/. Embryol. exp. Morph. Vol. 35, 3, pp. 559-575, 1976
Printed in Great Britain
559
The mechanism of chick blastoderm expansion
By J. R. DOWNIE 1
From the Department of Zoology, University of Glasgow
SUMMARY
At the time of laying, the domestic fowl blastoderm measures 4 mm across. After 4 days*
incubation, the extra-embryonic yolk-sac tissues have expanded to encompass the whole
yolk mass. This expansion involves the migration over the inner surface of the vitelline
membrane of a specialized band of 'edge cells' at the blastoderm periphery. As they move,
they pull out the blastoderm behind them, setting up a considerable tension. Expansion also
involves cell proliferation and changes in cell shape. This paper attempts to show how
locomotion, tension, proliferation and changes in cell shape all contribute to the orderly
process of expansion. As a simplification, only the extra-embryonic epiblast is considered
here.
The findings are:
1. Expansion does not occur at a constant rate, but starts slowly, rises to a peak (over
500 /*m/h) at around 3 days, and then slows as coverage of the yolk mass nears completion.
2. During the first day of incubation, edge-cell migration produces a tension in the
blastoderm. This rises to a peak at 20-24 h, then declines. This tension may be due to an
imbalance between expansion by migration and expansion by proliferation.
3. Migration of edge cells can be affected by tension in the blastoderm, i.e. very high tension
may hold them back. However, the tension level normally found in the blastoderm seems not
to do so. The low rate of expansion in the first day is therefore not due to the high level of
tension. It may instead be due to changes in edge-cell organization.
4. Proliferation occurs throughout the extra-embryonic epiblast during the expansion
period. It is not restricted to the blastoderm periphery. After the yolk has been covered, the
epiblast continues to grow, with proliferation restricted largely to a band just distal to the
advancing edge of the area vasculosa.
5. Cell shape and arrangement change considerably during expansion. The epiblast of the
unincubated embryo is a monolayer of tall cells. During expansion, these become considerably flattened so that each contributes a larger amount to yolk-sac surface area.
INTRODUCTION
One of the most impressive features of early avian development is blastoderm
expansion. At laying, the domestic fowl blastoderm measures about 4 mm across.
After 4 days' incubation, the extra-embryonic yolk-sac tissues have totally
encompassed the yolk mass, a more than 200-fold increase in tissue area.
The mechanism of blastoderm expansion has been investigated several times.
Schlesinger (1952) believed that the periphery was a syncytium, throughout
1
Author's address: Developmental Biology Building, Department of Zoology, University
of Glasgow, Glasgow G12 8QQ, Scotland U.K.
560
J. R. DOWNIE
expansion, and that expansion was the result of the growth of this syncytium
across the yolk surface, with individual cells being formed behind as the
syncytium spread outwards. Rather similarly, Haas & Spratt (1968) envisaged a
'ring blastema' zone just within the blastoderm periphery, where cells proliferate
very rapidly and move out centrifugally. However New (1959), without ignoring
the importance of cell proliferation, emphasized the role of active cell migration.
He noted that cells in a narrow band round the blastoderm margin attach to the
overlying vitelline membrane and, using this membrane as a substrate for
locomotion, move out centrifugally until the yolk mass is surrounded. The
properties of these specialized blastoderm 'edge cells' have been the subject of
several studies (Bellairs & New, 1962; Bellairs, 1963; Bellairs, Boyde & Heaysman, 1968; Downie & Pegrum, 1971; Downie, 1975). New (1959) also noted
that the blastoderm during expansion is under a tension, presumably generated
by the activity of the edge cells. He believed this tension essential for expansion,
the main effect being to maintain the sheet-like arrangement of the cells, and to
prevent their piling up.
This paper investigates the changing pattern of locomotion, tension and
proliferation in blastoderm expansion.
The details of individual experiments are described with the results. Hens'
eggs used were White Leghorn or De Kaalb.
EDGE CELL LOCOMOTION AND SHEET TENSION IN
THE EXPANDING BLASTODERM
(1) Rate of expansion
This was determined by measuring how far round the circumference of the
yolk mass the blastoderm edge had travelled after different times. This was not
an entirely simple matter since (1) there is considerable individual variation, but
any attempt to follow the whole expansion process in a single specimen involves
interference with the egg; and (2) it is difficult to make accurate measurements
on the surface of a soft wet sphere. The results shown in Fig. 1 were obtained by
taking samples from a single large batch of eggs at regular time intervals, removing the yolks and assessing the expansion distance by comparison with known
standards. These were drawings of the yolk mass as circles of 3 cm diameter
(close to the usual yolk-mass diameter) with the expanding blastoderm drawn
from above and from the side. Expansion was divided into 20 equal stages (in
terms of sphere surface area). It was always possible to assign any actual embryos
to one of these standards. Each standard is easily converted into blastoderm
radius as a proportion of the circumference of a circle of diameter 3 cm. Each
embryo is plotted in this way in Fig. 1. The broad pattern of results is consistent
with those from more direct measuring methods, and shows several points of
interest.
