J. Embryol. exp. Morph., Vol. 18,1, pp. 27-41, August 1967
Printed in Great Britain
27
The developmental potentialities
of regeneration blastema cell nuclei as
determined by nuclear transplantation
By ANN M. C. BURGESS 1
From the Department of Anatomy and Embryology,
University College, London
Formerly, many embryologists believed that the blastema cells in regenerating
amphibian tails and limbs arose from a reserve of undifferentiated cells, but
since the work of Hertwig (1927) and Butler (1935) few modern investigators
would support this view. Others have suggested that blastema cells are of
epidermal origin (Godlewski, 1928; Rose, 1948). However, this idea has largely
fallen into disrepute following the investigations of Karczmar & Berg (1951),
Chalkley (1954) and Hay & Fischman (1961). There is now abundant evidence
that blastema cells arise by the dedifferentiation of some of the stump tissues, in
particular the stump muscle and cartilage (Thornton, 1938 a, b; Hay, 1958,1959,
1962; Hay & Fischman, 1961), and that contributions may also arise from fibroblasts when they are present.
Unfortunately, there has been a great deal of confusion concerning the exact
meaning and implications of the term 'dedifferentiated'. The only definite
evidence is that during the process of dedifferentiation there is a loss of specialized
structural and functional characteristics. This evidence comes not only from the
work already mentioned (Thornton, 1938 a, b; Hay, 1958, 1959, 1962; Hay &
Fischman, 1961) but from many other investigators, notably Holtzer (1961),
who studied the problem of dedifferentiation by using the technique of fluorescein-labelled antibodies (Coons & Kaplan, 1950) on regenerating salamander
tails and limbs, and found that by 7 days the blastema cells no longer bind antimyosin. Perske, Parks & Walker (1957) have shown that liver cells which
dedifferentiate in tissue culture lose several enzymes and Holtzer, Abbott, Lash
& Holtzer (1960), using thionin to test for chondroitin sulphate, have shown that
chondrocytes from 10-day chick vertebrae, which had undergone rapid mitosis
in tissue culture, ceased to synthesize chondroitin sulphate and were unable to
resynthesize it and so redifferentiate either in vitro or when grafted into the
coelom of 4-day chick embryos or on to the chorioallantoic membrane.
1
Author's address: Department of Anatomy and Embryology, University College,
Gower Street, London, W.C. 1, U.K.
28
A. M. C. BURGESS
The simplification of cellular structure which occurs during dedifferentiation
has often given rise to the view that dedifferentiated cells become not only
morphologically similar to 'embryonic cells' but that there is also an accompanying reassertion of the wide developmental potentialities associated with
cells during the earliest stages of development. The purpose of the present
series of experiments has been an attempt to determine, by using the nuclear
transplantation technique, whether the dedifferentiated cells of Xenopus limb
blastemata actually do regain wider and more generalized developmental
potentialities.
MATERIALS AND METHODS
The South African clawed toad Xenopus laevis was used for all experiments
and all stages have been numbered according to Nieuwkoop & Faber (1956).
In order to induce amplexus and oviposition, adult female Xenopus were
injected with 340 i.u. and males with 160 i.u. each of chorionic gonadotrophins
(Pregnyl, Organon Laboratories). This supplied both the fertilized eggs used as
controls and for rearing the larvae, and, after removal of the male, the unfertilized eggs used as the recipients of the donor nuclei.
Nuclear transplantation. The method of transplanting single cell nuclei was
essentially the one adapted by Elsdale, Gurdon & Fischberg (1960) for use with
Xenopus eggs, from the original method of Briggs & King using Rana pipiens
(1952). The following slight modification was made to the method of Elsdale
et at. It was found that degassing the liquid paraffin under vacuum removed the
considerable quantities of dissolved air and, by reducing the compressibility of
the liquid paraffin, enabled the movement of fluid through the micropipette to
be more easily and accurately controlled. The method used for the transplantation of clumps of nuclei was that devised by King & McKinnell (1960). Ultraviolet light was used to inactivate the female pronucleus of the host egg according
to the method of Elsdale et al. A source of ultraviolet light was provided by an
80 W Mazda mercury street lamp from which the outer glass casing was removed.
This provided an ultraviolet source of 45 J/s or 56 % of the total energy output,
the other 44 % of the emitted energy being in the form of conducted and convected heat 24%, infra red 15% and visible light 5 % (Ruff, 1964). During
irradiation and nuclear transplantation, the eggs were maintained in a stable and
constant position by putting them into small hemispherical depressions cut in
solid 'Araldite' contained in a watch-glass.
