/ . Embryol. exp. Morph. Vol. 30, 2, pp. 283-300, 1973
Printed in Great Britain
283
Properties of the primary organization field
in the embryo of Xenopus laevis
V. Regulation after removal of the head organizer,
in normal early gastralae and in those already possessing
a second implanted organizer
ByJ. COOKE 1
From the Developmental Biology Group, University of Sussex
SUMMARY
Patterns of individuation occurring in the primary embryonic axis of Xenopus following
excision of the organizer region of the early gastrula are described. In some 70 % of cases the
information for induction of the complete head is qualitatively restored by the time of cell
determination, giving rise to an essentially normal embryo. In some 40 % of cases a second
posterior axis of bilaterality is formed, causing development of a secondary anus, tail-fin and
spinal cord, and often somites. The probabilities of twinning in the tailfield and of failure
to complete apical regulation (= head formation) are largely independent.
After such excision of the head organizer region, a delay of some 3 h in the schedule of
visible differentiation in the neurula/tail-bud embryo is commonly incurred, whether or not
apical regulation is successful.
When the apex is excised from a host embryo which has already contained for some hours
a second apex (= head organizer) as described in an earlier paper, that grafted apex then
captures a considerably increased territory in the host material, as seen from the size of the
individuation field finally caused by it. Such a shift across host cells, of the boundary between
fields of positional information due to two organizers, is not seen under any conditions where
these are left intact, or where host excision is carried out soon after implanting the donor
organizer.
In discussing the results and reconciling them with earlier observations, it is shown that
they strongly suggest the presence of local polar (i.e. vectorial) properties in the presumptive
mesoderm, due to signals from restricted regions which have achieved a special apical state.
Repolarization of cells by a new organizer is not very rapid, and may spread decrementally
from the source. Data on further delays in development, caused by the presence of the second
organizer during regulation in the host apex, suggest that one organizer may act directly on
cells elsewhere to delay or prevent the restoration of the apical state there.
INTRODUCTION
Hydroids have great merits for the study of pattern-forming mechanisms in
development, with their spatial simplicity as compared to the amphibian embryo
(Wolpert, Hicklin & Hornbruch, 1971). One disadvantage is that, generally,
1
Author's address: School of Biological Sciences, University of Sussex, Falmer, Sussex
BN1 9QG, England.
19
E M B 30
284
j . COOKE
only the status attained at boundaries of the field (hypostome and peduncle) is
visible in the results of experiments (see Wolpert, 1969). In the Xenopus embryo
each intermediate zone of positional value is made visible by a corresponding
histodifFerentiation. In the present paper, advantage is taken of this fact to explore
further the properties of pairs of dominant (organizer) regions existing in single
embryos, as begun in previous papers of this series (Cooke, 1972a, b).
In those papers an operation was described, closely similar to those of
Spemann and Mangold (see Spemann, 1938), which originally established the
organizing role of the dorsal lip in amphibian development. The present operation has shown that in Xenopus a plug-shaped mass of cells, incorporating the
earliest dorsal lip and some presumptive head endoderm/mesoderm, when
implanted into an early gastrula host at a maximal distance from its own
organizer in the marginal zone, can cause a complete second axial individuation
field to arise amongst host cells.
In the donor embryo following excision of the organizer, restoration of apical
(i.e. head structure and forebrain-inducing) values for the field will often occur
in the surrounding cells as the gastrulation movements continue after healing.
The final result is then a normal embryo, making the whole phenomenon a
paradigm case of regulation. In this paper other patterns that may result from
organizer removal are described. They suggest that the presumptively posterior
parts of the primary embryonic field are in a fairly labile condition at the time
of the operation, and that the normal configuration of positional information
there depends upon some fairly rapidly communicated influence along the
extent of the presumptive embryonic axis.
This tentative conclusion is reinforced by the results next described. Here, the
patterns of positional information finally attained in the whole mesoderm are
compared, as between control embryos having two organizers, and experimental
embryos where the host apical cells, homologous with the grafted plug, are
removed either synchronously with the initial implantation operation or else
some 3|-4 h later. The time delays occurring in the normal schedule of differentiation following some of the compound operations (though not following
simple implantations) are also described.
