48-2
770
D. A. EDE AND OTHERS
Electron microscopy of chick limb-bud
111
periderm layers of the general ectoderm cells but they occur less frequently
here than in the cells of the AER.
No differences are apparent between normal and talpid3 ectoderm in either
TEM or SEM pictures and none emerge from the quantitative data presented
in Table 1.
Apical ectodermal ridge
SEM pictures show that the cells of the AER are more irregular and tend to
appear larger at the surface than those of the adjacent ectoderm (Figs. 4, 5).
There appear to be fewer villi.
Figures 14 and 15 are typical thick sections cut through the apical region of
wing-buds of a normal and talpid3 embryo respectively, stained with toluidine
blue and viewed with the light microscope. They do not appear to differ greatly
from each other; in each the AER is a thickened region measuring about
25-35 //m at the apex, and though in some talpid3 specimens the ridge is flatter
and the central groove less conspicuous it is likely that this difference is due
merely to variations in the angle of sectioning.
In TEM pictures the basal layer usually gives the appearance of being several
cells deep because the nuclei lie at various levels. It has been possible, however,
to trace some cells through from the basal to the distal limits and this is confirmed in the SEM pictures which show that individual cells extend from the
basal lamina to the periderm, just as they do in the adjacent ectoderm. The
nuclei become displaced at different levels, giving a false appearance of a
stratified epithelium.
The medial part of the AER is the thickest and here the individual cells of the
basal layer are extremely narrow and elongated. They are characterized by
having many micron"laments, each about 5 run in diameter, which are orientated
mainly in the longitudinal direction (Fig. 23). By contrast, there were no bundles
of microfilaments running transversely along the base of the cells, whose orientation might have supported a 'purse-string' theory of AER formation similar to
that which is now widely held to explain the rolling-up of the neural tube (see
discussion by Bellairs, 1974). Microtubules are also present in these cells of the
FIGURES
12—17
Figs. 12, 13. SEM views of the cut surface of normal and talpicP limb-buds, respectively. Compare with Figs, land 2 in which similar limb-buds are seen from a different
orientation, x 370.
Figs. 14,15. Photomicrographs taken by light microscopy of sections through the tips
of the limb-buds of a normal and a talpid3 embryo, respectively. Stained with
toluidine blue. The arrow indicates the gap between ectoderm and irtesoderm in the
talpid3. x225.
Figs. 16, 17. SEM views of apical ectodermal ridge and underlying mesenchyme in a
normal and a talpid* limb-bud, respectively. Note the fine processes forming a web
between the ectoderm and the mesenchyme. x 1 350.
772
D. A. EDE AND OTHERS
19
Electron microscopy of chick limb-bud
113
apical ectoderm (Fig. 22). Each measures about 20 run in diameter and, like the
microfilaments, the tubules run predominantly in a longitudinal direction. There
appear to be considerably more microfilaments and microtubules present in the
ridge cells than in the cells of the general ectoderm. The mitochondria, Golgi
bodies and endoplasmic reticulum are all similarly extended along the long axis
in these middle cells. A further characteristic is that in many specimens the basal
lamina is interrupted in this region and is continuous with extracellular material
lying between the cells (Figs. 18, 19).
The basal layer cells on either side of the middle region of the AER radiate
fanwise (Figs. 16-17). Individual cells are broader than the middle cells in
section and their organelles do not show the same longitudinal orientation as in
the middle cells. The microfilaments are less easy to see; it is possible that fewer
of them are present but their random orientation renders them less conspicuous
and they are further obscured by the presence of many more ribosomes in these
cells.
SEM pictures of the AER show that the periderm cells become much less
flattened than in the general ectoderm (Figs. 4, 5). Many of them, equally in
both normal and taipicP limb-buds, become necrotic, with very electron-dense
cytoplasm and large Jysosomes. These cells have been described by Jurand
(1965) in the normal limb-bud.
Mesoderm
The characteristic appearance of the mesoderm in light microscope sections in
normal and talpid3 wing-buds is shown in Figs. 14 and 15. There is a tendency
for the cells immediately beneath the epidermis to be elongated and orientated at
right angles to it in the normal, but not so evidently in talpid3.
