/ . Embryol. exp. Morph. Vol. 23, 3, pp. 739-749, 1970
Printed in Great Britain
739
Differentiation potencies
of the young chick blastoderm as revealed
by different manipulations
II. Localized damage and hypoblast removal experiments
By H E F Z I B A H
From the Department
of Zoology,
EYAL-GILADI1
The Hebrew
University,
Jerusalem
SUMMARY
Transversely folded chick blastoderms of unincubated to primitive streak stages were used
in two different experimental series to check the following:
(a) The effect of local damage (right corner) on the embryo-forming potencies of the
blastoderm.
(b) The effect of the removal of the hypoblast on the stability of the embryo-forming potencies
of the primitive streak stage blastoderm.
The conclusions are:
(a) Intact, transversely folded, primitive streak stage blastoderms put on the culture medium
with their posterior half downwards are relatively stable and the embryo forms from
their posterior side.
(b) Younger blastoderms are less stable and reveal the inherent asymmetry of the blastoderm
by the formation of left-side embryos.
(c) Partial extirpation of the right corner in such blastoderms causes a remarkable local rise
in the embryo-forming potencies of the right side in both age groups, but the rise is more
pronounced in the unincubated group.
(d) Removal of the hypoblast from a primitive streak stage blastoderm reduces its developmental potencies to the level of those of the unincubated stage.
INTRODUCTION
The chick blastoderm when just being laid down is a very labile system. It
vaguely 'knows' its antero-posterior orientation imposed on it mechanically by
the rotation of the egg in the uterus of the mother hen (Vitemberger & Clavert,
1960; Clavert, 1960a, b). This orientation is probably superimposed on the
equipotentiality of the blastoderm which exists prior to its rotation.
The transformation from an equipotential into a bilaterally symmetrical
system, although starting in the uterus, goes on after laying and is a gradual
process, thefirstexpression of which is the loss of the embryo-forming potencies
of the central regions of the blastoderm (Eyal-Giladi & Spratt, 1965). This
leaves the whole ring of the marginal zone as the region capable of initiating
1
Author's address: Department of Zoology, The Hebrew University, Jerusalem, Israel.
740
H. EYAL-GILADI
embryonic development, with the strongest tendency for such a development
to occur at the future posterior side (Spratt & Haas, 1960A, 1967). Culturing
such blastoderms without disturbing their integrity results, therefore, in about
100 % of embryos developing from the posterior side (posterior embryos).
However, the presence of embryo-forming potencies of the marginal zone in
regions other than the posterior zone can be revealed by different manipulations (Spratt & Haas, 1960a). Eyal-Giladi & Spratt (1965) showed that a
transverse incision within the area pellucida tends to promote the formation
of an embryo along the incision and perpendicular to the original anteroposterior axis of the blastoderm. Similarly it was shown that transverse folding
of a blastoderm (lower side inside) and growing it on the culture medium with
its future posterior half down had the same effect as the incision (Eyal-Giladi,
1969): namely, both manipulations provoked a tendency for the development
of embryos from the lateral portion of the marginal zone (lateral embryos).
In both the above experiments several developmental trends were clearly
expressed.
The very young chick blastoderm has an asymmetrical pattern of developmental potencies, the left marginal zone possessing stronger embryo-forming
potencies than the right.
The imprinting of this developmental pattern is very labile at the early
stages of development. It was shown that it is possible to reverse completely
the developmental pattern of a folded blastoderm by merely putting it on the
culture medium in a reversed position, namely with its future anterior half
down. This reversal expressed itself in the formation of a remarkable percentage
of embryos which developed from both the original anterior and the right-side
regions of the marginal zone (anterior and right-side embryos respectively)
instead of from the posterior and left sides.
Older blastoderms are much more stable than younger ones. At an advanced
primitive streak stage there is almost no possibility of forcing the marginal zone
to form embryo-forming centres at positions other than the posterior.
Two questions are asked in this work: (1) Does an induced change in the developmental potencies of a blastoderm always involve a complete reorganization
of the blastoderm, or might there be a formation of embryo-forming foci
which do not interfere with the primarily imprinted pattern? (2) Can the growing
stability of the older blastoderm be attributed to some developing new structure,
and if so is it the hypoblast which is responsible for the fixation of the developmental pattern?
