Download Morphogenetic Interactions before Gastrulation in the

Morphogenetic Interactions before Gastrulation in the
Amphibian, Xenopus laevis—Regulation in Blastulae
by A. s. G. CURTIS 1
From the Department of Anatomy and Embryology, University College, London
WITH ONE PLATE
T H E site of the future dorsal lip of the gastrula is already determined to a considerable degree at the grey-crescent region by the 8-cell stage, probably by means
of interactions within the cortex (Curtis, 19626). But this determination is still
easily disturbed because excision of the grey-crescent cortex from the 8-cell stage
does not prevent the appearance of a dorsal lip. The embryo forms a new dorsal
lip by a process of regulation. Similarly, Votquenne (1933) destroyed the blastomere containing the grey crescent at the 8-cell stage in Ranafusca and nevertheless obtained nearly normal embryos; which result indicated that regulation had
happened. At a later stage, late blastula, excision of the cells of the presumptive
dorsal Up region does not prevent a dorsal lip forming and subsequent neural
induction occurring (Goerttler, 1926) although the embryos are of the abnormal
'ring' type. But though regulation in Goerttler's experiments can hardly have
occurred before gastrulation, due to the late stage used, it remains an open
question whether the regulation of the embryo after Votquenne's excision of a
micromere or my excision of cortex from the 8-cell stage embryo, happens soon
after the operation of excision or whether the necessary interactions amongst
cells are delayed until some later stage such as gastrulation. The answer to this
question will help to reveal the extent of developmental change before gastrulation. There is reason for thinking that a degree of morphogenesis of the various
presumptive regions occurs by late blastula stages because Dalcq (1933) showed
that considerable localization of organs had occurred by the late blastula stage
of R. fusca.
For these reasons experiments have been made on regulation of the presumptive dorsal lip region in blastulae. Early blastulae have been used since there is a
sufficiently long time before they become gastrulae to determine whether regulation occurs soon after removal of tissue or at gastrulation. In addition, since the
presumptive dorsal lip region plays a major part in the imagination process when
it reaches the gastrula stage (Schechtman, 1942) such experiments may throw
light on the question of how the morphogenetic movements of gastrulation
1
Author's address: Department of Anatomy and Embryology, University College, Gower Street,
London, W.C. 1, U.K.
[J. Embryol. exp. Morpb. Vol. 10, Part 3, pp. 451-63, September 1962]
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A. S. G. CURTIS—REGULATION IN BLASTULAE
are determined during development. In preliminary work I found that a
simple chemical treatment blocks regulation if applied just after excision of cells
from the blastula, but does not do so if applied slightly later. These experiments
will be described at length. The general deduction from them was that one essential step in regulation occurs fairly soon after excision. In consequence a series
of experiments designed to elucidate further the mechanism of this interaction
will be described.
A preceding paper (Curtis, 19626) showed that important morphogenetic
factors are located in the cortex. These experiments provide some suggestions
that these factors are still located in the cortex at blastula stages and interact
between various parts of the embryo during regulation.
METHODS
Xenopus laevis morulae were decapsulated and stripped of their vitelline
membranes by hand: they were cultured in Holtfreter saline buffered to pH 7-0
with 0 001 M 2-amino-2-hydroxymethyl-1, 3-propanediol-h ydrochloric
acid (tris-HCl). In experiments on regulation after excision, the whole of the
dorsal lip and much of the presumptive
nervous system were the regions excised.
The tissues were removed down to the
blastocoel roof. Since the exact extent
of these presumptive regions is uncertain,
more tissue than contains these regions
was cut out (see Text-fig. 1); in consequence it is unlikely that any tissue which
would normally invaginate and carry out
induction remained. The controls for
TEXT-FIG. 1. Side view of the embryo at early
blastula stage. The dashes indicate the limits of these experiments were unoperated emthe regions which were excised in these experi- bryos. Control and experimental embryos
ments. They include presumptive dorsal lip
and much of the presumptive nervous tissue. were treated with 0001 M tetra-sodium
The position of the lower limit was carefully ethylene-diamine tetra-acetate buffered
kept to in all excisions but the position of the
animal and anterior limit was somewhat vari- at pH 8-2 with 0-001 M 2-amino-2-hydroable, in many cases rather less presumptive xymethyl-1,3 -propanediol-hydrochloric
nervous tissue was excised.
acid, made up in calcium-free Holtfreter
saline. Embryos were treated with this
medium for either 1 hour with mechanical agitation when complete disaggregation was required or for half an hour when 'collapsed' embryos were required.