TEM micrographs show the mesenchyme cells in both to be of characteristically irregular shape (Figs. 20, 21) in section. The nuclei are about 4-8 /im in
diameter and usually of a relatively regular ovoid shape; they are smaller than
those of the ectoderm and usually contain several patches of nucleolar material.
The cytoplasm is rich in ribosomes but, like the ectoderm, contains little endoplasmic reticulum. The mitochondria are about 0-5-1 /im in diameter and are
usually round or oval in section. The nuclei are overlain over much of their
surface by a very thin layer of cytoplasm with large cytoplasmic extensions at
FIGURES
18-21
Figs. 18, 19. TEM views of the base of the apical ectodermal ridge of normal and
talpicP embryos, respectively. Note the whisps of granular material, collagen fibrils
and small cytoplasmic processes of the mesoderm (m). Not all sections through
normal limb-buds show the mesenchyme cells so closely pressed to the ectoderm (e)
in this region, but in lalpicP limb-buds such a close apposition has never been seen,
x 15000.
Figs. 20, 21. TEM micrographs of the mesenchyme from normal and talpicP limbbuds, respectively. x3000.
774
D. A. EDE AND OTHERS
775
Electron microscopy of chick limb-bud
Table 1. Variance ratio tests on grid sampling data from montages
of ectoderm (basal layer) and underlying mesoderm
Nuclei
Cytoplasm ic
processes
Mitochondria < -j- /im diam.
Remaining
cytoplasm
Intercellular
spaces
43-48
42-64
0070
40
P > 0-250
2108
2400
0-615
40
P > 0-250
No
No
45-25
38-15
8-461
31
0010 >
P > 0005
Yes
19-41
3406
24-422
31
P < 0005
Ectoderm (basal layer)
N
ta*
F
D.F.
P
Significant
TV
ta3
F
D.F.
P
Significant
26-38
2800
0-741
40
P > 0-250
No
23-42
20-98
1-1717
31
0-250 >
P> 0100
No
5-51
4-43
2-023
40
0-250 >
P > 0100
No
3-55
0-93
2-875
40
0100 >
P > 0050
No
Mesoderm
419
7-73
3-82
2-99
0-3250
22-657
31
31
P > 0-250
P < 0005
No
Yes
Yes
Abbreviations: N = average frequency of counts per 100 grid intersections in normal
embryos; tas = average frequency of counts per 100 grid intersections in talpid3 embryos;
F = variance ratio; D.F. = degrees of freedom; P = probability of chance value.
two or three points in the section, with much finer tapering extensions from the
surface in these regions, chiefly at the tips. It is of course impossible to say from
the sections whether these are thread-like or leaf-like, but thread-like cytoplasmic
extensions (filopodia), cut in cross-section are obvious in the intercellular spaces.
In the TEM micrographs it is impossible to visualize or estimate how long these
filopodia are, but the SEM micrographs (Figs. 24, 25) show that these very fine
cytoplasmic extensions are of considerable length, spanning the gaps between the
F I G U R E S 22-27
Fig. 22. TEM micrograph to show microtubules in the middle cells of the apical
ectodermal ridge of a normal limb-bud. Similar microtubules are present in talpid3
cells, x 45000.
Fig. 23. TEM micrograph to show microfilaments in the middle cells of the apical
ectodermal ridge of a talpid3 limb-bud. Similar microfilaments are present in the cells
of normal limb-buds, x 30000.
Figs. 24, 25. SEM views of distal mesenchyme from normal and talpid3 limb-buds,
respectively, x 3400.
Figs. 26, 27. SEM views of part of proximal region of limb-bud of normal and
talpid3 embryos, respectively. The mesenchyme (m) is pressed more closely beneath
the ectoderm (e) in the normal than in the talpid3. x 3400.
776
D. A. EDE AND OTHERS
cells and travelling in some cases over the surface of cells (though this particular
appearance may be an artefact of the preparative treatment) with fine arborizations at their tips. When stereopairs of mesenchyme cells are examined with
a stereo-viewer, it can be seen that each cell is in contact with many others.
