/. Embryo!, exp. Morph. Vol. 21, 1, pp. 177-92, February 1969
177
Printed in Great Britain
Differentiation potencies
of the young chick blastoderm as revealed by
different manipulations
I. Folding experiments and position effects of the culture
medium
By HEFZIBAH EYAL-GILADI1
Department of Zoology, The Hebrew University of Jerusalem
An earlier study has demonstrated both the equipotentiality and the lability
of the very young unincubated chick blastoderm (Eyal-Giladi & Spratt, 1965).
It was observed that when a central cut was made in a blastoderm of this age, an
embryo tended to form along the cut edge and the original embryo-forming
centre at the prospective posterior side was suppressed. As the blastoderms grow
older, the embryo-forming potency as expressed by the reaction to the cut is
gradually lost from the central area, becomes confined to the marginal zone, and
within it gradually to its posterior region.
It was thought that folding the blastoderm might create conditions along the
fold similar to those along the cut edge, and an attempt was made to follow the
results of such folding.
METHODS
Blastoderms ranging in age from unincubated to 16 h incubated with a clearly
defined postero-anterior orientation were used for this study. They were either
folded along their original longitudinal axis or perpendicular to it, with the lower
side of the blastoderm on the inside of the fold. The blastoderms were allowed to
develop on an enriched egg extract culture medium for three days, their development being recorded daily until fixation. The culture medium contained a 2:1
proportion of egg extract to chick Ringer solution as compared with the 1:1
ratio proposed by Spratt & Haas (19606), as well as a slightly lower concentration of agar.
The blastoderms were cultured for 3 days and then removed from the medium
and fixed in Lillie's modification of Bouin's solution. After being studied in toto,
they were stained with paracarmine, embedded in paraffin, and serially sectioned.
1
Author's address: The Department of Zoology, The Hebrew University of Jerusalem,
Israel.
12
JEEM2I
178
H. EYAL-GILADI
RESULTS
Blastoderms folded along the postero-anterior axis
This series, in which the original postero-anterior axis of the blastoderm
coincided with the folding axis, consisted of 17 blastoderms. Most of the blastoderms were very young (prior to the appearance of the hypoblast); a few showed
a rudimentary hypoblast. Six of the folded blastoderms were laid with their
right side on the culture medium, while eleven had their left side on it and their
right side up.
In both groups development proceeded according to the original posteroanterior axis (Text-fig. 1) and the respective embryos developed with either their
right or left side towards the culture medium.
Text-fig. 1. The upper row represents the folded blastoderm lying with its left side (/)
towards the culture medium. The lower row represents a folded blastoderm with its
right side (r) towards the culture medium. The outcome in both cases is identical.
a, Anterior; p, posterior.
Blastoderms folded perpendicular to the postero-anterior axis
These blastoderms formed the bulk of the experimental material and could
again be divided into two main groups: group AP comprised blastoderms placed
on the culture medium with the prospective anterior side of the blastoderm up
and the posterior side down, while group PA was put with the prospective
posterior side up. Only blastoderms whose orientation was clear were selected
from among the very many eggs opened and they were folded after their posterior
side was marked with either carbon or carmine. Sixty-eight blastoderms belonging to series AP and 61 blastoderms belonging to series PA developed satisfactorily and are included in this study.
Differentiation in chick blastoderm
179
Before starting to analyse the results, the different types of embryonic formation which developed in this experiment should be described (Text-fig. 2). They
fall into three main categories:
(a) The embryo or embryos which developed in conformity with the original
postero-anterior orientation of the blastoderm, i.e. along the original
longitudinal axis of the blastoderm.
(b) The embryo or embryos which developed perpendicular to the original
postero-anterior orientation.
(c) Blastoderms in which both tendencies were expressed. This resulted in
cross formations in which postero-anterior and perpendicular embryos transsected each other.
Text-fig. 2. The various types of embryonic formation as based on the AP folding
experiment series. Category A (Ax-Ag) embryos developed along the original
longitudinal axis of the blastoderm. Category B (Bi-Bi) types of lateral embryos.