Collapsed embryos are those in which most cells are only slightly disturbed from
their normal positions although the embryo has flattened down on the bottom
of the culture dish and the blastocoel has collapsed. In the various experimental
series this treatment was applied at varying times after the excisions were made.
A. S. G. CURTIS—REGULATION IN BLASTULAE
453
Embryos were returned to the normal culture medium after this treatment. In
three additional series grafts of either presumptive dorsal lip from blastulae or
part of the margins of the wound left in blastulae after excision of the presumptive dorsal lip, were placed in the embryos after treatment with the EDTA
solution.
All embryos were cultured in the normal Holtfreter saline at 19-21° C. until
they reached an age equivalent to stage 22 (Nieuwkoop & Faber, 1956). They
were then fixed in formol-bichromate, embedded in celloidin-paraffin, sectioned
at 8-9 fM, and stained with celestin-blue and eosin.
RESULTS
Regulation after excision
(a) Treatment with EDTA immediately after excision
This experiment forms the main series of the present work, for it was repeated
as a control whenever experiments on regulation were carried out. The embryos
had their presumptive dorsal lip removed in the early blastula stage, and were
then placed in the EDTA solution within 5 minutes. They were either treated
with this solution for 30 minutes without mechanical agitation to produce the
'collapsed' type of embryo, or for 1 hour with agitation which produces complete disaggregation.
Thirty-six 'collapsed' embryos were produced in six batches. At the end of
the treatment with the EDTA solution the embryos had partially flattened down
on to the bottom of the culture dish giving them a slumped appearance. Most of
the cells in these embryos were still in roughly the correct relationship to one
another. Their ectoderm was little damaged though the excision wounds had
opened out. After their return to the Holtfreter saline the embryos healed rapidly
and redeveloped their rigidity so that they rested on the bottom of the culture
dish in a normal (unslumped) condition. Thirty-three of these embryos showed
no definite sign of gastrulation, though three gastrulated normally. Unfortunately, it is hard to be certain whether or not slight invagination occurred in
these thirty-three embryos, but no definite sign of the positioning of the tissues
which would result from gastrulation was found. The ventral surfaces of these
embryos were composed of endodermal cells; this fact suggests that invagination
had failed. None of these thirty-three embryos showed any sign of neural differentiation, although all were composed of healthy cells. However, somite and/
or notochordal tissue lying dorsally in an unoriented mass appeared in fifteen
embryos (see Plate, fig. A); in only one of them was the mesodermal differentiation solely notochordal. No elongation of these embryos occurred. The three
embryos which had gastrulated developed nervous systems, somites, and notochord. In two embryos the nervous systems were neuroid in nature, but the
other embryo had a fairly normal nervous system.
When complete disaggregation was allowed to occur care was taken to mix
the various cell types thoroughly by mechanical agitation. Twelve embryos were
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A. S. G. CURTIS—REGULATION IN BLASTULAE
disaggregated, and after their return to the normal Holtfreter saline all reaggregated successfully. They were grown for 20 hours, a period of time more than
sufficient for complete cell sorting out to occur (Curtis, 1961). At the end of this
time they were fixed.
None of the twelve reaggregates showed any appreciable sign of the sorting
out of the various cell types, though the outermost cells were rather flattened in
an epithelial manner. Prospective ectodermal, mesodermal, and endodermal
cells were mixed up randomly in these embryos, see Plate, fig. C. The next series
forms controls with which this series may be compared.
(b) Treatment with EDTA 3 hours after excision
Twenty-eight early blastulae in four batches had their presumptive dorsal lips
removed and were allowed to heal in normal Holtfreter saline for 3 hours. Then
they were treated with the EDTA solution for 30 minutes to produce 'collapsed'
embryos; thereafter they were grown in normal saline. Twelve of these embryos
showed differentiation of all main cell types: they had well-organized neural
tissue, notochord, and somites which formed axial structures. Of the remaining
sixteen embryos seven differentiated mesoderm cells, but in nine embryos no
sign of morphogenesis was found.
