J. Embryol. exp. Morph. Vol. 33, 1, pp. 147-157, 1975
Printed in Great Britain
147
Local autonomy of gastrulation movements after
dorsal lip removal in two anuran amphibians
By JONATHAN COOKE 1
From Division of Development Biology,
National Institute for Medical Research, Mill Hill
SUMMARY
The time-course of the remaining gastrulation movements has been investigated after
removal of the dorsal lip, the presumptive foregut endoderm and the anterior mid-dorsal
mesoderm as a plug-shaped mass of cells from beginning gastrulae of Xenopus laevis and
Bombina orientalis. This embryonic region has been previously studied in its role as an
organizer, when grafted into a host gastrula marginal zone.
There is usually no effect of the removal of these cells, the first morphogenetically active
ones, either upon rate of subsequent completion of the external aspects of gastrulation, or
upon the internal evolution of the presumptive mesodermal mantle. This finding is discussed
in connexion with results of a previous paper on pattern formation in early Xenopus development, since it may help to distinguish between possible types of explanation for those results.
INTRODUCTION
A previous paper (Cooke, 1973 a) reported certain features of the results of
implanting second organizer regions into the marginal zone of Xenopus blastulae
and early gastrulae. The present paper begins to explore in greater detail the
anatomy of gastrulation and the causal relationships between movements in
parts of the embryo around the time of these organizer implantations. It reports
the results of removing a mass of cells, consisting of the surroundings of the
earliest visible organizer activity, together with the underlying bottle cells
(Holtfreter, 1943), endoderm cells and presumptive anterior mid-dorsal mesoderm, from early stage-10 Xenopus (Nieuwkoop & Faber, 1956) and the
equivalent stage in Bombina. This excision operation, the same as that reported
previously, has been followed by detailed observation of the completion of gastrulation in comparison with individually paired, precisely synchronous controls.
The rationale for including Bombina in the study was that due to the slower
time-course of its gastrulation, a greater chance was offered for observation
of possible retarding effects of the operation, since these might take some time
to set in if they were of a physiological rather than a mechanical nature.
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K.
148
J. COOKE
Gastrulation involves the formation of bottle cells, and neuroectodermal
spreading at the surface of the marginal zone, and the inrolling and animalward
migration of presumptive mesoderm cells internally (Nieuwkoop & Florschutz,
1950; Lovtrup, 1965). These activities spread with time along an axis, the dorsoventral, that corresponds with one of those for future pattern formation.
Furthermore, when sites of the initially localized mid-dorsal activity are grafted
to ventral regions of the margin, they can cause a similar spread of gastrulation
movements in the surrounding cells as well as a new field of axial pattern
formation subsequently (Cooke, 1972). It is thus likely that the time-course of
gastrulation is originally co-ordinated and controlled by a gradient system
identical with, or related to, that controlling the extent and proportions of the
final pattern of cellular differentiation in the mesoderm. At some unknown
early stage, this gradient system would then set, locally, the rate at which the
physiology of cells evolved towards the initiation of those mechanical behaviours
listed above as causing gastrulation.
The question remains, however, as to how immediately cells can affect their
neighbours during the actual progress of gastrulation. Are their interactions,
along the dorso-ventral axis, at least partially of a mechanical and orientating
or stretching nature, or are they solely physiological, reflecting the stability of a
gradient system set up by earlier intercellular communication? If the latter,
how stable is this gradient system? If a retarding effect were observed at any
subsequent time during gastrulation, on removing the first mechanically active
cells from the dorsal midline, it might have a mechanical explanation, showing
that the process is controlled or 'led' mechanically from the dorsal lip area.
Absence of such an effect would mean that control over the orderly dorsoventral sequence of gastrulation is in fact of a 'physiological' character, and
furthermore that by this time either the gradient or the cells' programmed
response to it is sufficiently stable to avoid any effect of apex removal upon the
schedule of morphogenetic movements.
In fact, it is shown that there are normally no interactional effects of removing
these first active cells, upon the subsequent cellular activities that complete the
gastrulation process throughout the rest of the embryo. The consequences of
this finding are discussed, for the interpretation of effects observed in the final
patterns of cellular differentiation, when organizer implantation is combined
in various ways with host organizer removal as described previously (Cooke,
1973 a).
