/. Embryol. exp. Morph. Vol. 26, 3, pp. 623-635, 1971
Printed in Great Britain
623
Organization of the chick blastoderm edge
By J.ROGER DOWNIE 1 AND SUSAN M. PEGRUM 2
From the Department of Zoology, University College, London
SUMMARY
A band of cells forming the edge of the chick blastoderm, and attached to the vitelline
membrane, causes the expansion of the blastoderm in the first few days of incubation by
active migration across the vitelline membrane.
The structure and organization of these cells was examined by light microscopy (both on
whole mounts and sections) and transmission electron microscopy. The account presented
differs markedly from previous descriptions of these cells.
The band of cells at the blastoderm edge is an association, between 90 and 130/tm wide, of
flattened, non-dividing cells forming a multilayer; some of these cells, and no other cells of
the blastoderm, are attached to the vitelline membrane.
Each attached cell has a thin flattened lamella, centrifugally oriented and underlapping the
next cell distally, except (1) the most distal cell, whose lamella is thick and long, though
tapering, and is not overlain by other cells; and (2) the most proximal attached cell which has
a short centripetally oriented lamella, as well as a centrifugal underlapping one.
The cells of the edge band not attached to the vitelline membrane also have flattened
lamellae attached to the cells below; these lamellae are, however, unoriented.
The cells of the edge band all have plentiful cortical filaments and cytoplasmic microtubules.
Specialized plaques are involved in the attachment of edge band cells to the vitelline
membrane.
The form of this edge structure is compared with the outgrowth edge of a chick yolk sac
epiblast explant cultured on vitelline membrane. It seems likely that the way the blastoderm
edge cells are organized may explain their prodigious migratory activity.
INTRODUCTION
In the first 3-4 days after laying, the chick blastoderm expands to encompass
the yolk mass, most of the vast expanse of tissue being the extra-embryonic
yolk sac. The work of New (1959) has established that expansion is brought
about by the active centrifugal movement across the vitelline membrane of a
narrow marginal band of cells, and that only these cells are normally attached to
the vitelline membrane.
Previous work on the structure of these cells has been confined to electron
microscopy. According to Bellairs (1963), the edge cell bodies are indistinguishable from those of surrounding cells. Their special feature is a long distal
process, up to 500 jtim long, tapering down at its tip to as little as 0-25 jtom thick.
1
Author's address: Department of Zoology, University of Glasgow, Glasgow W.2, U.K.
Author's address: Department of Zoology, University College, Gower Street, London,
U.K.
2
624
J. R. DOWNIE AND S. M. PEGRUM
At the extreme edge, the blastoderm is only as thick as the single process. As
one proceeds medially, processes overlap, and the edge increases in thickness.
These processes may have projections from both their upper and lower surfaces.
The processes appear internally devoid of endoplasmic reticulum, Golgi, yolk
droplets and mitochondria. With the scanning electron microscope (Bellairs,
Boyde & Heaysman, 1969), the blastoderm edge appears as a very thin
flattened cytoplasmic sheet with irregular outline.
Since normally only marginal cells attach to and move over the vitelline
membrane, an important question is how they differ from other cells in the
yolk sac. New (1959) suggested two hypotheses. Either 'the cells of the blastoderm edge are intrinsically different as regards their surface structure from those
of the rest of the blastoderm' or the cells are intrinsically the same 'but behave
differently as a result of their position'. On the basis of a number of experiments,
New favoured the former hypothesis. He was later supported in this by the work
of Bellairs & New (1962), Bellairs (1963) and Bellairs et al. (1969). However,
Downie (1971) has shown that the differences may have been overemphasized.
Given time and adequate culture conditions, non-edge yolk sac fragments (and
other cell types too: e.g. 9-day chick heart fibroblasts) are able to attach to and
move over the vitelline membrane, albeit more slowly than the normal blastoderm edge. This work began with the following aims:
(1) To reinvestigate the organization of the normal edge cells, and to compare
them with non-edge fragments grown on the vitelline membrane, using light
microscopy to get a better overall view than is possible with the electron
microscope.
