Download Morphogenetic Interactions before Gastrulation in the

Morphogenetic Interactions before Gastrulation in
the Amphibian, Xenopus laevis—the Cortical Field
by A. s. G. CURTIS 1
From the Department of Anatomy and Embryology, University College, London
WITH ONE PLATE
the main visible forms of morphogenesis commence at gastrulation it is of great interest to discover whether they are preceded by a series of
preparatory changes just as essential to development, although they have no
immediately visible effects. When Dalcq & Pasteels (1937) proposed the hypothesis that the cortex and yolk respectively contained morphogenetic factors
which determine the main structural features of the embryo, they did not suggest when or how these factors act. I have described a method (Curtis, 1960) for
grafting portions of the cortex of the Xenopus egg from one egg to another, and
by its use was able to confirm that parts at least of the cortex contain a morphogenetic factor. In the present work this technique has been used to make grafts
between the cortex of embryonic cells of varying ages in order to discover
whether the cortex alters its morphogenetic properties as development proceeds. A related question is whether the cortex brings its morphogenetic factors
into action only when gastrulation begins or does it act at an earlier stage.
A very similar question is posed by the regulation of blastulae from which the
presumptive nervous tissue has been removed for these embryos still produce
a nervous system (Bruns, 1931). Do some of the morphogenetic changes required
to establish a new nervous system occur immediately after the operation in the
blastula or are they all deferred until gastrulation begins ? Experiments to resolve
this question are described in a succeeding paper.
The main problem approached in this paper is whether or not morphogenetic
interaction occurs before gastrulation. Are the factors of morphogenesis stored
unused until gastrulation, when they start to act simultaneously or do they act
long before gastrulation initiating a sequence of operations through which the
developing embryo must pass?
In earlier work (Curtis, 1960) it was found that grafts of grey-crescent cortex
from uncleaved fertile eggs placed in the ventral margin of a second egg of the
same age would induce a whole second embryonic axis. I have repeated this
type of experiment, making grafts between embryos of differing age or of the
ALTHOUGH
1
Author's address: Department of Anatomy and Embryology, University College, Gower Street,
London, W.C. 6, U.K.
[J. Embryol. exp. Morph. Vol. 10, Part 3, pp. 410-22, September 1962]
A. S. G. CURTIS—THE CORTICAL FIELD
411
same age but at later stages, in order to discover whether this induction occurs,
or occurs in a different fashion. If the induction does not occur it would suggest that the inducing system has altered since the single-cell stage of the embryo.
In addition, closely related effects should be revealed by excision of the greycrescent cortex—a type of experiment closely parallel to the cytoplasmic excisions in the protozoon, Stentor, described by Tartar (1961). Since the subcortical cytoplasm may be of importance in this morphogenetic system excisions
and grafts of it have also been made.
METHODS
Uncleaved fertile X. laevis eggs were decapsulated and stripped of their vitelline membranes by hand and stored in Holtfreter solution buffered to pH 6-3
with 0001 M 2-amino-2-hydroxymethyl-l,3-propanediol-hydrochloric acid.
Cortical grafts were made by a technique largely the same as that described
previously (Curtis, 1960), with a number of modifications, which result in a
larger proportion of successful grafts. Chief amongst these is that many of the
grafts have been placed in the recipient cortex just after, instead of before,
calcium ions have been returned to the medium and its pH dropped back to
pH 6-3. In consequence the graft was placed in the wound just after it began to
heal instead of somewhile before: presumably this modification makes for
success because it obviates the danger of the graft being damaged while the
medium is changed. A second modification has been the abandonment of the
use of the supplementary medium containing heavy metal ions. A third change
has resulted from the finding that embryos stripped of their vitelline membranes
are very easily damaged if they are moved by pipette from dish to dish. This
damage is of a deceptive nature because it does not appear until some stages
later. In consequence once the embryos have been stripped of their vitelline
membranes they are not moved throughout all stages of the operation. Two
examples of cortical grafts soon after grafting are shown in Plate, figs. A, B.
Grafts were about 150 /ux 150 /x in area in all operations.