(1) Expansion does not start immediately, but after about 10 h. This period is
Mechanics of chick blastoderm expansion
561
PQ
20
30
40
50
60
Incubation time (h)
70
80
90
Fig. 1. Blastoderm expansion rate in ovo, plotted as blastoderm radius after different
incubation times. Each spot represents a different embryo. The line is drawn
through the mean radius for each incubation time.
much longer than the warming-up time of around 2 h. This pre-expansion
period has been previously reported (Downie, 1974).
(2) The rate of expansion is not constant. There is an initial slow phase to 16 h,
when the rate is 200 /*m/h. From 24-84 h, the average rate is much faster (555
/tm/h) and this slows again over the final phase, to 96 h at 292 /^m/h. The exact
time of completion of expansion is very variable, the last little patch of vitelline
membrane often taking a long time to cover.
(2) Blastoderm retraction
No method was found of measuring directly the tension generated by the
edge cells. The problem lies in holding the thin cell sheet without tearing it.
Only occasionally, as in the work of James & Taylor (1969), has this problem
been overcome in simple cell sheets. An indirect method was, however, successful in giving relative measurements.
Attached blastoderms of known incubation time were set up as New (1955)
cultures, using a glass ring of 25 mm internal diameter, staged (Hamburger &
Hamilton, 1951) and an outline drawing made using a Wild drawing tube and
microscope. The edge of each embryo was then detached from the vitelline
membrane and, after allowing a few seconds for any retraction to occur, a new
outline drawing was made. Since it is the centrifugal movement of the edge cells
which stretches the blastoderm, a comparison of the stretched and retracted
blastoderm size gives a relative measure of the tension exerted by the edge cells.
A difficulty in relating retraction directly to tension exerted by the edge cells is
that the tension required to stretch a cell varies with the mechanical properties
562
J. R. DOWNIE
Up to 30
31-3-8
3-9-4-5
4-6-6-0
61-7-5
Initial blastoderm radius (mm)
7-6-90
91 and over
Fig. 2. Blastoderm retraction after loosening of the edge. Retraction is given as the
percentage area reduction from the original (attached) area. Embryos are grouped
according to their original radius. The number of embryos in each group is given
in brackets. The histograms represent the mean retraction ± standard deviation
for each radius group.
of the cell. This may not alter much over the rather short incubation time covered
by these measurements, but is at present an indeterminate factor.
The results, involving measurements of 68 different embryos, are given in Fig.
2. Only embryos between 12 and 36 h incubation were used. Before 12 h,
embryos are rarely reliably attached, and after 36 h, embryos are too large and
too curved to be measured by this method.
From Fig. 2, we see that newly attached blastoderms retract little; retraction
rises to a peak when the blastoderm has a radius of around 6 mm (after 20-24 h
incubation), then falls again to a low level when the blastoderm radius passes
9 mm (after about 36 h incubation). A blastoderm area retraction of 30 % found when the radius is in the range 4-6-6-0 mm - implies that the cells are
each occupying 30/70 x 100 = 43 % more than their resting area when the edge
is attached. It is impossible to say what happens to tension later in development
but, as the blastoderm becomes more curved, it is difficult to imagine how a
tension generated at the edge could be transmitted throughout the cell sheet.
(3) The relationship between locomotion and tension
What might cause the changes in rate of movement of the edge cells during
the expansion period? There are two general possibilities: (1) external constraints, (2) changes in edge-cell organization.
Mechanics of chick blastoderm expansion
563
oq
Incubation time (h)
Fig. 3. Plots of expansion distance (given as blastoderm radius) against time,
comparing expansion by 'migration' (r = vt + r^ with expansion by 'growth'
(r = /*! «JetlT). Values used are T = 10 h, v = 0-3 ram/h, rx = 2 mm.
(a) External constraints
The most obvious external constraint (there could be others such as energy
supply, or the state of the substratum: one possible substratum effect is considered later) is the tension generated by the edge cells themselves. As they
stretch the sheet, this tension must act against further centrifugal movement.
Blastoderm spreading is the result of two processes: edge-cell migration and
growth of new yolk-sac tissue. The radius r of an expanding cell sheet (initial
radius r±) where the edge is moving with a velocity v is given by r = vt + rt at
any time t. Similarly, the radius of a flat cell sheet proliferating with a generation
time T (cell size constant and all maintaining their arrangement as a cell monolayer) is given by r = rx JetlT. Given constant v and T, these processes are in
imbalance; indeed, using the values of v, T and rx found in the 1-day chick
564
J. R. DOWNIE
blastoderm, expansion by migration runs ahead of expansion by growth for
about 38 h (see Fig. 3).