Comparing these results with those of the previous series suggests that the
time of treatment after excision affects the degree of regulation. It is necessary,
however, to discover whether these effects of ageing would appear in embryos in
which no excisions had been made.
(c) Intact blastulae treated with EDTA
Three groups of experiments were performed. Firstly, twenty-seven early
blastulae were placed in the EDTA solution for 30 minutes which brought them
to the collapsed state, returned to normal saline and cultured to fixation at stage
22. Of these embryos fourteen showed development of neural tissues. In eleven
of them the nervous structures were either unclosed neural tubes or neuroid
structures, or spina bifida. Four embryos were without any sign of tissue differentiation, but the remaining nine embryos produced somite and/or notochordal
tissue. The experiment was performed in four batches.
It was already known from earlier unpublished work that late blastulae could
be disaggregated with EDTA solution without affecting the subsequent sorting
out after reaggregation of the various cell types, so only a small confirmatory
series of twelve experiments on early blastulae was undertaken. This second series
forms a control to those disaggregates of excised embryos, see (a). These twelve
disaggregates reaggregated successfully, and sorting out of the presumptive cell
types occurred in them so that presumptive ectoderm lay at the outside, endoderm innermost, and mesoderm in between. But no differentiation other than
that pre-existing in yolk platelet size, cell size, and pigmentation was found in
these reaggregates, see Plate, fig. B.
A. S. G. CURTIS—REGULATION IN BLASTULAE
455
In the third series a dozen intact blastulae were treated with EDTA solution
for 30 minutes to produce 'collapsed' embryos, when they had reached an age
corresponding to that of the excised embryos given a 'delayed' treatment, see
(b), namely at mid-blastula stage. Eleven of these embryos developed all the
main tissue types, though bilaterally asymmetrical nervous systems and large
notochords were common. In one embryo somite tissue was the only cell type
differentiated.
Although statistical evaluation of these results is deferred to the next main
section it already appears that immediate treatment with EDTA probably has
more effect in stopping regulation than delayed treatment. The prime stimulus
to regulation may be the excision of cells. If so, the stimulus is transmitted over
a wide area producing a reaction in many cells. The failure- of regulation after
immediate EDTA treatment brings forward the question of whether its effect
is upon the reacting system or upon the system that transmits the stimulus. The
following experiments were designed to answer this question.
(d) Grafts placed in embryos treated with EDTA
The margins of the wound from which the excision has been made are presumably the first regions to bear the stimulus to regulate. In consequence grafts
of wound-margin made to embryos in which regulation has been blocked might
restart regulation. If so this result would suggest that EDTA damages the transmission system and not the reaction system. Likewise grafts of presumptive
dorsal lip might be able to induce fresh nervous systems in ectoderm if the EDTA
had not damaged its ability to react to a suitable morphogenetic stimulus.
Nineteen embryos had their presumptive dorsal lips removed as early blastulae.
They were treated with EDTA solution for 30 minutes. Meanwhile, the same
excision was carried out on other early blastulae to form wound-margins. One
graft from the wound-margin of these embryos was placed in each of the
treated embryos within 5 minutes of the end of the EDTA treatment. The grafts
were not treated with EDTA. They were placed in the wounded area of the
embryos. Of the twenty-one grafted embryos only two showed regulation to
form a nervous system; somite production alone occurred in three more embryos.
No morphogenesis appeared in the remainder. Furthermore, the grafts showed
no differentiation themselves, which suggests that the host embryos exert some
kind of suppression on the grafts.
In a subsidiary experiment the grafts were taken from the margins of excision
wounds which had healed for 3 hours, and the host embryos had just been treated
with EDTA solution. Of these nine embryos, only two showed signs of neural
differentiation or other morphogenesis.
Grafts of presumptive dorsal lip from early blastulae were placed in embryos
which had been treated with EDTA after excision. Of the six embryos receiving
this type of graft one alone showed development of neural structures; the remaining five were without signs of morphogenesis in either graft or host.