Donor cells. In order to obtain larvae for the production of blastemata,
fertilized eggs were reared until stage 59-60. At this stage, the hind limbs are
well developed and, although they are not quite identical with the adult limbs in
some minor respects, they are fully differentiated (Nieuwkoop & Faber, 1956)
and contain only a small population of fibroblasts (Dent, 1962). At this stage,
the larvae are undergoing metamorphosis but they will still form satisfactory
Blastema cell nuclear transplantation
29
regeneration blastemata whose general pattern of formation by dedifferentiation
is substantially the same as that found by other investigators (notably Thornton,
1938 a, b) using other species of Amphibia, although on regeneration a heteromorphic conical structure is formed instead of a normal limb. Blastemata
formed at this advanced larval stage are not so prolific as those developed after
amputation at earlier stages, but in the younger larvae the tissues are not fully
differentiated and there is a large number of fibroblasts present (Dent, 1962).
Blastema cells for transplantation were obtained by the following procedure of
amputation and culture: a larva of st. 59-60 was anaesthetized by immersion in
a 0015 % aqueous solution of M.S. 222 (tricaine methane sulphonate). When it
would no longer respond to tactile stimuli, it was taken dry on to a microscope
slide and one of its hind limbs was amputated with a scalpel. Transection was
at right angles to and approximately halfway along the tibio-fibula. The larva
was then put into approximately 250 cm3 of aerated tap water in a crystallizing
dish and left for 1 week. During this time it was maintained at room temperature
and fed on small quantities of powdered nettles (Urtica sp.). Previous experiments (unpublished) had shown that under these conditions dedifferentiation
was completed between 7 and 8 days. At the end of 1 week the larva was again
anaesthetized and the entire blastema was amputated and put into a cavity slide
prior to disaggregation. This formed the source of the blastema cells for the
transplantation experiments.
Disaggregation of donor cells. The excised blastema was first treated with
Barth's dissociating medium (Barth & Barth, 1959) containing 0074 % versene
(disodium ethylene-diamine-tetra-acetate), until the epidermis could easily be
stripped off with watchmaker's forceps, leaving the blastema intact. This
normally took approximately 4 min at room temperature. In the first series of
experiments involving the transplantation of single blastema cell nuclei, a
suspension of single cells was produced by treating the tissue with a 0-3 %
solution of trypsin in Niu and Twitty saline (Flickinger, 1949) followed by a
solution of 9 /tg/ml DNase in Niu and Twitty saline. Madden & Burk (1961)
used trypsin and DNase to produce a viable suspension of single cells from a
variety of solid tumours and in the present work the viability of the single
blastema cells was tested with Lissamine Green (Holmberg, 1961) and was
found to be at least 90 %. In the experiments involving the transplantation of
clumps of cell nuclei, clumps of a suitable size were obtained by using the same
procedure but omitting the treatment with DNAase.
Transplant controls. Three types of control transplants were carried out.
First, several single mid-gastrula endoderm nuclei were transplanted at the
beginning and at the end of each experiment in order to detect any errors in
the experimental procedure and also to determine whether the host eggs
were suitable recipients of donor nuclei. Gastrula endoderm nuclei have
been regularly used by other investigators (e.g. Briggs & King, 1953;
Elsdale et al. 1960; Gurdon, 1960 a, b, 1962) and their results provide a standard
30
A. M. C. BURGESS
of the development which can be expected using the nuclear transplantation
technique.
Secondly, a series of control experiments was carried out using dead blastema
cell nuclei transplanted by the 'clump' method. The excised blastema was
heated for 7 min in boiling Niu and Twitty saline; the epidermis was stripped
off following treatment with Barth's dissociating medium and the cells were
partially disaggregated by trypsin as described above. Staining with 1 %
Lissamine Green (after the method of Holmberg, 1961) in Niu and Twitty saline
showed that all the cells readily took up the dye, indicating 100 % mortality of
the heated cells. The use of dead blastema cell nuclei as controls was necessary
because of the low degree of development observed with living blastema cells
and also because of the findings of Briggs, Green & King (1951) which showed
that even an irradiated sperm could in some instances cause enucleate Rana
eggs to cleave. It was necessary to determine whether dead blastema cell nuclei
could give rise to normal development and until this had been ascertained, the
conclusion that any development which followed the injection of either blastema
or epidermal cell nuclei was due to the intrinsic properties of these nuclei was not
justified.