Category C (.Ci-Ci) some examples of cross formations. At the upper left corner a
schematic drawing of the inner side of a blastoderm before the folding process is
represented. The folding is done by putting point a (anterior) on point p (position).
/, Left; r, right.
12-2
180
H. EYAL-GILADI
(a) Embryos formed along the postero-anterior axis of the blastoderm
Here a distinction has to be made between three types:
(1) A single embryo which developed according to the fate of the blastoderm,
with its posterior part at the original posterior end of the blastoderm and its
anterior end pointing towards and pushing through the fold. In the series PA
such an embryo developed with its dorsal side up (Plate 1,figs.A, B). An embryo
of this category belonging to series AP developed with its dorsal side towards
the culture medium (Text-fig. 2, Ax).
(2) A single embryo with its longitudinal axis coinciding with the longitudinal
axis of the blastoderm but with a reversed orientation. In such cases the
posterior end of the embryo developed from the original anterior part of the
folded blastoderm with its head pushing towards the centre through the fold
area. An embryo of this type belonging to series PA developed with its dorsal
side down towards the culture medium, while if belonging to series AP its dorsal
side faced up (Text-fig. 2, A2).
(3) Two embryos which developed along the original longitudinal axis of the
blastoderm in the form of 'Siamese twins' with their ventral sides facing each
other and usually fused to different extents (Text-fig. 2, A3). In such cases the
posterior end of one of the twins coincides with the posterior part of the folded
blastoderm while that of the other twin with its anterior end. Both heads point
towards the original centre, and push through the fold (Plate 1, figs. C, D).
Such twins (Plate 2, figs. F, G) may share a common enteron but have
separate notochords and hearts. However, in many cases the twins are fused to a
greater extent and may even share a common notochord, on both sides of which
there are enteric tubes lateral to which hearts are formed. It is very probable that
both hearts developed from two rudiments each, one belonging to each twin.
PLATE 1
Figs. A, B. Blastoderm PA 200. The embryo was formed according to fate from the posterior
half of the blastoderm with its dorsal side up. The gut opens to the exterior on the upper
surface behind the embryo (fig. B). The lower surface of the folded blastoderm (fig. A) was
flattened on the culture medium with no obvious differentiation.
Figs. C, D. Blastoderm AP 252. Equally developed Siamese twins were formed, one being
the mirror image of the other. Each has its own somites and two ear vesicles (see Plate 2,
figs. F, G). The lower surface of the extra-embryonic region is fiat except for the peripheral
papilae which penetrated into the culture medium (fig. C). The upper surface shows the
characteristic crater-like gut opening posterior to the embryonic region (fig. D).
Fig. E. Blastoderm PA 217. A right-hand embryo demonstrates a reversal of orientation
caused by the position of the blastoderm on the culture medium. Here too a clear gut opening
exists on the upper surface (see also Plate 2, figs. D, E).
Fig. F. Blastoderm AP 254. A typical cross-formation. The posterio-anterior embryo
continued its development from the pre-existing streak with the embryo's head turned
around the fold to appear on the upper surface. In addition a left-hand lateral embryo was
formed. On the upper surface the orifice of the enteric cavity is also visible (ent.)
Differentiation in chick blastoderm
181
182
H. EYAL-GILADI
ent.
Differentiation in chick blastoderm
183
(b) Embryos perpendicular to the postero-anterior axis
These embryos which developed with their longitudinal axis along the fold
were of two main types:
(1) A left-hand embryo (Text-fig. 2, B^. The posterior end of these embryos
was located at the original left side of the blastoderm. Sometimes two parallel
laterally fused left embryos developed (Text-fig. 2, B2). Lateral embryos of this
type could not be detected macroscopically; each had its own neural plate and
notochord, but shared a common gut, heart, and coelom (Plate 2, fig. C).
Text-fig. 3. From left to right: three stages in the formation of a lateral embryo.
(2) A right-hand embryo (Text-fig. 2, B3). Similar to the former but with its
posterior end located at the original right side of the blastoderm (Plate 1, fig. E).