Portions of the cortex were excised from embryonic cells with fine tungsten
needles. The excisions were made with the embryos in 0-001 M tetra-sodium
ethylene-diamine tetra-acetate in calcium-free Holtfreter saline buffered at
pH 8-2 with 0-001 M 2-amino-2-hydroxymethyl-l,3-propanediol-hydrochloric
acid. The medium was replaced with normal Holtfreter saline buffered at pH 6-3
as soon as the excision was completed.
Simple excision of cytoplasm from beneath the cortex was carried out by lifting a small flap of the cortex at the site of excision with a needle: a micropipette
working by capillary action was inserted into the subcortical cytoplasm to suck
a small amount of cytoplasm out of the cell. These excisions were made with the
embryo in Holtfreter saline containing twice the normal calcium concentration.
A small pit remains for a short while after the excision, but it soon disappears
and the cortex heals over the excised region.
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A. S. G. CURTIS—THE CORTICAL FIELD
In order to make grafts of subcortical cytoplasm a site for the graft in the
recipient embryo was prepared by excising a portion of the existing cytoplasm.
The graft was quickly cut out of a second cell with needles and pushed into the
prepared site. These grafts were made in a Holtfreter saline containing four
times the normal calcium concentration. Under these conditions the cytoplasm
is sufficiently firm to be coherent during the brief process of grafting. The cortex
soon healed over the grafted material.
The embryos and unoperated controls were cultured in normal Holtfreter
saline pH 6-3 for several stages after operation: thereafter the tonicity of the
medium was slowly dropped until the medium was completely replaced by 0-5
Holtfreter saline. Embryos were grown at 19-21° C. until they reached Nieuwkoop stage 21-22 (late neurula) and were then fixed in formol-bichromate solution, embedded in celloidin-paraffin, sectioned at 8-9 /u, thickness, and stained
with celestin-blue and eosin.
In the present work all grafts were made with material from the grey-crescent
site placed in the ventral margin. Grafts were made between cells in these sites
in embryos of varying ages according to the following plan.
1. Donor stage 4; Recipient stage 4 (i.e. 8-cell stage).
2. Donor stage 4; Recipient stage 1.
3. Donor stage 1; Recipient stage 4 (Nieuwkoop stages).
In addition subcortical cytoplasm was grafted from the grey crescent of stage 4
to the ventral margin of stage 4. Grey crescent cortex was excised from stage 1
and 4 embryos and subcortical cytoplasm from the grey crescent of stage 4.
RESULTS
Cortical grafts
Stage 4 to stage 4
Grafts of grey crescent from stage 4 were made to the ventral margin of
embryos of the same stage. Of the ten grafts made all survived to fixation at
stage 21-22. The graft could be recognized for some stages after the operation,
see Plate, fig. A. During gastrulation slight loss of endoderm cells occurred from
the yolk plug in four embryos. Neurulation proceeded normally. Late cleavage
stages and blastula stages were entirely normal.
Examination of the sectioned material showed that a primary axis was established in the normal position in every embryo and there was no sign of the
induction of a second axis. The head structures of two embryos were slightly
distorted. In three embryos very small masses of pigmented material lying in the
anterior belly epithelium could be found in one or two sections. I think it possible that this material represents the remains of the graft.
Stage 1 to stage 4
Ten grafts were made in this series. The results were like those of the preceding series in the main respects: there being no sign of induction by the grafts
A. S. G. CURTIS—THE CORTICAL FIELD
413
in embryos which showed normal development till fixation. In one embryo a
slight thickening of the belly epithelium, so that it was 3-4 cell layers thick in
an area about 30 //. by 40 fx, was observed, but otherwise there was no sign of
the graft influencing the host.
Stage 4 to Stage 1
Thirteen embryos received this graft and developed well after the operation.
One such embryo is shown in the Plate, fig. B. Eleven embryos showed the
induction of a second axis complete with neural tube, notochord, and somite
material. The neural tubes were rather abnormal in the arrangement of tissue
around the central lumen; often being eccentric, see Plate, fig. C. In the remaining two embryos there was no proper induction but a large mass of rather disorganized epithelial material appeared on the ventral flank. Although only two
embryos were observed at the beginning of gastrulation, when two separate
dorsal lips formed in each embryo which had a common blastopore; it could be
deduced from observation of the remainder at the end of gastrulation that
separate dorsal lips had probably formed.