MATERIAL AND METHODS
A special experiment was performed in order to answer each of the above
questions.
Experiment I (localized damage)
Blastoderms of two age groups—unincubated and primitive streak stages
respectively—formed the two experimental groups of series AP". They were
Differentiation in chick blastoderm. II
741
transversely folded and put on the culture medium with their posterior side
down (expected to form left-side embryos). Next, the right corner of each such
folded blastoderm was cut off and removed to a certain distance from the rest
Series AP"
Fig. 1. Experimental procedure of Exp. I. (A) A blastoderm before folding with its
lower surface upwards. (B) A blastoderm folded transversely and cultured with its
anterior half down (series PA). (C) A blastoderm folded transversely and cultured
with its posterior half down (series AP). (D) Same as C but with the right corner of
the folded blastoderm extirpated (series AP"). # = anterior side of blastoderm; p =
posterior side of blastoderm; /= left; r= right.
Fig. 2. Experimental procedure of Exp. II. (A) A primitive streak stage blastoderm
with its lower surface up. (B) The hypoblast is cut free from the marginal zone. (C)
The blastoderm after complete removal of the hypoblast. (D) A hypoblastless blastoderm transversely folded and cultured with its anterior half down (series PA hyp).
(E) A transversely folded hypoblastless blastoderm cultured with its posterior side
down (series AP hyp). The control groups for this experiment are the same as shown
in Fig. 1, B and C. Key letters as for Fig. 1.
742
H. E Y A L - G I L A D I
of the blastoderm (Fig. 1D). Both pieces were allowed to develop for 72 hr
before fixation.
Two series, each composed of the same age groups as in the experimental
series, served as controls. In series AP the blastoderms after being folded were
cultured with their posterior half down (Fig. 1C), while in series PA they were
cultured with their anterior half down (Fig. 1B). They too were fixed after
72 h of culture.
Experiment II {hypoblast removal)
The blastoderms chosen for the two experimental groups were of the primitive streak stage (various lengths). After a complete removal of their hypoblast
(Fig. 2), the blastoderms were transversely folded and placed on the culture
medium in one of two ways: Group AP hyp (Fig. 2E) comprised those blastoderms which were cultured with their posterior half down; the blastoderms of
group PA hyp were cultured with their anterior half down (Fig. 2D). The
controls used for Exp. I served as controls for these experiments too. Each
experimental group was thus compared with two control groups: one of
unincubated and the other of primitive streak stage blastoderms, which were
merely folded and cultured in a similar fashion.
All the experimental and control blastoderms were cultured for 72 h, then
removed from the culture medium and observed macroscopically. Following
that, they were fixed, embedded, serially sectioned, stained with hematoxylineosin and studied microscopically.
RESULTS
Experiment I {localized damage)
The results are diagrammatically represented in Fig. 3. The embryos which
developed from the blastoderms of either the experimental groups or the
controls could be classified into four main categories according to their orientation. The first two categories are of lateral embryos, in which one includes
embryos which developed from the right corner of the folded blastoderms and
the other embryos developed from the left corner. The other two categories are
of embryos which developed along the original antero-posterior axis of the
blastoderm. One of them is composed of posterior embryos. This category
includes also a certain percentage of Siamese-twins, in which in addition to
the original posterior embryo there is another embryo : its mirror image. The
twins are fused belly to belly and the posterior embryo is supposedly responsible
for the induction of its twin (H. Eyal-Giladi, unpublished). Such twins were
regarded as a single axial system for the sake of calculations, and the twin embryo
which developed in a reversed position was projected in the diagram on top of
the 'correctly oriented' embryo. Only those embryos which developed from the
original anterior side of the blastoderm as a result of the reversal of the blasto-
Differentiation in chick blastoderm. II
743
derm's developmental tendencies, were separately represented as the fourth
category of anterior embryos.
