J. Embryol. exp. Morph. 88, 85-112 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
85
Dynamics of the control of body pattern in the
development of Xenopus laevis
1. Timing and pattern in the development of dorsoanterior and
posterior blastomere pairs, isolated at the 4-cell stage
JONATHAN COOKE AND JOHN A. WEBBER
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7,
U.K
SUMMARY
Xenopus embryos have been selected in which the second cleavage is occuring in a frontal
plane, i.e one tending to lie at right angles to the prospective plane of bilateral symmetry for the
body pattern. Some of these have been used to deduce a map of the disposition of materials for
the normal mesodermal pattern (the normal 'fate map') by injecting blastomeres to found
fluorescently marked clones from 4- to 32-cell stages. Other such 4-cell embryos have been
separated into two isolates across this second cleavage; in fate-map terms, prospective
dorsoanterior and posterior isolates. These have been allowed to develop to control axial larval
stages, with examination of the time schedule of their gastrulation movements in relation to
cofertilized whole controls. The patterns of mesoderm produced have been examined and
interpreted in the light of quantitative knowledge about the normal pattern, and our current
understanding of the map.
A meaningful fate map exists for the egg material even at this early, essentially acellular stage,
and it differs appreciably from what might have been expected in view of that traditionally
shown for early gastrula stages. The patterns developed in the isolates show that at least in many
eggs, widespread information that positively specifies material as to its body position is available
from at most 1 h after the events that give rise to bilateral symmetry upon fertilization. This
information usually leads to a mosaic development of the appropriate mesodermal part-pattern
in dorsoanterior isolates, and frequently allows development that approximates to this in the
reciprocal posterior part. Regulation, i.e. the replacement of removed information to specify a
development more complete than the normal contribution in isolates, is not observed. The
results suggest a revision of former claims for regulative ability in at least this amphibian
embryo. They also imply that systems for ascribing position value (positional information) to
early embryonic tissue can be diverse in dynamics, even among embryos whose body plans are
obviously homologous as are those of vertebrates.
INTRODUCTION
This is the first of a series of papers which will report a re-appraisal of pattern
formation in the development of an anuran amphibian, using the contemporary
techniques of cell lineage marking and the quantitative measurement of anatomy
at a particular early larval stage. These techniques have been used alongside
various traditional experimental manipulations, performed on newly fertilized
Key words: egg organization, pattern specification, gastrulation, fate mapping, non-equivalence,
Xenopus laevis.
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J. COOKE AND J. A. WEBBER
eggs and early embryos of the African Clawed Frog, Xenopus laevis. An overall
attempt to introduce these studies in terms of the inherited beliefs and concepts of
experimental embryology would result, rather, in a lengthy review. The reader is
referred instead to reviews by Holtfreter & Hamburger (1955), Nieuwkoop (1973)
and Gerhart (1980, especially relevant for the events before first cleavage). In each
of the present papers, revisions of particular previously held beliefs that are
indicated by results to be described will be emphasized in the appropriate
discussion sections. Attempts will be made at appropriate points to inter-relate the
findings as a whole. This and the next paper deal with the traditional separation of
the fertilized egg into defined fragments at very early cleavage stages, and their
development in isolation. The results are partly at variance with the traditional
reports, and resemble those recently documented by Kageura & Yamana (1983).
They may help in understanding the nature of the information that underlies
development of the body plan in embryos of this general type (Wolpert, 1971;
Cooke, 1983a, b).
Spemann and his students, working with urodele embryos, recorded that a
special limited region of the egg could be defined, at least from soon after
fertilization (Spemann, 1902, 1938). Constrictions were made, at times up to the
onset of gastrulation, to produce developing fragments each reasonably balanced
for constituents in the animal-vegetal axis. Any such fragment that included the
region destined to become the dorsal midline in the normal course of embryogenesis, could apparently develop an essentially complete, balanced body pattern
though of reduced tissue dimensions (i.e regulation). Fragments lacking this
region revealed, by their retention of radial symmetry and lack of dorsal axial
histogenesis, a profound loss of developmental information.
The crucial 'pre-localized' region was originally believed to be quite limited,
subtending some 60° of arc in the egg, but later experiments of similar sorts
revealed that the situation was more complex (Brice, 1958; Dollander, 1950;
Fankhauser, 1948) and that a wider region was capable of organizing at least a
significant partial degree of development. The extent to which the experimental
patterns were analysed histologically and quantitatively, to see if they accorded
with their 'gestalt' appearance, is also unclear. This work has nevertheless been
the classic evidence for the idea that prolonged interactions or signalling processes
across tissue, extending through blastula and into gastrula phases (104 cells), set up
the body pattern by progressive regionalization during the normal development.