The third control involved the transplantation of a nucleus of a living differentiated cell type in the form of epidermal cell nuclei taken from the hind limbs
of larvae at the same stage of development as those used for the production of
blastema cells. They were transplanted by the 'clump' method in the same way
as the blastema cell nuclei. Epidermal cell nuclei were used for the following
reasons. Practically, they are very convenient to use; the epidermis is easily
stripped from the underlying tissue following treatment with versene, and the
epidermal strips readily disaggregate to give clumps of a convenient size.
Furthermore, since it has been shown (Karczmar & Berg, 1951; Chalkley, 1954;
Hay & Fischman, 1961) to be extremely unlikely that epidermal cells contribute
to the formation of the blastema, they form a suitable control differentiated cell
type.
Quality and irradiation controls. In addition, control experiments were used
to give an indication of the quality of the eggs used as hosts and to determine
whether the process of irradiation had effectively destroyed the female pronuclei. Before a transplantation experiment was started, fertilized eggs were
collected and the percentage fertility determined. If this was sufficiently high
(see tables), the male was removed and the unfertilized eggs subsequently laid
were used as recipients of nuclei. Although these figures give an indication of
the quality of the host eggs, they are of course also influenced by the breeding
condition of the male. Fertilized eggs laid at the beginning of the experiment
were also used to test the efficiency of the enucleation method. The amount of
ultraviolet light needed for successful enucleation varies with the batch of eggs
and a subjective estimate was made according to the criteria of Elsdale et al.
(1960). Eight fertile eggs received the estimated correct dose and were left to
Blastema cell nuclear transplantation
31
develop in dilute Holtfreter saline. When a correct estimation of the dose had
been made the enucleated eggs developed as androgenetic haploids and exhibited
signs of the haploid syndrome (Hamilton, 1963). Experiments in which it was
subsequently shown by this method that the ultraviolet dose had been wrongly
estimated are not included in the results.
RESULTS
In Tables 1-5, percentage fertility refers to the fertilized eggs described as
'quality controls' in the Materials and Methods, and 'control transplants'
refers to the mid-gastrula endoderm cell nuclear transplants described under
'transplant controls' in the Materials and Methods.
Transplantation of single blastema cell nuclei. The results obtained from the
nuclear transplantation of individual single blastema cell nuclei are shown in
Table 1. The total number of transplants performed in this series of experiments
was 232. Of this number, the only signs of development which were observed
were in the 46 (19-8 %) specimens which showed irregular and abortive cleavages.
The control transplants of single gastrula endoderm cell nuclei gave rise to
development which resulted in an average of 28-6 % surviving to feeding stages.
A further 2-9 % were arrested during neurulation; 8-6 % were arrested during
gastrulation and 12-9 % were arrested as blastulae. Thus, 53-0 % of the control
transplanted gastrula cell nuclei gave rise to development at least as far as mid
to late blastula stages (st. 8-9).
Transplantation of blastema cell nuclei by the 'clump' method. The results
obtained from the nuclear transplantation of clumps of approximately 10-20
blastema cell nuclei are shown in Table 2. A total of 186 transplants were made
and of these, 32 (17-2%) of the host eggs showed abortive cleavages. In all,
10 (5-4 %) of the host eggs underwent initial normal cleavages, and of these one
was arrested at the 16-cell stage and one at approximately the 32-cell stage;
6 (3-2 %) were arrested as blastulae and 2 (1-1 %) started to gastrulate. There
had been no excessive leakage of cytoplasm, which sometimes occurs from the
host egg when the micropipette is removed after injection, and it is difficult to
account for this sudden arrest in development.