5584.10
Gg
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A. S. G. CURTIS—REGULATION IN BLASTULAE
Statistical testing of results
The incidence of neural induction and mesoderm differentiation in the various
experiments is displayed in Table 1. The effects of EDTA treatment applied to
embryos immediately after regulation began or applied 3 hours later can be
compared by the method of Cochran and Cox (see Snedecor, 1946) since the
experiments were done in six and four groups respectively, with five to seven
embryos in each group. There is significantly less neural induction in those
treated with EDTA to produce 'collapse' immediately after regulation began,
than in the other group, P < 0-05. Likewise there is significantly more mesoderm
differentiation than neural differentiation in embryos treated with EDTA immediately after regulation began, using the/-test, t = 22-56, d.f. = 5,P < 0-001.
TABLE 1
Summary of experimental results
Type
Excision of presumptive dorsal lip
Immediate treatment with EDTA
Collapsed embryos
Disaggregates
Delayed treatment
Collapsed embryos
No excision
Immediate treatment with EDTA
Collapsed embryos
Disaggregates
Delayed treatment
Collapsed embryos
Grafts into blocked embryos
Fresh wound-margin
3-hour-old wound-margin
Presumptive dorsal lip
Mesoderm differentiation alone
No. of embryos
Neural induction
6
6
5
6
7
6
12
1
1
0
1
0
1
0
2
3
0
4
1
5
0
7
7
7
7
4
3
2
3
2
2
1
2
3
5
2
4
2
1
2
0
0
12
11
1
19
9
6
2
2
1
2
5
0
10
6
6
5
12
4
Neural differentiation is affected by EDTA treatment to a much smaller extent
in unoperated embryos than in ones in which the presumptive dorsal lip has been
excised (using method of Cochran and Cox, P < 0-05). Mesoderm differentiation
is also more extensive in the unoperated embryos than in the excised ones,
P < 005.
A. S. G. CURTIS—REGULATION IN BLASTULAE
457
Comparisons with the remaining groups have been made by the 2 x 2 contingency test method. Despite the fact that there is more neural differentiation
when EDTA treatment after excision is delayed there is still significantly less
neural differentiation in the delayed group than in those unexcised embryos
treated with EDTA at a stage 3 hours later than the excision operation,
P < 0-005. Likewise there is more neural differentiation in unoperated embryos
treated with EDTA at a later stage (3 hours later) than an earlier one, P < 0-025.
These results suggest that the unoperated embryo becomes more resistant to the
effects of EDTA treatment through the blastula stage.
Grafts of wound-margin do not unblock differentiation in embryos treated
with EDTA immediately after excision, whether the wound-margin is fresh or
3 hours old, P > 0-1 in both cases, when compared with similar ungrafted
embryos. Likewise grafts of presumptive dorsal lip are without effect in restarting differentiation in similar embryos; P > 0-1 when compared with the ungrafted embryos. If they are compared with unexcised embryos treated with
EDTA to produce 'collapse' such grafted embryos show a significant reduction
in neural differentiation, P < 0-005.
It can be concluded that one essential step in regulation occurs soon after the
excision of the tissue, long before gastrulation, and that thereafter EDTA has
little effect on regulation in embryos in which the wound has healed. This step
is blocked by the action of EDTA. However, this behaviour is probably superimposed on a general increase in the resistance of the unoperated embryo to the
action of EDTA during the blastula stage.
It appears that the ability of cell types such as mesoderm, endoderm, and
ectoderm to sort out in reaggregation is destroyed by excision of the presumptive dorsal lip and surrounding regions if regulation cannot take place. But,
although failure of regulation occurs with EDTA treatment and though the dorsal lip has been removed, morphogenesis of mesoderm is not extensively suppressed. Grafts of untreated wound-margin or presumptive dorsal lip will not
restart regulation.
DISCUSSION
Goerttler (1926) had shown that excision of the presumptive dorsal lip from
blastulae did not prevent the formation of a regulated dorsal lip in gastrulae.