Furthermore, the contact is not confined to immediately adjacent cells, but may,
by means of these long thread-like processes, extend over several cells.
It is not always easy to decide from these pictures whether any individual
process extended from a cell is a true cytoplasmic filopodium or is composed of
extracellular strands of material; TEM micrographs enable us to see that the
processes are almost all cytoplasmic in the interior of the mesoderm but that
both types are present at its junction with the ectoderm. Both microfilaments and
microtubules are present in the cell cytoplasm and can be seen extending along
the cytoplasmic processes.
From inspection of the TEM micrographs we gained the impression that the
spaces between talpid3 mesenchyme cells were slightly larger than those between
normal cells and also that they were much emptier, in the sense of their being
occupied by fewer sectioned areas of the filopodial extensions, especially the very
fine ones of less than 0-5 /on diameter. This impression is confirmed in the
quantative data presented in Table 1. No significant difference is obtained for
nuclei and mitochondria, suggesting that these cover an equal area and are
found equally frequently in normal and talpid3. On the other hand, the figures for
cytoplasmic processes less than 0-5 /mi across, remaining cytoplasm and intercellular spaces are all significantly different, the greatest difference being in the
relative numbers of the very fine cytoplasmic processes (2\ x greater in normal
than in talpid3 montages). These figures suggest that the nuclei and main cell
bodies are not much, if any, more widely spaced in talpid3 than in normal
mesenchyme but that the difference lies in the distribution of the cytoplasm, i.e.
that where in talpid3 the space between cells is relatively empty and more likely
actually to be scored as intercellular space, in normals it is more often traversed
at grid points by a cytoplasmic extension, especially by a fine filopodium. This
would suggest that either talpid3 cells are less endowed with fine filopodial
processes than normal cells, or that the filopodia are shorter and therefore
occur in fewer sections. It may also be significant that the origins of filopodia in
talpid3 cells often appear to be 'bunched' at the ends of the larger cell extensions, giving the appearance of a sea-anemone with tentacles (Fig. 21); this
would mean (see Fig. 1) that where one cytoplasmic extension was scored the
chances of scoring its close neighbours would be reduced. From this analysis,
therefore, it appears that talpid3 wing-bud mesenchyme cells differ from normals
in having fewer or less extensive fine filopodial cytoplasmic processes.
More light is thrown on these differences by a detailed examination of the
SEM pictures. These show, especially in the normal embryos (Figs. 8, 16, 24,
26), that the mesenchyme cells are flatter than might be expected. In the mass,
they give somewhat the appearance of a heap of cornflakes, each mesenchyme
Electron microscopy of chick limb-bud
Normal
111
lulpid*
Fig. 28. Diagrammatic comparison of normal and talpicP wing-bud mesenchyme
cells, partly in section (see text).
cell having extensive flattened surface areas, bounded by long edges of lamellar
cytoplasm, from which most of the filopodia arise either directly or from larger
cytoplasmic extensions. Our impression is that normal cells tend to be elongated,
with more filopodia arising from the lamellar edges at each end. There appear
to be usually 3-4 flattened surfaces in most cells, reflected in the characteristic
preponderance of cells appearing triangular or quadrilateral in section (Figs.
14, 20).
The general impression given by the talpid3 mesenchyme cells in SEM pictures
is clearly different and may be described by saying that they appear, en masse
(Fig. 13) and individually (Figs. 9, 17, 25), more 'ragged' than normal cells.
There are fewer of the very fine filopodial extensions lying over their surface,
but there are more filopodia arising from the lamellar cytoplasmic boundaries
and their extensions all around the cell instead of predominantly at leading or
trailing lamellae, with rather long uninterrupted lamellar regions between, as in
the normal cells (compare Figs. 24 and 25). The 'bunching' of filopodial origins,
which would account for the appearance mentioned in TEM micrographs, can
bz clearly seen in Fig. 25. Our impression is that the larger cytoplasmic extensions in normal cells are more finely drawn out than in talpid3 cells, with the
filopodia branching off in a tree-like manner, whereas these extensions in talpid3
are shorter, with the filopodia arising closer together. These differences are
summarized in diagrammatic form in Fig. 28. Taken in conjunction with the
quantitative evidence obtained from the TEM micrographs it appears that
talpid3 cells have a larger number of filopodial extensions than normal cells and
that these arise more generally around the edges of the cell, but that they do not
extend so far through the intercellular spaces or produce such fine terminal
arborizations as do the filopodia in the normal cells.
