J. Embryol. exp. Morph. 74, 221-234, 1983
Printed in Great Britain © The Company of Biologists Limited 1983
221
Pattern regulation in isolated halves and
blastomeres of early Xenopus laevis
By H. KAGEURA AND K. YAMANA 1
From the Department of Biology, Faculty of Science, Kyushu University
SUMMARY
Xenopus embryos at the 2-cell stage were cut into right and left halves, those at the 4-cell
stage into dorsal and ventral halves or individual blastomeres, and those at the 8-cell stage into
lateral, animal and vegetal halves. Defect embryos, that is, 8-cell embryos from which a
particular pair of blastomeres had been removed, were also prepared. These halves, blastomeres and defect embryos were cultured in 50 % Leibovitz (L-15) medium supplemented with
10 % foetal calf serum and then in 10 % Steinberg solution. Their development was determined from their macroscopic appearance when controls reached stage 26 (early tailbud stage)
or later.
The only halves that could develop into normal larvae or frogs were lateral ones of 2- and
8-cell embryos. An interesting finding was that these halves of 2-cell embryos developed into
only half-embryos when cultured in the above Leibovitz medium beyond the beginning of
gastrulation. On the other hand, most or all the dorsal and ventral halves at the 4-cell stage
and the animal and vegetal quartets at the 8-cell stage did not form normally proportioned
embryos. Defect embryos lacking any two blastomeres of the animal half gave rise to nearly
normal embryos, whereas those lacking two dorsal or two ventral blastomeres of the vegetal
half did not.
From the present results and those of studies now in progress, it is concluded that development of blastomeres and halves from these early embryos, except lateral halves from 2- and
8-cell embryos, is not regulative as expected earlier, and that a certain combination of blastomeres is essential for complete pattern regulation.
INTRODUCTION
Isolation and defect experiments on early amphibian embryos provide much
information on pattern regulation, which is of importance in understanding the
influences of different parts of the animal body on one another during embryogenesis. Many isolation and defect experiments have already been carried
out, but many problems still remain, because of the difficulty in preparing and
culturing isolated blastomeres or defect embryos.
Various kinds of media are now available for culture of isolated blastomeres
and defect embryos. Moreover, we have devised new methods for dissociating
embryos into intact individual blastomeres and for removing a particular blastomere(s) from many embryos. Our methods make use of the newly forming
1
Author's address: Department of Biology, Kyushu University, Fukuoka 812, Japan.
EMB74
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H. KAGEURA AND K. YAMANA
cleavage furrow and do not interfere with furrow formation (Shiokawa &
Yamana, 1979). Thus, they cause slight local damage of the outermost layer of
the blastomere, but no damage of the main body of the blastomere. Therefore,
our methods are more gentle than earlier ones, such as constriction with a hair
noose or a glass rod, and seem to cause little perturbation of the embryo structure. In the present study we prepared isolated blastomeres and defect embryos
of Xenopus laevis at the 2-, 4- and 8-cell stages, cultured them until they became
normal or abnormal tailbud embryos, and in some cases, tadpoles or frogs, and
examined their development by external observation.
The results confirmed that the lateral halves of 2- and 8-cell embryos can
completely regulate their own pattern to become normal tadpoles or frogs. The
dorsal and ventral halves at the 4-cell stage and the animal and vegetal
hemispheres of 8-cell embryos usually gave rise to abnormal embryos, though
the degree of abnormality varied markedly in different series of experiments
and in different individual embryos. This shows that there is little or no
regulation in these halves. Results obtained with individual blastomeres of
4-cell embryos and defect embryos at the 8-cell stage were complementary to
the above ones.
From these and other unpublished data the developments of blastomeres and
halves, except lateral ones, of these early embryos are concluded not to be so
regulative as has been believed, and a certain combination of blastomeres is
essential for complete pattern regulation.
