/. Embryol. exp. Morph. Vol. 64, pp. 11-22, 1981
Printed in Great Britain © Company of Biologists Limited 1981
\\
A SEM study of cell shedding during the
formation of the area pellucida in the
chick embryo
By BARRY FABIAN 1 AND HEFZIBAH EYAL-GILADI2
From the Department of Zoology, Hebrew Univerity of Jerusalem
SUMMARY
Intrauterine eggs at different stages of development were retrieved from hens and the
blastodiscs were dissected out and prepared for scanning electron microscopy. Our observations trace the polarized shedding of cells from the central area of the lower layers of the
blastodisc, which leads to the formation of the area pellucida. Prior to the shedding, single
central blebs appear on the ventral side of the cells facing the sub-blastodermic cavity. We
also noted the presence of lobopodia which establish contact with nearby cells. The possible
function of the lobopodia is discussed. The cells of the blastodisc are at the same time (stages
IV and V, E.-G.&K). also intimately interconnected via a profusion of threadlike cytoplasmic processes (filopodia). When shedding starts the blebs as well as the lobopodia disappear, and thefilopodiaundergo typical withdrawal steps such as showing a beaded stage and
appearing drawn out or taut, before they finally disrupt to leave behind a smooth round cell
which is detached from its neighbours. The beading phenomenon points to the possibility of a
disassembly of the microtubular skeleton of the filopodia operated by some physical force
influenced by gravity.
INTRODUCTION
At the end of cleavage, the first obvious morphogenetic event in the development of the chick embryo is the shedding of cells from the central area of the
blastodisc into the subgerminal cavity, where they then degenerate. The shedding
process proceeds as a wave from the future posterior end to the future anterior
end of the embryo and reduces the thickness of the central area of the blastodisc
from a stratified epithelium five to six cell layers thick into a single-layered
epithelium. The resulting thin epithelial layer is the area pellucida; it is transparent and is surrounded by the multilayered area opaca (Eyal-Giladi &
Kochav, 1976). This polarized formation of the area pellucida through a directional shedding of cells is thus seen to anticipate the polarity of the future headto-tail axis of the embryo.
The polarity of the head-to-tail axis in the chick embryo is determined by the
1
Present address: Zoology Department, University of the Witwatersrand, Johannesburg,
Republic of South Africa.
2
Author's address: Department of Zoology, Hebrew University of Jerusalem, Jerusalem,
Israel.
12
B. FABIAN AND H. EYAL-GILADI
force of gravity (Kochav & Eyal-Giladi, 1971). These authors found that if intact
eggs which had been in the uterus for 10 h are removed and further incubated in a
vertical position, with the pointed end facing upwards, then the blastodisc is
found to orientate in an inclined position. Thereafter the upwards facing end of
the blastodisc will develop into the tail-end while the downwards facing end will
develop into the head-end, of the embryo. The polarity of the embryo can be
completely reversed by inverting the 10 h aborted egg during the first 3 h of
incubation in vitro, whereas by 6-7 h of incubation the polarity becomes completely fixed and can no longer be reversed whatever the position of the egg. In
between these two limits a series of intermediate positions of the embryo are
expressed (Eyal-Giladi & Fabian, 1980). It is of interest that the latter experiment
shows that the establishment of the head-to-tail polarity of the blastoderm in the
different orientations, is indeed preceded by a posterior-anterior process of cell
shedding which brings about the formation of the area pellucida.
In vivo, the shedding of cells from the blastodisc during the formation of the
area pellucida is accomplished during the final 6-8 h of the eggs' sojourn in the
uterus (stage VII to stage X; Eyal-Giladi & Kochav 1976). Thereafter the egg is
laid at a stage in its development which overlaps with the beginning of the formation of the hypoblast.
In the present investigation the process of cell shedding during the formation
of the area pellucida is examined with the scanning electron microscope.
MATERIALS AND METHODS
Eggs were removed at various times from the uteri of hens by the application
of external pressure, as described by Eyal-Giladi & Kochav (1976). The eggs were
opened under Ca 2+ enriched chick saline (115 g NaCl, 5-92 g KC1, 2-88 g CaCl2
anhydrous, per 16 1 distilled water). The blastodisc together with both a covering
of vitelline membrane and about 5 mm of adhering yolk were removed and
placed in fresh chick saline. The ventral surface of the blastodisc was carefully
ABBREVIATIONS FOR FIGURES
ao
ap
be
bl
bdf
area opaca
area pellucida
border between cells
bleb
beaded drawn-out or taut
(filopodium)
fibre
dc
df
f
h
lp
detaching cell
drawn-out or taut fibre (filopodium)
filopodia
emerging hypoblastic cell
lobopodium
Figs. 1-4 General view of the ventral suiface of blastodiscs at stages IV, VI, VIIVIII, and IX-X respectively, x 40. Note that the stage V1I-VI1I blastodisc (Fig. 3)
has an eccentric patch from which cells have been cleared. This area is the posterior
part of the area pellucida which is forming and represents a limited area up to stage
VII-VIII from which cells have been already shed into the subgerminal cavity, as the
wave of cell shedding proceeds from the future posterior to the future anterior end of
the embryo (Eyal-Giladi & Kochav, 1976; Kochav, Ginsburg & Eyal-Giladi, 1980).