Junctions between mesenchymal cells are sometimes seen in both normals and
778
D. A. EDE AND OTHERS
Table 2. Variance ratio tests on data from montages of
ectoderm I mesoderm boundary
Normal embryos
talpid3 embryos
Variance ratio
Degrees of freedom
Probability
Significant
No. of mesodermal
processes approaching
within 5-6 /mi of
ectoderm basal lamina
No. of mesodermal
processes touching
ectoderm basal lamina
104-76
2810
20-927
27
P < 0005
Yes
33 03
8-53
18-355
27
P < 0005
Yes
talpid3, similar to those in the ectoderm, but no desmosomes have been seen
connecting them in this region of the limb-bud.
Ectodermaljmesenchymal boundary
SEM micrographs (Figs. 12, 13, 16, 17), TEM micrographs (Figs. 10, 11) and
light microscope sections (Figs. 14,15) all reveal that there is a slight gap between
the ectoderm basal lamina and the underlying mesoderm in both normal and
talpid3 embryos, but that this gap is much more extensive in the mutant, in which
it frequently measures as much as 2 / m . In both normals and talpids the mesenchyme cells make contact with the basal lamina by means of many fine processes
(Figs. 16, 17); some of these are as thin as 10 nm and they form a web between
the mesenchyme and the ectoderm. Many of these processes are obviously
cytoplasmic but the finest of them show a periodic structure in TEM micrographs
at high magnification and almost certainly consist of collagen. Thus TEM
micrographs show that the mesenchymal cells are separated from the ectoderm
basal layer cells by the basal lamina, but also, beneath that, by strands of
extracellular material, including collagen fibres. In Fig. 27 this extracellular
material occupies much of the large gap between ectoderm and mesoderm in
a talpid3 wing-bud. In the region immediately beneath the AER (Figs. 18, 19),
the space contains wisps of granular material resembling the substance of the
basal lamina (which, as already noted, is not continuous in this region) as well as
fine collagen fibrils and small cytoplasmic processes.
The visual appearance of a much more extensive gap between ectoderm and
mesoderm in talpid3 than in normal limb-buds is confirmed by the data presented
in Table 2, where both the number of mesodermal cell processes approaching
close to (within 5-6 /<m) the basal lamina and the number actually touching it is
much greater in normal than in talpid3 embryos. SEM micrographs suggest that
in normal embryos contact with the basal lamina (Figs. 16, 26) is often made by
an extensive region of one of the lamellar cytoplasmic cell edges mentioned
Electron microscopy of chick limb-bud
779
above, and this is confirmed in TEM micrographs (Fig. 10). Such extended
connexions do not appear to occur in the talpid* wing-buds. In both normal and
talpid* embryos, though much more numerous in normal limb-buds (see Table 2,
and compare Figs. 26 and 27), are found the fine cytoplasmic processes which
connect the basal lamina with the underlying mesoderm cells and which appear
to be drawn taut, like guy ropes. Though the cell processes are in close contact
with the basal lamina, none have been seen to extend through it.
DISCUSSION
Structure of the ectoderm and formation of the AER
Amprino (1965) and Amprino & Ambrosi (1973) have presented clear evidence
that the ectoderm slides in a proximo-distal direction over the underlying
mesoderm towards the apex of the limb-bud; they believe that the formation and
maintenance of the AER depends, at least in part, on packing of the cells at the
distal tip where cells from the dorsal and ventral sides meet, after the manner
outlined, explicitly by Ede (1971).