MATERIALS AND METHODS
Embryos and blastomeres
Fertilized eggs were obtained from Xenopus laevis, which had been reared in
our laboratory. Eggs with regular and symmetrical patterns of cleavage and
pigmentation were selected. Blastomeres were named as shown in Fig. 1.
Procedures for preparations of isolated blastomeres and defect embryos and
their culture
Eggs were dejellied with 2-5 % sodium thioglycollate, and then sterilized with
0-1 % sodium p-toluenesulphone chloramide for 1 min. Glassware, instruments
and solutions were all autoclaved. The operation was done on a clean bench.
Dejellied eggs were put into 50 % Leibovitz (L-15) medium supplemented
with 10 % foetal calf serum (L-15 FCS) in a Petri dish that had been coated with
2 % agar. Under a binocular microscope, the vitelline membrane was removed
with a pair of forceps. The egg became dumb-bell shaped during the first
cleavage. When the cleavage was almost complete, one of the resulting blastomeres was rotated twice or three times in one direction around the connecting
part with a hair loop. Then the blastomeres were stood for a while, and the
223
Pattern regulation in Xenopus laevis
(ventral)
1/8 VL
1/8VR
1/8DR
Fig. 1. Designation of blastomeres. Viewed from the animal pole (x). Normally,
pigment distribution in the animal hemisphere is bilaterally symmetrical to the first
cleavage plane. The densely pigmented portion is shown above. (I) 2-cell embryo;
the two halves were designated as right (1/2RH) and left (1/2LH), respectively.
(II) 4-cell embryo; the blastomeres were 1/4DR, 1/4DL, 1/4VR and 1/4VL. The
pairs of dorsal and ventral blastomeres were 2/4DH and 2/4VH, respectively.
(III) 8-cell embryo; the inner and outer circles represent the animal (4/8AQ) and
vegetal (4/8VQ) quartets, respectively. The blastomeres of each quartet are designated as in the 4-cell embryo, except that 1/4 is replaced by 1/8. Underlines indicate
vegetal blastomeres.
cytoplasmic thread that still linked them together was cut with a glass needle.
The separated blastomeres were 1/2RH and 1/2LH (Fig. 1). They then divided
and gave rise to 1/4DR, 1/4DL, 1/4VR and 1/4VL when separated again.
A particular pair of blastomeres was removed directly from the demembranated embryos by repeatedly flipping the cleavage furrow with a glass needle.
The blastomeres are tightly bound to each other only at the cortex after cleavage,
and can easily be separated by this manipulation. Embryos from which a pair of
blastomeres had been removed served as 'defect embryos'.
The embryos and their halves were kept in a vertical orientation with their
animal pole up, except during operations. Halves and blastomeres derived from
the same embryos were arranged so that they could be distinguished throughout
the experimental period. The halves, blastomeres and defect embryos were put
into holes of a tissue-culture plate (Falcon-3034), the surface of which was coated
with 2 % agar. They were cultured in L-15 FCS, which was gradually changed to
10 % Steinberg solution. The former medium is favourable for culture of blastomeres and defect embryos just after operation, but prevents wound healing and
often induces exogastrulation. When control embryos reached stage 26 (early
tailbud stage, about 30 h after fertilization) (Nieuwkoop & Faber, 1967), the
embryos that had developed from isolated blastomeres and defect embryos were
examined macroscopically. Some of them were then allowed to develop further,
and examined occasionally.
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H. KAGEURA AND K. YAMANA
RESULTS
First and second cleavage planes and future body axes
In Xenopus, the first and second cleavage furrows have been shown to correspond roughly to the planes that cut the embryo or larva into right and left or
anterior and posterior halves (Kirschner, Gerhart, Hara & Ubbels, 1980). This
was confirmed in the present study by a different technique. Chimaeras were
formed by fusing a right (1/2RH) and a left (1/2LH) half or a dorsal (2/4DH)
and a ventral (2/4VH) one. The blastomeres used for this fusion were derived
from two different parents: wild-type animals and mutant ones which were
homozygous for periodic albinism (ap) and heterozygous for anucleolate mutation (O-nu). The procedure for making chimaeras and the results obtained will
be published elsewhere.