Cortical excisions
Large portions of the cortex can be excised from early embryonic stages; the
wound heals over within 10 minutes in normal Holtfreter saline. However, if
more than about 10 per cent, of the cortex is removed, although healing occurs
it is followed by the appearance of damage to the cells some few cleavages later.
The time of appearance of this damage seems to depend upon the size of the
excision to some extent, for large excisions result in rapid damage but smaller
ones cause damage which may not appear until blastula stages. The effect of
large excisions from fertile uncleaved eggs appears at about the 16-cell stage;
from 8-cell stages at about the 32-cell stage. Large blebs of cytoplasm protrude
from cells in the vegetal region: apparently these blebs have no normal cell
surface over them because they are very fragile by comparison with the normal
embryonic cells. Simultaneously, small deeply pigmented bodies about 50 p in
diameter are budded off the cells at the animal pole, one cell producing several
such bodies. These bodies remove most of the pigmentation from the animal
pole cells and it is conjectured that they consist of cortex. Cells which form either
blebs or pigmented bodies soon cytolyse completely but adjacent cells may
remain unaffected if the excision was not large. The damage at the vegetal pole
resembles that described (Curtis, 1960) when animal pole cortex was grafted into
the vegetal pole.
If an extensive piece of cortex was removed these processes develop to such
an extent that the embryo is completely cytolysed. But if less cortex was excised
the damage is less extensive and an embryo containing many cells survives. Such
damage occurred in about one-half of all the excisions attempted, but the following results refer alone to those embryos in which no such damage appeared-
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Ten excisions of grey-crescent cortex from uncleaved fertile embryos were
made. Cleavage continued unimpaired in all these embryos. Two formed normal
neurulae. The remaining eight embryos showed no sign of morphogenesis or
gastrulation at stage 22. In these embryos mitoses were still happening at fixation and it appeared that mitosis and cleavage had continued unimpeded. The
cells were of increasing size from animal to vegetal pole, see Plate, fig. D. It is
difficult to excise all the grey crescent without producing the damage described
above; this may mean that not all the crescent was excised in those operations
which produced normal embryos.
Eight stage-4 embryos had their grey-crescent cortex excised. The cortex was
removed from either cell on each side of the midline. The wounds healed well
and development continued normally. All the embryos showed no abnormality
at stage 21.
Excisions of sub-cortical cytoplasm
About 50-100 fj? of subcortical cytoplasm was removed from beneath the
grey-crescent cortex of each of ten stage-4 embryos (8 cell). The wounds healed
very rapidly. Development continued normally through blastula and gastrula
stages. All the embryos showed differentiation of the main cell types. Seven
embryos were normal in all major respects at stage 22 though three of these
showed slight signs of bilateral asymmetry in the brain region. The remaining
three embryos had neuroid types of nervous systems.
Grafts of subcortical cytoplasm
Grafts of cytoplasm from beneath the grey crescent of stage-4 embryos were
placed in the ventral margin region of embryos of the same stage. Unfortunately,
a certain amount of cytoplasm tends to spill out of the grafting site during the
operation, in consequence of which it is impossible to be certain how much
material was grafted into the embryo. The cortex soon healed over the graft
and development continued normally. Of the ten embryos so grafted none
showed any sign of an induction by the graft. Four developed entirely normally;
in one embryo the host nervous system only reached the neuroid stage of
development. The remaining five embryos showed slight defects in their nervous
systems: in three the nervous system was abnormal in the head region so that
the neural lumen was split into several in the front of the head. In two embryos
a slight splitting of the nervous tissues occurred posteriorly so that spina bifida
developed. These results are rather similar to those of Pasteels (1932) who
obtained defects in the head region from embryos which had been wounded at
the uncleaved stage in the ventral margin region.
These results are summarized in Table 1; and some are shown in diagram in
Text-fig. 1.
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TABLE 1
Summary of main experimental results
Experiment
Grafts of grey-crescent cortex to
ventral margin
Donor stage 4, recipient stage 4 .
Donor stage 1, recipient stage 4 .
Donor stage 4, recipient stage 1 .