This development has in its turn been treated as a model system in which to study
early pattern formation in vertebrates generally. The mechanism has been thought
of sometimes as a hierarchy or succession of specific inductions (Spemann, 1938;
Nieuwkoop, 1973; Slack, 1983; see Cooke, 1983a) and sometimes as the setting
up of a physiological gradient interpreted as positional information of some kind
(Cooke, 1912a, 1973; Wolpert, 1971).
We have re-investigated the states of specification for patterned development
across the egg by capitalizing upon the recently expanded knowledge of the
immediate postfertilization events in anuran amphibians, particularly Xenopus
Pattern in Xenopus blastomere pairs
87
(Gerhart et al. 1981; Kirschner, Gerhart, Hara & Ubbels, 1980). By appropriate
early manipulation and selection of eggs, preparations can be made in which the
two pairs of blastomeres separated at the 4-cell stage will constitute, respectively,
that part of the egg surrounding the presumptive dorsal midline and the
remainder. It is known that the system organizing the future pattern is first
orientated during the precleavage period by reorganization of plasms within the
egg. This normally occurs in relation to the position of sperm entry which can be
used as a marker. At second cleavage (orientated vertically like the first but
orthogonal to it), we have selected eggs with a bilaterally symmetrical cleavage
pattern lying orthogonal to the presumptive plane of symmetry of the embryo.
Some were then used to fill particular bilateral pairs of blastomeres with a celllineage tracer (Gimlich & Cooke, 1983), and thus trace their relative contributions
to mesodermal body pattern in normal development. Those contributions turn out
to be quite characteristic and constant, so that a fate-mapping exercise has been
performed, and the deduced map for mesoderm differs from what might be
expected in view of previously published fate maps for amphibian early gastrula
stages. The remaining eggs were used to perform separations into the two
presumed bilaterally symmetrical pairs of blastomeres, followed by their development as isolates. The partial body plans developed by these reciprocal isolates
were observed and representative samples examined histologically. Their mesodermal component was studied in relation to our understanding of the fate map for
whole development, and our knowledge of certain quantitative regularities in the
normal pattern (Webber & Cooke, in preparation).
We conclude that, by onset of the 4-cell stage, the informational system across
the egg is frequently in such a condition as to lead to stable retention, by the
separated parts, of the ability to develop in a near-mosaic way their reciprocal
contributions to the normal whole mesoderm. Regulative behaviour - that is,
production by a fragment of a greater part of the pattern than its presumptive
contribution in the intact embryo - was not observed. Mosaic behaviour is not the
invariable outcome however, and the whole spectrum of results is discussed in
terms of the possible nature of the primary system of spatial information in the
egg. Our understanding of the flow of developmental information that sets up and
co-ordinates the principal layers of the body structure (germ layers) is also
discussed in relation to the present results, in order to justify our concentration
upon mesoderm as the most direct expression of the original system coding for
'body position'.
We stress that the results reveal an early specification for position within the
body plan (see Slack, 1983 for a defined usage of this term), and in no sense a set of
determinations for tissue types. The behaviour which is described indeed precedes
by several hours the origin of the mesoderm-forming tissue by cell interactions in
the animal-vegetal dimension of the embryo (Sudarwati & Nieuwkoop, 1971).
There is no incompatibility between a demonstration of specification, however
early, and other findings that the normal fates of moderate-sized fragments of the
material can be altered at much later stages by their experimental re-positioning
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J. COOKE AND J. A. WEBBER
within the embryo or in culture (Smith & Slack, 1983; Forman & Slack, 1980).
But when the present results are related to those for other types of miniature
embryonic body, as in the discussion of paper II in this series, they cause us to
question previous interpretations that true regulation of pattern can occur in this
system (Cooke, 1975, 1979, 1981, 1982).