We formed about 600 right-left and 200 dorsal-ventral chimaeras, and about
80% of each neurulated. The point of relevance here is that in the right-left
chimaeras, the boundary between the two regions occupied by the two different
types of cells ran straight backwards along the median plane from the rostal end to
the caudal end of the embryo. In the dorsal-ventral chimaeras the boundary plane
separated the head from the rest of the animal body at a ventrodorsal angle inclined
towards the tail, its position and inclination varying from chimaera to chimaera.
Thirty-one right-left and 15 dorsal-ventral chimaeric larvae were obtained, and
about one-third of the former and only one of the latter succeeded in metamorphosis. The present results, in addition to earlier ones, are clear-cut evidence for
the validity of our designation and positioning of blastomeres (Fig. 1).
Development of embryonic halves of2-cell embryos
Of the right (1/2RH) and left (1/2LH) halves prepared, 5-10 % died during
cleavage and blastulation, mainly because of abnormal divisions. When controls
reached stage 26, the survivors showed no significant retardation of development. Of the 212 survivors, 154 (about 73 %) developed into quite normal embryos (Table 1). On day 3, tadpoles (stage 41) appeared nearly normal in proportions and were almost the same length as controls, though much more slender
(Fig. 2A). Their eyes were smaller and their fin was somewhat under-developed.
These normal tadpoles were derived from 74 right and 80 left halves of embryos,
including each of the two halves of 48 embryos. There was no difference in the
developmental capacities of the right and left halves with respect to either the
frequency at which normal tailbud embryos arose as indicated above, or the
general appearance of the embryos. Nearly 50% of these .normal embryos
metamorphosed to reach adulthood.
In addition, about 5 % of the survivors exogastrulated. About 13 % showed
some abnormality in their head region, and a few formed double embryos
(Table 1).
Pattern regulation in Xenopus laevis
225
Table 1. Development of right and left halves of2-cell embryos
Normal
Abnormal
Permanent gastrula
Exogastrula
Double embryo
Enlarged belly
Microcephaly and others
Left half
74 (70 %)
80 (75 %)
0
7 (7%)
1 (1%)
8 (8%)
15 (14 %)
105
Total
• » « ? - .
Right half
-•
1 (1%)
3 (3%)
2 (2%)
9 (9%)
12 (11%)
107
••
Fig. 2. Embryos derived from right and left halves (1/2RH and 1/2LH) at the 2-cell
stage. (A) A normal tadpole developed from 1/2RH (lower) and a control (upper).
After 3 days. (B) A half-embryo that developed from a 1/2LH in L-15 FCS. The
cement gland, the left eye and a part of the right eye, and a half-neural tube-like
structure are shown. Right view. After 2 days. (C) The same half-embryo as in (B).
Left view. The left side of the embryo is covered by epidermis, and the tail is curved
dorsally. The left eye can be seen.
At stage 47, we examined the positions of the stomach and intestine and the
coiling of the intestine in a total of 80 normal tadpoles (39 derived from the right
half, and 41 from the left half). Viewed from the ventral side, the stomach of the
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H. KAGEURA AND K. YAMANA
Table 2. Development of dorsal and ventral halves of 4-cell embryos
Dorsal half
Large head and small tail
Near-normal
Exogastrula
63 (83 %)
5 (6%)
8 (11 %)
24 (31 %)
41 (53 %)
12 (16 %)
Vesicle
Vesicle with axial structures
Embryo lacking a head, otherwise nearly normal
Total
Ventral half
76
77
tadpoles was on the left of the intestine, and the intestine coiled clockwise,
irrespective of whether the tadpoles were derived from the right or left half.