The structural features of the non-ridge ectoderm and of the AER described
here support this interpretation. The basal layer of the non-ridge ectoderm, with
its columnar cells separated by wide gaps except at their base, forms in effect an
extended caterpillar track which is well adapted to flow smoothly over the limb
contours. The thin periderm layer, with its closely interdigitating pavement-like
cells, forms a flexible cover over this. The deformation of the basal cells at the
collision line would give the appearance we find in transverse sections of the
AER: tall wedge-shaped cells, arranged fan-wise, which retain their connexion
with the basal lamina, i.e. pseudo-stratified rather than stratified as suggested by
Jurand (1965). The elimination of the large spaces between the cells of the basal
layer and the compression of the cells would lead to the rounding off of the
peridermal cells which we observed. The necrosis of many of these cells may be
connected with their being forced out of their previous cell contacts. The gaps
which occur in the basal lamina beneath the AER might also be due to compression of the ectodermal basal layer cells in this region.
Mesoderm
Structure of individual cells
The descriptive term generally applied to the mesenchyme cells in the chick
has been 'stellate'; e.g. this has been used to describe the primary mesenchyme
of the early embryo (Trelstad, Hay & Revel, 1967) and the undifferentiated
mesenchyme of the early limb-bud (Gould et al. 1972). It implies a more or less
radially symmetrical cell with pointed cytoplasmic projections radiating from it
and this is indeed the impression given by the appearance of the cells in section.
It is perhaps the chief contribution of the SEM technique in this study to show
that it is misleading: normal wing-bud mesenchyme cells, at any rate in the
780
D. A. EDE AND OTHERS
region we examined, appear to be rather flattened, often longer than broad and
presenting extensive flat surfaces bounded by long edges of lamellar cytoplasm
from which fine filopodial extensions arise. The picture is closer to that presented
by limb-bud mesenchyme cells in culture (Ede & Flint, 1974#) than has been
supposed, and it is possible that valid comparisons may be drawn between the
lamellar edges of the cells in vivo and the lamellipodia (ruffled membranes) of the
same cells in vitro.
Connexions between cells
The filopodia form an extremely complex system, connecting each cell not only
with its immediate neighbours but with others several cells away and, in the case
of cells near the surface of the limb-bud, with the basal lamina of the ectoderm.
It is not possible to be quite clear where the terminations of the filopodia fall on
the contacted cell, but many of them appear to be on the flat surfaces of the
main cell bodies.
Activity and motility of the mesoderm cells
The cells in SEM pictures give the appearance of being in a highly active state,
for it is unlikely that either the filopodia, or the fine lamellar edges of the cells
from which they arise, are rigid or static structures. The question as to what
part cell motility plays in the developing limb-bud has been raised by Ede &
Agerbak (1968), Ede & Law (1969), Ede (1971) and subsequently by Gould
et ah (1972). The appearance of the cells in SEM suggests that at least the peripheral regions of the cytoplasm are in a state of dynamic activity. The absence of
desmosomes in these cells lends support to this interpretation.
Ectodermal'/mesodermal junction
The two layers are completely separated by the basal lamina except at the
region immediately beneath the AER where there is a number of perforations.
Beneath the basal lamina there is a mat of very fine fibres, of which some
at least have the characteristic banded appearance of collagen. Fine filopodial cell extensions are also present, many of them attached or at least closely
juxtaposed to it, and they appear to be stretched taut between the basal lamina
and their mesodermal cell bodies. In normal embryos the connexion, as seen in
both TEM micrographs (Fig. 10) and SEM pictures (Fig. 26), is often along
a considerable length of a lamellar edge, but in talpid3 embryos the connexion is
in almost all cases only by the tips of the filopodia. In the normals particularly,
it appears that the filopodia are making contact with the basal lamina, adhering,
and then increasing the contact beyond the original point. We are reminded of the
inference drawn by Trelstad et al. (1967) from observations on primary mesenchyme cells in the early embryo, that 'filopodia extending from the advancing
edge of the uppermost and lowermost mesenchymal cells seem to attach to the
basement epithelial lamina (of the epiblast and hypoblast) as if they were feeling
Electron microscopy of chick limb-bud
781
3
their way along this substratum'. In both normal and talpid wing-buds there is
a small gap of the order of 4-6 jum in depth immediately beneath the central cells
of the AER. Elsewhere in the parts of the limb-bud which we have examined, the
gap is much narrower and often almost absent in normal embryos, but a clear
gap between ectoderm and mesoderm is a marked feature of talpid3 and explains
why, in experiments involving removal of the ectodermal cap from the underlying mesoderm following trypsinization, separation has always been found to
occur more easily in the mutant.