MATERIALS AND METHODS
Preparation of isolated blastomere pairs
Females were injected with 350i.u. of human chorionic gonadotrophin (TregnyF Organon
Ltd) and held at 20°C overnight. Batches of 1-200 eggs were stripped from them at intervals,
between 15 and 20 h after injection, into wet glass Petri dishes. Each batch was immediately
covered with fresh testis suspension in 30 % Ringer. Full-strength Ringer in this work is 0-1 MNaCl, 2mM-KCl, 2mM-CaCl2 and lmM-MgCl2, buffered to pH7-3 with NaHCO?. Newly
fertilized eggs were flooded with 20 % Ringer within lOmin. Within 30min after fertilization at
20 °C, eggs were de-jellied by standing for 10 min and then gentle agitation in 2 % cysteine HC1 in
distilled water, brought to pH7-9 with NaOH. They were then washed and allowed to fall,
through a layer of 5 % Ficoll (Sigma - type 400) in 20 % Ringer, onto a perspex grid of wells,
each of about 70 % of typical egg diameter. Examples with prominent sperm entry points well
down from the animal pole were rapidly selected and aligned under the dissecting microscope,
so that animal poles faced upward and sperm entry meridians (see Fig. 1A) were all aligned as a
record of the presumptive midline for pattern formation. Such eggs rapidly became pinned
against any free-floating rotations in gravity, due to collapse of the perivitelline space by the
Ficoll. Each egg was then manually rotated by some 30° in the sperm entry meridian so as to
lower the s.e.p. in gravity, as this is understood to increase the accuracy with which the planes of
sperm entry and bilateral symmetry co-incide by re-inforcing the shifts of egg plasm that are
organized in relation to the spreading sperm aster during the remaining hour before first
cleavage. In a pilot batch of 50 eggs treated in this way, over 80 % developed their dorsal
midlines within 20° of the position opposite sperm entry, while none showed a deviation of as
much as 45°.
In each co-fertilized batch, when the second vertical cleavage furrow had progressed some
halfway around the eggs from their animal poles (some l h and 45min after selection and
orientation), a set of up to ten was re-selected in which the first cleavage had co-incided most
closely with that plane originally established by sperm entry. Such eggs were demembranated
with finely ground forceps while still in Ficoll, and immediately transferred to loosely fitting
wells in 2% agarose under 66% Ringer which had been buffered to pH6-4 with 20mM-Na
phosphates. Over the following 20min the products of the new cleavage plane could be
separated to give two bilaterally symmetrical but different pairs (see Fig. 1C), by gentle blowing
of the solution from a fine pipette against the new membrane in the deepening furrows. As the
eggs stayed essentially vertically orientated in their wells throughout this process, undue
distortion or tipping of the material in either of the separating pairs was minimized. Examples in
which such noticeable distortion or tipping occurred were discarded. Some sperm-entry side
isolates were made with even less disturbance, by awaiting the completion of cleavage, cutting
away the material of the other pair with tungsten knives and then gently blowing off the residual
membrane and cytoplasm. Neither procedure for creating isolates involved tipping for more
than a minute at any point, whereas tipping for many minutes is believed to be required for
artificial dorsalization or significant activation of pattern formation.
Isolates were placed, as reciprocal pairs whenever possible, in smaller wells to continue
development without the excessive flattening or monolayering of cleavage products that occurs
on a plane surface. The solution was changed gradually to 20 % Ringer, pH 7-0-7-3 during early
blastula stages when abnormal 'internal' type cell surface was no longer visible (see
Observations). Controls within each synchronous batch were eggs (not necessarily so highly
selected for direction of first cleavage) that had been demembranated and processed along with
the experimental individuals until only the spindle mid-bodies appeared to join the blastomeres
Pattern in Xenopus blastomere pairs
89
Fig. 1. Preparation of dorsoanterior and posterior isolates. (A) View from the animal
pole at the 4-cell stage emphasizing the second cleavage plane in which the separation
is made. Third cleavage is horizontal, thus in the plane of the page, and the normal
partitioning of the egg by the vertical fourth cleavage planes is indicated dashed (see
Fig. 2, the 16-cell stage). Sperm entry and dorsoanterior meridians are indicated by
bracket and arc, respectively. Scale bar equals 0-75 mm approx. (B) A pair of isolates
and a control in their wells at the morula stage. Positions of cleavage planes usually
deviate from those that would normally have occurred, in control as well as experimental eggs after treatment. Areas of transient exposure of new, 'inner' type membrane are shown stippled.
from each pair, but which were then left in full-size wells to re-adhere and develop as whole
embryos. Early observation had revealed that the process of blowing for several minutes upon
new cell surface to separate blastomeres causes, in itself, a relative delay of some 20-25 min in
the earliest dorsal lip appearance many hours later. Embryos treated as above were therefore
the correct controls for the following comparative observations.
Observation of comparative time courses of gastrulation
Development of each matched set of embryos and isolates was slowed by cooling to 15 °C
overnight, commencing at control 64-cell stage. Fifteen hours later, on return to 20°C, the late
blastulae could be inverted in their wells in time for the onset and progression of the external
pattern of gastrulation to be monitored by observation and drawing every 20 min (see Cooke
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J. COOKE AND J. A. WEBBER
1972ft; Nieuwkoop & Florschutz, 1950; Keller, 1976). Under the conditions of equivalent
treatment given here, co-fertilized whole embryos commence dorsal lip formation (stage 10 Nieuwkoop & Faber, 1967) within at most 20 min of each other, and the later time of mid-ventral
completion of the blastoporal ring is only a little more variable.