In discussing possible mechanisms involved in the build-up and maintenance
of positional information in the primary organization field of Xenopus, these
observations as well as those from previous papers in the series must be incorporated. The theory that has been evolved will be discussed only in outline in this
paper, in order to show how it differs qualitatively from some others that have
seemed appropriate to other systems (Wolpert et al 1971).
Primary organizationfieldoj"Xenopus. V
285
MATERIALS AND METHODS
Details of solutions used, handling of blastulae and gastrulae during and
after operations, and of operations themselves have been presented in full in a
previous paper (Cooke, 1972«). The apical excision operation, always made at
stage 10, the earliest gastrula, is identical with that performed on the donor in
an organizer transfer, many of the subjects of these experiments being the
actual donors for operations of the second type described in this paper. After
excision and implantation operations, gastrulae were left in half- or thirdstrength Holtfreter for about 35 min, for healing, before transfer to 1/10
strength solution on glass for storage. The incidence of exogastrulation was low,
and such embryos were discarded.
Careful pretreatment with EDTA in the manner described elsewhere (Cooke,
\912a) enabled the host stage for early implantation of an organizer to be
lowered to 8~, in which case almost 4 h sometimes elapsed, at 21 °C, before
onset of host stage 10 (the visible onset of dorsal lip - see Nieuwkoop & Faber,
1956).
Embryos for histology were fixed overnight in Bouin, washed for 24 h in
several changes of 70 % ethanol, dehydrated and wax-embedded, and finally
sectioned at 12/<m. Staining was with haematoxylin/eosin.
RESULTS
The head organizer excision operation causes, at most, a \ h delay in completion of the gastrulation movements as judged by time of reaching stage 12. By
stage 28 in initially synchronous controls, however, there is usually delay of
between 2 and 5 h in the schedule of differentiation as judged by tail-fin and
somite development, and by headparts when present.
The actual pattern of primary embryonic axes, resulting from the operation,
presumably represents the configuration of positional information obtaining in
the mesendoderm by the time of initial cell commitment, whereupon mosaic
rather than field properties would supervene (see Cooke, 19726). The spectrum
of results is variable between experiments, due principally to the varying properties of the eggs laid by individual females. However, all successful operations
over a series of some ten experiments gave rise to one of the nine categories of
result shown in Table 1. These categories are themselves an expression of two
variables: the degree of restoration of apical properties around the excised
region, and thus of induction of head structure; and the degree of destabilization
of bilaterality in the trunk/tail inducing part of the field, resulting in a more or
less profound accessory tail individuation. To a first approximation, these two
features of the results vary independently. However, some egg-batches give
much higher incidence of successful apical restitution, or regulation, than others,
and a low incidence at least of profound doubling in the tailfield is slightly
19-2
7
14
3
21
3 (0-5) h
9
85
3(0-5)h
4 (2-6) h
2
5
22
5
Additional small, usually
isolated, tailfield
Two profound tail and trunk
fields of similar dimensions
coalescing anteriorly
Totals
13
No levels anterior
to ear vesicle
54
Isolated cementgland with little
or no other head
structure
Single normal tailfield
No. of examples observed.
Regulation to give
essentially complete
head structure
T= 120
4+ (3-6) h
19
3(0-5)h
29
3(0-5)h
72
Totals
(See text for detailed morphology of the results. Each class represents the total number of embryos observed to show a particular
combination of morphogenesis over the whole series of organizer excision operations. Also given are the mean and spread, for
each total category of head and tailfield morphogenesis, of hcurs delay in differentiation of tail and (where present) headparts,
at 21 °C, by the time unoperated controls have reached stage 28, when embryos are about 33 h old.)
Table 1. The relation between bifurcation in the tailfield and degree of apical restoration
{ = head induction) following head organizer excision from stage-10 gastrulae
o
o
C
/!*~\
oo
Primary organizationfieldof Xenopus. V
287
Cement
gland
Fig. 1. The organizer excision operation, and examples of the morphogenesis
resulting by stage 28 approx., ventrolateral view.
(A) The operation. The boundary of the region removed at stage 10, including
presumptive head endoderm and mesoderm, is shown dotted.
(B) Complete apical regulation, though with the small head structures frequently
seen. A small, isolated supplementary tail structure is also present.