This theoretical analysis could explain the observed tension and expansion
rate results. At the start, migration is expanding the sheet faster than can be
accommodated by the increase in cell numbers. The result is that cells are
stretched to occupy more space. This generates a tension in the sheet which holds
back the migrating edge, reducing expansion rate. As expansion proceeds,
growth catches up, allowing edge-cell migration to proceed up to its maximal
rate. When this happens, tension in the sheet drops as the cells cease to be
stretched.
The plausibility of this explanation is enhanced by the close coincidence of the
theoretical time when growth catches up with migration (38 h) and the actual
time when tension appears to drop to a low level (around 36 h).
This explanation of the changes in tension and edge-cell migration rate rests
on two ideas: (1) edge-cell migration can be restrained by tension within the
blastoderm and (2) it is so restrained in the first 36 h of development, leading to
a reduction in the potential expansion rate. The first idea receives support from
experiments already reported (Downie, 1975). Colchicine slows down and may
stop blastoderm expansion, but the effect is not on the locomotory abilities of
the edge cells, but rather on the remaining cells of the blastoderm. Dissolution
of their microtubules by colchicine reduces their ability to maintain a flattened
shape, thereby increasing the tension on the attached edge cells and halting
migration. Indeed, this tension may be so great that the whole blastoderm
retracts, with its edge cells still attached to the vitelline membrane.
This does not necessarily mean that the tension normally generated during
expansion restrains the edge-cell migration rate. If it does, then reduction of the
normal tension should increase the rate. Two attempts were made to reduce the
tension:
Experiment 1. Eleven 1-day incubated embryos were set up as New cultures
and most of the blastoderm (about 80 %) excised, leaving a ring of tissue with
the edge cells intact and attached. The expansion rate of these edge cells over a
4 h period was compared with 14 controls. With most of the blastoderm removed,
tension on the edge should be considerably reduced, but controls and experimentals expanded at indistinguishable rates.
Experiment 2. As we have seen, when the edge of a 1-day embryo is detached
from the vitelline membrane, the blastoderm retracts by as much as 30 % of its
original area (Fig. 2), releasing all the tension in the blastoderm. If edge-cell
migration is slowed down by tension, the edges of these blastoderms, on reattaching, should move quickly until they approach their previous attached
position. Since re-attachment time is variable, these embryos, again set up as
New cultures, were recorded by means of time-lapse filming. The results, shown
in Table 1, were precisely the opposite of the prediction. On re-attaching, expansion rate was at first slow, increasing later and then stabilizing.
Mechanics of chick blastoderm expansion
565
Table 1. Expansion during the period immediately after edge re-attachment
Distances moved calculated from time-lapse films.
Incubation time
before expansion
Embryo
re-starts (min)
1
2
3
4
5
Mean
5
18
18
53
27
24
Distances (>m) moved in successive time periods
(min) after start of expansion
0-25 25-50 50-75 75-100 100-125 125-150 150-175
153
82
163
52
87
107
204
109
224
61
104
140
214
133
211
61
104
145
214
150
228
70
113
155
228
160
245
87
113
167
238
156
245
87
113
168
224
156
228
104
131
169
The results of expts 1 and 2 suggest that though edge cells may be responsive
to tension within the blastoderm, the actual tension generated during early
expansion is not responsible for the low initial expansion velocity. The work of
Curtis & Buultjens (1973) makes this result less unexpected. They showed that
very large differences in surface adhesiveness had no effect on the rate of cell
(fibroblast) movement and suggested a bulldozer analogy - that within rather
wide limits of environmental conditions, rate of movement remains constant.
This may well be the case for blastoderm edge cells. Over the range of tension
normally experienced, rate of movement of edge cells is unaffected; but when
tension is drastically increased (by treating with colchicine (Downie, 1975))
movement is inhibited.
This argument may also apply to another possible external constraint - the
tautness of the substrate. The vitelline membrane of the unincubated egg is rather
slack. During the period of blastoderm expansion it becomes taut, due to pumping of water into the sub-blastodermal space (New, 1956). Bellairs, Bromham &
Wylie (1967) noticed that in New cultures, blastoderms cannot expand on a very
loose vitelline membrane. This factor needs further investigation, but could well
be an all-or-none one: once the edge cells can get a grip, they move at their
maximal rate.
The most likely alternative explanation of the changes in the migration rate of
edge cells is a change in the edge cells themselves.