But this result was regarded with some doubt until Dalcq (1933) found that a
fairly ventral level in the future blastopore region could still form dorsal lip
when the ventral half of the blastula was rotated under the dorsal half, and
Brice (1958) observed that the isolated ventral half of a blastula might, 'develop
a dorsal lip'. In the present experiments the same type of regulation of cells to
form dorsal lip has been found many times. To a large degree the evidence for the
formation of a dorsal lip has been the discovery of signs of invagination and
induction. But, although this is not wholly conclusive evidence for regulation,
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accepting any alternative effect would involve proposing new theories about the
nature of gastrulation and neural induction.
The results show that one step in the morphogenetic process which reforms
the dorsal lip occurs soon after the excision of the cells. This step is susceptible
to damage by the action of EDTA. In addition this part of the process appears
to be related to the mechanism whereby cells acquire those properties which
allow them to sort out in reaggregates. Sorting out of the various cell types does
not occur in reaggregates from embryos in which regulation has been blocked.
Although this might be explained on the hypothesis that since induction at
gastrulation is inhibited by blocking regulation these properties of the cells are
normally developed at gastrulation, there are several objections to such a
hypothesis. First, disaggregates of normal blastulae do sort out correctly into
the various cell layers, as observed in this work, and appreciable separation of
the cell types occurs although none of the neural differentiation associated with
gastrular induction happens. It is difficult to imagine how induction would
occur in a disaggregate before sorting out when the various cell types are mixed
up randomly. In a recent review (Curtis, 1962a) it was pointed out that the sorting out of cell types in reaggregates involves differences in both motile and
adhesive properties between the various cell types. Gastrulation is a similar
process in that considerable directed movement of the cells is involved. Thus it
is reasonable to expect that such adhesive and motile properties of the cells
would be differentiated before gastrulation, and the results of the present work
suggest that this differentiation can occur anew soon after excision of cells from
the early blastula.
In the preceding paper (Curtis, 19626) it was suggested that important morphogenetic factors responsible in part for determining the site of the future dorsal
lip were localized in the cortex during cleavage stages. These factors were organized in such a way that excisions were regulated for and additions by way of
grafts were of no effect. It appears that during early blastula stages at least, the
same type of system operates, since excisions of presumptive dorsal lip cells are
regulated for. This correspondence suggests either that regulation for operations
done on cleavage stages does not occur till the early blastula stage or that the
same system operates at both stages. Since some of the properties affected by
regulation appear to be cell surface properties it is possible that regulation is a
phenomenon involving the cortex alone even during blastula stages. In this
connexion it is of interest that once the wound caused by the excision has
healed the process of regulation can no longer be affected by treatment with
EDTA. This result implies that very extensive areas of cells in contact may be
a necessary condition of regulation.
Grafting untreated wound-margin to blastulae in which regulation has been
blocked did not restart regulation. This result suggests that it is not the system
which transmits the stimulus to regulation which is damaged by EDTA, but
that it is the reacting system. The reason for such a conclusion is that the graft
A. S. G. CURTIS—REGULATION IN BLASTULAE
459
from an embryo which had just been wounded would be expected to contain or
develop the stimulus when it was placed in the blocked embryo. However, it is
possible that transmission and reaction are carried out by the same system. In
addition not only are grafts to blocked embryos without effect on regulation but
there appears to be an active suppression of the development of the graft itself:
for instance, grafts of presumptive dorsal lip fail to undergo self-differentiation
even though they contain presumptive mesodermal and neural elements.
Furthermore, the treatment with EDTA only affects morphogenesis extensively
in embryos which have had cells excised from them. EDTA has little effect on
the development of whole embryos. This result implies that regulation in some
way alters the cells so that they are much more sensitive to the action of EDTA,
Such an action indicates the spread of a stimulus amongst the cells as a result of
the excision. The grafting experiments mentioned above suggest that it is the
reaction to this stimulus which is affected. The reaction is not merely temporarily
prevented but appears to be in some way directed into the wrong channel so
that active suppression of morphogenesis occurs. EDTA tends to separate cells
even when the treatment is of short duration. Thus its effect has a considerable
similarity to that of wounding, and therefore EDTA might be expected to
produce a stimulus similar to that occurring in regulation. The fact that intact
embryos show some degree of susceptibility to damage of their differentiation
mechanisms by EDTA at least in the early blastula stage may reflect this possibility. Presumably the lesser degree of inhibition of development found in
such embryos compared with that in regulating embryos treated with EDTA,
may be explained as being due to the chemical acting simultaneously at many
points to separate the cells. Thus any stimulus to regulate the cells might produce on partial loss of contact, can only spread amongst a few cells before it is
barred by a gap between cells. In the embryo in which an excision has just been
made by operation cell contact is good on either lateral side of the excision so
that the stimulus to regulate can be easily transmitted to many cells before the
embryo is placed in EDTA.