Fate mapping by lineage tracing
Embryos to be used for this work were prepared and individually selected as for the
blastomere separations. Single blastomeres or more usually bilateral pairs, corresponding to the
isolates, were injected with fluoresceinated lysine-dextran (FLDX) as a lineage-linked tracer
(see Gimlich & Cooke, 1983, for injection concentration and procedures for fixation,
embedding, sectioning and visualization of the descendant cells within the structure of the
tailbud larva). Additional cases were also analysed where bilateral pairs of blastomeres
contributing to mesoderm from 8-, 16- or 32-cell stages had been filled. This extended our
understanding of the geographical distribution of the presumptive regions within the egg, to
enable the deduction of the fate maps of Fig. 2. Reconstruction of the contributions of
blastomeres to the mesodermal layer was carried out by sampling the larval transverse section at
twelve equal intervals between anterior notochord tip and tailbud, and recording the FLDXfilled domain on a form containing a standard series of sectional profiles of the normal stage-30
body. Somite numbers for filled domains were checked by horizontal sectioning.
Quantitative comparison of the mesodermal part-patterns produced by isolates with
those of whole siblings
Some representative reciprocal pairs of isolates, and synchronous controls, were fixed for
quantitative analysis (see Cooke, 1979, 1981, 1983b for details of fixation, staining with
Feulgen/light green/Orange G, serial sectioning at 1 pan and cell counting from regularly
distributed sections). The standard stage chosen for examination of pattern throughout this
Fig. 2. Egg regions contributing to mesoderm from early cleavage stages. (A)
Distribution of materials contributing to mesodermal pattern, superimposed on a
lateral view of the idealized 16-cell partitioning of the egg. The second cleavage plane is
emphasized in this and the following diagram. The future left side of the body is
represented (cp. 2B), as the involution movements of gastrulation turn the mesoderm
inside out while extending it to give the long cylinder of the larval mesodermal mantle.
Mesoderm is never at the surface, and should be thought of as deriving from the inner
parts of marginal blastomeres by the tangential divisions that begin at blastula stages.
Cleavage lines are continued off the egg outline to distinguish them from fate map
lines. (B) Formalized plan of the stage-30 larval mesoderm. Its partitioning by the
second (2-4 cell) cleavage plane is indicated by the heavy line, while lighter lines
indicate typical partitioning by the fourth (8-16 cell) planes that establish four sectors
on each side of eggs. The somitogenic columns are considered as simple strips of parachordal mesoderm, as they are in reality before myocoel formation. Precise contributions to mesodermal pattern made by marked clones vary according to precise
positions of planes in individuals, and in particular the number and extent of somite
segment contributions lying between planes, though truly represented, is an average
from the studied material. The coherence and order of the mapping, however,
diminish only where very early-founded marked clones participate in tail mesoderm,
whose pattern is probably the latest specified and where much growth then occurs
before morphogenesis. (C) A large marked clone extending from ventral mesoderm
into primitively lateral somite domain, before and after the dorsal convergence and
cavitation that accompany somite formation. In this way it can be understood how a
'posterior' blastomere makes a pair of discrete contributions to each somite as seen in
Fig. 3, but also how the size of those contributions increases for successively more
posterior somites as shown in Fig. 2A,B. Neural plate omitted from left diagram, s,
somite segment; n, notochord; pn, pronephros; Ip, lateral plate; pc, prechordal.
Pattern in Xenopus blastomere pairs
91
work, as in previous studies, was the late tailbud, stage 29-30. For most of the work in this and
the subsequent paper on 2-cell separations the previously standard, transverse sectional series
was used for cell counting. Certain experimentals and controls were sectioned horizontally
however, to aid in comparison of the real size of notochord cell populations. Somite numbers
segmented at the stage of fixation were usually directly countable in oblique lighting under the
dissecting microscope.
D
pc
pn
B
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J. COOKE AND J. A. WEBBER
OBSERVATIONS
The fate map by lineage tracing
As compared with the traditional in situ vital staining experiment, the use of a
satisfactory injected lineage tracer provides superior information regarding
normal contributions to the body, and degrees of intermingling of descendant
cells, from each part of the early cleaving egg. Single cells descended from labelled
blastomeres can be distinguished unequivocally from neighbours in the early larva
where multiplication to the order of 105 cells has occurred with as yet little true
growth. There are interesting differences in the degree of cell mixing occurring during morphogenesis, among cells destined for the different germ layers
(Smallcombe & Cooke, in preparation), but here we are concerned only with
normal contributions to mesoderm.