Control series AP includes two age groups. The younger group (AP bhf)
contains 23 blastoderms folded prior to hypoblast formation. The folding
resulted in equal percentages of left-side and posterior embryos. In four
blastoderms both a posterior and a left-side embryo developed and formed a
cross-formation. Moreover, in two of the four the posterior embryo had a
Siamese-twin ventrally attached to it. Siamese-twins developed also in 3 additional
blastoderms of this group. No right-side embryos developed here.
bhf
pss
AP
bhf
pss
AP"
bhf
pss
PA
Type of manipulation
Fig. 3. Differentiation potencies of chick blastoderm. A diagrammatic representation
of the data of Exp. I. Every series (control and experimental) is comprised of two age
groups: bhf = folded prior to hypoblast formation; pss = folded at primitive streak
stage. The total number of embryos developed in each age group belonging to a
certain experimental series was regarded as 100% with the exception that a pair of
Siamese-twins was regarded as a single embryo.
In the 24 blastoderms folded at the streak-stage (AP pss) the tendency
towards formation of left-side embryos almost disappeared, 96 % of the
embryos being posterior ones. The only left-side embryo appeared together
with a posterior embryo as a cross-formation. In five of the blastoderms there
was twin-formation and in no case was there any right-side embryo formation.
In the experimental series AP" the removal of the right corner of the folded
blastoderms had a very remarkable effect on the developmental tendencies of
the damaged blastoderm. In many of the cut-off triangular pieces minute
embryos formed. These embryos also originated from the cut edge.
In the youngest group of 22 blastoderms (AP" bhf) up to 70-8 % of the embryos
744
H. EYAL-GILADI
developed from the damaged corner. These right-side embryos equally inhibited
the development of either left-side or posterior embryos. In two blastoderms
there was, however, a simultaneous formation of a right- and a left-side embryo.
In another blastoderm there was a simultaneous formation of a right-side
embryo and antero-posterior Siamese-twins.
In the 30 streak-stage blastoderms (AP" pss) the removal still had an effect,
but only 30 % of the embryos developed from the right side. Of these only one
was the sole embryo in the blastoderm. Another right-side embryo shared the
blastoderm with a left-side and a posterior embryo. Seven additional rightside embryos participated in cross-formations with rudimentary posterior
embryos.
In both AP and AP" series, all the embryos which developed from the original
anterior side of the blastoderm were twin embryos, which are interpreted as the
result of secondary inductions. Thus in control series AP only two categories of
embryos of the above four exist, namely, posterior and left-side embryos. In
experimental series AP", however, there are three categories of embryos;
posterior, left-side and right-side embryos.
In control series PA the additional fourth category also exists, namely, that of
anterior embryos. Of the 29 blastoderms comprising the youngest group
(PA bhf), one third of the embryos developed from the left side, another
third from the right, some 23 % developed along the original antero-posterior
axis from the posterior side of the blastoderm, whereas 13-2 % of the embryos,
although longitudinally oriented, developed from the original anterior side
of the blastoderm without having a normally positioned posterior twin. The
same tendency persists at the primitive streak stage (pss) of series PA (21
blastoderms), although the percentage of both lateral and anterior embryos is
smaller.
Experiment II {hypoblast removal)
The results are presented in Fig. 4 in two separate sets of columns. One set
represents the AP type series (posterior half of the blastoderm facing the culture
medium) and the other set represents the PA type series (anterior half of the
blastoderm facing the culture medium).
In each set the experimental group (either AP hyp or PA hyp) is compared
with its two control groups : one control group of blastoderms folded before
hypoblast formation (bhf), and the second at the primitive streak stage (pss).
The first set is comprised of: experimental group AP hyp pss (25 blastoderms)
(Fig. 2E), control group AP bhf (23 blastoderms), and control group AP pss
(24 blastoderms). In this set only two kinds of embryos developed, left-side
and posterior embryos (Fig. 4). On comparing the percentages of the left-side
embryos in the three groups, it becomes obvious that the removal of the
hypoblast resulted in about 52 % of left-side embryos, exactly the same percentage as in the unincubated control group (bhf). In the second control
Differentiation
in chick blastoderm. II
745
group of primitive streak blastoderms (pss) only a single left-side embryo was
formed (4 %), which participated in a cross-formation with a posterior embryo.
As to the group of posterior embryos, the percentage of those having a twin
embryo (Siamese-twin formation) as a result of secondary induction is twice
that in the control groups.