(C) Presence of a small, isolated apical region (= cement-gland induction) only,
due either to partial regulation or to incomplete excision of original material.
(D) No apical regulation evident; absence of structure from anterior to approx.
ear vesicle level. Profound doubling in tailfield has given rise to two complete sets
of tail/trunk structures at a wide angle, coalescing anteriorly.
correlated with this. The overall percentages of successful apical regulation and
of all degrees of tail doubling are, respectively, 73 % and 40 %, taken from the
pooled data of Table 1.
Fig. 1 shows a selection of typical individual results together with a representation of the excision operation. Fig. 2 is a photograph of a particular configuration of the result which is prominent in making up the bottom right-hand
category of Table 1. Here two almost equally profound sets of somites, trunk
nervous systems and tail-fin structures run ventro-ventrally opposed, coalescing
over the anterior end where information has not been restored to a more apical
level than to give rise to a neural vesicle underlain by notochord. In one of these
examples, histology revealed continuity of both sets of somites on either side of
the vesicle.
In cases where regulation has succeeded qualitatively the complete head is
288
J. COOKE
Fig. 2. Two examples of an extreme type of result, from organizer excision. In each
case a second posterior axis, at about 180° to the original one and having its own
anus, tail, small somites and neural tube, meets the original directly in an anterior
neural vesicle with complete absence of head structure. Development was here
delayed some 5 h by operation, as judged by tail-fin morphogenesis, x -2.
often, though by no means always, significantly smaller than that of controls.
Qualitative failure in regulation is expressed in two forms. In the first, induction
of a small cement gland, often isolated on a proboscis-like termination of the
axis, is accompanied by no other head structure or by a small neural vesicle
representing the position of the forebrain field above the gland, and perhaps a
small monophthalmic but recognizable eye. In the other form the axis is completely truncated apically, in which case the pattern ends at or just behind ear
vesicle level.
A series of excisions was made in which special care was taken to cause no
disruption of the actual presumptive trunk/tailfield material or of the neurectoderm lying behind the site of excision across the exposed blastocoel, and to
leave no loose cells in the embryo. The results confirm the initially surprising
conclusion that the formation of accessory tail and trunk structures is genuinely
due to the necessity for apical regulation, and not to mechanical splitting of the
presumptive material concerned. The fate map of Xenopus is not known in
great detail so far as the author is aware, but the probability of accessory tail
formations did not correlate with the cleanness or otherwise of operations. Also,
it seems unlikely that under conditions where presumptive areas immediately
around the wound regularly heal to give unitary, even if otherwise deficient
patterns, those more distant should be mechanically split. Configurations typical
of double organizer embryos (see below, and Cooke, 19726) were never seen
following excisions.
The smaller, more isolated secondary tail individuations are often revealed by
histology to consist only of a small-lumened neural tube, underlying a fin-like
Primary organizationfieldof Xenopus. V
289
Neural tube
00 °o
Notochord
Normally organized mesoderm
Disorganized mesoderm
Ectoderm
Yolky endoderm
Fig. 3. Outline figures of two transverse sections of stage-28 embryos, showing
additional tailfield structures following organizer excision.
(A) Small, isolated secondary axis showing at a posterior level, with neural tube
and fin but no proper somites or notochord in underlying mesoderm. Corresponds
to Fig. 1B.
(B) Point of anterior coalescence of two well-developed trunk level fields, where
the anterior-most level of neural development is simply a wide lumened vesicle. Two
well-developed notochords and somite-rows (outer only) are visible here. Corresponds to Fig. 1 D. x f.
specialization of the ectoderm with a local anus-like ingression into the yolky
endodermal mass at some distance from the main axis of the embryo. There is
then little evidence of shape-changes in the vicinity of the second anus and the
field is isolated, anteriorly, from the main neural axis. At the other extreme,
however, it is difficult to ascribe priority to one or the other of two equally profound, histologically complete sets of trunk structures, each with anus, tail-fin,
notochord and somites forming an independent axis of elongation. In such
cases the co-presence of a complete head is relatively rare and this is always of
the abnormally small variety. Fig. 3 shows outlines of transverse sections
demonstrating each type of secondary trunk field.