Differences between normal and talpid3 cells
Two differences have emerged from this study: (a) in the morphology of
individual cells; (b) in the extent of the gap between ectoderm and mesoderm.
The distinct morphology of the talpid3 cells is presumably a direct genetic
effect. Other differences have been discovered and have been or will be described
elsewhere: (i) in cell-to-cell adhesion (Ede & Agerbak, 1968; Ede & Flint,
1974/?); (ii) in rate of locomotion on plastic (Ede & Flint, 1914a); (iii) in
resistance to injury and death (Ede & Flint, 1972). Such genetic evidence as is
available suggests that, though no fine genetic analysis has been possible, talpid3
represents a mutation of a single gene; our assumption is therefore that all of
these effects at the cellular level are related to each other and to the effect of the
mutation on morphogenesis. The relation between the morphological differences
and the differences in cell adhesion and rate of locomotion is beginning to
emerge, appearing to depend upon the differences in the number, structure and
distribution of fine filopodial cytoplasmic extensions, and is discussed in detail
elsewhere (Ede & Flint, 1974a, b).
The relatively more prominent ectodermal/mesodermal gap in talpid3 wingbuds must be a more indirect effect of the gene and there appear to be two
possible explanations. (1) Ede & Kelly (1964) described a generalized oedema in
later talpid3 embryos which developed from the 8th day. No oedema was
observed before this stage but it is possible that a fluid pressure, sufficient to
force the ectoderm away from the underlying mesoderm, has already built up at
the stage we have examined here. (2) Perhaps the more extensive gap in talpid3
is caused by differences in the nature of the connexion of the mesodermal cells
with the basal lamina, depending upon the differences in cellular morphology
which we have observed.
We believe that the second hypothesis is more likely, since no obvious oedema
has been observed until a much later stage, and since a gap is as much a feature
of the normal wing-bud and is only exaggerated in the mutant. Gustafson &
Wolpert (1961) described a mode of cell locomotion in sea-urchin morphogenesis: the mesenchyme cells concerned threw out fine cytoplasmic processes,
up to 30 [im long, which exhibited random exploratory movements; if they did
not make contact with the ectodermal body wall they collapsed, but if a successful contact was made the processes shortened and pulled the cell bodies towards
782
D. A. EDE AND OTHERS
uilpitl3'
Normal
Peiklerm
Basal
layer of
ectoderm
Basal lamina
Mcsenchyme
Collagen
web
Fig. 29. Diagrammatic representation of the ectoderm and underlying mesoderm in
the region of the AER, with a comparison of the ectoderm/mesoderm boundary in
normal and talpid3 wing-buds. 'The collagen web' contains collagen fibres embedded in additional extracellular material.
the point of attachment. Essentially similar forms of cell locomotion have
subsequently been found to occur in a wide variety of developmental situations
(reviewed by Trinkaus, 1973) and the taut appearance of the filopodial processes
connecting the mesenchymal cells with the basal lamina across the ectodermal/
mesodermal gap which we have found in normal embryos, together with the
orientation of many subectodermal cells towards the ectoderm, makes it seem
likely that the same activity occurs in the limb-bud. If this were so, the more
extensive gap found in talpid3 embryos might again be attributed to differences
in the structure and distribution of the filopodia referred to above, e.g. that since
they were less extended they might be less effective in making connexions with
the basal lamina, as suggested diagrammatically in Fig. 29. For whatever reason,
it appears that in normals the mesenchymal cells approach closely to the ectoderm through out-reaching filopodial and lamellipodial processes, whereas in
talpid3 wing-buds this close connexion fails to become established.
