Differentiation potencies of the young chick blastoderm as revealed by different manipulations: II Localized damage and hypoblast removal experiments

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, the first expression 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 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?

A special experiment was performed in order to answer each of the above questions.

Blastoderms of two age groups—unincubated and primitive streak stages respectively—formed the two experimental groups of series AP″. They were 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 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.

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 hematoxylin-eosin and studied microscopically.

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 blastoderm’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.

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 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.

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 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 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.

Fig. 4. A diagrammatic representation of the data of Exp. II. 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.

The chick blastoderm has already been found to be an asymmetrical system by Rudnick (1932) and Rawles (1936, 1943). They found that at the head-process stage grafts from the left side of the blastoderm possessed a superior 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 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, 1960b, 1961) of the lower layer during hypoblast formation?

Spratt & Haas (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.

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 fountain-like 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.

On a utilisé des blastodermes de poulet pliés 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 l’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 pliés 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 l’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.