An interesting finding was that there was only very limited regulation when the
right and left embryonic halves were cultured in L-15 FCS at least up to the end
of gastrulation. While most of the embryos underwent exogastrulation, about
20% reproducibly developed into half-embryos (Fig. 2B, C). Such embryos
would not have arisen from the embryonic halves if they had been transferred to
10 % Steinberg solution before the beginning of gastrulation. In these halfembryos one of pairs of organs, such as an eye, was missing, and no segmentation
of somites occurred. A still unhealed wound made by separation of a blastomere
was seen on one side of some of these embryos, while in others it remained often
until the beginning of gastrulation.
Development of dorsal and ventral halves of 4-cell embryos
Development of about 5 % of the dorsal (2/4DH) and ventral (2/4VH) halves
of 4-cell embryos was arrested at early stages (Table 2). About 10% of the
survivors of embryos derived from the dorsal half exogastrulated. Most of the
rest were smaller than controls, and had a strikingly large head and a small,
slender tail, the posterior part of which was curved dorsally (Fig. 3A); on day 3
they developed into small tadpoles with a swollen abdomen and slender tail (Fig.
3B). The posterior part of the tail was frequently curved dorsally, like the tail of
tailbud embryos, and always lacked a fin. In addition to these embryos, there
were a few neurulae that seemed to be much more normal (Fig. 3C); on day 3
Fig. 3. A series of embryos developed from dorsal (2/4DH) and ventral halves (2/
4VH) at the 4-cell stage. (A), (B) Embryos derived from dorsal halves and having
an extraordinarily large head and trunk and a slender tail. After 2 days (A) and 3 days
(B). (C) A nearly normal embryo derived from a dorsal half. After 2 days. (D)
Nearly normal tadpoles derived from dorsal halves (middle and lowest). A control
(top). After 3 days. (E) A 'belly piece' derived from a ventral half. After 2 days. (F),
(G) Embryos derived from ventral halves and lacking the head region. After 2 days
(F) and 3 days (G).
Pattern regulation in Xenopus laevis
i
D
Fig. 3
227
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H. KAGEURA AND K. YAMANA
they were normally proportioned, their tail was much wider and they lacked only
the ventral fin (Fig. 3D).
About 80 % of the embryos derived from the ventral half gave rise to cell
masses or vesicles that were covered with epithelium and lined with mesoderm
(Table 2). About one-third of these vesicles had no head or axial structures (Fig.
3E), and seemed to correspond to Spemann's 'belly piece' (Spemann, 1938, cited
by Gerhart, 1980). The other vesicles had small axial structures, the development of which differed greatly in different embryos. Embryos derived from onefifth of the ventral half lacked a head region, but other parts of the body appeared
quite normal (Fig. 3F). After culture for 3 days, tadpoles from these embryos had
no head region, but their trunk and tail were almost normal or rather well
developed (Fig. 3G). Thus, embryos in this group were characterized by lack of
a head region, and incomplete development of axial structures.
Development of single blastomeres of 4-cell embryos
The developments of the two dorsal blastomeres (1/4DR and 1/4DL) were
essentially the same (Table 3). Exogastrulation occurred more frequently in
these dorsal single blastomeres than in the dorsal halves (2/4DH). The
exogastrulae had small axial structures. About half the other embryos had a
disproportionately large head and trunk, and a narrow tail. These embryos
resembled those derived from the dorsal halves (2/4DH), though they were
about half the size of the latter, and their degree of development was much less.
Most of the other embryos had a mass of endoderm.
Embryos derived from ventral blastomeres (1/4VR and 1/4VL) were very
similar to one another (Table 3). Six to 8 % of the embryos exogastrulated, and
the rest became cell masses or vesicles without typical axial structures. However,
half of them had a slight dorsal-ventral axis manifested by localization of a
mesoderm cell mass on the suspected dorsal side. Some embryos had small axial
structures.