Of the 6 (3-2 %) specimens that were arrested as blastulae, 4 showed normal,
regular and complete cleavage up to the late blastula stage (st. 9) but were
arrested before the onset of gastrulation. The other 2 were arrested as mid
blastulae (st. 8); one was apparently normal but in the other case there had
been a rather excessive leakage of cytoplasm from the host egg at the time of
transplantation and it was probably this factor which led to an arrest in development. Three host eggs gave rise to partial blastulae.
The 2 (1-1 %) embryos that were arrested as gastrulae developed only as
far as st. 10J, that is to say, there was a concentration of pigment in the region
of the presumptive blastopore and the beginning of the formation of the dorsal
60
80
80
60
70
80
80
70
% Fertile
6
5
7
5
7
6
7
3
46
19-8%
100%
Abortive
cleavage
22
26
30
32
31
27
31
33
232
Total
transplants
•«•
Normal
cleavage
Single blastema cell nuclei transplants
12-9%
100%
1
2
1
1
2
9
1
1
1
8-6%
2
6
1
1
1
Arrested
gastrulae
Arrested
blastulae
7
8
8
8
8
12
7
12
70
Total
transplants
2-9%
1
2
1
Arrested
neurulae
28-6%
2
1
2
2
2
4
3
4
20
Normal
larvae
Control transplants (single gastrula endoderm cell nuclei)
Figures showing survival refer to the numbers which were arrested at a particular stage in development. Percentage survival is
given in terms of the total number of transplants. 'Percentage fertile' refers to the fertilized eggs described as 'quality controls' in
the Materials and Methods.
Table 1. Results from the blastema cell nuclei transplanted singly and the control single gastrula endoderm cell nuclei
i—i
to
c;
O
en
p
•
to
Blastema cell nuclear transplantation
33
lip, which, however, never became crescentic or started to extend ventrally.
Both specimens appeared normal with the exception of the presence of the small,
plug of cytoplasm which generally remains on the surface at the site of
injection.
The single gastrula endoderm cell nuclei which were used as control transplants gave a total of 16 (29-1 %) embryos which survived to feeding stages.
A further 2 (3-6 %) were arrested as neurulae; 7 (12-7 %) were arrested during
gastrulation and 7 (12-7%) were arrested as blastulae. In all, 32 (58-1 %) of
the control gastrula endoderm cell nuclei resulted in development to at least
mid to late blastula stages.
Transplantation of epidermal cell nuclei by the 'clump'' method. The results
from this series of experiments are shown in Table 3. The total number of
transfers was 155 and the number of host eggs which showed abortive cleavages
subsequent to transplantation of the epidermal cell nuclei was 24 (15-5%).
Nine (5-8 %) of the host eggs underwent normal cleavage in the initial stages but
one was arrested at the 16-cell stage, although up until this time it appeared
normal. Of the remaining 8 specimens 7 (4-5 %) developed to apparently a
normal late blastula stage (st. 9), although one of them was observed to have
leaked cytoplasm to a more than usual extent, so that quite an appreciable bleb
was present between the surface of the blastula and the vitelline membrane.
All 7 of these specimens ceased development at the late blastula stage (st. 9)
and eventually became necrotic, but the eighth specimen showed signs of early
gastrulation, which did not, however, advance beyond the stage of pigment concentration in the region of the presumptive blastopore and the formation of the
dorsal lip of the blastopore which, as in the case of the gastrula derived from the
blastema cell nuclei, did not become crescentic. This early gastrula showed no
abnormalities other than the small bleb of cytoplasm which frequently remains
as a result of piercing the host egg with a micropipette. Six of the host eggs
developed into partial blastulae.
The single gastrula endoderm cell nuclei which were used as control transplants gave a total of 14 (29-8 %) embryos surviving to feeding stages. A further
4 (8-5 %) were arrested as neurulae; 4 (8-5 %) were arrested during gastrulation
and 5 (10-6 %) were arrested as blastulae. A total of 27 (57-4 %) of the control
gastrula endoderm cell nuclei resulted in development to at least mid to late
blastula stages (st. 8-9).
Transplantation of dead blastema cell nuclei by the 'clump'' method. The results
from this group of experiments are shown in Table 4. The total number of
transfers in this group was 176. Of this number, none of the host eggs showed any
signs of development, or even initial cleavage of either a normal or an abnormal
nature.
The single gastrula endoderm cell nuclei which were used as control
transplants gave a total of 13 (28-3%) embryos surviving to feeding stages.