(2) To reinvestigate the fine structure of the edge cells.
Since the work of Bellairs (1963), it has been established that the conventional
osmium fixation methods she employed often fail to preserve delicate cytoplasmic structures, such as microtubules, particularly in early embryonic
tissues (Porter, 1966). This may account for the 'emptiness' of the edge cell
processes she observed.
MATERIALS AND METHODS
One-day incubated chick embryos were prepared for light microscopy (both
whole mounts and sections), and transmission electron microscopy as follows.
In order that the embryos might be easily cleaned of yolk, and fixed as flat as
possible (for precise orientation of sections, and easy examination of whole
mounts), they were removed from the egg and mounted on glass rings, still
attached to the vitelline membrane, according to the culture method of New
(1955). Yolk particles and hypoblast cells could then be blown off using a jet of
saline from a fine pipette, leaving cleaned epiblast and edge region, still attached
to the vitelline membrane. The embryos were then fixed and dehydrated on the
rings so that they remained flat. For whole mounts, overnight treatment in
Organization of the chick blastoderm edge
625
xylene tautened and further flattened the embryo so that, on mounting in
Canada balsam, large areas of the embryo were in almost the same focal plane,
even at high magnifications. For light microscopy, several fixatives were tried Bouin, Zenker, Susa, Smith's and Formol-saline. All gave similar results, though
Formol-saline proved a poorer fixative for this material. Bouin was used
routinely. Specimens for sectioning were embedded in paraffin wax and cut at
5 or 10 /<m. Thick (1 /mi) Araldite sections of material fixed for electron
microscopy gave similar results when viewed in the light microscope with phase
contrast optics.
Photographs were taken on a Zeiss Photomicroscope with Ilford Pan F film.
Drawings were made using a Wild M20 microscope and Zeichentubus.
Embryos were fixed for electron microscopy using 2 % glutaraldehyde in
buffered salt solution (pH 7-4) for 30 min, followed by 1 % osmium tetroxide in
buffered salt solution (pH 7-4) for 2 h. After dehydration through a graded
ethanol series, selected areas of the edge of the blastoderm were embedded flat
in a shallow layer of Araldite.
The embedded material could be viewed under phase-contrast light microscopy.
Suitable areas for cutting were selected, photographed and marked using a
Zeiss diamond scriber. By this procedure the precise position and orientation
of the sections was known. Sections were cut using a diamond knife, mounted
on uncoated copper grids, stained with uranyl acetate (Gibbons & Grimstone,
1960) and lead citrate (Reynolds, 1963) and viewed with an AEIEM6B
electron microscope.
Fragments of 1-day non-edge yolk sac epiblast were grown for 24 h on
vitelline membrane set up as for normal New cultures, but with a medium of
90% 199 (Glaxo Laboratories) and 10% chick serum (Flow Laboratories).
These were fixed and examined for electron microscopy in a similar way to the
whole embryos.
OBSERVATIONS
(1) Whole mounts
At the extreme edge of the blastoderm is an easily distinguishable band,
50-80 jtim wide, of rather dispersed (as much as 60 jam centre to centre) large
flattened nuclei which are elliptical in shape with a long axis of around 15 /«n.
These are situated at different levels, and occasionally overlap, showing the
edge area to be multilayered. Because the vitelline membrane stains as strongly as
cytoplasm, the processes of the cells to which these nuclei belong are difficult to
see: but, in favourable specimens, typical leading lamellae (Ingram, 1969;
Abercrombie, Heaysman & Pegrum, 1970a) of an epithelial rather than
fibroblastic type can be seen extending distally from the band of nuclei. The
lamellae stretch about 40-60 /.im beyond the nuclei, and have an irregular
outline, similar to that of an epithelial outgrowth on glass (e.g. Lewis & Lewis,
1912). In surface view, the leading lamellae of epithelial cells appear to form a
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J. R. DOWNIE AND S. M. PEGRUM
flat coherent sheet. Cell boundaries are not distinguishable. Nowhere can be
seen the 500 jLtm processes described by Bellairs (1963).