I do not think one should conclude that since EDTA treatment blocks regulation, calcium ions must be involved causally in the regulatory system. Not only
does this chemical have effects other than the chelation of calcium but it is also
possible that the important effect is a physical one, such as a temporary loss
of contact between cells or alteration of the mechanical properties of the cell
surface. Shoger (1960) suggested that the mechanical interaction of cells may
be of importance in the regulation of the chick primitive node region. He treated
whole nodes isolated from the rest of the embryo with EDTA and observed
considerable though not total suppression of morphogenesis; the experiment is
not exactly comparable with the present ones, for the node most nearly represents the dorsal lip region, which was the excised area in my experiments,
whereas in his the node was the only remaining area.
A theoretical mechanism which appears to account for these observations is
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A. S. G. CURTIS—REGULATION IN BLASTULAE
one based on a' field' organization of morphogenetic factors originally developed
by Dalcq & Pasteels (1937). For the purpose of this paper this 'field'will be
defined as that network of spatial and temporal interactions involved in producing the dorsal lip of the blastopore and the related invagination. The ideas
advanced in the previous paper (Curtis, 19626) to explain the organization of
such factors in the cortex of the 8-cell stage will be extended. In the intact
embryo the main arrangement of the morphogenetic field has been determined
by the 8-cell stage, and the field may be centered on the future site of the dorsal
lip. One particular structure, the dorsal lip, appears at the 'highest' value of this
field. If this region is cut out another nearby region becomes that of highest
value and thus becomes the dorsal lip. But how does this new region have the
information that it alone is the now 'highest' region? This problem occurs in
considering both the regulation of cortical excisions and of blastular excisions.
Since regulation involves a change in function for many cells in the embryo it
seems that the information must be transmitted between them. In addition the
apparent necessity for cell contact to complete regulation, the behaviour of
grafts in blocked embryos, and the sensitivity of regulation to the action of
EDTA, all argue that a transmission system is involved in readjusting the field
after excisions of cells have been made. It seems that this system for transmission
of the first stimulus to regulation and the reaction to it only comes into existence
after an excision has been made, because EDTA damages it then but is without
great effect on the morphogenesis of embryos in which no excision has been
made. In other words, there is no transmission of information between the
cells of the undamaged blastula, but excision activates the morphogenetic field
so that interaction takes place and a new field is established in the regulated
embryo. The activation is not susceptible to the effects of EDTA treatment but
the reaction, namely the interaction between parts of thefieldto establish a new
field, is affected. In consequence it would be expected that EDTA would have
no large effect on the subsequent morphogenesis of whole unoperated blastulae
not allowed to disaggregate, which of course is so. If a new field is established,
which apparently requires that extensive cell contact be present, the embryo
again is not greatly damaged by EDTA treatment. So it appears that the morphogenetic field in the blastula is normally arranged in an inactive manner so
that no information is exchanged between cells, and regulation is a process in
which it becomes activated and its various parts interact with one another. Since
the blocked embryo has apparently some suppressive action on grafts of normal
tissue it is possible that this influence may be organized in a fashion like a morphogenetic field.
It is usual to presume that the properties in a field system are graded evenly
from one part of thefieldto another. In consequence it may seem a little illogical
that it should apparently be involved in determining the cell properties responsible for sorting out in reaggregation, for there are three distinct types of cell,
ecto-, meso-, and endoderm, and there appear to be no intermediate types of cell
A. S. G. CURTIS—REGULATION IN BLASTULAE
461
reflecting a graded property. However, if these cell types represent various phase
states of the cell surface as I suggested (1962a) grading of the chemical properties
of the surfaces of various cells in accordance with the field would produce only
a few different phase states and thus types of cell.