Inspection during the process of induction following excision operations
290
J. COOKE
reveals that the accessory anus-like site, which is the most constant feature of
extra tailfields, arises neogenously in the ectoderm at a point discrete from the
closed blastopore, and thus by a process unlike that in normal morphogenesis.
Any subsequent neural induction in the area between it and the main neuraxis
is delayed relative to the main neural plate, although the folds may coalesce
before closure.
Many of the classes shown in Table 1 are too small for significance-testing
despite the overall sample of 120 operations. However, it is apparent that only the
extreme class of the bottom right, incorporating profound doubling and the
least successful apical regulation, is greater than would be expected from a
chance association of the two features that define it. Thus we may tentatively
conclude that within individuals following organizer excision, there is no strong
association between conditions leading to extra axes of bilaterality in subapical
areas of the field and those decreasing the chance of successful apical restoration
of positional information. Such conditions in their more extreme forms may be
correlated, however. Inspection of the means and spread of developmental
delays, entered with the totals for each class of tail and head configuration,
suggests also that conditions associated with profound tailfield doubling and
lack of apical regulation may both also be associated with the greater delays.
Examination of the data for individual egg-batches (i.e. individual females)
shows that in this case, lower probability of apical regulation is found in types
of eggs where greatest developmental delays are observed. But these delays
exhibit great variability always, and the numbers of operations that can be
performed in one experiment preclude a statistical demonstration of this
relationship.
A preliminary conclusion from these observations is that conditions obtaining
at or near the apex of a field (apex, because the area excised and it alone can
potentially organize a whole secondary field) are necessary in some rather
immediate way for the stability of the positional information in regions far
from the apex. It thus became of great interest to study the effect, upon the final
pattern obtained in host material, of excising the host's organizer after implanting a second one.
Table 2 gives the final results of two series of operations, where the effects of
excising the host stage-10 organizer were compared undertwo sets of conditions.
In the first series the second organizer had been in position, with proper cellcontact, for over 3 h at time of host excision, having been implanted at stage 8~.
In the other series, healing in of the second organizer coincided closely in time
with removal of the host's own. A small control series confirmed that without
host excision, essentially similar configurations were seen after each timing,
consisting (see Cooke, 1912b) of dual anterior axial structures, merging together
into a unitary axis at some anteroposterior level dependent upon interorganizer
angle. The clear effect of host organizer excision, but one seen only when the
implant has been in position for some hours beforehand, is a considerable
A. Implantation of
organizer at host stage 8~.
Host excision after
3|-4 h
B. Implantation of
organizer at host stage 9 + .
Host excision within 20 min
12
< lh
29
< 1h
3(2-5)h
0
Host axis fails
to regulate. Sec.
axis smaller,
subsidiary
Host axis regulates
-» head differentiation.
Sec. axis smaller,
subsidiary
6
2
1
A
3
0
6 (4-7) h
27
Host axis regulates
-> head differentiation.
Sec. axis as massive
as host's
Class of result
1 and 2 h
2
6h-*
unmeasurable
delay
20
Host axis fails
to regulate. Sec.
axis as massive
as host's
4
(For details of operations, and variability, see text. Apical morphogenesis due to implanted organizers occurred in some 80 %
of cases overall, and similarly in series A and B, as well as in control operations without host excision. Also given for each class
are the mean and spread of hours delay in differentiation at 21 °C, in addition to that due simply to host excision. Thus comparison was made at the time of stage 28 in initially synchronous control operations having simple excision of their own organizers
at stage 10. Class A 4 is very variable and often uninterpretable in this respect.)
Table 2. The morphogenesis resulting from combined organizer implantation and host organizer excision
3
to
0
o
><
a
292
J. COOKE
Fig. 4. Results of operations combining organizer implantation with host organizer
excision. Drawings made at stage 28, approximately, in initially synchronous unoperated controls.
(A) Control operation. Organizer implanted into host stage 9+, with no subsequent excision. Results for stage 8~ implantations are similar.
(B) Organizer implanted into stage 9+, followed within 20 min by host excision.
The implanted organizer has caused apical individuation of a very subsidiary axis.
Host regulation happens to be of the incomplete type, see Fig. 1 C.