The second set is comprised of: experimental group PA hyp pss (23 blastoderms) (Fig. 2D), control group PA bhf (29 blastoderms), and control group
PA pss (21 blastoderms). In this set the altered relations of the folded blastoderms to the culture medium resulted in the formation of four different kinds of
bhf
pss
AP
pss
bhf
AP hyp
pss
PA
.pss
PA hyp
Type of manipulation
Fig. 4. A diagrammatic representation of the data of Exp. TI. The controls are the
same as for Exp. I. They are of two types, AP and PA, and each type is comprised
of two age groups: bhf = folded prior to hypoblast formation; pss = folded at primitive streak stage. The two experimental groups, AP hyp and PA hyp (see Fig. 2),
were at the primitive streak stage (pss) when folded. The total number of embryos
developed in each age group belonging to a certain experimental series was regarded
as 100% with the exception that a pair of Siamese-twins was regarded as a single
embryo.
embryos, namely : left-side, right-side, posterior and anterior. However, here too
the same trend is seen as in the first set. Experimental group PA hyp pss even
shows a slight increase in the percentage of lateral embryos (left- and rightside) as compared with the younger age group PA bhf.
DISCUSSION
The chick blastoderm has already been found to be an asymmetrical system
by Rudnick (1932) and Rawles (1936, 1943). They found that at the headprocess stage grafts from the left side of the blastoderm possessed a superior
48
E M B 23
746
H. EYAL-GILADI
developmental capacity to those from the right side when transplanted onto the
chorioallantois.
Mulherkar (1958) described another aysmmetrical character of the definitive
streak blastoderm. She found that pieces taken from the left side of the blastoderm, adjacent to the streak, had stronger inducing potencies than similar
pieces from a corresponding region on the right side of the streak. These
observations were made on relatively 'old' blastoderms whose developmental
pattern is remarkably stable.
Eyal-Giladi & Spratt (1964, 1965) and Eyal-Giladi (1969) showed that the
dominance of the left side of the blastoderm already exists in the earlier and
more labile stages of development. This dominance is expressed by the frequent
formation of embryos from the left marginal zone, following certain manipulations which affect both the left and the right side of the blastoderm equally.
It must therefore be assumed that during the time of the egg's rotation in the
uterus, the establishment of the plane of bilateral symmetry of the blastoderm
and the stabilization of one end of it as the posterior side are only two events
in a series. Another event is the establishment of the dominance of the left
side of the blastoderm, probably due to the development of unequal conditions on either side of the blastoderm during this critical period.
However, although unequally distributed, the embryo-forming potency is
still present in the entire marginal zone. At least until the stage of a definitive
primitive streak, there is a possibility of an organized remoulding of the entire
fate map of a blastoderm provided that the external conditions involved
affect large areas of the blastoderm (control series PA). In cases in which the
external intervention is localized, as in the AP" series, another possibility
exists, namely, that of a local rise in the embryo-forming capacity at one point
of the marginal zone without interference with the original pattern. A new
embryo developing from this newly formed centre might coexist with the
normally predicted embryo. In many cases, however, especially in younger
(more labile) blastoderms, the new embryos might compete with the original
embryos and suppress their development.
One can therefore deduce that in the cases in which embryos were formed
simultaneously by two or more points of the marginal zone of a certain blastoderm, those points must have gained an equally high level of embryo-forming
potency. This is probably the explanation for all the cases of embryonic crossformations and of the three blastoderms from series AP" in which both a
left- and a right-side embryo were formed. In other cases in which only a
single embryo was formed from an atypical embryo-forming centre our interpretation would be that the embryo-forming potency of the marginal zone
at the responsible point was raised above that of the other potential embryoforming centres and subsequently suppressed them. One can take the unincubated blastoderms as an example. In such blastoderms, when merely explanted
on a culture medium, the posterior point of their marginal zone has the highest
Differentiation
in chick blastoderm. II
141
level of embryo-forming potency and from them a single posterior embryo will
always form.