13
SEM study of area pellucida in chick embryo
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B. FABIAN AND H. EYAL-GILADI
exposed using steel needles, and a small quantity of glutaraldehyde fixative
solution was gently introduced with a pipette over the blastodisc. Glutaraldehyde
fixative is 2 % glutaraldehyde in Ca2+-enriched phosphate buffer; phosphate
buffer is 41-5 ml of 2£% NaH 2 PO4.H2O and 8-5 ml of 2\% NaOH (pH 7-27-4), including 90 mg CaCl2. 2 % glutaraldehyde in chick saline (adjusted to
pH 7-2) was also used as a fixative with no observable change in the quality of
fixation. After introducing fixative at room temperature above the blastodisc,
the latter was promptly dissected away from the membrane and placed for
2 h or overnight in fresh fixative at 4 °C. The blastodisc was then washed
three times in buffer or saline and dehydrated in ethanol (15 min in 50%, 15 min
70%, 15 min in 90%, three times 30 min in 100%) and stored overnight in fresh
ethanol. Still under ethanol the blastodiscs were then placed into small envelopes
prepared by stapling together non-fluffy tissue paper such as Velin Tissue
(General Paper and Box Co., Glam., U.K.). After changing the ethanol, the
blastodiscs in their envelopes were transferred in ethanol for critical-point
drying to a Polaron E 3000. The blastodiscs were orientated under a dissecting
microscope with their ventral (inner) sides facing upwards, mounted on doublesided sticky tape attached to stubs, coated with gold in a Polaron E 5000 and
viewed in a Cambridge Stereoscan S410.
RESULTS
The SEM observations of the lower, inner side of blastodiscs at different
stages of development prior to and during the formation of the area pellucida are
presented below. The stages (Roman numerals) are according to Eyal-Giladi &
Kochav (1976), as is the determination of elapsed time in hours from when the
egg enters the uterus.
Stage IV (8-9 h)
Cleavage is proceeding in the blastodisc. At the periphery there are still some
very big blastomeres, and some uncleaved cytoplasm (Fig. 1). The cells are
intimately connected by cytoplasmic processes - filopodia (Fig. 12) and their
lower surface is relatively smooth.
Stage V(10h)
Cleavage of the blastodisc is almost complete. The filopodia look more
organized, like ropes (Fig. 13). The lower surface of many cells shows a big
Fig. 5. The fracture area of a stage VI blastodisc (ventral side up) demonstrating that
it is 5-6 cells in thickness, x 200.
Fig. 6. The fracture area of a stage IX-X blastoderm (ventral side up). The cell
shedding is almostfinishedand the blastoderm is now one cell thick, x 200.
SEM study of area pellucida in chick embryo
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15
16
B. FABIAN AND H. EYAL-GILADI
central bleb (Fig. 7). Some cells have formed an elongate process, or lobopodium
(Fig. 8). A few lobopodia seem to have extended over the ventral surface and
have established contacts with nearby cells (Fig. 7).
Stage VI (12 h)
The cleavage process is complete and the blastodisc is five to six cells in
thickness (Fig. 5). The arrangement of cells in the lower surface of the blastodisc
appears looser as some cells seem to have withdrawn their fiolopodia and have
rounded up. In most of the lower layer cells the filopodia appear to be drawn out
or taut. Occasionally these processes show bead-like thickenings (Fig. 9).
Stage VIT-VIII (16 h)
Lower layer cells are falling off the central area at the posterior side of the
blastodisc (Fig. 3) resulting in the formation of a rudimentary area pellucida
(See also Kochav, Ginsburg & Eyal-Giladi, 1980). At a more anterior region
erosion is just beginning and the still attached cells are interconnected by thin
stretched out or taut fibres (Fig. 10), often decorated by one or more beads
(Fig. 14). Figure 10 also shows vestiges of fibres on the now smooth 'erupting'
cells. It would appear that the interconnecting fibres have been disrupted and are
possibly retracted.