The first noteworthy aspect of the results is that it is indeed meaningful to speak
of a fate map at pre- or early cleavage stages. This is not the case for mammalian
development, and very unlikely to be so for that of other amniotes or for teleosts.
But the relative constancy of the observed contributions from particular
blastomeres in our selected cleavage pattern is evidence only that the materials of
the egg are used in a consistent way relative to the 'reference' plane of bilateral
symmetry and to gravity. In individuals with various other perfectly viable
cleavage patterns there are correspondingly different sets of clonal contributions
to an identical body pattern. There is thus no suggestion, in development of this
type, of the kind of fixed lineage or cleavage programme that builds up the body in
certain invertebrate embryos (e.g. Sulston, Schirenberg, White & Thomson,
1983).
Fig. 2A, based on some 40 embryos with blastomeres filled at 4- to 32-cell stages,
shows the distribution of materials contributing to the mesodermal pattern
superimposed upon the cleavage planes of the ideal 16-cell stage as seen from the
future left side (the movements of gastrulation invert the head-tail axis of the
embryo by turning the mesodermal material 'inside-out'). 2nd and 3rd cleavage
furrows have usually been so placed as to cause this stage to consist of a somewhat
smaller dorsoanterior and larger posterior octet, and a distinctly smaller animal
than vegetal octet. The vertical second cleavage is most relevant as this gives rise
to the isolates whose developmental capacities are studied here. It separates off a
sector containing the precursor of the classical 'organization centre' (Spemann,
1938; Nieuwkoop, 1973) from the remainder of the egg. Figs 2B and 2C display
principally the partitioning of the mesodermal pattern of the tailbud larva by this
cleavage, although the contributions of the four sectors between sperm-entry and
mid-dorsal meridians, segregated at the later 16-cell stage, are also indicated in
Fig. 2B.
It can be seen that cellularization, epiboly, gastrulation and dorsal convergence
successively bring about a great geometrical transformation as between the
egg/morula and the larva. Great coherence of neighbour relationships among
cellular relatives is nevertheless preserved during recruitment, migration and
Pattern in Xenopus blastomere pairs
3A
93
B-'
Fig. 3. Fluorescently marked clones in transverse sections of the larval structure, as
used in fate-mapping. (A) T.S. of somite, notochord and epidermis at mid-trunk level
(Somite 10-12), after filling a posterior 4-cell blastomere. Note the unfilled notochord
and adjacent domain in the somite, with relatively little intermixture of descendant
cells. The more brightly fluorescing profiles in somite are sections through nuclei,
which also show up in the adjacent yolky endoderm cells derived from the same
blastomere. (B) T.S. through postblastoporal tailbud region after filling both posterior
4-cell blastomeres, but in a case where the first cleavage had deviated appreciably from
the sagittal plane for pattern. Thus the unlabelled juxtachordal somite contribution
from the dorsoanterior egg region, normal for posterior somites, is much reduced to
the right of the notochord and unusually expanded on the left. From this we can deduce
that one posterolateral boundary of the notochord anlage reached almost to the
cellular domain deriving from the posterior side of the second cleavage.
morphogenesis throughout the mesoderm, diminishing only in the posterior
tailbud where morphogenesis follows a great deal of growth from a very small
region of the original material. Sectional appearances of particular fluorescently
marked domains in larvae are shown in Fig. 3. In undisturbed development the
majority of mesodermal cells descended from an individual blastomere, from the
4-cell even up to the 64-cell stage, are in contact with one another and form
essentially a solid patch within the larval tissue. Such patches show no evidence,
however, of relating to boundaries between anatomical domains or structures. The
patch deriving from a single 'dorsal' 32-cell blastomere of the second tier from the
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J. COOKE AND J. A. WEBBER
animal pole, for instance, may contribute to prechordal plate, to notochord, and
to every somite on one side of the body. The distances across which cell intermingling has given rise to peripheral clusters and isolated members surrounding
main clonal patches are small in relation to the principal dimension of a patch
originating at, say, the 8-cell stage.