Right-hand twin embryos developed very rarely (Text-fig. 2, B4).
Whenever a lateral embryo started to develop the folded blastoderm acquired
a characteristic notch indicating the posterior side of the forming embryo. This
was probably caused by the unusual direction of the morphogenetic movements
(Text-fig. 3). Only somewhat later the future anterior end of the embryo bulged
out of the blastoderm and acquired the form of a head.
PLATE 2
Figs. A, B. Blastoderm AP 254 (see also Plate 1, fig. F). Here a posterior and a left-hand
embryo formed a cross. A, Cross-section through the junction area of both embryos where
both neural plates are fused; B, a cross-section through the left-hand embryo lateral to the
junction area.
Fig. C. Blastoderm AP 211. Cross-section through twin left-hand embryos. The neural
plates are fused laterally and under each one there is a notochord. The enteric cavity and heart
are common to both embryos.
Figs. D, E. Blastoderm PA 217 (see also Plate 1, fig. E). A typical cross-section through a
lateral embryo. In fig. D the external opening of the enteric cavity is seen on the upper
surface. This wide cavity is seen in fig. E to be continuous with the embryonic gut.
Figs. F, G. Blastoderm AP 252 (see also Plate 1, figs. C, D). Cross-sections of a blastoderm
with twins. Each of the twins has its own ear vesicles (ear ves.) but they share a common
gut (ent.) and a notochord (ch.). Two lateral hearts (hrt.) were formed, each belonging to
both embryos.
184
H. EYAL-GILADI
Table 1. Embryos which developed in each of the folded blastoderms
of age-groups a and b {Table 1) and c and d {Table 2).
(In cases of a cross-formation the two embryos are connected by a line, as are twin embryos.
A relatively well-developed embryo is marked by a plus sign and a rudimentary embryo by a
star. A scar marking the position of a primitive streak formed after folding and which
subsequently degenerated, is indicated by two stars. Two pluses in the same row indicate
twins.)
Series AP (anterior half up)
Embryo according to p-a axis
Embryo perpendicular to p-a axis
Serial no.
4
6
7
28
37
351
353
354
350
352
154
302
305
150
1
101
151
153
155
54
24
27
33
Left embryo
Right embryo
Dorsal
side up
Dorsal
side down
Group a (no sign of hypoblast yet)
+
+
+
4.
4.
+
+
+
+
+
I (one third to fully developed hypoblast)
Group b
356
323
100
251
103
104
357
353
3
30
32
26
256
325
359
360
361
362
363
364
365
+_
4.
4.
•+
Differentiation in chick blastoderm
185
Table 1. {continued)
Series PA (posterior half up)
Embryo perpendicular to p-a axis
Serial no.
3
6
8
9
10
21
153
0
1
20
22
27
32
51
53
102
103
52
7
Left embryo
Right embryo
Group a (no sign of hypoblast
+
*
+
+
+
+
+ —+
.
+
+
+
+
+
+
+
+
.
.
.
+
+
+
+ —+
Embryo according to p-a axis
,
*
,
Dorsal
side up
side down
yet)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+
54
+
101
150
.
.
.
.
+
*
*
+
33
5
154
152
30
31
.
.
.
.
*
.
.
.
.
.
+
+
+
+
+
+
.
.
.
210
34
4
207
217
203
35
25
200
201
204
265
+
Group b (one third to fully developed hypoblast)
+
.
.
+
.
.
.
+
.
+
+
*—*
+
.
.
.
.
.
+
.
+
h
.
+
+
+
+
+
.
186
H. EYAL-GILADI
Table 2. Embryos which developed in each of the folded blastoderms
of age-groups a and b (Table 1) and c and d (Table 2).
(In cases of a cross-formation the two embryos are connected by a line, as are twin embryos.
A relatively well-developed embryo is marked by a plus sign and a rudimentary embryo by a
star. A scar marking the position of a primitive streak formed after folding and which
subsequently degenerated, is indicated by two stars. Two pluses in the same row indicate
twins.)
Embryo perpendicular
to p-a axis
Stage
Serial no.