In one embryo three of four blastomeres showed dorsal development.
Table 3. Development of individual blastomeres of 4-cell embryos
Dorsal
Vesicle:
lacking a dorsoventral axis
with a dorsoventral axis
with small axial structures
Embryo:
with a large head and a small tail
with a mass of endoderm
Exogastrula
Total
Right
Left
Ventral
Left
Right
1 (2%)
1 (2%)
0
1 (2%)
0
0
15 (31 %) 21 (43%)
18 (37 %) 20 (41 %)
8(16%)
4 (8%)
12 (24 %) 13 (27 %)
10 (20 %) 5 (10 %)
25 (51 %) 30 (61 %)
49
49
2 (4%)
2 (4%)
4 (8%)
49
1 (2%)
0
3 (6%)
49
Pattern regulation in Xenopus laevis
229
Development of left halves of 8-cell embryos
Of the 50 left halves (1/8DL + 1/8VL + 1/SDL + 1/8VL) of 8-cell embryos
prepared, 47 survived beyond cleavage and blastulation. Although 21
gastrulated abnormally, 25 (50 % of the total) became normal or nearly normal
neurulae, which were essentially the same as those derived from the lateral
halves of 2-cell embryos. Thus, lateral halves at the 8-cell stage formed normal
neurulae much less frequently than those at the 2-cell stage, but the halves
derived from the two stages showed no significant differences in their developmental capacities.
In five of the normal or nearly normal neurulae the right half of the head was
less developed than the left half, and the right eye was smaller. Such unbalanced
growth was restored by stage 47. No abnormalities were found in other parts of
the body.
Development of animal and vegetal quartets of 8-cell embryos
In all, 73 animal (4/8AQ) and 83 vegetal (4/8VQ) quartets were obtained
from embryos in the late 8-cell stage, when the third cleavage plane was complete . One of the former and three of the latter were arrested during cleavage and
blastulation.
Embryos derived from the animal quartets were of two types: 'vesicles' and
'vesicles with a long projection' (Fig. 4A). The frequencies of these two types
were similar (38 vesicles and 34 vesicles with a projection). The vesicle type
resembled embryos derived from the ventral halves (2/4VH), except for the
presence of a cement gland. The projection of the vesicles consisted mainly of
muscle, melanophores, and a cement gland, in addition to epidermis, arranged
in this order from the top of the projection to its bottom (Fig. 4B). The projection
grew out of the vesicle at about the time when invagination of mesoderm took
place in controls.
The embryos derived from the vegetal quartets died after 2 or 3 days in 10 %
Steinberg solution. However, when kept in L-15 FCS they continued to develop
further. They were capable of forming a blastopore, but none of them showed
normal invagination, perhaps owing to lack of an expanding animal hemisphere
surface. The embryos had a neural-plate-like structure on their dorsal side,
which then developed into a small axis (Fig. 4C). On day 3 the heart was seen
beating in the ventral position. Epidermis covered the tail, but not other areas
of the embryo. The embryos derived from the vegetal quartets showed no further
development on longer culture, and did not differ significantly in external appearance.
Development of defect embryos
The blastomeres removed from 8-cell embryos were either one of the following
230
4>
•
' ' • *
H. KAGEURA AND K. YAMANA
Me
Fig. 4. Embryos derived from animal (4/8AQ) and vegetal quartets (4/8VQ) at the
8-cell stage. (A), (B) Animal quartet-derived vesicles with a projection. After 2 days
(A) and 3 days (B). C, cement gland. E, epidermis. Me, melanophores. Mu, muscle.
(C) An exogastrula derived from a vegetal quartet. After 2 days.
four pairs (Fig. 1): (a) 1/8DR + 1/8DL, (b) 1/8VR + 1/8VL, (c) 1/8DR + 1/
8DL, (d) 1/8VR + 1/8VL. The numbers of relatively normal neurulae obtained
with defect embryos from which pairs (a), (b), (c) and (d) had been removed
were 20 (40 %), 21 (42 %), 3 (6 %) and 5 (10 %), respectively.