(b) Edge-cell organization
It seems possible from the above that the changes in the rate of blastoderm
expansion in ovo reflect real changes in the capabilities of the edge cells. To test
this, edge-cell fragments with attached vitelline membrane were isolated from
1-, 2- and 3-day incubated embryos, set up as New cultures and the expansion
rate assessed over a 5-h incubation period. The results (Table 2) indicate that
36
EMB 35
566
J. R. DOWNIE
Table 2. Expansion rates in New culture of blastoderm edges, isolated from
embryos at different stages
Expansion is given as the distance covered in a 5-h test period.
Incubation
time in ovo
(h)
Proportion of
circumference
covered (approx.)
Number of
edge pieces
tested
Distance (/*m) moved
in 5 h test period
(mean ± standard)
deviation)
24
48
66
I
i
±-f
11
5
5
169-3 ±38-8
285-7 ±43-7
383-8 ±41-7
3-day edge cells are capable of moving faster than 1-day edge cells, with 2-day
cells intermediate.
Downie & Pegrum (1971) found that the attached blastoderm edge in 1-day
incubated embryos was a band of cells between 90 and 130 /im wide. Preliminary
results from embryos of different ages suggest that this is variable, from around
70 jam when the edge cells first attach to the vitelline membrane, to as much as
260 (im at 3 days' incubation, when the blastoderm is expanding at its fastest.
Though the details of this change must await further work, it seems likely that
recruitment to the population of attached edge cells, making the edge a more
powerful motile unit, is responsible for the increasingly rapid rate as expansion
proceeds.
TISSUE GROWTH IN THE EXPANDING BLASTODERM
Blastoderm expansion involves tissue growth as well as edge-cell migration.
Growth is analysed here in terms of cell proliferation, shape and size.
(1) Cell proliferation - the pattern ofmitotic index
During blastoderm expansion, proliferation rate might vary (i) in different
parts of the expanding tissue, (ii) at different developmental stages. Both New
(1959) and Haas & Spratt (1968) envisaged a peripheral ring of rapidly proliferating cells, with mitosis absent, or at a low rate elsewhere.
Classically, comparison of proliferation rates in embryos has been by means
of the mitotic index, i.e. the proportion of cells in mitosis at any one time. This
is used here to compare proliferation in different parts of the extra-embryonic
epiblast at different stages. Though mitotic index is usually calculated from
serial sections, this is not easy since a correction factor is necessary for nuclear
size (Abercrombie, 1946; Simnett, 1968). Fortunately, extra-embryonic yolk-sac
can be stripped of its yolk and endoderm, and the epiblast fixed as a flat sheet,
allowing counting of the cells as if in a monolayer culture.
Extra-embryonic tissue was stripped of hypoblast and yolk by means of a fine
Mechanics of chick blastoderm expansion
567
Table 3. The mitotic index (%) in the extra-embryonic epiblast at different stages
and in different positions
Stages are given as defined by Hamburger & Hamilton (1951) (HH); incubation
time in hours (inc) and yolk-sac coverage proportion as a fraction (cp). Positions
are given as distances (mm) distally from either the area pellucida margin (apm)
or area vasculosa margin (avm).
Stage
HH2-3
inc 15
cp start
HH4-6
inc 24
cpi
HH 12
inc 48
cpi
HH 17-18
inc 72
cpi
HH 19-23
inc 90
cp complete
HH 25-26
inc 120
cp complete
No. of
embryos
5
Mean mitotic index (%) at different positions
All counts made near the blastoderm margin
(see text)
apm
2
A\
4-2
position
5
2-7
2-7
avm
20
avm
3- 2
mitotic index
8
2
4
6
2-2
2-6
2-6
2
1-2
1-8
avm 2
4
4
21
6
6
30
21
8
10 12
0-4 1-4 1-7 1-7 10
avm 2
4
6
11
8
2-4
10
12
1-6
21
14 16 18
20
22
24
10 0-8 0-3 0-2 0 1
10
12
14 16
01
0
>.
0-4
0-9
1-6
1-2
0-6
0-2
0
0
0
jet of chick saline (Britt & Herrmann, 1959). To keep the large pieces of tissue
flat, they were fixed in Bouin on a coverslip, with the edges held by the corners
of the coverslip. They were then stained in Ehrlich's haematoxylin and mounted
in Canada balsam.
Cell counts were made at a 400 x magnification, using a counting grid that
divided the field into 180 x 900 /tm2 squares. The mitotic index for each position
in each embryo is the mean number of mitotic cells found in five complete
microscope fields involving a total cell population of 10 3 -1-5 x 104, depending
on cell density. Table 3 gives the means of these mitotic indices for each embryonic stage examined.