Excision of grey-crescent cortex
Stage 1
Stage 4
Excision of subcortical cytoplasm
from grey crescent
Stage 4
Grafts of subcortical
from grey-crescent
.
.
margin
cytoplasm
to ventral
.
.
.
No. of
embryos
Primary
differentiation
Secondary
neural induction
and axis
Weak
induction
10
10
13
10
10
13
—
—
11
—
—
2
10
8
2
8
—
—
—.
10
10
—
10
10
—
—
Interpretation of the results
Grey-crescent cortex from stage-4 embryos (8 cell) grafted to the ventral
margin of uncleaved fertile eggs induces a second main embryonic axis, but does
not do so when grafted to the same site in stage-4 embryos. This difference is
statistically significant (P < 0-01) when a 2 x 2 contingency test is applied to the
data of the results. A similar result is that there is no induction when greycrescent material from uncleaved fertile embryos is grafted to stage-4 embryos,
although it is known from earlier work (Curtis, 1960) that it contains this active
inducing factor. Comparing the results of this series with stage-4 to stage-1
grafts the difference is again significant (P < 0-01).
It is known that the grey-crescent cortex in the uncleaved fertile egg possesses
the potentiality to induce an embryonic axis and that its ventral margin can
accept this induction. But the stage-4 cortex, although still containing the
inducing influence in the grey-crescent region, is unable to accept an induction.
In other words, by stage 4 there has been a change in the system leading to the
induction so that no secondary axis is produced.
These conclusions are extended by those derived from the experiments on
cortical excision. The excision of grey-crescent cortex from uncleaved eggs results
in a significant effect (P < 0-05), the arrest of morphogenesis, when they are
compared with the effects of excisions from stage-4 embryos, whose morphogenesis was unaffected. Again a change in cortical properties is indicated between
stages 1 and 4. These results also suggest that by stage 4 those parts of the
morphogenetically important factors which are actually going to act are either
no longer in the cortex or that they can in some manner be regenerated or
regulated for when part of the cortex is removed. It may be felt that the failure
of excision of cortex from stage-4 embryos to affect morphogenesis is due to the
heals
heals
TEXT-FIG. 1. A diagrammatic summary of the main results, a, excision of the grey-crescent cortex
from a stage-4 embryo results in a normal embryo being formed (compare with d). b, grafting greycrescent cortex from stage 1 to the ventral margin of stage 4 does not result in the induction of a
second embryonic axis, c, grafts of grey-crescent cortex from stage-4 embryos to the ventral margins
of stage-1 embryos induce secondary embryonic axes, d, excision of grey-crescent cortex from stage-1
embryos prevents morphogenesis though cleavage and mitosis continue.
A. S. G. CURTIS—THE CORTICAL FIELD
417
presence of cortical material in the cleavage furrows. This is, however, very unlikely, for the furrows open wide during the operation and cortex can be and
was removed far into the furrows. In addition no pigmented cortex is found in
the furrows, whereas it is the pigmented cortex which contains the factor at
earlier stages. Selman & Waddington (1955) suggest that the cell surface in the
furrow is synthesized de novo and is not formed by stretching the existing cortex.
In consequence if the cortex of the furrow contains a morphogenetic factor this
has been formed by the action of some transfer system from existing cortex,
and represents a morphogenetic interaction.
Are the factors then present in the subcortical cytoplasm? If so the inducing
ability of the grey-crescent cortex of stage-4 cells would be viewed as a sort of
leftover part of the inducing system. However, excision of the cytoplasm from
the subcortex of the grey crescent of stage 4 is without effect on morphogenesis,
the results being without significant difference (P > 01) from those of the
cortical excisions from this stage. Although this negative result suffers from the
defect that it is always possible that I have not yet removed the 'right' piece of
cytoplasm which would affect morphogenesis, it indicates that the inducing
system has not moved into the subcortical cytoplasm.
This conclusion is in agreement with the result that grafts of grey-crescent
subcortical cytoplasm do not induce second embryonic axes from the ventral
margins of stage-4 embryos (P > 0-1) when compared with the effects of similar
grafts of cortex. Obviously the subcortical cytoplasm does not contain such
factors at stage 1 for excision of the cortex stops morphogenesis. It seems unlikely that in the short space of time between stages 1 and 4 the subcortex
acquires the factors from the cortex and also becomes able to block the inductive
effects of addition of subcortex (such as a graft) bearing the factors and yet
neighbouring subcortex is able to acquire the factors again if it is excised.