Defect embryos that lacked the ventral blastomeres of the vegetal hemisphere
gave rise to embryos with an extraordinarily large head and a slender tail. These
characters were the same as those of embryos obtained from the dorsal halves.
The frequencies at which normal and nearly normal embryos appeared in these
defect embryos and the dorsal halves were also comparable. The embryos obtained on removal of the vegetal dorsal blastomeres had a small head or
sometimes no head. Some of them had small axial structures. These morphological features were also similar to those of embryos originating from the ventral
halves (2/4VH) as stated above, although no normal or nearly normal embryos
were obtained from these halves.
Removal of blastomeres of the animal hemisphere had much less effect on
development than removal of vegetal blastomeres. Few vegetal blastomeredeflcient embryos reached adulthood, whereas many animal blastomere-deficient
ones succeeded in metamorphosis. Embryos lacking animal blastomeres
Pattern regulation in Xenopus laevis
231
produced rather similar embryos, irrespective of whether dorsal or ventral
blastomeres were removed. In some cases, embryos lacking the dorsal blastomeres appeared similar to those from the ventral halves and their individual
blastomeres.
A full series of defect experiments is now in progress and results will be
described elsewhere.
Blastopore formation and invagination
Blastopores formed in all the cases studied, except animal quartets (4/8AQ)
of 8-cell embryos, from which vegetal material had been excluded. This suggests
that the ability to form a blastopore is localized in the vegetal part of the embryos. This is consistent with the results of Ruud (1925, cited by Gerhart, 1980)
and Grunz (1973, 1977).
Some experimental conditions, such as the osmotic pressure of the medium
and the timing of medium exchange greatly influenced the extent of invagination. Above all, the amount of presumptive ectoderm covering the endoderm
and mesoderm was critical. This has been inferred from the fact that the dorsal
halves could gastrulate normally, but most individual dorsal blastomeres and all
the vegetal hemispheres could not.
The dorsal lip was situated on the side corresponding to the dorsal side of the
original egg, in embryos derived from the right and left halves, the dorsal halves
and their individual blastomeres, and the vegetal hemispheres. The blastopore
was C-shaped (or two blastopores often appeared opposite to each other), but
its orientation was not known in embryos originating from the ventral halves and
their individual blastomeres. The area surrounded by the blastopore was very
wide in vegetal quartets but narrow in ventral halves and their individual blastomeres.
DISCUSSION
In Xenopus, the right and left blastomeres of a 2-cell embryo will normally
form the right and left halves, respectively, of the animal body. However, when
these blastomeres are separated, each of them produces a complete animal. This
was confirmed in the present experiments, it should be stressed that either
blastomeres of more than 60 % of the 2-cell embryos used gave rise to normal
tailbud embryos or tadpoles, and nearly 50 % of them metamorphosed normally.
Thus, either blastomere can compensate for the other and complete pattern
regulation, and there was no detectable difference in the developmental
capacities of the two blastomeres. No inversion of internal organs was observed.
This is not compatible with the finding of Spemann (1938, cited by Mangold,
1953) of inversions of internal organs in about 50 % of his embryos of Triturus
derived from the right half.
Also lateral halves from the 8-cell stage were found to be able to regulate their
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H. KAGEURA AND K. YAMANA
pattern. This excludes the possibility that the only halves that can regulate their
pattern are single blastomeres, and shows that the lateral half of 2-cell embryos
or a set of blastomeres equivalent to it, is essential for complete pattern regulation. We will present direct evidence for this view elsewhere.