(C) Organizer implanted into stage 8", followed only after 3& hours by host
excision. In this result, of the first type discussed, the two axes (both apical) are well
individuated and of almost equal size.
(D) Operation as in (C), but result of the second type, with the developmental
delay very great, and neither of the two massive axes well-organized or of apical
development, x f.
encroachment of the territory of the secondary organized field into that of the
host, at the boundary joining them. These are the classes 3 and 4 in Table 2.
The increases in size of secondary axes in host material are absolute in these
cases, not just the small, apparent increases that might be expected from the
simple removal of presumptive head material from the host. The intermediate
interorganizer angles used in these operations normally cause joining of axes
just to the rear of the head region, and it should be realized that there is no
evidence that host excision leads to change in this level, i.e. the value for
Primary organizationfieldof Xenopus. V
293
Fig. 5. Drawings of (A) control stage 8 implantation without host excision, (B) late
stage 9 f and (C) early, stage 8~ implantations combined with subsequent host
organizer excision, ca. stage 32. The dynamics of the particular egg-batch giving
these results was such that for three parallel series, of six operations each, no
secondary axis was more individuated or massive than that shown in (A) within
either series A or B. In each embryo of series C, however, paired anterior axes of
approximately equal size, as shown, were developed. Examples shown were matched
for an original inter-organizer angle of ca. 110° in the marginal zone, x f.
positional information at the boundary between fields. The proportion of the
total host material devoted to the secondary field is simply increased.
As mentioned but not stressed in a previous paper (Cooke, 1912b), anterior
axial structures due to the influence of implanted organizers tend to be smaller
than those of the host, although complete back to the level of fusion. Also they
are usually eccentrically placed and subsidiary, i.e. it is evident to external
inspection (and checked by vital staining) which axis is primary in the host and
which due to the newly arisen field. There is variability in this respect among
material from different females. Table 2 is composed only of results from eggbatches where even in the control unexcised operations the implants caused
secondary axial fields of fair size in host material. Such secondary individuation does not always reach fully apical values, but this is considered not to
prejudice the important feature of the results. Fig. 4 shows representative
examples of results using such eggs.
Fig. 5 shows examples from a particular experiment where the effect of host
organizer excision after early implantation is particularly clearly seen. This is
294
j . COOKE
due to the rather homogeneous dynamics of the eggs, where in the control and
the simultaneous implantation/excision situations, fields due to implants were
almost too small to give recognizable individuations.
The means and spreads of delay time given in Table 2 are measured against
controls, having excisions only, performed synchronously with excisions in the
experimental hosts reaching stage 10 at the same time. Thus we have the situation that:
(a) delay averaging some 4 h in differentiation by tail-bud stages is incurred
by the requirement for apical regulation begun at stage 10,
(b) a second organizer, provided that it is present for a few hours before host
excision, causes a pronounced additional delay averaging some 6-7 h, and
(<:) the presence of a second organizer for up to 4 h before onset of host stage
10 causes no delay (or advance) in the schedule of subsequent differentiation in
the absence of host organizer excision.
Included among the results of class 4 in Table 2, and represented in Fig. 4 D, are
embryos whose morphogenetic delay cannot be estimated, being altogether
greater than that for any other type of result. These embryos characteristically
show two massive but ill-defined axes, with little appearance either of anteroposterior or of dorso-ventral differentiation within the material between them, and
with greatly delayed neural folds. Neither axis shows apical individuation as
evidenced by a cement-gland induction, even if the neural fold closure finally
reaches a stage where this would normally be visible. Disintegration sets in
before muscle-cell differentiation or fin formation and tail-bud extension occur.
These results following the early implantation/excision operation are relatively
distinct from all others, including those where either the graft or the host has
failed to achieve fully apical development but where the two axes are well
defined, and the schedule of differentiation is not much more than 6 h delayed
by stage 28 in simple excised controls. When they are excluded from class 4 it
appears that, within the limits of the small numbers involved, presence of a
second organizer for several hours beforehand does not greatly alter the chance
of complete apical regulation in the host axis following excision, which is found
from the protocols to be 27/39 (= 69 %).