MATERIALS AND METHODS
Eggs were obtained from matings induced by subcutaneous injection of
human chorionic gonadotropin ('Pregnyl', Organon Laboratories Ltd) for both
Xenopus and Bombina. 250 i.u. per female and per male was satisfactory for the
latter species. Groups of embryos were dejellied and demembranated manually
at the late blastula stage (Nieuwkoop & Faber stage 8 in Xenopus) and stored
Autonomy of gastrulation after dorsal lip removal
Dorsal
ca. 1 mm
149
(C)
Surface zone
of marginal
activity
Internal
D.L.
Line of progress
of internal
/ >
mesodcrmal
mantle
Archenteron
Fig. 1. The excision operation. A, Vegetal surface view of stage-10 gastrula. B,
Sagittal section. Heavy dashed boundaries indicate region removed, shown as an
isolate in centre. C, Vegetal surface view of the healed embryo some 2h later
(Xenopus) on completion of ventral blastoporal lips. D, Combined lateral surface
view and sagittal section of same stage as C.
vegetal surface uppermost in black wax-bottomed dishes under two-thirdsstrength Niu-Twitty solution (see Rugh, 1962) whose Ca2+ and Mg 2+ concentration had been doubled by means of the chlorides, and the pH brought to 7-0
by HC1. This solution frequently provides perfect healing and morphogenesis
after operations in the present material. By inspection at 15 min intervals, pairs
of embryos were selected whose activity of the bottle-shaped cells (surface
pigment gathering and dimpling) at the incipient dorsal lip had started synchronously within this margin of error. They were placed side by side, and the
organizer region cleanly removed with tungsten needles from one of them.
To control for a possible physiological effect of laying open the blastocoel to
the culture fluid, a small tear was also made in the animal region to open the
blastocoel in each embryo. Between \ h and 45 min after this operation, the
strength of the solution was lowered to 0-1 with glass-distilled water to avoid
the exogastrulation which often occurs without this. Observation, with camera
150
J. COOKE
Table 1. Time-course of completion of gastrulation movements after organizer
excisions in Xenopus
Experiment
1
3
(low
temperature)
Pair no.
Time to completion of marginal zone
activity, i.e. lips of blastopore
Yolk plug closure
to stage 12\
0*
+1
+1
0
-1
-1
-1
+1
0
+1
-2
-1
+2
0
0
-1
-1
+1
0
+1
-2
-2
0
+1
+2
+1
-1
-2
+2
+2
-2
0
* 0, 1 or 2 indicates synchrony, delay ( + ) or advance ( - ) of the time of completion of the
stated phase of gastrulation in the experimental as compared with its initially synchronous
control, the unit inter-observational period being 20 min.
lucida drawing, was made at 10-20 min intervals thereafter for each synchronous
pair.
Between observations, pairs of embryos were lying on the laboratory bench,
away from intense lighting and at an even temperature of between 21 and 23-5 °C
(as between different experiments). On one occasion, Xenopus embryos were
kept between observations in a cooled incubator between 16-5 and 17-5 °C.
The group of cells removed was that described in previous papers (Cooke,
1972, 1973 a), laying open the blastocoel and leaving initially an embryo in
which none of the marginal cells were visibly different from those of stage-9
blastulae. The operation is shown in Fig. 1. The wound, which was cleared of
cell debris by gentle aspiration with a micropipette before the first observation,
had usually closed off the blastocoel within 30 min in Xenopus and 1 h in
Bombina, at which times the first new gastrulation activity was beginning. The
excised plugs of cells were used as implants into other embryos, where in
Xenopus they often caused complete secondary axes as described previously.
Selected embryos were fixed for 10 min in 4 % formalin in two-thirds-strength
Niu-Twitty, halfway through gastrulation, before examination of the internal
course of mesoderm morphogenesis by bisecting in the frontal plane and then
chipping away the endodermal blastocoel floor.
Autonomy of gastrulation after dorsal lip removal
151
Table 2. Time-course of completion of gastrulation movements after organizer
excisions in Bombina
Experiment
1
2
3
Pair no.
Time to completion of marginal zone
activity, i.e. lips of blastopore
Yolk plug closure
to stage 12$
2
3
4
5
1
2
0*
0
-1
-1
+1
+1
0
+1
0
0
0
+1
+2
+1
3 + 1
4
5
1
2
3
4
5
-1
-1
+1
0
+2(35min)
+2(35min)
+2(25min)
0
-1
-2
+1
+1
+2
+2
+2
1
* 0, 1 or 2 indicates synchrony, delay ( + ) or advance ( —) of the time of completion of the
stated phase of gastrulation in the experimental as compared with its initially synchronous
control, the unit inter-observational period being 20 min.