During this study we found that in L-15 FCS about one-fifth of the isolated
halves of 2-cell embryos developed into half-embryos, whereas this did not occur
in 10 % Steinberg solution. This is the first report that embryonic halves give rise
to half-embryos even when cultured separately. The fact that the wound made
on separation of the blastomere was still open may be related to the formation
of half embryos. Wound healing may also not occur when a blastomere remains
in contact with an inactivated neighbour in Roux's experiment (1888, cited by
Gerhart, 1980) and this absence of healing of the wound may prevent pattern
regulation in some way. Therefore, this observation is of interest when considering the reason for the discrepancy between Roux's findings and those of others.
Unlike the right and left halves, the dorsal and ventral halves are not capable
of completing pattern regulation: the dorsal halves almost always developed into
embryos with a disproportionately large head and trunk and a slender tail,
whereas the ventral halves gave rise to vesicles without a head region but with
poorly developed axial structures. Essentially the same results were obtained
with individual blastomeres of the dorsal and ventral halves. Development of the
individual blastomeres was definitely inferior to that of the halves, that is, a pair
of blastomeres. This duplication effect suggests that the more material there is
available for development, the further development proceeds. The present
results with the dorsal and ventral halves are in part compatible and in part
incompatible with those of Ruud (1925, cited by Gerhart, 1980). She reported
that two dorsal blastomeres of 4-cell embryos of Triturus developed to quartersized gastrulae and partial neurulae, whereas ventral ones developed to only
ventral pieces.
Defect experiments provide complementary results to those obtained in isolation experiments. Embryos that retain dorsal blastomeres of the vegetal
hemisphere but not ventral blastomeres gave rise to similar, though much more
normal, embryos, like the dorsal halves and their individual blastomeres.
Likewise, embryos from which dorsal blastomeres had been removed developed
into embryos resembling those of ventral halves and their individual blastomeres. In either type of defect embryo, the degrees of abnormality were greater
when the vegetal blastomeres had been removed than when the animal blastomeres had been removed, suggesting that the vegetal blastomeres are more
important in pattern regulation. Furthermore, the dorsal and ventral blastomeres of the animal hemisphere seem to be interchangeable to some extent.
These facts provide further support for the view of Boterenbrood and Nieuwkoop(1973).
The development of the animal and vegetal quartets were also studied. These
quartets were shown to produce vesicles of epidermis (with or without a
Pattern regulation in Xenopus laevis
233
projection) and exogastrulae, respectively. Thus, neither the animal nor vegetal
halves of 8-cell embryos are able to complete pattern regulation. Our conclusion
on the development of animal quartets agrees in general with that of Ruud (1925)
and Vintemberger (1934) (both cited by Gerhart, 1980). More recently Grunz
(1977) isolated animal quartets from 8-cell embryos of Triturus alpestris and
found that they developed to ciliated epidermis in agreement with our present
findings. However, unlike us, Grunz (1977) obtained few nearly complete embryos with all brain regions from vegetal quartets. Perhaps, the difference
reflects some difference in the experimental materials and manipulations used.
From these results we conclude that the separation of blastomeres of Xenopus
embryos along the second and third cleavage planes restricts the developmental
capacity of the isolated halves and blastomeres so much that their development
is no longer regulatory, i.e. they form mainly what they would have formed when
left in place. The only blastomere or half in which regulation is complete is the
lateral one, that is, the half obtained by cutting 2- and 8-cell embryos along the
first cleavage plane. In the present study we did not examine the development
of lateral halves of 4-cell embryos, but they can be expected to regulate their
pattern. Defect experiments are now in progress to test the developmental
capacities of 'embryos' of every possible combination of blastomeres. Results of
these experiments and the present results show that for normal or nearly normal
embryogenesis either one of the dorsal and either one of the ventral blastomeres
of the vegetal hemisphere and any two of the four blastomeres of the animal
hemisphere are essential.
We are grateful to Dr J. B. Gurdon for a generous supply of the mutant animals used in the
present study, and to Dr K. Shiokawa for helpful discussion.
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{Accepted 22 October 1982)