Immediately proximal to the edge band is a region of densely packed cells with
smaller overlapping nuclei, and more proximal still, cell density decreases, the
cell sheet thinning out to a monolayer - the epiblast of the yolk sac. Since the
areas described have cells at different planes, photographs are unsatisfactory. A
Zeichentubus drawing is shown in Fig. 1.
•<
Unattached extra-embryonic epiblast
>-<
Attached edge
^
100/mi
Fig. 1. Zeichentubus drawing of a whole mount of the edge area of a chick blastoderm, showing the form of the leading lamella and the variation in size and density
of nuclei, proceeding from the edge medially into the embryo.
The area of dispersed flattened nuclei corresponds to the attached region:
cells here are termed 'edge cells'. No mitotic figures have been found among
these cells. The other regions described are not attached to the vitelline membrane, and have abundant mitotic cells.
(2) Sections
(Sections parallel to the main axis of the edge cells, i.e. cut as a radius of the
embryo, are termed longitudinal. Those at right angles to this axis are transverse.
The lower surface of a cell is that nearest to the vitelline membrane.)
Light microscopy. In longitudinal sections, a band round the edge of the
blastoderm, between 90 and 130/mi wide, corresponding exactly with the edge
cell nuclei and cell processes seen in whole mounts, is seen to be attached to the
vitelline membrane. Proximally to this, the cells are not attached. The attached
edge is of the order of three cells wide.
The attached area is made up of:
(1) A very thin lamella. With the x 100 objective, this can be resolved right
to its tip (Fig. 2). A representative length for these lamellae cannot be given
Organization of the chick blastoderm edge
627
since in life they are continually shortening and expanding, but they are between
40 and 60 /«n long. The underside of each lamella has an undulating profile, as
if attachment were only at a few points along its length. Each lamella increases
in thickness from the tip.
(2) A region of both cell bodies and lamellae, proximal to the distal lamella
described above. This is an area of overlapping cells. Detail is difficult to make
out at light microscope resolution but cells attached to the vitelline membrane
each appear to have a distal lamella underlapping the next cell. The orientation
of the processes of the overlapping cells in this region (i.e. those in the edge not
attached to the vitelline membrane) is impossible to determine. As an exception
10//m
Fig. 2. High-power light micrograph of the blastoderm edge. This shows the
undulatory appearance of the region attached to the vitelline membrane; and the
under- and overlapping of cells in the edge region. The most distal leading lamella
is resolved to its tip.
to the centrifugal orientation of most of the attached edge cells, the most
proximal of these, at the junction between attached edge and unattached
epiblast, appears to have a short lamella pointing centripetally (Fig. 7).
Electron microscopy. Both longitudinal and transverse thin sections of the
blastoderm edge have been examined. The material fixed for electron microscopy
does not differ markedly at light microscope resolution from Bouinfixedmaterial.
Since a montage of sections (either longitudinal or transverse) across the whole
edge would have to be reduced too much to show useful detail, we have sketched
one of each in Figs 3 and 4. Electron micrographs of discrete areas are shown in
Figs 5-7.
The area close to the vitelline membrane is mainly composed of thin
lamellae averaging 0-12 jum in thickness, arising from proximally situated cells,
and underlapping those situated laterally and distally to these cells. The lamella
of the most distal cell is not overlapped by other cell bodies for much of its
length, and tapers from 3-0/«n to as little as 0 1 /tm in thickness. It is underlapped though not as far as the tip, by proximal lamellae. The most proximal
attached cell has a short thick lamella pointing centripetally, but also a thin
centrifugal underlapping lamella, so that all the cells attached to the vitelline
membrane are centrifugally oriented, except the most proximal one, which is bidirectional.
From the transverse sections, it appears that the upper layer cells in the edge
40
E M B 26
628
J. R. DOWNIE AND S. M. PEGRUM
are truly unattached to the vitelline membrane. They have thin flattened processes closely apposed to the cells below them, but have no specific orientation
(Fig. 4).