In early embryos (15 h, Hamburger & Hamilton stage 2-3) there is no clear
distinction between intra- and extra-embryonic tissue. Counts were made near
the margin. In 24-h embryos (stage 4-6), the densely packed cells of the area
pellucida give a boundary, and counts were made at 2 mm intervals outwards
from the area pellucida/area opaca margin. In later embryos, the area vasculosa
mesoderm remains attached to the epiblast when the hypoblast is removed. The
area vasculosa/vitellina margin was therefore used as a starting point, with
36-2
568
J. R. DOWNIE
counts again made at 2 mm intervals outwards, to near the blastoderm edge.
After most of the yolk has been covered by the yolk-sac, the vitelline membrane
ruptures. The yolk-sac continues to grow in total volume, and the area vitellina
eventually becomes obliterated by the invasion of area vasculosa mesoderm.
Although no longer relevant to the inter-relationship of active edge cell migration and yolk-sac tissue growth, it was of interest to discover whether tissue
growth continued in the epiblast after rupture of the vitelline membrane. One
group of embryos (stage 25/26) represents the post-rupture situation. Proliferation clearly continues but in a restricted band. Since most of the epiblast at this
stage is in fact in the area vasculosa, an estimate of mitotic index in this area
was made. Serially-sectioned material from a single stage-25/26 embryo, where
the area vasculosa epiblast was cut obliquely, laying it out flat, gave a mitotic
index of 1 % (on 2 x 103 cells). Proliferation obviously still continues in the area
vasculosa epiblast.
Two general points emerge from the counts.
(1) Mitotic index is highest at the earliest stages examined: a mean of 4-2%
at stage 2-3, declining to a maximum of 2-1 % in the latest embryos examined
(stage 25-26).
(2) The mitotic index remains fairly uniform throughout the epiblast until
stage 17-18. Thereafter, mitosis becomes progressively restricted with two
low points - near the area vasculosa, and towards the edge - and a broad high
band between. Mitotic index towards the periphery becomes low only around
the time blastoderm expansion is completed. After this, mitosis is largely
restricted to an area between 2 mm and 10 mm from the margin of the area
vasculosa.
This pattern is unlike the 'ring blastema' of Haas & Spratt (1968), envisaged
as a narrow peripheral ring of ' undifferentiated' and rapidly dividing cells.
During the expansion period, mitosis occurs at a high level throughout the
extra-embryonic epiblast, rather than being restricted to a peripheral ring.
(2) Cell proliferation {doubling time)
Though mitotic indices are the easiest data to collect on proliferating tissues,
they may be difficult to interpret, since they can vary with both proliferation
rate and mitotic time (the time spent in the process of mitosis). A low mitotic
index may therefore indicate a low proliferation rate or any unusually short
mitotic time. Because of this, some workers, (e.g. Woodard, 1948; Dondua,
Efremov, Krichinskaya & Nikolaeva, 1966) have suggested that mitotic index is
a useless measure, at least in comparing different tissues.
A better measure of proliferation rate is doubling time (the time for the cell
population to double in numbers), equivalent to the generation time in a population where all cells are dividing with cell cycles of equal duration. Doubling time
can be calculated from the proportion of the cell population entering a fixed
point in the cell cycle in a specific time. In practice, this proportion is often
Mechanics of chick blastoderm expansion
569
Anterior
Left
Right
Posterior
B
Edge
109 ±1-2
Outer
12-2 ±0-4
|;-Vv;"'!f| Middle 11-6 ± 1 0
Inner
12-3 ±0-9
Fig. 4. Metaphase index (%) after 2 h Colcemid block in different parts of 1-day
epiblast. (A) for regions anterior, posterior, left and right. (B) for zones edge, outer,
middle and inner. Each index is given as the mean ± standard deviation of 11
embryos.
determined from the number of cells entering metaphase during a specific period
of treatment with colchicine or one of its derivatives. This has often been done
to chick embryos (see for example Woodard & Estes, 1944; Emanuelsson, 1961;
Pearce & Zwann, 1970) but usually to investigate embryonic rather than extraembryonic tissues, and with rather variable success. The problem lies in treating
all the cells at as near the same time as possible. Preliminary experiments in
which the drug was injected into either the yolk, the albumen, or both together
gave very variable results. The most reliable method was to set up embryos as
New cultures, adding the drug in a few drops of medium 199 (Biocult) both to
the albumen and directly on top of the embryo. Unfortunately, this worked well
with 1-day embryos only. All attempts to obtain reliable metaphase blocking
with later (3-day to 5-day) material failed, with 2-h metaphase indices sometimes
lower than simple mitotic indices for the same tissue. The reasons for this are
unknown.