RESULTS
Twelve pairs of Xenopus embryos, from three different egg-batches in
separate experiments, were observed at laboratory temperature (21-23 °C), and
four more pairs in another experiment at 16-17 °C. At the latter temperature,
the total course of gastrulation between the operation and stage 12^ was prolonged from the normal 4^- h to some 8 h. Pairs of Bombina embryos (15 from
three different egg-batches) were observed only at laboratory temperatures
where the equivalent morphogenetic stages last some 8f h.
The results are presented in Tables 1 and 2, where (+1) and ( - 1 ) signify that
in the experimental embryo completion of the annular zone of marginal cell
activity (blastoporal lips), or reduction of the yolk plug to stage 12^ size, either
followed ( + ) or preceded ( - ) that in the control by one inter-observation
period. These unit periods were of about 10 min for the Xenopus experiments,
and 20 min for Bombina. Apart from those of Bombina experiment 3, the values
of 2 which appear in each set of results were in fact determined by extra observation to be more nearly a difference of one period than of two whole periods, so
that the course of the gastrulation movements is seen to be rather precisely
timed, and the relative degree of precision to be equivalent when the whole
process takes longer.
The variation in absolute gastrulation rate between experiments, due to
varying laboratory temperature and, it is suspected, to characteristics of eggs
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J. COOKE
ca. 2 mm
Fig. 2. The normal lack of timing effect after excision. Camera lucida of vegetal
surfaces of four pairs of synchronous Bombina gastrulae, at various times beginning
1 h after organizer excision in the left of each pair. Short heavy arrows indicate
mid-dorsal lines (i.e. early organizer sites), long fine arrows link the three successive
observations of each pair. In the three left-hand pairs, gastrulation remained
synchronous, whilst in the right-hand pair a delay arose and was maintained following organizer excision.
laid by particular females, renders a population comparison of these meaningless. For Xenopus, there is no evidence that the excision of the entire stage-10
dorsal lip, and its surroundings, influences at all the subsequent sequence of
completion of the gastrulation movements. This holds true even when gastrulation is artificially slowed relative to the species norm by low temperature.
In two out of three egg-batches of Bombina, the result was similar. In three
out of the five pairs in the remaining experiment a delay close to 30 min was
found in the operated member by blastoporal lip completion, and this delay
was then maintained unchanged through yolk-plug closure. The laboratory
temperature was then at its lowest during the experimental series, and due
either to this, to the particular egg-batch characteristics, or to both, the total
absolute time from stage (early 10) to (12|) was in these embryos somewhat
over 9 h. In view of the decisive lack of an effect in the other experiments, where
operated embryos are as likely as not to complete each gastrulation phase
some minutes ahead of controls, this delay is probably significant.
Autonomy of gastrulation after dorsal lip removal
153
Of the 16 experimental Xenopus, 13 remained uninfected until tail-bud stages,
and of these 10 were normal whilst the other 3 showed deficient head patterns
of types discussed previously (Cooke, 1973 a). All of the ten surviving Bombina
experimental embryos showed qualitatively normal patterns at tail-bud stages,
though two had small heads.
Fig. 2 shows camera lucida drawings at four points during gastrulation in
four pairs of Bombina embryos. Whenever final completion of well-marked
stages in the process was essentially synchronous, as in the first three pairs
shown, the intervening stages of spread of the lateral blastoporal lip showed no
consistent trend of retardation or advance in experimentals as compared with
their controls. This was easiest to confirm in Bombina, where gastrulation is
itself more consistent in the sense that the lateral and ventral parts of the
blastopore are formed by a rather regular progression of onset of mechanical
activity at the surface. In Xenopus, although the lip is completed ventrally with
great consistency between embryos, this is done in some egg-batches by a
secondary zone of activity spreading to meet the first, dorsal one. The whole
lip, apart from the organizer, often forms in a somewhat 'patchy' manner in
this species.
In the cases from the third Bombina experiment where a significant delay did
occur, it appeared to be incurred at the start of the progression, i.e. during the
hour after operation in which the spread of new movements actually started in.
controls, and then to be maintained unchanged during the completion of the
process. This delay may be related in some way to the unusually slow progression of gastrulation on this particular occasion (see above).
Within 20 or 30 min after operations (or 1 h in Bombina) when cell debris
overlying the wound was cleared away, the pit of the dorsal lip in the control
was beginning to deepen. It could often be seen that the surroundings of the
wound, now consisting only of uninjured cells, were performing mass invagination movements in time with those of the homologous area in the control, and
distinct from the surface contraction accompanying healing after a wound
elsewhere in the embryo. In several cases, these movements were seen whilst the
cleaned wound was still just open through to the blastocoel beneath, and in
these cases lines of invagination activity at the outer cell surface, the beginning
extension of the blastoporal lips, were seen leaving the wound edge, having
appeared since operation. This local autonomy with respect to early gastrulation
movements is similar to that noted in other work (e.g. Spemann, 1938;
Waddington, 1941).