Cell structure. Both the edge cells and those cells near the edge but not
attached to the vitelline membrane are epithelial, with little specialization of
internal organization. The endoplasmic reticulum is poorly developed; a few
cells are rich in Golgi cisternae and mitochondria, and microtubules are
plentiful.
cf
Fig. 3. Diagrammatic longitudinal section through the whole edge showing its
organization. The cells have been expanded along the vertical axis for clarity, (cf,
cortical filaments; d, desmosome; j , specialized junction; /, lacuna; p, plaque; vm,
vitelline membrane; vv, villous projection into vitelline membrane; vy, villous projection into yolk; y, yolk.)
Fig. 4. Diagrammatic transverse section through the middle of the edge. Again, the
cells have been expanded along the vertical axis.
The edge cells show specializations related to active movement on, and
attachment to, the vitelline membrane. The most distal edge cells, as noted above,
have large leading lamellae, containing mostly free polysomes, with occasional
smooth endoplasmic reticulum profiles and some mitochondria. There are also
microfilaments and microtubules showing a distribution similar to that in the
lamellae of actively moving chick heart fibroblasts (Abercrombie, Heaysman &
Pegrum, 1971). Large vesicles containing yolk and lipid droplets are frequently
Organization of the chick blastoderm edge
629
found at the base of a lamella and in the perinuclear zone; also there are
Golgi bodies and mitochondria.
More proximal edge cells have a less regular outline, but contain similar
organelles. The cell surface is frequently extended into villous projections, which
penetrate into the spaces between the thick fibres of the vitelline membrane
inner surface (Fig. 8). On the upper surface where the yolk would normally be,
similar but much longer and often branching projections are found. These
Fig. 5. Longitudinal section through the leading cell of the edge, with overlapping
and underlapping lamellae.
Fig. 6. Longitudinal section through mid-region of edge with nuclear
overlap and lacunae (L).
40-2
630
J. R. DOWNIE AND S. M. PEGRUM
Fig. 7. Longitudinal section through the most proximal cell of edge, showing
centripetal lamella.
Fig. 8. Villous projection from cell of blastoderm edge penetrating into
vitelline membrane.
Organization
of the chick blastoderm
631
projections appear to have no organized filamentous sub-structure (Cornell,
1969; Follett & Goldman, 1970).
Locomotory and attachment specializations
(1) Cell-cell relationships
Two common types of relation are found among the edge cells, (i) Large
areas of the surfaces of two cells running parallel to each other are found, with
the plasma membrane profiles 150 A apart. In these areas, there is usually no
specialization of the cell inner surface, but, occasionally, condensations of 50-60
A filaments are found in one or both of the cells, (ii) Between these areas are
enlargements of the intercellular space, or 'lacunae'; these may be bridged by
narrow processes from one or both adjacent cells, the tips of the processes being
separated by a gap of only 60-70 A.
Less commonly desmosomes are seen. They are, however, prominent in the
region proximal to the edge.
Fig. 9. Dense condensations of filaments adjacent to plasma membrane forming a
plaque approximately 70 A from the vitelline membrane.
(2) Cell-substrate relationships
Close apposition of cell surface to substrate is restricted largely to points on
the lower surfaces of the thin underlapping lamellae, and towards the tip of the
thick distal cell lamella. At these points, the plasma membrane is only 60-70 A
from the substrate, and the inner cell surface is organized into a dense, apparently fibrous plaque. These attachment plaques also occur (Fig. 9) where the
lamellae penetrate into the vitelline membrane, and appear similar to the
attachment plaques found near the leading end of chick heart fibroblasts
moving on an Araldite surface (Abercrombie et al. 1971).
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J. R. DOWNIE AND S. M. PEGRUM
(3) Locomotory specializations
Cortical filaments, about 60 A in diameter, oriented parallel to the direction
of movement, and forming a layer about 0-15 /im thick, are present in large
areas near the upper surface of cells in the edge, and smaller areas near the
lower surface (Fig. 10). Filamentous tracts running throughout the cytoplasm
Fig. 10. Approximately 60 A thick filaments forming an organized cortical layer
at the upper surface of a blastoderm edge cell.