The fate map of Fig. 2, essentially from the egg stage, is strikingly similar to that
ascertained by a new labelled-grafting technique at the onset of gastrulation
(Webber & Cooke, in preparation). These maps differ in some important respects
from previously published and 'textbook' interpretations for the respective stages
of anuran amphibian development (e.g. Nakamura & Kishyama, 1971). The horizontal dimension around the pregastrular marginal zone is traditionally thought of
as a 'dorsal-to-ventral' dimension for future pattern, but Fig. 2 shows that the
mapping of larval body position onto this dimension, from the meridian of the
presumptive dorsal lip to that of sperm entry, is in many respects a head-to-tail
one. The lateral plate territory is distributed in this way as a belt around the
vegetal marginal zone, and above it, the 'average' final body position for material
contributed to somites passes from the head towards the tail end of the series as
one passes from what will be the first-invaginating region (Keller, 1976) towards
the sperm-entry meridian. Considerable numbers of posterior somites are derived
principally from material in the 'ventral' 90° sector segregated at the 16-cell stage,
and considerably more than the posterior 50% of the total somite cells are
descended from the sperm-entry side of the second cleavage, i.e. the material of
our 'posterior' isolates.
The blastomeres opposite the original s.e.p. at the 4-cell stage produce the
entire notochord, and make some contribution to all the somites, but quantitatively their derivatives are overwhelmingly anterior. They will thus be referred
to as the dorsoanterior pair (or isolate). A specific profile of relative tissue mass
per somite, in different regions of the somite series, characterizes the normal body
(Webber & Cooke, in preparation), and the differential contributions to this from
the various sectors of the early material are also characteristic. But as seen from
the animal pole aspect, the derivation of each somite is so widespread that material
for most individual segments of the series is gathered from around much of the
equatorial region on each side. Only the anterior 4-8 somites (dependent upon
the degree of eccentricity of second cleavage) are entirely derived from the
dorsoanterior pair, whereas even tail somites behind the 30th receive a juxtanotochordal contribution from those same blastomeres. In passing away from the
sperm entry meridian around the belt of the somite territory one encounters
material that will contribute to the lateral edges of increasingly anterior somite
segments, reaching on average somite 6 in crossing from the original posterior to
the dorsoanterior side of the second cleavage plane.
Comparison of the schematic larval body plan of Fig. 2B with the more realistic
block diagrams of sectors of the dorsal axis in Fig. 2C shows how the somite
material becomes arranged. A clonal patch in the primitive mesodermal mantle
which reaches only to the lateral edge of any particular somite territory (i.e. that
Pattern in Xenopus blastomere pairs
95
furthest from the notochord anlage within the original cell sheet), comes to reside
in two populations at dorsal and ventral 'wings' of that somite's cross section (see
also Fig. 3). This is due to the later morphogenesis (Hamilton, 1969) in which a
split, the derivative of the primitive myocoel, appears in the somitogenic columns
during dorsal convergence movements.
In the sample of 4-cell reciprocal fills used to establish these maps, the restricted
territory of the pronephros bulb lies athwart the second cleavage plane, with its
material often coming almost entirely from the posterior and never so much as half
from the dorsoanterior cell pair.
Gill and other head mesenchymes which push ventrally in the larval head, are of
neural crest, and not primary mesodermal origin. This has been confirmed in
embryos where animal cap blastomeres were filled at 32-cell stage, and primitive
prechordal true mesoderm (unlabelled) could be distinguished from crest-derived
mesenchymes (labelled and adjacent to labelled brain) that were intimately
associated with them (Gimlith & Cooke, unpublished).
Patterns of mesoderm developed by dorsoanterior and posterior isolates
Development in isolation of dorsoanterior cell pairs was relatively invariant,
provided that good early restoration of the blastula configuration had occurred. 47
such successful isolates were observed. The mesodermal patterns achieved
corresponded, qualitatively and quantitatively, to the normal contribution of these
blastomeres to the whole pattern. There was full prechordal head structure and a
notochord whose size (cell population) was indistinguishable from that in withinset controls. The series of somite segments was usually numerically complete, but
its major, posterior part was represented only by small, shallow blocks of cells
alongside the notochord (cp. Figs 2 and 3). The lateral plate tissue was substantial
but entirely anteriorly positioned. The yolky endoderm took up a shape and
distribution conforming to the partial mesodermal pattern - and indeed to the
normal endodermal contribution from this blastomere pair - giving the characteristic 'pigeon-chested' configuration with very slim trunk and small tailbud with
near-terminal blastopore, illustrated in Fig. 4B. Histological analysis of this body
form confirmed that the anterior 4-6 somites, relatively few-celled and shallow in
the complete larva and derived entirely from the relevant cell pair, retained
approximately their normal construction. The remainder of the complement
however, normally of somites progressively more massive in cross section, consisted instead of a set of reduced cell populations, comparable at most to those
normally contributed to each segment by the blastomeres in question.