For the metaphase blocks on 1-day embryos, Colcemid (Ciba) was used at a
concentration of 0-l^gm/ml ( 2 - 7 X 1 0 ~ 7 M ) , the lowest concentration to give
570
J. R. DOWNIE
reliable results. The blocking period was 2 h since after longer periods, cells
sometimes appeared abnormal. After 2 h incubation, the embryos were cooled,
the hypoblast removed as before and the epiblasts prepared for examination.
The epiblast of 11 embryos treated in this way was divided into zones as
shown in Fig. 4, and each zone counted separately as described before. The zone
marked 'edge' does not include the 'edge cells', but is a distinctive zone, about
90/*m wide, of densely packed cells adjacent to the edge. The 'edge cells'
themselves do not appear to divide at all. Colcemid treatment for as long as 6 h
failed to reveal any mitotic figures amongst them.
The results (Fig. 4) showed no significant differences in metaphase index (using
Student's t test) between any of the epiblast zones, confirming that in the 1-day
embryo, the whole of the extra-embryonic epiblast is proliferating at about the
same rate. The overall mean 2-h-blocked metaphase index was 11-6 ± 0-7 % (±
standard deviation). This can be converted to doubling time (T) using the
formula
lne2 x block duration (h)
T =
metaphase index (as a fraction)'
giving a doubling time of 11-9 ±0-7 h. Mitotic index is higher at 15 h (4-2%)
than at 24 h (2-7-3-2 %) and to deduce from this a higher proliferation rate
would fit in with Wylie (1972) and Emanuelsson (1965), who found a doubling
time in the earliest stages (after laying) of 7-8 h.
After 1 day, mitotic index declines and cell division becomes restricted in
area. The precise effect of this on proliferation rate depends on what happens to
mitotic time. Few seem to have studied this in detail. Dondua et ah (1966) found
variation from 34-90 min in different regions of stage 3-5 chick embryos, but
did not study later ones. Perhaps more suggestively, Fujita (1962) found a mitotic
time in 1-day neural tube of 24 min, lengthening to 60 min in the same tissue at
6 days. If a similar lengthening occurs in chick epiblast, then the drop in mitotic
index may actually underestimate the drop in proliferative activity.
In summary, at the start of expansion, all cells of the extra-embryonic epiblast,
apart from the edge cells, are proliferating at a rapid rate. As expansion proceeds, proliferation becomes progressively restricted in area, and probably very
reduced in rate.
(3) Cell size and shape in the expanding blastoderm
The yolk-sac epiblast stretches from the area pellucida to the blastoderm edge.
It is generally a single epithelial layer, though at some points, e.g. the epiblast
of the area vasculosa and its periphery, there is enough overlapping to consider
it a bilayer.
Cell packing density was determined from the Bouin-fixed material used for
mitotic index calculations. Wax sections turned out to be unsuitable for epiblast
height determinations. Instead, yolk-sacs were prepared as for transmission
Mechanics of chick blastoderm expansion
571
Table 4. Cell diameter (/*m) in the extra-embryonic epiblast at different stages and
in different positions
Diameters are calculated from cell density counts: where there is significant overlapping (marked *), these figures do not accurately reflect the yolk-sac surface area
occupied by each cell. Abbreviations as in Table 3.
Stage
HH 1
unincubated
cp start
HH 1
inc 6-5
cp start
HH2-3
inc 15
cp start
HH4-6
inc 24
cpi
H H 12
inc 48
cp|
HH 17-18
inc 72
cpi
HH 17-23
inc 90
cp complete
HH 25-26
inc 120
cp complete
No. of
embryos
Mean cell diameter (/tm) at different positions
7
Counts made near the blastoderm margin
13-4
3
Counts made near the blastoderm margin
10-2
5
Counts made near the blastoderm margin
14-6
apm
2
4
16-3
avm
19- 7
2
16-6
4
cell diameter
6
8
17 6
12
18-9
14 0
avm
^
15- 9
4
8
11-1*
avm
12-3*
4
16-3
8
200
12
114*
avm
130*
4
16-2
8
18-9
12
10-8*
12-0*
14-6
180
position
16-:
16
20
21-6
21-8
electron microscopy, sections cut at 1 /*m and stained with methylene blue.
Epiblast height was then measured from camera lucida drawings.
The mean area of epiblast surface occupied by individual cells (ignoring overlapping) at different stages and in different positions is given in Table 4. Some
of these figures, along with epiblast height determinations, are used in Table 5
to calculate cell volume at three stages. Figure 5 shows camera lucida drawings
from epiblast transverse sections to indicate the appearance of the cells at
different positions and stages.
Two general points emerge from this analysis:
(1) Extra-embryonic epiblast cell volume is halved during the first day's
incubation, and thereafter remains fairly constant. A reduction in cell size has
been noted by Bancroft & Bellairs (1974), though they give no measurements.