Fig. 11. Section through an outgrowth from an explant of non-edge yolk sac
epiblast on vitelline membrane, showing closely packed cells underlapped by several
layers of thin lamellae.
Organization of the chick blastoderm edge
633
have not been found. Microtubules are scattered around singly in lamellae and
cell bodies, but with no specific orientation. They are occasionally found in
lamellae distal to the most distal attachment plaque.
Non-edge yolk sac epiblast explanted on vitelline membrane
The outgrowing cells have a similar general organization and appearance to
the attached edge cells, with centrifugally oriented lamellae underlapping the
distal cells. One difference in overall organization from the in vivo situation
is that the whole area of explant and outgrowth has adhesions to the vitelline
membrane, attachments not being restricted to a special edge group. A monolayer 5-6 cells wide surrounds an area of more densely packed cells, piled up
six cells deep, and underlapped by several layers of thin lamellae (Fig. 11).
Few desmosomes or lacunae are found between the cells of the outgrowth.
DISCUSSION
Bellairs (1963) found at the blastoderm edge a single layer of highly specialized
cells with distally pointing processes up to 500 /mi long. Figs 3 and 4 show
in summary our finding; an association 90-130jam wide of flattened cells
forming a multilayer. Some of these cells, and no other cells of the blastoderm,
are attached to the vitelline membrane by lamellae 40-60 jtim long. Individual
edge cells do not differ markedly in appearance from ordinary yolk sac cells
allowed to attach to and move over the vitelline membrane. Reasons for the
differences in view are obscure. In favour of our results is the fact that they are
based on observations of three different kinds (light microscopy of sections and
whole mounts; electron microscopy), and on a sectioning technique which
allows precise orientation of the specimen.
As expected, the attached cells showed many structures characteristic of
actively moving cells - an abundance of microtubules, bundles of cortical
filaments (see Baker & Schroeder, 1967; Spooner & Wessels, 1970) and thin
oriented lamellae with substrate attachment plaques (Abercrombie, Heaysman
& Pegrum, 1971).
Projections from both upper and lower surfaces are frequently found. The
significance of these projections is uncertain; they may be involved in engulfing
yolk particles (Bellairs & New, 1962) and some may be 'ruffles' (Abercrombie
et al. 19706). Bellairs (1963) also noted the lower surface projections into the
vitelline membrane and felt these must be important in giving the cells a grip
on the substrate. The so-called 'attachment plaques', found both at the ends of
these projections, and at other points along the lower surface of those cells
attached to the vitelline membrane, may contribute to this.
The organization of the edge region of a spreading epithelium is crucial, as
discussed by Abercrombie (1961). Downie (1971) has argued, on theoretical
grounds, that an epithelial edge a few cells wide, all these cells being attached to
634
J. R. DOWNIE AND S. M. PEGRUM
the substrate and motile, should be more capable of effective oriented movement
than a similar edge, many cells wide. In the latter case, the many attached
cells would lack co-ordination, and tend to move in all directions at once,
resulting in slow overall spreading of the epithelium. These two conditions are
seen in the normal blastoderm edge and in the yolk sac epiblast culture edge
respectively, and may explain why the former spreads much the quicker of the
two (Downie, 1971).
In both cases, the cells attached to the substrate have centrifugally oriented
leading lamellae underlapping the next more distal cells; these lamellae indicate
the direction of active locomotion of the cells.
The most proximal edge cell of the normal blastoderm is exceptional in
having both centrifugally and centripetally oriented lamellae. Presumably, the
free substrate proximally and distally allows these cells to extend lamellae in
both directions.
How the normal edge comes to be organized in the way described and why
explanted yolk sac cells, though capable of attachment to and movement
across the vitelline membrane, fail to organize themselves similarly, requires
experimental treatment and will not be discussed here.
We should like to thank Mr M. Abercrombie for reading and criticizing the manuscript. The
work was carried out while J.R.D. was in receipt of a Science Research Council Studentship.
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