In consequence it appears that the cortex undergoes some change between
stages 1 and 4 which makes it impossible for fresh inductions to be made by
grafting whenever it is the receptor of a graft. Nevertheless, the morphogenetic
system does not leave the cortex but alters so that considerable restoration of
excised parts can be made.
DISCUSSION
The mechanism which suggests itself as an explanation of these results is one
modelled on the development of a gradient system. There are reasons for thinking that the morphogenetic influences in the cortex are organized as a field as
Dalcq & Pasteels suggested (1937). The behaviour of the cortex in the 8-cell
stage is such that additions to the cortex are without effect but excisions are
replaced. This can be pictured as happening in a system in which the main
features of the cortical field have been laid down. In consequence removal of
a fairly large part of the cortex does not damage the system seriously because
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A. S. G. CURTIS—THE CORTICAL FIELD
enough of the gradient remains to control development. For example, if the
gradient works so that some part with the highest concentration of a substance
or structure forms one definite tissue and the other parts form tissues in relation
to this, the excision of the part of highest value will leave surrounding regions
as those of highest value on which development will now centre. There is one
objection to this hypothesis, which is that it may not seem to explain how grafts
of stage 1 grey crescent into stage-4 embryos fail to produce an induction. An
answer to this objection will be given a little further on.
In contrast the cortex in the uncleaved fertile egg may be pictured as containing no established gradient system, but only a morphogenetic factor centred in
one place, the grey crescent. At some time between the first and third cleavages
this grey crescent acts as a centre which initiates and activates the establishment
of a cortical field. In consequence a graft of grey-crescent cortex into the uncleaved egg acts as a centre for activation. Hence in an embryo with such a graft
the cortical field will spread from the host grey crescent and from the graft. This
embryo with two separate cortical fields will, of course, produce a double
embryo.
A hypothesis to meet the objection suggested two paragraphs previously can
now be given. A graft of grey-crescent cortex made into a stage-4 egg is made
after activation occurs, in consequence this graft cannot establish its own
cortical field and hence it cannot affect development.
This hypothesis also explains how it is that excision of the grey crescent from
uncleaved eggs prevents morphogenesis, because no activating centre is left.
Even in the absence of this hypothesis the experimental results form a direct
parallel with those of Seidel (1929) on Platycnemis from which he claimed that
one part of the egg acted as an activating centre for development, for isolation
of this region from the rest of the Platycnemis egg prevented development as
does excision of one region of the Xenopus cortex.
In this discussion I have rather ignored the influence of the yolk gradient
which Dalcq & Pasteels (1937) showed to be another important factor in morphogenesis. However, all grafts have been to the same part of the yolk gradient
(ventral margin) as the grey crescent so that it does not become a direct factor
in these experiments. But the yolk gradient plays an important part in activation since Dalcq & Pasteels showed that it was the interaction of this with the
cortical factors that helped determine the formation of two embryonic axes in
certain experimental situations. It might seem at first sight that the present interpretation is difficult to reconcile with Dalcq & Pasteels's hypothesis about the
interaction of these two morphogenetic factors. Their inversion or centrifugation
experiments resulted in new regions of the cortex (sometimes two simultaneously) taking part in the formation of the dorsal lip of the blastopore. However,
this result does not imply that the grey crescent is no longer the activating
centre in such embryos. Their interpretation comes into complete agreement
with the present hypothesis if we take their suggestion that the yolk gradient has
A. S. G. CURTIS—THE CORTICAL FIELD
419
some value at the junction of light and heavy plasms allowing the interaction
which determines the site of the blastopore and add the following condition:
that this interaction fixes and establishes the cortical field. If this is so the
activation in inverted eggs would spread from the original grey crescent forming a rather labile system; on reaching those regions with the right value of the
yolk gradient it would become fixed and irreversibly determined. Thus it would
spread in this fixed condition outwards from one or two new centres forming
one or two new morphogenetic fields.
The most important conclusion that can be drawn from these results is that
a morphogenetic interaction occurs within the cortex before the 8-cell stage.