(2) The shape taken up by the epiblast cells shows a definite pattern of
572
J. R. DOWNIE
Table 5. Derivation of extra-embryonic epiblast cell volume at different stages and
in different positions
Cell area (/*m2) calculated from density counts on Bouin-fixed flat-mounted
epiblasts. Epiblast height (/im) measured from 1 [im araldite sections.
Stage
(incubation
time)
Unincubated
24 h
72 h
Position
Mean cell
area Om2)
Mean epiblast
height (/*m)
141-9
19-8
Near edge
221-3
5-3
Near edge area opaca
248-2
4-8
Not near edge
186-6
7-5
Area vitellina near edge
Area vitellina near area
106-5
12-3
vasculosa*
465-5
3-3
Area vasculosa*
* Positions with significant overlapping.
Mean cell
volume (/Mm3)
2809-6
1172-9
1191-4
1399-5
13100
1536-2
development. In the unincubated blastoderm, they are close-packed and tall. Soon
after the onset of expansion, they become considerably flattened. This persists
throughout expansion, though flattening seems less pronounced in the 3-day
than in the 1-day epiblast. A new feature in the later stages (continuing after the
yolk mass is covered) is the appearance of a zone of high cell overlap in the
epiblast around the periphery of the area vasculosa. Epiblast cells within the
area vasculosa are in a bilayer and are very flattened.
We can perhaps regard the unincubated blastoderm partly as a reservoir of
tissue, compactly stored as tall cells. Once expansion begins, these cells assume
a shape that covers a large area. This flat shape is partly intrinsic, maintained to
some degree by microtubules (Downie, 1975), and partly produced by the
stretching effect of edge-cell tension, at least during the early stages of expansion.
Later, just before vitelline membrane rupture, the yolk-sac is very tightly
opposed to the vitelline membrane, due to the large volume of sub-blastodermal
fluid (New, 1956). Any edge-cell tension that persists until then could hardly be
transmitted throughout the yolk-sac. This may partly account for the considerable variations in cell density and arrangement that are then apparent.
The role of tension in blastoderm expansion
New (1959) first noticed the tension in early blastoderms, his observation being
confirmed by Bellairs et al. (1967); but in neither study was there any attempt to
measure changes in blastoderm tension as expansion proceeds. New found that
edge-cell attachment is essential if expansion is to occur, and suggested that this
is because the tension created by the edge cells is in some way essential for
expansion. It is difficult, however, to imagine how blastoderm expansion could
occur in any way other than by active outgrowth, involving cell migration at the
Mechanics of chick blastoderm expansion
573
B
D
50 urn
Fig. 5. Camera lucida drawings of epiblast cells, all to the same scale and all from
1 /Am araldite sections. (A) Unincubated embryo, near the blastoderm periphery,
(B) 23-h incubated embryo, area opaca. (C) 74-h embryo, near blastoderm periphery. (D) 74-h embryo, area vitellina near the area vasculosa; a region of high cell
overlap, (E) 74-h embryo, area vasculosa.
periphery. Alternative mechanisms such as appositional growth or cell spreading
are not real alternatives since they are likely also to require peripheral attachment and cell migration.
How essential then is the tension created by the edge cells ?
New suggested that tension is necessary to maintain the epiblast as a flattened
monolayer. His test was, however, not entirely unequivocal. He showed that
when yolk-sac epiblast is prevented from expanding by anchoring its edge, it
continues to grow, inevitably forming a multilayer. This does not show that the
574
J. R. DOWNIE
rather high tension found in the 1-day blastoderm is necessary to prevent this
multi-layering. Epiblast cells are well able to maintain a flattened shape (Downie,
1975) and post-mitotic cells would have little resistance to overcome in assuming
this shape: they would certainly not require the assistance of a tension capable
of stretching them to 40 % more than their resting area.
I would like to suggest that the high level of tension around 20-24 h is the
result of an imbalance between active migration and proliferation. At the start
of expansion, cells are columnar and tightly packed, giving considerable 'slack'
that the edge cells pull out in the first few hours. Tension in the sheet is therefore
rather low. Around 20-24 h, this 'slack' has been used up and, as expansion
continues, tension increases. Thereafter, the amount of new tissue produced by
proliferation catches up with edge-cell migration (as suggested in Fig. 3) and
tension is reduced. It seems likely that the fall in proliferation activity in the
later stages of expansion is correlated with this 'catching-up' process.
I should like to thank Professor D. R. Newth and Professor Ruth Bellairs for reading the
manuscript and suggesting several improvements. This work was started while I was in
receipt of an S.R.C. studentship and under the guidance of Professor M. Abercrombie at
University College London, and continued in the Department of Zoology, University of
Glasgow.