A further 3 (6-5%) were arrested during gastrulation and 7 (15-2%) were
3
J E E M l8
3
6
5
7
6
5
32
17-2%
26
31
33
30
35
31
186
100%
Abortive
cleavage
Vl°/t;
2
1
1
Arrested
during
early
cleavage
i-6%
3
2
1
Partial
blastulae
3-2%
2
6
1
1
1
1
Arrested
blastulae
2
1-1%
1
1
Arrested
gastrulae
55
100%
12
12
7
8
8
8
Total
transplants
2
3
7
12-7 %
2
1
7
12-7 %
1
1
2
1
1
Arrested
gastrulae
Arrested
blastulae
3-6%
2
2
#
Arrested
neurulae
29-1%
2
3
3
2
4
2
16
Normal
larvae
Control transplants (single gastrula endoderm cell nuclei)
80
70
80
60
70
%
fertile
6
6
3
5
4
24
15-5%
100%
Abortive
cleavage
32
34
30
27
32
155
Total
transplants
0-65 %
1
1
Arrested
during
early
cleavage
3-9%
1
1
2
6
2
Partial
blastulae
4-5%
2
1
1
3
7
Arrested
blastulae
Epiderma 1 cell nuclei transplants; (clumps)
1
0-65%
1
Arrested
gastrulae
o
d
(30
n
•
1
5
10-6 %
47
100%
3
9
1
Arrested
blastulae
12
8
10
8
Total
transplants
2
4
8-5 %
2
Arrested
gastrulae
29-8%
o.c
2
1
4
4
3
4
2
1
14
Normal
larvae
1
0/
Arrested
neurulae
Control transplants (single gastrula endoderm cell nuclei) oo
Table 3. Results from the epidermal cell nuclei transplanted in clumps and the control single gastrula endoderm cell nuclei
70
80
80
60
90
70
fertile
/o
Total
transplants
Blastema (;ell nuclei transplants (clumps)
Table 2. Results from the blastema cell nuclei transplanted in clumps and the control single gastrula endoderm cell nuclei
Conventions as in Table 1.
70
60
80
70
80
80
% fertile
100%
Total
transplants
100%
.Normal
cleavage
8
7
8
8
8
7
46
Abortive
cleavage
20
27
31
24
33
41
176
Total
transplants
15-2%
7
1
1
2
1
2
1
6-5%
1
1
3
Arrested
gastrulae
Arrested
blastulae
6-5 %
1
3
1
1
Arrested
neurulae
28-3%
2
2
3
2
2
2
13
Normal
larvae
Control transplants (single gastrute1 endoderm cellnuclei)
U)
a
^
Conventions as in Table 1.
Dead blastema cell nuclei
transplants (clumps)
^
.
Table 4. Results from the dead blastema cell nuclei transplanted in clumps and
the control single gastrula endoderm cell nuclei
36
A. M. C. BURGESS
arrested as blastulae. A total of 26 (56-5 %) of the control endoderm cell nuclei
resulted in development to at least mid to late blastula stages (st. 8-9).
DISCUSSION
The main finding from these experiments is that blastema cell nuclei have not
been demonstrated to regain such a wide spectrum of embryonic potentialities
as has been supposed by many investigators. Before considering the results to
support this rinding it is first necessary to consider the controls. As has been
mentioned in the Materials and Methods section under 'quality controls', the
percentage fertility was estimated on all egg clutches used for experiment and
any found to be of poor quality were not used; the low level of development
obtained with the blastula and epidermal cell nuclei is therefore not due to the
use of inferior eggs. The ultraviolet irradiation of control fertilized eggs ('irradiation controls') ensured that there was always a check on the efficiency of the
destruction of the female pronucleus in spite of the variation in dose needed for
different batches of eggs. Any development could therefore not be attributed to
the egg nucleus.
In considering the figures from each group of experiments, two methods have
been used. In Table 5, which compares all the relevant final results, the figure
appearing in each column represents not only the numbers of embryo arrested
at a particular stage of development but also those that went on to develop
further. In the other tables, which present the results as they were recorded, only
the numbers arrested at a particular stage are shown in each column.