This interaction is one in which the cortical field is determined. Since the cortical
field helps control the future site of the dorsal lip it can be regarded as determining the main axes (anterior-posterior, lateral, and dorso-ventral) of the
embryo. Because the cortical field acts in determining where the dorsal lip of
the blastopore forms, it can be said to determine the main geometric axes of the
embryo. Once the field is determined these axes are fixed. Some pertinent
suggestions can be made in relation to this conclusion. The morphogenetic processes which happen in the cortex during activation occur in a very thin membrane. Whether the activation is a chemical process or a physical one its spread
across the cortex is considerably impeded by the geometry of this membrane.
The thinness and great extent of the cortex in the uncleaved egg would tend to
slow down the spread of any change in the cortex. But once cell-division has
occurred the diffusion of any change in the cortex becomes much easier because
interaction can occur across the small gap separating apposed cell surfaces. The
process of interaction is vastly aided because the diffusion process can occur
over the wide area of contact between cells and across the narrow gap between
them. Once three cleavages have been completed enough cell surfaces exist for
these interactions to be made across apposed cell surfaces in each of the three
axes of symmetry of the embryo. In this very tentative hypothesis I have assumed
that the cortex or at least active derivatives of it are present in the cleavage
furrows. The attraction of this hypothesis is that it does suggest how it is that
the establishment of the cortical field occurs when it does.
The general argument given in this discussion is in agreement with the results
of Dollander (1950), who found that the cortical field can be easily altered by
ligaturing operations at the 2-cell stage. His experiments suggest that the cortical
field has not been fixed in position at this stage. Votquenne (1933) found that
removal of a micromere containing the grey crescent at the 8-cell stage in Rana
fusca did not affect development: this result is paralleled in the present work by
the excision of grey-crescent cortex at the same stage. Both results agree with
the hypothesis in that once the field is established parts which are removed can
be regulated for.
The damage following extensive excision of cortex in the 8-cell stage occurs
not only in those cells which are derived from the original cell operated upon
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A. S. G. CURTIS—THE CORTICAL FIELD
but also in other cells. This result implies that there is interaction between cells
even in these early stages.
The absence of any large effect when cytoplasm was excised may at first seem
to be in contradiction with the results of Milaire (1961) who found that various
abnormalities developed in the nervous system after excision of subcortical
cytoplasm. However, it is probable that much more cytoplasm was removed in
his experiments than in mine.
One other general conclusion can be drawn from these results. This is that
the next series of problems to be attacked are the mechanisms which establish
the grey crescent in the fertile uncleaved egg. Presumably some of these are connected with the formation of the egg and others perhaps with fertilization; the
function of the latter process in this respect has, of course, been extensively
studied by Ancel & Vintemberger (1948).
SUMMARY
1. Grafts of grey-crescent cortex were placed in the ventral margin of cleavage
stages of X. laevis embryos. Grafts from 8-cell embryos placed in uncleaved
fertile eggs induced secondary embryonic axes, but such grafts placed in 8-cell
embryos had no effect on morphogenesis. Grafts of cortex from uncleaved fertile
eggs were without effect on the morphogenesis of the 8-cell embryo when placed
in it.
2. Excisions of cortex from the grey crescent of uncleaved fertile eggs prevented morphogenesis although cleavage and mitosis were unimpeded. Excisions
of such cortex from 8-cell stages were without effect on the normal morphogenesis of the embryos.
3. Excisions of sub-cortical cytoplasm from the grey-crescent cortex of 8-cell
embryos do not affect development.
4. Grafts of subcortical cytoplasm from the grey-crescent region of 8-cell
embryos were placed in the subcortex of the ventral margin of embryos of the
same age. These grafts were without effect on morphogenesis other than slightly
damaging brain formation.
5. These results taken in conjunction with those of previous work indicate
that though the cortex contains morphogenetic factors determining the embryonic
axes and the site of the dorsal lip at gastrulation, the nature of these factors
undergoes some change between the beginning of the first and the end of the
third cleavages. The change is such that the embryo becomes able to regulate
for excisions or additions of cortex. The grey-crescent cortex appears to act as
an activating centre in producing this alteration in the morphogenetic system
of the embryo. There is no evidence that the cytoplasm beneath the cortex is
directly involved in this interaction.