DISCUSSION
1. Apical regulation
Variability in the results of organizer excision may be largely due to inevitable
variation in the amount and values of the presumptive material removed. It can,
however, be stated that organizer plugs, whose removal has caused each of the
degrees of regulation observed, are seen to cause apical individuations in hosts
following implantation. Also there does appear to be a slight correlation
between failure in anterior regulation, profound doubling in the tail/trunk
Primary organization field oj'Xenopus.
V
295
field, and the greater delays in schedule of differentiation after gastrulation,
particularly when the properties of individual female's eggs are compared in
protocols. Earlier work (Cooke, 1912b) provides evidence that the mesendoderm
causing cement-gland induction is at the apex of the primary embryonic field,
in positional information terms, followed by forebrain-inducing material.
Doubling in the tail/trunk field is most generally interpretable as a destabilization of the bilaterality component of positional information there, somehow
caused by removal of the apex of the field and occurring before any restoration
of the special conditions there that constitute its dominance and organizing
power. It would appear that maintenance of the labile gradient in the mesendoderm at this stage depends rather immediately upon the presence of the material
normally at its apex. Thus following the temporary removal of this, the bilaterality component of the gradient might become so shallow as to be below some
threshold for reliable perception by the cells. There might then be a probabilistic
process whereby, due to temporary loss of cells' memory as to their 'polarity
potential1 (see Wolpert, 1969), the gradient field becomes reorganized around
two axes of bilaterality, following restitution of the organizing power of the
apex. Each of these would then become the basis for its own tail and trunk
individuation and induction. It is much easier to understand the possibility of
this doubling of a distant, bilaterally symmetrical region of a gradient landscape
on the supposition that cells create substance gradients by actively assuming
locally vectorial properties (e.g. polarized uphill transport of the substance),
rather than existing in a gradient caused only by passive diffusion from a local
source at the head region.
The delay of a small number of hours caused by the necessity for apical
regulation, parallels that observed during an equivalent situation in the slime
mould Dictyostelium (Robertson, 1972), then the anterior tip of a newly formed,
developmentally labile slug is removed. There too, such a tip is capable of
organizing a new slug, whilst the remainder of the old slug experiences a delay
in its differentiation after regulation. In the present system, delay is found both
when regulation finally succeeds and in cases where it fails qualitatively to
restore all zones of anterior information, although the range of delays is greater
for the extreme examples of the latter type. Thus we have the following combination of conclusions:
(a) The final time of onset of differentiation, as visible in morphology and
histology, depends upon the prior operation of a condition which is lost on
apical excision and restored some 3 or 4 h afterwards. It is not set by some
developmental clock within cells which is independent of the state of the morphogenetic field, with the latter merely determining the pattern of differentiation
produced.
(/?) Nevertheless, the restoration of that condition in the field which leads in
due course to differentiation does not by itself guarantee restoration of the whole
pattern of positional information (e.g. the height of a gradient) apically.
296
J.
COOKE
Particularly in cases more delayed than average as judged by the setback in
schedule of differentiation, such regulation may not have been achieved by the
time cell commitment supervenes and development hence becomes mosaic.
2. The effect of a second apex during regulation
Reference to Fig. 6A defines the bilateral positional information profile
normally caused by the presence of two head organizers during development.
The horizontal axis defines actual extent of territory (essentially, numbers of
cells) in the host material, the level of the curve the anteroposterior and/or
dorsolateral levels of the positional gradient (whatever its nature) as registered
in the final differentiation pattern. The level of the valley floor, representing
level of fusion of fields due to the two organizers, is roughly a constant for
organizers initially separated a given distance in the marginal zone of gastrulae.
Levels of both graft and host apices must be set similarly, as both may lead to
full head individuation. The steepness of the valley sides, however, differs,
preserving approximate proportionality within the patterns of the axes despite
their occupation of differently sized territories among the host cells. The
asymmetry of this first profile, found when neither organizer is excised, is an
equilibrium position in some sense, stable over time and under varying conditions of temperature and relative ages of graft and host organizers as discussed
in a previous paper (Cooke, 1972/?). This fact by itself, i.e. the different sizes of
axes due to secondary and host original organizers, suggests that any substance
gradient involved is a result of active properties of the host cells, which either
try to maintain a 'remembered' position as a scalar quantity locally, or else
maintain a direction of active transport of substance. Theories utilizing only
passive diffusion gradients, with local sources and cells being non-vectorial
sinks, would all predict an equilibrium in the present instance consisting essentially of an equal division of the host territory, in terms of area, back to the
point of field fusion. The phase-shift model of Goodwin & Cohen (1969) in its
original form, which does not utilize large-scale transport of substance, would
also predict equal partition of the territory by two organizers, and has already
been found inadequate for the present system on grounds given elsewhere
(Cooke, 1972c).
