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 the filopodia undergo 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.

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 head-to-tail axis of the embryo.

The polarity of the head-to-tail axis in the chick embryo is determined by the 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.

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 Ca2+ 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 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 % NaH2 PO4.H2O and 8·5 ml of % NaOH (pH 7·2–7·4), 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.

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 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 VII-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.

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 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 developmental fate of these Tobopodial’ 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, 1972a, b;Lesseps, van Kessel & Denucé, 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 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. Eyal-Giladi is supported by a research grant from the Israeli Academy of Science. B. Fabian is supported by a C.S.l.R. South African overseas bursary and travel grant.

Abercrombie
,
M.
,
Heaysman
,
J. E. M.
&
Pegrum
,
S. M.
(
1971
).
The locomotion of fibroblasts in culture
.
Expl Cell Res
.
67
,
359
367
.
Albrecht-Buehler
,
G.
(
1976
).
Filopodia of spreading 3T3 cells
.
J. Cell Biol
.
69
,
275
286
.
Albrecht-Buehler
,
G.
(
1978
).
The tracks of moving ceils
.
Sci. Amer
.
238
,
68
76
.
Chernoff
,
A. G.
&
Overton
,
J.
(
1977
).
Scanning electron microscopy of chick epiblast expansion on the vitelline membrane
.
Devi Biol
.
57
,
33
46
.
Downie
,
J. R.
(
1974
).
Behavioural transformation in chick yolk-sac cells
.
J. Embryol. exp. Morph
.
31
,
599
610
.
Downie
,
J. R.
(
1976
).
The mechanism of chick blastoderm expansion
.
J. Embryol. exp. Morph
.
35
,
559
575
.
Downie
,
J. R.
&
Pegrum
,
S. M.
(
1971
).
Organisation of the chick blastoderm edge
.
J. Embryol. exp. Morph
.
26
,
623
635
.
Dyson
,
R. D.
(
1978
).
Cell Biology. A molecular approach
. 2nd ed.
Boston
:
Allyn and Bacon, Inc
.
Eyal-Giladi
,
H.
&
Fabian
,
B. C.
(
1980
).
Axis determination in uterine chick blastodiscs under changing spatial positions during the sensitive period for polarity
.
Devi Biol
.
77
,
228
232
.
Eyal-Giladi
,
H.
&
Kochav
,
S.
(
1976
).
From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. 1. General morphology
.
Devi Biol
.
49
,
321
337
.
Gonda
,
M. A.
&
Hsu
,
Y.
(
1980
).
Correlative scanning electron, transmission electron and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage
.
J. Embryol. exp. Morph
.
56
,
23
39
.
Kitching
,
J. A.
(
1957
).
Effects of high hydrostatic pressures on Actinophrys sol (Heliozoa)
.
J. exp. Biol
.
34
,
511
517
.
Kochav
,
S.
&
Eyal-Giladi
,
H.
(
1971
).
Bilateral symmetry in chick embryo determination by gravity
.
Science
171
,
1027
1029
.
Kochav
,
S.
,
Ginsburg
,
M.
&
Eyal-Giladi
,
H.
(
1980
).
From cleavage to primitive streak formation: a complimentary normal table and a new look at the first stages of the development of the chick. 11. Microscopic anatomy and cell population dynamics
.
Devi Biol
.
79
,
296
308
.
Kubota
,
H. Y.
&
Durston
,
A. J.
(
1978
).
Cinematographical study of cell migration in the opened gastrula of Ambystoma mexicanum
.
J. Embryol. exp. Morph
.
44
,
71
80
.
Lesseps
,
R. J.
,
Van Kessel
,
A. H. M. G.
&
Denucé
,
J. M.
(
1975
).
Cell patterns and cell movements during early development of an annual fish, Notabranchius neumanni
.
J. exp. Zool
.
193
146
.
Overton
,
J.
(
1977
).
Response of epithelial and mesenchymal cells to culture on basement lamella observed by scanning microscopy
.
Expl Cell Res
.
105
,
313
323
.
Revel
,
J. P.
,
Hoch
,
P.
&
Ho
,
D.
(
1974
).
Adhesion of culture cells to their substratum
.
Expl Cell Res
.
84
,
207
218
.
Sanders
,
E. J.
,
Bellairs
,
R.
&
Portch
,
P. A.
(
1978
).
In vivo and in vitro studies on the hypoblast and definitive endoblast of avian embryos
.
J. Embryol. exp. Morph
.
46
,
187
205
.
Taylor
,
A. C.
&
Robbins
,
E.
(
1963
).
Observations on microextensions from the surface of isolated vertebrate cells
.
Devi Biol
.
7
,
660
673
.
Tilney
,
L. G.
&
Gibbins
,
J. R.
(
1969
).
Microtubules and filaments in the filopodia of the secondary mesenchyme cells of Arbacia punctulata and Echinarachnius parma
.
J. Cell. Sci
.
5
,
195
210
.
Tilney
,
L. G.
,
Hiramoto
,
Y.
&
Marsland
,
D.
(
1966
).
Studies on the microtubules in Heliozoa. III. A pressure analysis of the role of these structures in the formation and maintenance of the axopodia of Actinosphaerium nucleofilum (Barrett)
.
J. Cell Biol
.
29
,
77
95
.
Tilney
,
L. G.
&
Porter
,
K. R.
(
1967
).
Studies on the microtubules in Heliozoa. II. The effect of low temperature on these structures in the formation and maintenance of the axopodia
.
J. Cell Biol
.
34
,
327
343
.
Trinkhaus
,
J. P.
&
Lentz
,
T. L.
(
1967
).
Surface specializations of Fundulus cells and their relation to cell movements during gastrulation
.
J. Cell Biol
.
32
,
139
153
.
Trinkhaus
,
J. P.
(
1973a
).
Surface activity and locomotion of Fundulus deep cells during blastula and gastrula stages
.
Devi Biol
.
30
,
68
103
.
Trinkhaus
,
J. P.
(
1973b
).
Modes of cell locomotion in vivo
.
From: Locomotion of Tissue Cells Ciba Foundation Symposium
14
(new series)
233
249
.
Trinkhaus
,
J. P.
(
1976
).
On the mechanism of metazoan cell movements
.
In: The Cell Surface in Animal Embryogenesis and Development
(ed.
G.
Poste
&
G. L.
Nicolson
.
Amsterdam, New York, Oxford
:
North Holland
.
Vakaet
,
L.
&
Hertoghs-De Maere
,
E.
(
1973
).
Jonctions intercellulaires dans le feuillet inférieur du jeune blastoderme de Poulet, étudiées au microscope électronique à balayage
.
C.r. Séanc. Soc. Biol
.
167
,
1300
1303
.
Wourms
,
J. P.
(
1972a
).
Developmental biology of annual fishes. I. Stages in the normal development of Aust r of undulas myersi Dahl
.
J. exp. Zool
.
182
,
143
168
.
Wourms
,
J. P.
(
1972b
).
Developmental biology of annual fishes. II. Naturally occurring dispersion and reaggregation of blastomeres during the development of annual fish eggs
.
J. exp. Zool
.
182
,
169
200
.