Group c*
254
255
366
262
263
307
309
313
318
367
212
217
205
213
214
265
268
269
270
271
272
308
310
311
Group d-\
Left embryo Right embryo
Embryo according to
p-a axis
Dorsal
side up
Dorsal
side down
Series A P (anterior half up)
H
+ •
Series PA (posterior half up)
Group c*
Group
212
234
216
220
222
232
270
223
268
233
269
01
261
228
263
264
266
267
231
* Short to half primitive streak.
t Half to complete primitive streak.
+
1
• : •
. * .
• ! •
+
I
+
+
+
+
+
+
+
T
:|:
+
Differentiation in chick blastoderm
187
(c) Cross formations
In each blastoderm at least two embryos developed at an angle of 90° to each
other, one in conformity with the original postero-anterior axis of the blastoderm and the other perpendicular to it. Here almost all the possible combinations existed between the single longitudinal and single perpendicular embryos
(Text-fig. 2, Cl5 Co) and even between twin and single embryos (C3) and twins in
both directions (C4).
Blastoderm AP 254 (Plate 1, fig. E) is a very good example of a crossformation. When folded the blastoderm showed the first signs of a primitive
streak so that there could be no doubt about the original orientation. After
being folded the original streak continued to develop into an embryo with a
neural plate but an even better embryo developed from the left side of the
blastoderm. The blastoderm was fixed earlier than usual (after 48 h). In the
sections (Plate 2, figs. A, B) both neural plates seem to be confluent at the
crossing-point of the two embryos.
The results of all the experiments are summarized in Tables 1 and 2. They
are arranged in the tables according to the series to which they belong, and
within the series according to age groups of the blastoderms at the time of
folding. A general review of the tables reveals that lateral embryos are very
frequent in the youngest age groups (Table la) of both series (AP and PA) and
are more rare in the older age-groups. However, there seems to be a very conspicuous difference between the two series as to the relative frequency of lateral
embryos and their direction. From a total of 68 folded blastoderms in series AP,
18 lateral embryos developed, whereas from a total of 59 folded blastoderms in
series PA, 33 lateral embryos were found. All the 18 lateral AP embryos
developed from the left corner of the folded blastoderm with their heads
pointing towards the former centre of the blastoderm. The 33 PA lateral embryos
are almost equally divided, 16 being left-hand embryos and 17 right-hand.
The frequency of the postero-anterior embryos is inversely related to the
frequency of the lateral embryos in each of the series. In series AP, 56 of the 68
blastoderms developed a postero-anterior embryo as compared with 18 lateral
embryos in the same series, whereas in series PA, comprising 61 blastoderms,
only in 32 was a postero-anterior axis developed, as against 33 lateral embryos.
The sum of lateral and postero-anterior axial developments cited here for
each series is larger than the total number of blastoderms, as in some cases the
same blastoderm contained both a lateral and a postero-anterior axial formation. Series AP includes seven such formations and series PA five.
In 13 folded blastoderms of series AP and in 11 blastoderms of series PA twin
embryos developed along the original longitudinal axis with their ventral parts
fused. In ideal cases the two embryos formed an exact mirror image of each
other, in others the duplication was only partial, one embryo being complete
and the second only a partial image of either its anterior or posterior part.
188
H. EYAL-GILADI
In the majority of the 13 pairs of twins of series AP, the embryo formed from
the original posterior half of the blastoderm facing the culture medium developed
far better than its upper twin. In series PA the development of the twins appears
to be more labile and there seems to be an equal number of twins representing
each of the three possibilities: upper or lower one better developed than its twin
or both of them equally developed.