An implanted organizer takes at least \ h to achieve full, normal cell contact
with its host, so that for one of the two present series of operations, introduction
of the new apex and removal of that of the host are essentially contemporaneous.
Under these conditions the final profile (see again Fig. 6 A) is not affected. That
is, restoration of dominance properties at the host apex has occurred, restoring
stability in non-apical parts of the host's original field, before the new organizer
can influence and thus capture as part of its field more than the normal equilibrium number of cells.
In the other series, where we should expect the normal equilibrium to have
been attained by the time of host apex excision, the new organizer is able to
Primary organizationfieldof Xenopus. V
297
w
B _L_
I
C _L
f---
f —
Iz.%. ?
t
Fig. 6. Hypothetical representation of the effects of operations combining organizer
implantations and excision.
(A) Control operation with no excision, leading to a small secondary field in host
tissue. Implantation and simultaneous excision lead to a similar final result.
(B) Operation with excision following some 4 h after early (stage 8~) implantation. Two well-individuated but almost equally large axes.
(C) Operation as in (B), but axes ill-individuated, neither being apical. Developmental delay considerably greater than in cases of other operations.
Dashed line, profile of positional information landscape (including that following
host apical regulation in (B)). Hatched, implanted head organizer. Stippled, homologous area, removed from host. Arrows, presumed directions of induced morphogen transport (= local polarity) within host territory.
capture, before restoration of organizer properties at the host field apex, an
increased territory such that the two axes finally individuated are about equally
massive (Fig. 6B). A significant additional time delay is also incurred by this
last situation, and is in some cases very great, and associated with imperfect
pattern formation in both the two equal axes. Experiments discussed in another
paper (Cooke, 1973) involving mitotic inhibition, show that, as has been suspected by embryologists, differential cell division can play little or no part in
regulating the overall size of primary embryonic axes.
298
J. COOKE
Gradient models utilizing diffusion as the only type of global interaction
within fields, but proposing that cells act as local homeostats for remembered
concentrations without locally polar properties, seem also to be inadequate to
explain these results. On such a model the region of the lower interface between
fields due to rival upper boundaries should be the least subject to disturbance
when one of these boundaries is temporarily removed. Also, if such cells are
subject to readjustment between remembered values and those actually obtaining due to diffusion (as they should be to allow smooth regulation), then the
normal equilibrium would again be expected to tend towards equal field division.
It is perhaps significant that the only atypical results from the double series of
96 implantation/excision operations were seen in egg-batches with somewhat
atypical properties. Thus in six cases only, early implantation and later excision
caused secondary axes of normally small size, i.e. class 1 results of Table 2.
There was independent evidence of rapid and reliable apical regulative ability,
as judged by results of the first type given in this paper, in the egg-batch concerned. In the two cases of class 4 results, i.e. enhanced size of secondary axes
following simultaneous implantation/excision, deriving from separate experiments, other embryos in the same egg-batches similarly gave evidence of little
apical regulative ability, with greater time delays caused by excision operations
per se.
A model suggested by these data proposes, in outline, that regions having
achieved organizer status, through prior chemodetermination or by subsequent
regulation, become apical in a substance gradient by the emission of signals,
which polarize the transport of the substance within the material around them.
The substance may itself be made at a uniform rate in all cells. Alteration of
transport polarity towards an implanted organizer, within regions already
controlled by the host's organizer, is difficult unless the new one is much nearer
to them, or unless the influence of the original one is removed. Such repolarization and thus redirection of transport may then spread rather slowly outward
from the new organizer until such time as the dominance properties are restored
around the host excision site by local regulation.