In five pairs of Bombina gastrulae dissected after 4 % formalin/saline fixation
at the stage of ventral completion of the blastoporal lip, no evidence could be
found that the stage of evolution of the internal mesoderm (Nieuwkoop &
Florschutz, 1950; Lovtrup, 1965) had been altered by the excision operation.
This sleeve, or ring of cells was distinguished by grey/brown, translucent appearance and homogeneous, small cell-size in this species. It rolls up from the
154
J. COOKE
vegetal margin of the inner (sensorial) layer of neurectoderm to lie between
this and the invaginating yolk mass, and was well formed at mid-lateral levels
and just beginning in the mid-ventral region in all cases. It was not detectable
as such in the latter region at the early gastrula stage 10 in this species. As
visualized under x 50, after this admittedly inadequate fixation technique, this
sleeve of cells is in distinct, though loose, mutual contact as a sheet with no
detectable cell orientation in either the animal-vegetal or dorso-ventral axes of
the embryo.
Loose cellular structure and a slightly less deep invagination of the archenteron, mid-dorsally, were the only signs of the excision operation by this time,
the amount of dorsal mesoderm appearing normal even though some of these
cells are distinctly removed in the stage-10 operation. Thus there is some
evidence that the removed cells may be replaced early on by spreading, but none
that there is any mechanical orientating or attracting force to the dorsal midline
in the normal mesoderm at this stage, which would be destroyed by the
operation.
DISCUSSION
The results allow two conclusions. First, there is no evidence suggesting that
either the external or the internal gastrulation activities are controlled in their
dorso-ventral sequence by a transmission of mechanical traction forces that act
across distances large compared with cells. Secondly, any physiological gradient
underlying the time-course of gastrulation, or at least the local expression of an
earlier response to the values of such a gradient by cells, is normally sufficiently
stable by stage 10 to avoid any alteration of the programme upon removal of
what is presumably the organizing region.
There is other evidence, however (Cooke, 1972), that this organizer region,
implanted sufficiently early into a blastula, can initiate a small field of gastrulation activity of its own in host cells. But the observations suggest that the erection
of the gradient, and the cellular response to it, is slow in relation to the timecourse of development as a whole in Xenopus. It is thus interesting to compare
the lack of any immediate effect of apex removal, shown here, with the definite
delaying effect that this operation has upon the much later pattern of differentiation within the late neurula (Cooke, 1973 a). It may be that the little understood
force, which normally causes dorsal convergence of cells within the definitive
mesodermal mantle at this latter stage, is missing or reduced after organizer
excision, because cells originally adjacent to the excised mid-dorsal ones have
not yet regulatively acquired their properties. This is a separate question from
the one addressed in this paper, however, and will be investigated in the future.
The present results tell us simply that during the few hours of gastrulation,
following the removal of the first mechanically active cells which would have
formed the anterior archenteron roof and dorsal midline of the mesoderm
(Lovtrup, 1965), the balance of forces causing movement elsewhere in the
embryo is not modified.
Autonomy of gastrulation after dorsal lip removal
155
Preliminary observation of sectioned paraformaldehyde/glutaraldehyde-fixed
material in this laboratory has confirmed the finding (Nieuwkoop & Florschutz,
1950) that the mesoderm in the Anura is internal throughout gastrulation. It
also suggests, together with the dissection of gastrulating material described
earlier, that presumptive mesoderm cells, before the late yolk-plug stages,
migrate only parallel to the animal-vegetal (future antero-posterior) axis, and
are not visibly stretched or orientated in the dorso-ventral plane at this time.
This would render comprehensible the lack of mechanical interaction in this
axis.
In the one instance where an apparent delaying effect of the operation was
observed, it is interesting that the absolute rate of development was at its
slowest in the whole series of experiments. It may be that for a very few minutes
after it is first visible, the organizer is indeed in the process of exerting a necessary effect on the cells lateral to it, and that on this one occasion, this effect
was interrupted. In Xenopus, on which the pattern formation results discussed
below were obtained, there is no evidence that the operation, as performed here,
ever exerts a timing effect.