Stage IX-X (18 h)
The area pellucida and area opaca are clearly demarcated (Fig. 4). Nevertheless the process of cell shedding is continued in some areas, while other areas are
seen to be almost entirely 'clean'. This is well seen in Fig. 6, which shows the
epithelial arrangement of the blastoderm. Among the detaching yolk-laden cells
there begin to appear a number of small round hypoblastic cells (Fig. 11). The
lower surface of the blastoderm is characterized by the formation of a dense
arrangement of intercellular connections, giving it a woven carpet-like appearance.
Figs. 7-11. Ventral surface of blastodiscs. x 750.
Figs. 7 and 8. Stage V. The cells show ventral blebs and lobopodia and are
intimately connected by filopodia.
Fig. 9. Stage VI. Blebs and lobopodia disappear and filopodia of the most ventral
cells appear stretched or taut and beaded.
Fig. 10. Stage VII-VIII. The most ventral cells have been shed and cells of the
deeper layers are now becoming involved in cell shedding. Note the presence of
drawn-out or taut fibres (filopodia).
Fig. 11. Stage IX-X. Only a few large cells are still to be shed. The epithelial cells of
the blastoderm are intimately connected with one anothei. Single small round hypoblastic cells appear amongst them.
SEM study of area pellucida in chick embryo
17
18
B. FABIAN AND H. EYAL-GILAID
Figs. 12-15. Filopodia at different stages representing degrees of detachment, x 3750.
Fig.
Fig.
Fig.
Fig.
12 Stage IV. Cells closely connected by short filopodia.
13. Stage V. Filopodia appear somewhat taut.
14. Stage VII-VIII. Beaded fibres.
15. Stage X. Cells of the one-cell-thick blastoderm are intimately connected.
DISCUSSION
This study has employed the scanning electron microscope to follow the
changes in the ventral side of the uterine chick blastodisc during the formation of
the area pellucida.
The general view with the SEM at low magnifications is very similar to the
pictures of the normal stages as shown by Eyal-Giladi & Kochav (1976). The
SEM study of area pellucida in chick embryo
19
process of cell shedding has recently been followed with the use of thin plastic
sections, and these findings are complementary to and supportive of our SEM
observations (Kochav et al. 1980). The pictures of the fracture area of a stage VI
blastodisc (Fig. 5) and a stage IX-X blastodisc (Fig. 6) reveal however with
greater clarity than a microscopic slide the morphological details of the sloughing
of the ventral cells and the transformation of the multilayered embryonic disc
into a single-layered epithelial blastoderm.
The new data in this study concern the rapidly changing appearance of the
ventral cells and their cytoplasmic processes. At the end of cleavage (stages IV
and V), two kinds of cellular outgrowths were encountered, the short thread-like
processes - filopodia, by which the blastomeres are interconnected and a single
much wider ventral outgrowth (per cell) typical of the above stages only. The
latter appears as a bleb and might elongate to form a rootlet-like protrusion or
lobopodium which was sometimes seen stretching out to attach to a nearby cell.
A similar phenomenon has been described by Trinkhaus (1973a, b) in the late
cleavage stages of Fundulus, based on a time-lapse cinematographic study, in
which both blebs and lobopodia were observed. In these in vivo observations on
Fundulus, Trinkhaus described how the blebs were seen to give rise to finger-like
lobopodia. The tips of the lobopodia appeared to adhere to the surface of
adjacent cells and to spread lightly on them, and sometimes became stretched
thin and taut. Furthermore the lobopodia were able to shorten and pull their cell
bodies forward. During this locomotory activity the cell bodies remained
rounded and were rarely seen to flatten (see Trinkhaus, 1976, for review).
The above observations on Fundulus fit very well from a morphological standpoint, with the situation in the uterine chick, as presented through our SEM
analysis. From the nature of an SEM analysis it is not of course possible to
conclude that the blebs actually give rise to lobopodia, as concluded by Trinkhaus
from his in vivo observations on Fundulus. While our SEM observations cannot
record cell motility, the actual presence of lobopodia on the chick blastomeres
does suggest, based on the work of numerous other investigations, that such
cells may indeed be motile (see Trinkhaus, 1976; Dyson, 1978, Chapter 9).
If the latter proposal is accepted then two alternate conclusions may be drawn;
firstly that the manifestation of motility by these blastomeres may merely
represent a typical 'at rest' state of these cells in which the cells are continually
probing the microenvironment, and may thus be described to be in a state of
dynamic equilibrium. Alternatively, the presence of the extended lobopodia in
some blastomeres may be an expression of a directed cell movement or rearrangement and may be indicative of a particular morphogenetic event that is
taking place, which may or may not be coupled to the shedding process. Perhaps
the lobopodia serve a tactic function, as could occur in a chemotactic response of
the blastomeres to a special microenvironment (see Dyson, 1978, Chapter 9).