The transplantation of controls of single gastrula endoderm cell nuclei gave
results comparable with those obtained by other workers, thus establishing
the adequacy of the technique. By contrast the lack of development in host
eggs that had been transplanted with single blastema cell nuclei was striking
(Table 1). The only exceptions were the 46 eggs (19-8 %) which showed abortive
cleavages. It has been shown that contact with the saline medium in which
transplants are carried out has an adverse effect upon the nuclei (Comandon &
de Fonbrune, 1938; Briggs & King, 1953; Gurdon, 1960a, b). Blastema cells
are small and have little cytoplasm, and therefore it seemed possible that the
transplanted nuclei were being damaged by contact with the saline or by the tip
and sides of the micropipette and that the lack of development did not necessarily reflect a complete lack of potency of the nuclei. This conclusion was
borne out by the results obtained from the blastema cell nuclei transplanted in
clumps.
The remaining four groups of results which are pertinent to this discussion
are those from the blastema cell nuclei and the three groups of control transplants, i.e. the dead blastema cell nuclei and the epidermal cell nuclei, transplanted by the 'clump' method, and the already mentioned gastrula endoderm
cell nuclei which were transplanted singly and used throughout as controls.
Blastema cell nuclear transplantation
37
From Table 4 it will be seen that in no case did the transplantation of multiple
dead blastema cell nuclei result in any cleavage of either a normal or an abnormal
nature, so that the idea that development could commence in the absence of an
active living nucleus can be dismissed. This series of experiments may be compared with those performed by Briggs & King on Rana (1953), in which they
found that granules from tail-bud embryos, when injected into enucleate eggs,
gave a complete lack of development. In view of the findings of Briggs et ah
(1951) on Rana, it is perhaps surprising that not even abortive cleavages were
observed. It might have been expected that dead blastema cell nuclei would
form sufficient cleavage centres to give rise to at least initial development. As
Briggs et ah used Rana and these experiments have been done with Xenopus,
it is possible that this is due to a basic species difference.
Table 5. Comparison of the results obtained from the transplantation of blastema
cell nuclei and epidermal cell nuclei {^clump"1 method) and the single gastrula
endoderm cell nuclei
Percentages calculated not only from the numbers of embryos arrested at a particular
stage of development but also those that went on to develop further.
Type of development
Multiple
blastema
cell
nuclei
Total % cleavages including abortive cleavages
24-2
and partial blastulae
5-4
% cleaving normally
% forming blastulae
4-3
1-1
% forming gastrulae
1-6
% forming partial blastulae
% forming abortive cleavages
17-2
% forming neurulae
% giving feeding stages
* Not recorded.
Multiple
epidermal
cell
nuclei
25-2
5-8
5-15
0-65
3-9
15-5
Single
gastrula
control
cell
nuclei
*
57-3
57-3
44-5
*
*
35-3
291
Table 5 shows in a comparative form the results which were obtained from
the transplantation of blastema cell nuclei and epidermal cell nuclei by the
' clump' method, and the gastrula endoderm cell nuclei which were transplanted
singly. The total percentages of cleavages of any type which were observed (the
figures include all abortive cleavages and cleavages which resulted in partial
blastulae) are found to be very similar. When the figures are broken down into
their constituent types, it is seen that there is a close agreement between the
percentages obtained from the blastema nuclei and from the epidermal nuclei.
For instance, blastema cell nuclei caused a total of 5-4 % of the host eggs to
cleave normally and regularly and this is very close to the percentages found
with the epidermal nuclei (5-8 %), but much less than the numbers of gastrula
endoderm nuclei which gave normal cleavages (57-3 %).
38
A. M. C. BURGESS
Surveying the results in Table 5 as a whole, it is apparent that the figures from
the blastema nuclei are similar to the results from the epidermal nuclei. On the
other hand, the differences in the results from the blastema nuclei and the
gastrula endoderm nuclei are extremely marked. The conclusion may be drawn
that, as the results from the blastema cell nuclei so closely resemble those
obtained from the differentiated cell type and differ by such a high degree from
those obtained from the undifferentiated cell type, the blastema nuclei are still
differentiated in spite of the fact that their cytoplasm has lost its specialized
structural and functional characteristics.