In considering various current theories of the nature of cellular interactions
controlling morphogenesis and the pattern of cellular differentiation (Crick,
1970; Wolpert, 1971; Wolpert, Clarke & Hornbruch, 1972; Lawrence, Crick &
Munro, 1972), it is necessary to put these results beside those of another paper
(Cooke, 1973 a), dealing with the establishment of double axial patterns by
competing pairs of organizers under various conditions. They are among the
few recent results that seem inconsistent with the theory that the actual cellular
communication mediating pattern formation is via diffusion according to a
concentration gradient only (though see Wilby & Webster, 1970).
When a second organizer is implanted into a blastula of Xenopus at a wide
angle from the host presumptive dorsal midline, a conspicuous asymmetry is
typically observed in the sizes of the two axial mesodermal fields and their
induced structures created in the host material. Since cell division is known to
be essentially similar throughout the mesoderm at these stages, and plays no
necessary role in morphogenesis around this time (Cooke, 19736), this result
implies an asymmetry in the steepness, in space or on a per cell basis, of the
gradients embodying positional information (Wolpert, 1971) for cellular
differentiation, with mirror image polarity on either side of the boundary
between the two fields. This asymmetry, whereby the grafted cells are at the
head of a shorter, steeper gradient than that remaining due to the host's field,
would not be troublesome for a diffusion theory of gradient control per se.
However, it is maintained to a constant degree after implantations into hosts
of a wide range of ages up to stage-10 gastrulae, and after post-operative
development at widely differing rates due to experimentally varied ambient
temperatures, suggesting that this position of the boundary between fields
represents a stable state of some sort. It is obliterated, frequently giving equal
156
J. COOKE
partition of the host mesoderm between the axes, under one experimental
circumstance; that of the excision of the host's own group of cells, homologous
with those used as an implant, but only when that group becomes visibly active
some two or more hours after the implant has been healed in position elsewhere
in the embryo.
Two classes of explanation appeared possible for such a pattern of results.
One of these supposed that the head-organizer region, which appears to initiate
gastrulation, in fact leads to the formation of the definitive mesoderm by traction,
and/or results in a stretching of the surface and deep cells of the marginal zone
towards itself, due to some special adhesive-locomotory properties of its own
cells. Thus, its removal, in delaying the process of gastrulation, was assumed
to allow an organizer implanted elsewhere to continue and alter mechanically
the balance between numbers of cells under its own influence and those remaining to the host field. From time-lapse studies on sea urchin embryos (Gustafson
& Wolpert, 1967), where the mechanical activity initiating gastruJation can
actually be seen, it would be plausible to expect this sort of interaction between
pairs of early organizer regions in the present situation, where one of them is
temporarily removed (i.e. until regulative restoration of its activity in neighbouring cells). Because we find, however, as shown here, that the local mechanical situation in more ventral parts of the gastrula remains unchanged following
organizer excision (especially in Xenopus, the species on which the earlier
experiments were done), it is very difficult to suppose that the boundary shifting
results are due to mechanical competition between dorsal lips during gastrulation as such. Interactions between these local organizing regions, determining
the relative position of the boundary between positional-information fields due
to them, probably occur later, after gastrulation is completed in at least its
external aspects. For the early morphogenetic movements, the host organizer
seems to have no immediate controlling function for the cells around it, and the
ventrally grafted one at most (i.e. when implanted early into a blastula) a local
inducing one.
The other class of explanation for the earlier results requires that organizer
regions, in controlling the final gradients for positional information, affect
cells around them in some other or additional way than by diffusion (restricted
or otherwise) or mechanical stretching. The asymmetrical balance of these
influences, coming from one recently established and one much older organizer,
may then only be shifted to symmetry by the temporary removal of the special
activity of cells at the host's own, more deeply established site. Another way of
expressing such an idea is to say that one effect of a dominant region is to
imbue individual cells elsewhere with a property that can be described as a
polarity, or as vectorial, as opposed merely to causing a gradient in a scalar
property whose slope automatically defines a polarity locally.
The results of the present investigation do seem to mean that an adequate
explanation for the pattern formation results would fall into this second class,
Autonomy of gastrulation after dorsal lip removal
157
which challenges entirely diffusion-based models because of the normally stable
maintenance of an asymmetry, under the influence of two organizers. Even if
the symmetrical or asymmetrical distribution into morphogenetic fields within
the mesoderm cell population is at some later time expressed mechanically, e.g.
by cell migration towards each dorsal midline, it seems not to be caused
mechanically.
Future analysis will involve dissection and histology of paraformaldehyde/
glutaraldehyde fixed material at various stages following operations of the types
discussed here and in the previous paper.
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