Outgrowth of the limb-bud
These observations may throw some light on the problem of limb-bud
outgrowth. Ede & Law (1969) drew attention to the parameters at cell level
affecting changes in shape of a developing organ, particularly the limb-bud, in
developing a computer simulation programme. These were eight in number:
Electron microscopy of chick limb-bud
783
cell number, proliferation, position, movement, size, shape, packing density and
the constraint produced by the ectodermal layer on the underlying cell mass.
Amprino (1965, 1973) has suggested that the main role in shaping limb-bud
outgrowth is played by the last of these factors and Hornbruch & Wolpert
(1970) have also taken this view; all of them consider that the main shaping
force is the relatively rigid structure of the apical ectodermal ridge.
This is probably an over-simplification; the shaping of the limb must be due to
a balance between the forces exerted by all of the factors enumerated above. The
distribution of cell divisions within the developing limb-bud might obviously be
important, and Amprino (1965), Ede & Law (1969) and Hornbruch & Wolpert
(1970) have found evidence for a gradient of mitosis within the limb-bud and
used it in their models. Summerbell & Wolpert (1972) have proposed a hypothesis relating this gradient to the outgrowth of the ectoderm, which is controlled
- i.e. given a firm shape - by the AER, thus giving the AER the key role.
It is certainly the case that the ectoderm, supported by the AER, does provide
a relatively rigid jacket in which, in fact, dissociated cells can be packed to
produce a fairly well-formed limb. But when ectoderm and mesoderm are
dissociated after trypsin treatment, the mesoderm also maintains its shape and
continues to do so until cells begin to migrate away from it. We may suppose
that in the course of evolution, ectodermal outgrowth and underlying mesodermal growth have come to be correlated so as to act harmoniously without one
being necessarily the sole driving force. Indeed, according to Amprino's
hypothesis of ridge formation by ectoderm sliding from two directions and
colliding, the sliding must take place over a rigid determined form, provided
presumably by the underlying mesoderm. In the earliest stages of limb-bud
formation no ridge exists and we may suppose that in this case the limb-bud
form is determined principally by the mesodermal cell behaviour, particularly
by mitosis and possibly by orientated movement. The possibility that movement
is important in limb-bud outgrowth was suggested by Ede & Agerbak (1968) and
shown by Ede & Law (1969) to produce, in conjunction with a mitotic gradient,
a good approximation to the limb-bud shape in computer simulations. Mitolo
(1971) and Wilby (personal communication) have since shown by computer
simulation that it is possible to produce a limb-bud-like outgrowth in the absence
of cell movement, providing that the mitotic distribution is very precisely controlled in a particular way. But it is certainly much simpler and more flexible to
produce outgrowth if movement is included. Moreover, exclusion of movement
also eliminates the necessity, by implication, of postulating any modelling role
for the ectoderm, which would produce outgrowth by forcing cell movement in
certain directions by the constraints its structure imposed on simple expansion
through cell division. On the whole it seems likely that cell movement is a factor
in limb outgrowth, and the question is really whether the movement is active or
passive, i.e. whether the modelling role of the ectodermal jacket is so predominating that the cells are squeezed forward like toothpaste in a tube, or whether
49
EM 13 31
784
D. A. EDE AND OTHERS
they move actively towards the distal tip. The contribution of the present study
to this problem is that the cells do not give the appearance of either static
immobilized bodies or passively flowing particles; on the contrary, they appear
to be in a state of considerable activity in the production of cytoplasmic processes, and in the regions we have looked at there is evidence that cells near
the surface are attaching themselves to the basal lamina and hauling themselves
towards it. This movement might then be transmitted to cells lying deeper
within the mesoderm, producing the slight shifting movement required in the
original computer model.
One of us (D. A. E.) wishes to acknowledge support in the course of this work by grants
from the Agricultural Research Council, the Science Research Council and Unilever Ltd.
We are grateful to Mr Raymond Moss for technical assistance, and to O. P. Flint and
O. K. Wilby for critical discussion of the manuscript.
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