The incidence of mesoderm differentiation in embryos in which regulation and
gastrulation have been blocked suggests that mesoderm is predetermined before
gastrulation to a considerable extent. Evidence for such a possibility was found
by Dalcq (1933).
SUMMARY
1. After the presumptive dorsal lip is excised from early blastulae of Xenopus
Iaevis the tissues surrounding regulate to reform the dorsal lip. This regulation can be prevented by treatment of the embryos with the chelating agent,
EDTA, provided that treatment is carried out fairly soon after excision. If
regulation is prevented there is no differentiation of nervous tissue, but, nevertheless, there is a considerable mesodermal differentiation. If embryos whose
presumptive dorsal lips have been excised are completely disaggregated with
EDTA, reaggregation occurs but no sorting out of the cell types is found. Disaggregates of whole embryos reaggregate and show sorting out of the cell types
though no further differentiation occurs.
2. These effects appear to be superimposed on a slight sensitivity of the intact
embryo to damage to its neural differentiation by EDTA in the early blastula
stage. But this sensitivity is greatly increased if excision of presumptive dorsal
lip is carried out before treatment with EDTA.
3. Grafts of wound-margin material from embryos whose presumptive dorsal
lips have been excised will not restart regulation in embryos blocked by EDTA
treatment, whether the grafts are from freshly wounded embryos or from embryos wounded some hours previously. Likewise grafts of presumptive dorsal
lip material will not restart regulation or differentiation in blocked embryos,
nor do the grafts themselves differentiate.
4. The results suggest that one step at least in regulation occurs soon after the
excision of tissue, long before gastrulation. This step is sensitive to the action of
EDTA. It appears to be connected with the determination of those cell properties
responsible for the sorting out of cell types in reaggregation, and there is considerable evidence that these properties are determined in the blastula stage.
It seems probable that EDTA acts on the system which reacts to the stimulus to
regulate and not on the actual transmission of this stimulus. Blocked embryos
appear to contain a system such that additions, e.g. grafts, have their morphogenetic potentialities suppressed. Regulation in intact blastulae acts to replace
missing tissues. The regulative properties of blastulae are probably the same as
those to be found in mid-cleavage stages, save that there is some evidence of
partial determination of mesoderm by the blastula stage. These results are
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discussed in terms of a field theory, and it is suggested that regulation requires an
intercellular communication system.
RESUME
Interactions morphogenetiques pre-gastruleennes chez VAmphibien
Xenopus laevis. Regulation chez les blastulas
1. Apres excision de la levre dorsale presomptive de jeunes blastulas de
Xenope, les tissus environnants reforment cette levre dorsale par regulation.
Celle-ci peut etre empechee en traitant les embryons par l'ethylene diamine
tetra-acetate (EDTA), pourvu que ce traitement soit accompli assez tot apres
l'excision. Si on empeche la regulation, le tissu nerveux ne se differencie pas,
mais il se produit neanmoins une differenciation mesodermique considerable.
Si on desagrege completement au EDTA des embryons dont la levre dorsale a
ete excisee, la reagregation se produit mais on n'observe pas de segregation des
types cellulaires. Les desagregats d'embryons entiers se reagregent et montrent
une segregation des types cellulaires bien qu'il n'y ait pas de differenciation
ulterieure.
2. Ces effets paraissent etre surimposes a une legere sensibilite de l'embryon
intact aux effets nocifs de l'EDTA, au stade de la jeune blastula, envers sa
differenciation nerveuse. Mais cette sensibilite est tres augmentee si on pratique
l'excision de la levre dorsale presomptive avant le traitement a l'EDTA.
3. Des greffons de materiel marginal de la blessure, pris sur des embryons
dont la levre dorsale presomptive a ete excisee, ne reprendront pas leur regulation chez les embryons bloques par le traitement a l'EDTA, — que ces greffons
proviennent d'embryons fraichement blesses ou d'embryons blesses quelques
heures avant. De meme, des greffons de materiel de la levre dorsale presomptive
ne retablissent pas la regulation ou la differenciation des embryons bloques, et
les greffons eux-memes ne se differencient pas.