REFERENCES
M. (1946). Estimation of nuclear population from microtome sections. Anat.
Rec. 94, 239-247.
BANCROFT, M. & BELLAIRS, R. (1974). The onset of differentiation in the epiblast of the chick
blastoderm. Cell and Tissue Res. 155, 399-418.
BELLAIRS, R. (1963). Differentiation of the yolk sac of the chick studied by electron microscopy. /. Embryol. exp. Morph. 11, 201-225.
BELLAIRS, R., BOYDE, A. & HEAYSMAN, J. E. M. (1968). The relationship between the edge of
the chick blastoderm and the vitelline membrane. Wilhelm Roux Arch. EntwMech. Org.
163, 113-121.
BELLAIRS, R., BROMHAM, D. R. & WYLIE, C. C. (1967). The influence of the area opaca on
the development of the young chick embryo. /. Embryol. exp. Morph. 17, 195-212.
BELLAIRS, R. & NEW, D. A. T. (1962). Phagocytosis in the chick blastoderm. Expl Cell Res.
26, 275-279.
BRITT, L. G. & HERRMANN, H. (1959). Protein accumulation in early chick embryos grown
under different conditions of explantation. / . Embryol. exp. Morph. 7, 66-72.
CURTIS, A. S. G. & BUULTJENS;, T. E. J. (1973). Cell adhesion and locomotion. In Locomotion
of Tissue Cells, pp. 171-9. Ciba Foundation Symposium 14 (new series).
DONDUA, A. K., EFREMOV, V. I., KRICHINSKAYA, E. B. & NIKOLAEVA, I. P. (1966). Mitotic
index, duration of mitosis and proliferation activity in the early phases of the development
of the chick embryo. Ada bioi, Szeged. 17, 127-143.
DOWNIE, J. R. (1974). Behavioural transformation in chick yolk-sac cells. /. Embryol. exp.
Morph. 31, 599-610.
DOWNIE, J. R. (1975). The role of microtubules in chick blastoderm expansion - a quantitative study using colchicine. /. Embryol. exp. Morph. 34, 265-277.
DOWNIE, J. R. & PEGRUM, S. M. (1971). Organisation of the chick blastoderm edge.
J. Embryol. exp. Morph. 26, 623-635.
EMANUELSSON, H. (1961). Mitotic activity in chick embryos at the primitive streak stage.
Actaphysiol. scand. 52, 211-233.
ABERCROMBIE,
Mechanics of chick blastoderm expansion
575
H. (1965). Cell multiplication in the chick blastoderm up to the time of laying.
Expl Cell Res. 39, 386-399.
FUJITA, S. (1962). Kinetics of cellular proliferation. Expl Cell Res. 28, 52-60.
HAAS, H. & SPRATT, N. T. (1968). Studies on growth polarity in the ring blastema (germ wall
ring) of young chick embryos. Physiol. Zool. 41, 129-148.
HAMBURGER, V. & HAMILTON, H. L. (195.1). A series of normal stages in the development of
the chick. /. Morph. 88, 49-92.
JAMES, D. W. & TAYLOR, J. F. (1969). The stress developed by sheets of chick fibroblasts in
vitro. Expl Cell Res. 54, 107-110.
NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro.
J. Embryol. exp. Morph. 3, 326-331.
NEW, D. A. T. (1956). The formation of sub-blastodermic fluid in hens' eggs. /. Embryol.
exp. Morph. 4, 221-227.
NEW, D. A. T. (1959). The adhesive properties and expansion of the chick blastoderm.
/. Embryol. exp. Morph. 7, 146-164.
PEARCE, T. L. & ZWANN, J. (1970). A light and electron microscopic study of cell behaviour
and microtubules in the embryonic chicken lens using Colcemid. /. Embryol. exp. Morph.
23, 491-507.
SCHLESINGER, A. B. (1952). Analysis of growth of the chick marginal blastoderm. Science,
N. Y. 116, 64-5.
SIMNETT, J. D. (1968). The measurement of mitotic incidence and radioautographic labelling
index from tissue sections: some mathematical considerations. // R. microsc. Soc. 88,
EMANUELSSON,
371-382.
T. M. (1948). The mitotic index in the chick embryo. Am. Nat. 82, 129-136.
T. M. & ESTES, S. B. (1944). Effect of colchicine on mitosis in the neural tube of
the 48-hr chick embryo. Anat. Rec. 90, 51.
WYLIE, C. C. (1972). The appearance and quantitation of cytoplasmic ribonucleic acid in the
early chick embryo. /. Embryol. exp. Morph. 28, 367-384.
WOODARD,
WOODARD,
{Received 19 November 1975; revised 23 January 1976)