DISCUSSION
The unincubated chick blastoderm is known to be a labile system. If its
integrity is not disturbed, the embryo tends to develop according to the labile
postero-anterior axis determined in the uterus of the mother hen during the
rotations of the egg (Vintemberger & Claver, 1960; Claver, \960a, b). When
cultured on a solid culture medium it was observed (Spratt & Haas, 1960#) that
development of an embryo occurs only if the blastoderm is put on the medium
in an inverted position, allowing the morphogenetic movements at the lower
surface of the blastoderm to take a normal course. Experimental cutting of such
blastoderms into several fragments, exchanging of parts of different blastoderms,
and the combining of several blastoderms, have enabled their regulative
properties to be demonstrated (Spratt & Haas, 19606, 1961 a, b, 1962). These
properties were attributed by Spratt & Haas (19606) to the diffuse embryoforming potency of the entire marginal zone which gradually tends to concentrate in its posterior region only.
Eyal-Giladi & Spratt (1965) were able to show by explanting a semicircular
central piece that in very young blastoderms even the central part possesses an
embryo-forming potency similar to the unincubated duck blastoderm (Lutz,
1949; Lutz, Departout, Hubert & Pieau, 1963), and in addition that the blastoderm is asymmetrical, its left side having stronger embryo-forming potencies
than its right.
In the present investigation experimental series AP leads to exactly the same
conclusions. In very young blastoderms a mere folding of the blastoderm is
frequently able to cause the formation of a new embryonic centre at the folding
point of the marginal zone. Without any exception this centre is formed on the
left side of the blastoderm. In most cases the primary posterior embryonic
centre is suppressed. In other cases (probably a little more differentiated) both
tendencies are expressed resulting in a cross formation. In blastoderms a little
older the ability to form lateral embryos is probably much weaker and is
suppressed by the increasing potency of the posterior marginal zone. Most of the
embryos of the AP series folded at the primitive streak stage thus gave rise to a
single embryo which developed normally.
Series PA, however, leads to somewhat different conclusions. The results
seem more variable and even in blastoderms folded at the primitive streak stage
one can still find lateral embryos. Here, and especially in the younger stage
Differentiation in chick blastoderm
189
groups, there is an obvious tendency towards a change in the equilibrium, as
shown by a doubling of the percentage of lateral embryos. In addition the
lateral embryos do not show a tendency to develop from the left side of the
blastoderm, but are equally divided into left- and right-hand embryos. These
findings seem to be connected with the position of the folded blastoderm on the
culture medium. The fact that the blastoderm has to rest in an inverse position
on the culture medium in order to enable embryonic development to proceed
(Spratt & Haas, I960 a) is probably not only concerned with the facilitation of
morphogenetic movements. There seems to be an additional factor which
encourages the development of an embryo from the side facing the culture
medium. This is the reason that in series AP which is 'correctly' placed on the
culture medium, as far as the posterior half of the blastoderm is concerned, the
results are identical with those of a former publication (Eyal-Giladi & Spratt,
1965), i.e. whenever a single postero-anterior embryo developed its origin was in
the pre-existing posterior centre. In most cases when twins developed, the lower
(posterior) twin was more fully developed. All the lateral embryos were lefthand.
In the youngest age-group of the AP series (Table 1, group a) the folding
caused a formation of a new left-hand centre in 50 % of the cases, whereas in the
other 50 % the original posterior centre continued to give rise to the embryo.
The inversion of the position of the folded blastoderms of the same age-group
on the culture medium (series PA) had an unfavourable effect on the posterior
region which now faced up, and as a result the percentage of lateral embryos
increased to about two-thirds of the cases in this age-group. At the same time
the embryo-forming potency of the anterior region, which under normal
conditions is not expressed, was strengthened and in a few additional cases
single embryos developed from the side facing the culture medium (original
anterior half of the blastoderm). This last fact clearly indicates a tendency
towards a reversal of the postero-anterior orientation of the blastoderm. On
examining the lateral embryos the same tendency could be seen. Whereas in
series AP only left-hand embryos developed, in the confused PA series in which
two contradictory tendencies (original orientation and relation of blastoderm to
the culture medium) affect the blastoderm, the lateral embryos are divided into
equal right- and left-hand groups.
Another very pronounced position effect is revealed in connexion with the
gut. In the normal development of the chick, the hypoblast forms the basal
layer concerned with the formation of the gut. First, the primary endoblast is
formed, and then, as indicated by several authors (Fraser, 1954; Modak, 1965,
1966; Nicolet, 1967; Vaket, 1962), cells from the primitive streak invade this
layer and form the secondary hypoblast, which contributes to the formation of
the gut.