4. Les resultats suggerent qu'au moins une etape de la regulation se situe tot
apres l'excision du tissu, longtemps avant la gastrulation. Cette etape est sensible
a Faction de l'EDTA. Elle apparait en liaison avec la determination de celles des
proprietes cellulaires responsables de la segregation des types cellulaires au
cours de la reagregation et il existe de nombreux faits montrant que ces proprietes sont determinees au stade blastula. II semble probable que l'EDTA
agisse sur le systeme qui reagit au stimulus en regulant, et non sur la transmission
reelle de ce stimulus. Les embryons bloques paraissent renfermer un systeme
tel que des additions, c'est-a-dire des greffons, voient leurs potentialites morphogenetiques supprimees. La regulation dans les blastulas intactes agit pour
remplacer des tissus manquants. Les proprietes regulatives des blastulas sont
probablement les memes que celles observees aux stades moyens de la segmentation, sauf qu'il existe des indices d'une determination partielle du mesoderme
au stade blastula. Ces resultats sont discutes sous forme d'une theorie de champ,
J. Embryol. exp. Morph.
Vol. 10, Part 3
A. S. G. CURTIS
A. S. G. CURTIS—REGULATION IN BLASTULAE
463
et on suggere que la regulation requiert un systeme de communications intercellulaires.
ACKNOWLEDGEMENTS
I thank Professor M. Abercrombie, F.R.S., and Professor J. Z. Young,
F.R.S., for their advice and encouragement, and Mr. J. M. Pettitt, B.Sc, for
his able assistance. The work was carried out during the tenure of grant
C-4847 from the National Cancer Institute, U.S.A.
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BRICE, M. C. (1958). A re-analysis of the consequences of frontal and sagittal constrictions of newt
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107-22.
(1962a). Cell contact and adhesion. Biol. Rev. 37, 82-129.
(19626). Morphogenetic interactions before gastrulation in the amphibian, Xenopus laevis—the
cortical field. / . Embryol. exp. Morph. 10, 410-22.
DALCQ, A. (1933). Experiences de transplantation, de translocation et d'ablation relatives a la determination du systeme nerveux primitif chez les amphibiens. C.R. Soc. Biol. Paris, 114, 159-63.
& PASTEELS, J. (1937). Une conception nouvelle des bases physiologiques de la morphogenese.
Arch. Biol., Liege et Paris, 48, 669-710.
GOERTTLER, K. (1926). Experimentell erzeugte 'spina bifida' und 'ringembryobildungen' und ihre
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Publishing Co.
SCHECHTMAN, A. M. (1942). The mechanism of amphibian gastrulation. Univ. Calif. Publ. Zool. 51,
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SNEDECOR, G. W. (1946). Statistical Methods, pp. 485. Iowa: State College Press.
VOTQUENNE, J. (1933). La disposition generale des ebauches presomptives dans Fceuf de Grenouille
divise en huit blastomeres et les consequences de la destruction d'un micromere dorsal. C.R. Soc.
Biol. Paris, 113, 1531-3.
E X P L A N A T I O N OF PLATE
FIG. A. Section through an embryo near its anterior end from which the presumptive dorsal lip
had been excised at early blastula stage. The embryo was then treated with EDTA. The section is
mainly transverse but somewhat tilted so that the dorsal half of the embryo is anterior to the ventral
half. Note the absence of neural differentiation and the failure of any definite structures to form.
Ectoderm cells lie towards the dorsal side of the embryo, mesoderm beneath, and endoderm bottommost. The vacuolation of the mesoderm cells, indicative of some degree of differentiation, is just visible
in the middle of this section.
FIG. B. Section through the reaggregate from a whole blastula which had been disaggregated. The
reaggregate is positioned in this figure with its 'ventral' side on the left-hand side. Sorting out of the
presumptive cell types into three roughly concentric layers has occurred, though mesoderm fails to
surround the endoderm completely. Ectoderm covers the whole surface of the reaggregate. No further
differentiation of the cells has occurred, e., ectoderm; m., mesoderm; end., endoderm.
FIG. C. High-power view of intermingled cell types in the reaggregate obtained from an embryo from
which the presumptive dorsal lip had been excised. Ectoderm cells, recognizable by their numerous
pigment granules, lie side by side with endoderm cells, which can be identified by their large yolk
platelets. Stain, celestin-blue and eosin.
{Manuscript
received
9:1:62)