The transverse folding of such a blastoderm raises the embryo-forming
potencies of both corners (left and right) of the marginal zone. As the initial
value of the left side was originally higher than that of the right, the left
corner reaches a value of embryo-forming potency equal (cross-formation)
or superior (left-side embryos only) to the unchanged value at the posterior
side. The embryo-forming potency of the right corner, although raised, in
series AP always remained lower than that of the left corner and was thus
suppressed. A local damage, however, to the right corner of such a folded
blastoderm raised the embryo-forming potency at that point to a level equal to
(left- and right-side embryos) or higher than that of the left corner (rightside embryos only).
This system of folded blastoderms might therefore be used as a system for
measuring the relative local effect of certain manipulations, and perhaps also
substances, on the local intensification of the embryo-forming potencies of the
marginal zone.
The first experimental series was concerned with the effect of local damage on
the potencies of the blastoderm. The second series dealt with the limitation of
the embryo-forming centre to the posterior side of the blastoderm. It was
designed to check the possible role of the hypoblast as a stabilizing factor
as against the possibility of this state of differentiation being achieved autonomously by the blastoderm independently of hypoblast formation. The background of exactly the same control groups (AP bhf and pss, and PA bhf and
pss) was therefore used to check the importance of the hypoblast in this process.
Waddington (1930, 1932, 1933) noticed long ago that the hypoblast has an
epigenetic influence on the epiblast. Summarizing his earlier work in The
Epigenetics of Birds (1952), he proposes that there is an inherent polarity in
the hypoblast as a whole in the form of a gradient field, which gives it an
inductive effect. This is responsible for the formation of embryos in conformity
with the polarity of the hypoblast, following rotation of the hypoblast in relation
to the epiblast.
Also Spratt & Haas (1961) postulate that: 'the upper layer (epiblast) is
originally neither morphologically nor potentially polarized but depends upon
an influence of the polarized lower layer movements for its later axiation
(antero-posterior polarization) '.
The question therefore arises as to when this axiation of the upper layer
takes place. Is it a process occurring at the time of, or shortly after, the fountain-like movement (Spratt & Haas, 19606, 1961) of the lower layer during
hypoblast formation?
Spratt & 7Taas (1960 A) and Eyal-Giladi & Spratt (1965) tried to analyse the
embryo-forming potency of successive stages of young blastoderms from the
unincubated stage up to blastoderms incubated for several hours.
48-2
748
H. EYAL-GILADI
The potencies of the older blastoderms, in the above studies, were determined at a double-layered stage (the blastoderms being already comprised of
epiblast and hypoblast). The conclusions of the above authors were that while
the embryo-forming potency of an unincubated blastoderm is diffuse and is
at most confined to the entire marginal zone, it tends to be concentrated in
the posterior marginal zone during the first 12 h of incubation.
The experiments in the present work, which were designed to test the embryoforming potency of a blastoderm whose hypoblast has been removed, prove
that the naked epiblast is at least as labile as an unincubated blastoderm.
It must therefore be concluded that during the period of a blastoderm's
development from the unincubated stage till the formation of a full length
primitive streak, the epiblast and its surrounding marginal zone do not undergo
a process of irreversible differentiation on the one hand, and do not lose even
slightly the potency for the initiation of heterotopic embryo-forming centres on
the other.
It is the influence of the polarized hypoblast which creates the environmental
conditions promoting the formation of an embryo-forming centre at the
posterior marginal zone. This effect is not limited to the period of the fountainlike movement of the hypoblast, but is exercised as a long-term continuous
influence. It is probably firstly concerned with the formation of the primitive
streak, which again needs further influencing by the hypoblast for its own
normal development.
RÉSUMÉ
Potentialités de différenciation du jeune blastoderme de Poulet, révélées par diverses manipulations. II. Expériences de lésions localisées et d'exérèse de I'hypoblaste.
On a utilisé des blastodermes de poulet plies transversalement, depuis le stade non incubé
jusqu'à celui de la ligne primitive, pour vérifier les données suivantes:
(a) l'effet d'une lésion locale (coin droit) sur les potentialités embryogènes du blastoderme;
(b) l'effet de l'exérèse de I'hypoblaste sur la stabilité des potentialités embryogènes du
blastoderme au stade de la ligne primitive.