This body form can be understood by imagining the dorsoanterior mesodermal
contribution (Fig. 2) in isolation, with appropriate co-ordination of neurectoderm
and yolky endoderm, and from comparing Fig. 5A and 5B. Only three of the nine
such isolates examined histologically had developed appreciable pro-nephroi.
Those without pro-nephric formations included four examples of a version of the
isolated development showing a more extreme 'jug handle' body form in which the
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J. COOKE AND J. A. WEBBER
deep and very fully developed head and chest region, of near normal size, is
followed only by a dorsally curling appendage lacking endoderm or fin and with
entirely notochord and somite mesoderm, ending in an entirely terminal
blastopore (Fig. 4C). Quantitative comparison of these particular bodies with the
Fig. 4. Partial body forms developing from isolates made across the second cleavage
plane. (A) Typical control development at the stage used for examination of pattern.
(B) Less extreme and (C) more extreme (commoner) dorsoanterior part patterns. B
probably represents mosaic and C, a slightly more restricted than mosaic development
of the normal blastomere contributions to the mesodermal pattern. (D) The fully axial
development that can frequently be seen from isolates reciprocal to those giving B or
C. Such a body represents mosaic development of the blastomere contributions to
mesoderm, though with a nervous system part-pattern derived from quite other
precursor cells than normal. (E) Significantly axial development of the posterior
isolate, but containing a lower percentage of somite cells, and contributions to fewer
segments of the plan, than the normal contribution. (F) Development corresponding
to the 'belly-piece' of Spemann, containing lateral plate and blood-island mesoderm
with little or no somite and no neural induction, fb, forebrain; ev, hindbrain level and
ear vesicle; eg, cement gland; g, gill structure; e, eyecup; pn, pronephros; bp,
proctodaeum (original blastopore). Somite segment numbers indicated approximately
in relation to other structure. Scale bar represents 1 mm approx.
Pattern in Xenopus blastomere pairs
Fig. 5. Internal pattern in development of isolates. (A) Trunk level in typical control
development. Note the large yolky endoderm profile and deep somite segments. (B)
Trunk level of pattern as in Fig. 4B, with slender yolky mass and small, shallow somites
in relation to the full-sized notochord. (C) T.S., passing to horizontal section
posteriorly, of extreme body form as in Fig. 4C. Note the abrupt posterior termination
of the massive, fully differentiated notochord and disorganized somite. (D) Anterior
level of a fully axially developed posterior isolate as in Fig. 4D. Note the well-formed
pro-nephroi and small anterior-most somite mass, but absence of notochord. (E)
Trunk level of body shown in (D). Note the more massive somite profile at the more
posterior axial position. (F) Tailbud region of an isolate showing a small notochord
structure absent from all more anterior levels. See text for probable explanation of
such development. Scale bar represents 0-5 mm. s, somite; sp, spinal cord; y, yolky
endoderm; n, notochord. Other labelling as in Fig. 4.
97
98
J. COOKE AND J. A. WEBBER
normally extended pattern was difficult because of the variable plane of section
involved, but careful comparison with both transversely and horizontally sectioned material suggested that as in dorsoanterior isolates generally, their notochord cell populations were not distinguishable from those produced in whole
embryos. In the most extreme cases the segmentation in the rear somite material,
which was well histodifferentiated and ensheathed the massive notochord, was
unclear. Both differentiated notochord and somite masses terminated abruptly
near the blastopore without the posterior immature or predifferentiated zone seen
in normal or other experimental bodies (Fig. 5C). More than half of all these
isolates (26/47) developed part-body patterns tending towards the more extreme
version associated with the foregoing internal features.
The numerical proportion of cells in lateral plate of dorsoanterior isolates was
little, if any, less than that normal for controls (30-42%, mean 38-5% control
36-48 %, mean 44-2 %). But this was almost confined to levels opposite the first
few somites, a distribution understandable in terms of a near-normal contribution
according to the fate map (Fig. 2B).
Pattern in the posterior isolates was much more profoundly variable, even
among those with normal blastocoel closure after isolation. 48 such isolates were
observed, and their mesodermal morphology varied between that suggesting once
more a mosaic part-pattern according to the normal contribution, and the radially
symmetrical cylinder of lateral plate differentiation which is the classical
'bauchstucke' (belly-piece) of Spemann. 37 examples were members of successful
reciprocal pairs of isolates, and no correlation was apparent between their modes
of development within the spectrum and the classification (extreme 'jug handle'
configuration or simple mosaic dorsoanterior development) of their other halves.