A. S. G. CURTIS—THE CORTICAL FIELD
421
RESUME
Interactions morphogenetiques avant la gastrulation chez VAmphibien Xenopus
laevis — le champ cortical
1. Des fragments de cortex du croissant gris ont ete greffes dans la region
ventrale de germes de Xenope en cours de segmentation. Des fragments preleves
au stade des 8 blastomeres et greffes sur des ceufs fecondes insegmentes y ont
induit des axes embryonnaires secondaires, mais de tels greffons places sur des
germes au stade 8 bl. n'ont pas eu d'effets sur leur morphogenese. Des fragments
de cortex d'oeufs fecondes insegmentes sont restes sans effet sur la morphogenese
de germes pris au stade 8 bl., quand on les y a greffes.
2. L'excision du cortex du croissant gris d'oeufs fecondes insegmentes a
empeche la morphogenese, bien que le clivage et la mitose n'aient pas ete
inhibes. Mais l'excision de ce cortex pratiquee sur des germes au stade 8 bl. est
restee sans effet sur la morphogenese normale des embryons.
3. L'exerese de cytoplasme sous-jacent au cortex du croissant gris n'a pas
affecte le developpement.
4. Du cytoplasme subcortical de la region du croissant gris de germes au
stade 8 bl. a ete greffe en position subcorticale dans la region ventrale d'embryons
du meme age. Ces greffes n'ont eu d'autre effet sur la morphogenese qu'une
legere alteration de la formation de l'encephale.
5. Ces resultats, groupes avec ceux d'un travail anterieur, indiquent que, bien
que le cortex contienne des facteurs morphogenetiques determinant les axes
embryonnaires et l'emplacement de la levre blastoporale a la gastrulation, la
nature de ces facteurs subit des modifications entre le debut du premier clivage
et la fin du troisieme. La transformation est telle que l'embryon devient capable
de regulation apres excision ou adjonction de cortex. Le cortex du croissant gris
parait agir comme un centre activant en produisant cette alteration dans le
systeme morphogenetique de l'embryon. Rien ne permet de conclure que le
cytoplasme subcortical soit implique directement dans cette interaction.
ACKNOWLEDGEMENTS
I thank Professor M. Abercrombie, F.R.S., and Professor J. Z. Young,
F.R.S., for their advice and encouragement. Mr. D. West and Mr. J. M. Pettitt,
B.Sc, have given able technical 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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l'oeuf des amphibiens. Bull. Biol. Suppl. 31, 1-182.
BRUNS, E. (1931). Experimente iiber das Regulationsvermogen der Blastula von Triton taeniatus und
Bombinator pachypus. Arch. EntwMech. Organ. 123, 682-718.
CURTIS, A. S. G. (1960). Cortical grafting in Xenopus laevis. J. Embryol. exp. Morph. 8, 163-73.
DALCQ, A., & PASTEELS, J. (1937). Une conception nouvelle des bases physiologiques de la morphogenese. Arch. Biol. 48, 669-710.
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A. S. G. CURTIS—THE CORTICAL F I E L D
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E X P L A N A T I O N OF PLATE
FIG. A. Graft of grey-crescent cortex (gr) placed in the ventral margin of an 8-cell embryo seen at
the 16-cell stage. Graft clearly visible.
FIG. B. Graft of grey-crescent cortex (gr) placed in an uncleaved embryo at the lateral ventral
margin seen at 32-cell stage. Graft beginning to merge into surrounding cortex.
FIG. C. Embryo, which had received graft of grey-crescent cortex at stage 1 from stage-4 embryo,
seen in section after fixation at stage 21. The graft, which was placed in the ventral margin, has
induced a second axis on right-hand side of section.
FIG. D. Section through an embryo which had its grey-crescent cortex excised at stage 1. Cleavage
and mitosis have continued but there is no sign of morphogenesis in this embryo which was fixed at
an age corresponding to stage 22. A.P. andKP. refer to animal and vegetal pole positions respectively.
{Manuscript received 20 : xii: 61)
J. Embryol. exp. Morph.
Vol. 10, Part 3
A. S. G. CURTIS