The data require that the repolarization process be rather slow, and spreading,
since even where the second organizer remains in situ for some hours before
removal of the host influence, the boundary between fields of graft and host
only advances to be roughly equidistant between them. Results given in Paper
III (Cooke, 1972c) also demonstrate the retention, for some 2 h, of original
polarity in small subapical squares from early gastrulae, when implanted with
reversed orientation some distance from a host organizer.
Any complete theory must also explain quantitatively the delays seen under
all these circumstances, in the final schedule of local differentiation. Delays can
be caused even when apical positional information is finally restored completely
(see Tables 1 and 2), whilst on the other hand, severe delays are often associated
with failure of such restoration before cell commitment. This logically implies
Primary organizationfieldof Xenopus. V
299
that two processes or aspects of the field can be distinguished. One, temporarily
abolished by organizer removal but later reinstated, is required for cell differentiation and ultimately determines the time of its onset. The second, the
gradient of positional information itself, starts to be degraded in the remaining
host material at the time of organizer excision, and to be built up again when
the organizer process is reinstated after regulation. Thus, the longer the delay
before organizer reinstatement, the less the chance of recovery of a complete
gradient before the onset of differentiation itself, some unit time after this
reinstatement.
The delay in recovery of organizer properties and thus rebuilding of the
gradient may be very variable if direct competition between the graft and the
region around the host excision occurs via some signalling system, which may
also be the one finally polarizing the cells and thus building the gradient. Fig.
6C is a hypothetical representation in the terms of the previous two diagrams,
of the ill-differentiated, highly delayed type of result from an early implantation/
excision operation. Such a result might follow an instance of extra prolonged
competition between graft and host (regulating) organizer regions, and thus
delay in setting the polarity of the cells between them.
Detailed treatment of such a model, inappropriate to an experimental
paper, will be presented elsewhere, and an alternative class of explanation for
the phenomena reported here will now briefly be considered. This second type
of explanation, essentially mechanical in nature, has been suggested by Professor Lewis Wolpert during discussion of these results. It supposes that cell
stretching within the mesodermal mantle plays a dominant role in establishing
the extents of fields for final axial individuation, and that such stretching, i.e.
the assumption of elongated shapes by cells, is initially caused by pulling forces
exerted due to cell-locomotory activity at the beginning organizer region only.
Most of the tightly cohering cells of the sheet are then stretched passively, and
in early phases of gastrulation their shape, and various properties depend upon
it, is itself assumed to be continuously dependent upon the special status and
activities of the immigrating cells near the apex of the field. Such an interpretation emphasizes the homology between amphibian gastrulation and that of the
sea urchin, whose mechanics are relatively easier to study (Gustafson & Wolpert, 1967).
During the period before regulative restoration of apical status to cells,
following excision of the original apex in early gastrulae, opportunity would
arise for mechanical reorientation of the axis of passive stretching in cells, due
to the established presence elsewhere of a second implanted group of cells with
apical properties. Such an implanted organizer might require to be resident in a
cell-sheet for some hours before achieving a state of graft/host contact, or graft
cell activity, which allows the re-alignment and thus capture of host cells
following host organizer excision.
On such a theory, the final boundary between fields due to two head
20
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J. COOKE
organizers, in positional information terms, would result from the divide between
zones of mechanical cell orientation due to each organizer, rather than from
any 'valley' in an initial gradient landscape. This could remain true even if
positional information within each field of orientated cells were subsequently
to be established as a gradient with upper and lower boundaries.
In the normal equilibrium situation, without any excisions, fields due to
implanted organizers are normally small in extent relative to the host's original
field. Thus the feature must remain, in this alternative mechanical theory, that
cells once oriented by one organizer are relatively difficult to realign even by a
new organizer situated closer to them.
Further close observation of gastrulae containing two organizers, and following organizer excision, may help in assessessing the two alternative conceptions
of axial field initiation outlined here. The use of larger, slower-developing
urodele species of amphibian may facilitate such work.
I am grateful for frequent discussions, during the work described in this and the previous
paper, with Professor J. Maynard Smith and Drs Brian Goodwin and Stuart Newman, all of
the University of Sussex. I thank Ann Blanshard and Rosi Tucker for skilled technical work.
The research was supported by the Science Research Council.
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{Received 31 October 1972, revised 7 March 1973)