In our experiments we did not label the various parts of the folded blastoderms, so that no comment can be made on the cellular contribution to the gut
190
H. EYAL-GILADI
in general. However, two very interesting phenomena have been observed. A gut
formed in a folded blastoderm is always open to the outside. This is true also in
older blastoderms, in which the pre-existing hypoblast was enclosed inside the
folded blastoderm. It was observed that on the upper surface of such folds one
or more invaginations appeared which seemed to sink deep into the developing
blastoderm (Plate 1, figs. B, D-F). In sections these invaginations were seen to
lead all the way to the region where the embryo developed and to be continuous
with the embryonic gut (Plate 2, figs. D, E). The impression obtained was that
the gut was at least partially formed by this invagination. No invagination of this
nature was ever seen on the side of the blastoderm facing the culture medium.
Thus the gut of an embryo which developed with its dorsal side towards the
culture medium opened in a manner resembling its appearance in a normal
embryo, whereas the gut of an embryo which developed with its dorsal side up
opened somewhere dorso-lateral or posterior to the embryonic axis (Plate 1,
figs. B, D). The gut of a lateral embryo always opened on that side of the embryo
which pointed upwards (Plate 1, fig. E).
In some cases in which no embryo was formed the upper surface of the
folded blastoderm tended to turn into a typical endoderm-like epithelium
without any relation as to whether this side was originally the anterior or
posterior part of the blastoderm. The impression was therefore gained that
whereas the contact with the culture medium is favourable for axial organ
formation, the contact with the air phase enhances the development of invagination in relation to gut formation.
The above results may serve as a warning that the interpretation of experimental data gained from culturing chick embryos in vitro should take the
specific environmental conditions into strict account. It is also thought that the
analysis of the different factors influencing the formation of an atypical embryonic centre might prove useful in the understanding of embryonic determination.
SUMMARY
Unincubated blastoderms up to primitive streak stage were folded in two
along or perpendicular to their longitudinal axis. While no change in the main
differentiation potencies was caused by longitudinal folding, interesting
features were observed in blastoderms folded perpendicularly:
1. The mode in which the folded blastoderm was put on the culture medium
seemed to have an important influence on the location of the embryo-forming
centre.
2. It also seemed to influence the formation of the embryonic gut which in
these blastoderms always opens on the side opposite the culture medium.
3. Former observations which indicated a stronger tendency of the left side
of the young blastoderm towards embryonic development were confirmed.
4. This tendency can be altered by changing the position of the blastoderm on
the culture medium.
Differentiation in chick blastoderm
191
RESUME
Possibilites de differentiation du jeune blastoderme de Poulet revelees par
differentes manipulations. I. Experiences de plissements; effets de la position
sur le milieu de culture
Des blastodermes non incubes et jusqu'au stade de la ligne primitive ont ete
plies soit le long de leur axe longitudinal soit perpendiculairement a cet axe.
Alors qu'aucun changement du pouvoir de differentiation n'a ete provoque par
le plissement longitudinal, des faits dignes d'interet ont ete observes sur des
blastodermes plies perpendiculairement a l'axe.
1. Le mode dont le blastoderme pile a ete place sur le milieu de culture semble
avoir une influence importante sur la localisation du centre formateur de
l'embryon.
2. II a egalement semble influencer la formation de l'intestin embryonnaire,
lequel dans de tels blastodermes, s'ouvre toujours sur la face opposee au milieu
de culture.
3. Des observations anterieures qui indiquaient une plus forte tendance de
developpement embryonnaire du cote gauche du jeune blastoderme ont ete
confirmees.
4. Cette tendance peut etre modifiee en changeant la position du blastoderme
sur le milieu de culture.
This work was begun in 1964 during the authors stay at Dr Spratt's laboratory in Minneapolis and was then partially supported by an N.S.F. grant no. 18689.
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{Manuscript received 14 August 1968)