While the manifestation of lobopodia in the early blastomeres is of interest, it is
premature to propose a function for them until more is known about the precise
20
B. FABIAN AND H. EYAL-GILADI
developmental fate of these 'lobopodial' blastomeres. We do however agree with
Trinkhaus that * contrary to popular notions' the blebbing is an expression of an
early cellular differentiation. In the case of the chick we can even pinpoint with
certainty that the blebbing precedes the determination of the anterior-posterior
axis.
At stage VI the area pellucida starts to form, the blebbing phenomenon is over,
and the filopodia interconnecting the ventral cells become involved in a rapid and
very characteristic process of disruption. The filopodia do not differ morphologically from the thin cytoplasmic processes of a great variety of cells ranging
from protozoa to higher vertebrates. Attempts have been made by a number of
workers to investigate the role of such processes in cell movement in culture
(Abercrombie, Heaysman & Pegrum, 1971; Overton, 1977; Albrecht-Buehler,
1976, 1978), in the expansion of chick blastoderm (Downie & Pegrum, 1971;
Downie, 1974,1976; Chernoff & Overton, 1977), during the atypical aggregation
of the embryonic shield of annual fishes (Wourms, 1972«, b; Lesseps, van Kessel
& Denuce, 1975), in gastrulation movements in different groups (Tilney &
Gibbins, 1969; Vakaet & Hertoghs-De Maere, 1973; Kubota & Durston, 1978;
Sanders, Bellairs & Portch, 1978) and in the in vitro development of the mouse
embryo at the implantation stage (Gonda & Hsu, 1980).
We feel, however, that the papers relevant to our study are the ones dealing
with the role of filopodia in cell detachment when exposed to changing environmental conditions. Axopods of Heliozoa were described to become beaded after
exposure either to high hydrostatic pressures or to low temperatures (Kitching,
1957; Tilney, Hiramoto & Marsland, 1966; Tilney & Porter, 1967) and consequently to gradually disappear. The process was shown to involve the dismantling of the axoneme formed of bundles of microtubules.
Similar phenomena were described for vertebrate cells in culture (Taylor &
Robbins, 1963; Revel, Hoch & Ho, 1974) exposed to low temperature, high pH
or trypsin. The morphological changes which the filopodia undergo during the
different treatments are identical and involve their elongation and thinning out
to form so called retraction fibres which finally disrupt and get absorbed into the
rounding up cells.
In our present SEM observations we assume that the appearance of beads on
the filopodia and the disruption of the filopodia, followed by the formation of
smooth round erupting cells are the morphological transformations that lead to
cell shedding. The fact of cell shedding implies the loss of some sort of'adhesiveness' between the cells concerned. As a consequence of the directional formation
of the area pellucida (see Introduction) it is possible to postulate the existence of
a wave of filopodial disruption based perhaps on a gradient of decreasing cellular
'adhesiveness' from the future posterior to the future anterior side of the blastodisc. Furthermore, as the directional formation of the area pellucida (correlating with the determination of the anterior-posterior polarity in the chick) is
gravity dependent (Kochav & Eyal-Giladi, 1971; Eyal-Giladi & Fabian, 1980) it
SEM study of area pellucida in chick embryo
21
would appear that the proposed wave of 'filopodial disruption' would in some
manner be driven by the force of gravity.
In conclusion we therefore suggest that in the symmetrizing blastodisc
(which is obliquely orientated in the egg) there exists a gradient which is somehow
connected with the blastodisc's position in space (gravity) and which causes the
orderly disassembly of the microtubules within the filopodia interconnecting
the lower layer cells (Eyal-Giladi, unpublished results). This idea is corroborated by the reaction of stage VI uterine embryos (prior to cell shedding) to
storage at 15 °C for 5 days (Eyal-Giladi, unpublished results) which results in a
massive nonpolarized cell shedding reminiscent of the reaction to cold treatment
of the variety of cells described above.
Our appreciation and thanks to Professor B. T. Balinsky for his critical reading of this
manuscript. We are grateful to Mr A. Niv for the skilled printing of the pictures. H. EyalGiladi is supported by a research grant from the Israeli Academy of Science. B. Fabian is
supported by a C.S.I.R. South African overseas bursary and travel grant.
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(Received 21 August 1980, revised 16 March 1981)