When we come to consider the morphology of dedifferentiated cells and
undifferentiated embryonic cells, it is not surprising that they should show a
close resemblance if only for the fact that they both lack any of the well marked
cytological specializations which would enable them to be distinguished. It is
not difficult to see, when the problem is viewed superficially, how the idea has
arisen that because of the morphological similarities there should also be a
similarity of development potentialities. However, when the question is considered further, it would seem unlikely that the function of dedifferentiated
blastema cells should closely resemble the function of undifferentiated cells
when their immediate environments are so dissimilar. To take one example,
amphibian embryonic cells are largely concerned with the breakdown of yolk
and the utilization and re-synthesis of its products. This function of yolk breakdown must be absent from the cells of the regeneration blastema. This is only
one extreme example of the many differences which may be assumed to exist
between undifferentiated and dedifferentiated cells.
Even if the process of cellular dedifferentiation does not involve a return to
the embryonic state, yet none the less there is a suspension of the mechanisms
which lead to the production of specialized cytoplasmic structures. This suspension is reflected in the fact that there is a loss of structural and functional
characteristics and an apparent simplification of structural organization during
dedifferentiation.
SUMMARY
1. Dedifferentiated cell nuclei of the regeneration blastema cells of the toad
Xenopus laevis have been transplanted into enucleate eggs of the same species in
an attempt to determine the developmental potentialities of the blastema nuclei.
2. It was necessary to transplant the blastema nuclei in clumps rather than
singly because of their small size and paucity of cytoplasm. Control nuclear
transplants consisted of an undifferentiated cell type in the form of gastrula
endoderm cells and a differentiated cell type in the form of epidermal cells, and
also nuclei from dead cells. Additional controls were used to check the egg
quality and the efficiency of the enucleation technique.
3. The very poor level of development which resulted from the transplantation
of multiple blastema cell nuclei was similar to that obtained with multiple
Blastema cell nuclear transplantation
39
epidermal cell nuclei but much less than that obtained with single gastrula
endoderm cell nuclei.
4. It is concluded that the blastema cells more closely resemble the differentiated than the undifferentiated cell type in their developmental potentialities
and that the loss of specialized morphological and functional characteristics
which occurs during dedifferentiation does not imply a return to a more 'embryological' state with the regaining of full developmental potentialities.
RESUME
Transplantation de noyaux cellulaires provenant d'un blasteme de regeneration,
pour Vetude de leurs potentialites embryogeniques
1. Des noyaux de cellules dedifferenciees du blasteme de regeneration de
Xenopus laevis ont ete transplanted dans des oeufs enuclees de la meme espece,
pour essayer de determiner les potentialites embryogeniques de ces noyaux.
2. II a ete necessaire de transplanter les noyaux de blasteme en petits groupes
plutot qu'isolement, a cause de leur petite taille et de leur pauvrete en cytoplasme peripherique. Les temoins ont consiste en transplantations, d'une part de
noyaux d'un type cellulaire indifferencie (cellules endodermiques de gastrula),
d'autre part de noyaux de cellules differenciees (cellules epidermiques), et aussi
de noyaux de cellules mortes. Des temoins supplementaires ont ete realises pour
verifier la qualite des oeufs et Pefficacite de la technique d'enucleation.
3. Le degre de developpement maigre atteint apres transplantation des
noyaux de cellules blastematiques a ete semblable a celui qu'on a obtenu avec
des noyaux de cellules epidermiques mais tres inferieur a celui qu'ont realise
des noyaux d'endoderme gastruleen.
4. On conclut que les cellules de blasteme ressemblent davantage a un type
cellulaire differencie qu'a un type cellulaire indifferencie, par leurs potentialites
embryogeniques, et que la perte des caracteristiques specialisees, morphologiques
et fonctionnelles, qui survient lors de la dedifferenciation, n'implique pas un
retour a un etat plus 'embryonnaire', avec recuperation de potentialites
embryogeniques completes.
I wish to thank Dr E. M. Deuchar for her advice and encouragement throughout every
stage of this work. I should also like to thank both Professor J. Z. Young, in whose department this project was carried out, and Dr R. Bellairs, for their helpful criticism of the manuscript. My thanks are also due to Miss S. Bevan and Miss J. Weedon for technical assistance
and to the Medical Research Council for financial support.
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{Manuscript received 19 December 1966, revised 13 February 1967)