Les conclusions sont les suivantes:
(a) les blastodermes au stade la ligne primitive, intacts et plies transversalement, placés sur le
milieu de culture avec leur moitié postérieure vers le bas, sont relativement stables et
l'embryon se forme à partir de leur coté postérieur.
(b) les blastodermes plus jeunes sont moins stables et révèlent, par la formation d'embryons
du côté gauche, l'asymétrie inhérente au blastoderme.
(c) l'extirpation partielle du coin droit dans de tels blastodermes provoque un accroissement local remarquable dans les potentialités embryogènes du côté droit, dans les
deux groupes d'âge, mais est plus prononcé dans le groupe non incubé.
(d) l'exérèse de I'hypoblaste d'un blastoderme au stade de la ligne primitive ramène ses
potentialités de développement au niveau de celles du stade non-incubé.
Most of the operations of the AP" series were done by Mrs S. Eshel, to whom I am very
much indebted.
Differentiation in chick blastoderm. II
749
REFERENCES
CLAVERT, j . (1960#). Déterminisme de la symétrie bilatérale chez les oiseaux. Archs Anat.
microsc. Morph, exp. 49, 22.
CLAVERT, J. (19606). Déterminisme de la symétrie bilatérale chez les oiseaux. III. Influence
de la position de l'axe de l'œuf dans l'utérus sur l'orientation de l'embryon. Archs Anat.
microsc. Morph, exp. 49, 207-28.
EYAL-GILADI, H. (1969). Differentiation potencies of the young chick blastoderm as revealed
by different manipulations. I. Folding experiments and position effects of the culture
medium. J. Embryol. exp. Morph. 21, 177-92.
EYAL-GILADI, H. & SPRATT, N. T. (1964). Embryo formation in folded chick blastoderms.
Am. Zool. 4, 428.
EYAL-GILADI, H. & SPRATT, N. T. (1965). The embryo forming potencies of the young chick
blastoderm. J. Embryol. exp. Morph. 13, 267-73.
MULHERKAR, L. (1958). Induction by regions lateral to the streak in the chick embryo.
J. Embryol. exp. Morph. 6, 1-14.
RAWLES, M. E. (1936). A study in the localization of organ-forming areas in chick blastoderm of the head-process stage. / . exp. Zool. 72, 271-315.
RAWLES, M. E. (1943). The heart forming areas of the early chick blastoderm. Phvsiol. Zool.
16, 22-42.
RUDNICK, D. (1932). Thyroid-forming potencies of the early chick blastoderm. J. exp.
Zool. 62, 287-313.
SPRATT, N. T. & HAAS, H. (1960a). Integrative mechanisms in development of the early
chick blastoderm. I. Regulative potentiality of separated parts. / . exp. Zool. 145, 97-138.
SPRATT, N. T. & HAAS, H. (19606). Morphogenetic movements in the lower surface of the
unincubated and early chick blastoderm. J. exp. Zool. 144, 139-57.
SPRATT, N. T. & HAAS, H. (1961). Integrative mechanisms in development of the early
chick blastoderms. II. Role of morphogenetic movements and regenerative growth in
synthetic and topographically disarranged blastoderms. / . exp. Zool. 147, 57-94.
SPRATT, N. T. & HAAS, H. (1967). Nutritional requirements for the realization of regulative
(repair) capacities of young chick blastoderms. / . exp. Zool. 164, 31—46.
VINTEMBERGER, P. & CLAVERT, J. (1960). Sur le déterminisme de la symétrie bilatérale chez
les oiseaux. C. r. Séanc. Soc. Biol. 154, 1072-6.
WADDINGTON, C H. (1930). Developmental mechanics of chick and duck embryos.
Nature, Lond. 125, 924-5.
WADDINGTON, C. H. (1932). Experiments on the development of chick and duck embryos
cultivated in vitro. Phil. Trans. R. Soc. B 221, 179-230.
WADDINGTON, C. H. (1933). Induction by the primitive streak and its derivatives in the
chick. J. exp. Biol. 10, 38-46.
WADDINGTON, C. H. (1952). The Epigenetics of Birds. Cambridge: University Press.
{Manuscript received 4 July 1969)