16 of the most highly organized 'posterior' bodies displayed a pattern approximating closely to the reciprocal of the typical dorsoanterior isolate pattern in
terms of mesoderm. These contained well-formed pro-nephroi, slightly fewer than
the normal body complement of well-developed somite segments, and about 50 %
of their mesoderm cells as lateral plate. In the total absence of notochord, somite
bodies were bridged across the midline and the pronephroi were situated at the
anterior end of the series (Figs 4D, 5D,E). Somite number was three or four less
than that formed in synchronously fixed controls, and the tailbud was of control
size and developmental stage. The presence of a pair of ear vesicles flanking the
widened, hindbrain-like anterior end of the induced neural tube, and just ahead of
the first clearly segmented mesoderm, was an additional marker for pattern as
judged by the field of inductive influence in mesoderm. 12 isolates were classified
as radially symmetrical lateral plate only. The remaining 20 formed a range of
intermediate morphologies in which there was no pro-nephric or ear vesicle
development, and where a progressive diminution in the proportion of mesoderm
as somite cells in relation to lateral plate was accompanied by production of
progressively smaller numbers of segments, always followed by a terminal
predifferentiated zone behind blastopore level and corresponding to tailbud
(Fig. 4E).
Pattern in Xenopus blastomere pairs
99
Fig. 6 shows how, in embryos of this series, the profiles of somite sizes
(maximum cell counts in T.S.) against position in the body are interestingly related
to the profile seen in the whole body pattern. As already mentioned, somites from
different positions in the normal series incorporate characteristic relative amounts
of the total somite tissue, as well as drawing characteristic proportions of their
material from different sectors of the embryonic somite territory. The spatial
patterns of somite contributions seen in these posterior part-bodies are
reminiscent of the specific contributions to be expected from variously restricted
'posterior' (near the s.e.p.) egg sectors to the complete development (see Fig. 2B).
The complete absence of notochord appeared not to affect axial elongation as
such.
100-
80-
60-
40-
20-
12
16
20
23
Segment no.
Fig. 6. Relative sizes and numbers of somite contributions in development of posterior
isolates. Horizontal bars represent the largest nuclear counts in each segment (back to
somite 26) in three control embryos. From this the shape of the normal profile whereby
somite cell populations differ along the series can be seen. Normal somitogenic
columns have a particular shape, being at their most substantial between the positions
of segments 15 and 20. The four histogram outlines (two heavier and two finer lined)
show the same measurements in two fully axial (A,B) and two less complete (C,D)
posterior isolates. If the ear vesicles (where present, in cases A and B) and largest
somites seen are lined up in the plots as shown, the experimental profiles resemble
quantitatively the sets of contributions to somite morphology that would be expected
from sub-regions of the egg extending outwards from the meridian of sperm entry, in
cases (A,B) as far as the second cleavage plane used to make the isolates. This can be
understood by reference to Figs 2A,B, 3, 7A,B. Maximum somite numbers in
posterior isolates are thus three or four less than in the complete pattern, and in those
cases are accompanied by a terminal ear vesicle and well-developed pronephros.
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J. COOKE AND J. A. WEBBER
The qualitative and numerical observations are all consistent with the following
anatomical interpretation. The part-pattern represented in Figs 4B and 5B probably corresponds most nearly with mosaic dorsoanterior contribution to mesoderm, in relation to the normal map. The extreme 'jug-handle' form of Figs 4C and
5C probably corresponds with a pattern contribution slightly more restricted, at
its posterior boundary, than that which the blastomeres would normally make.
The posterior somite and notochord zone which is scheduled still to be predifferentiated at stage 30 is absent. The most highly axial morphology achieved by
the posterior isolates also represents a mosaic contribution to mesoderm according
to fate, whereas the rest of the series would represent production of part-patterns
corresponding to the normal contributions from successively more restricted
'posterior' sectors of the egg. In the limiting case, information for somite tissue
participating in all parts of the segment series is lost, or has never been acquired,
by the isolate. The highest grade posterior isolates achieved a pattern of somite
contributions corresponding at least to the 'highest' encroachment into anterior
somites ever seen after a 4-cell embryo had been symmetrically filled in the
posterior blastomeres.
In three additional isolates designated as posterior, the body that was developed
included a very short, thin but unmistakable notochord region in the tailbud (less
than 1 % of the cells), dividing somites that were elsewhere bridged across as usual
(Fig. 5F). Other features were as in the most highly axial notochordless partpatterns. The reciprocals of these cases were unfortunately not survivors, but they
could perhaps be assumed to be examples where the initial cleavage plane had in
fact deviated sufficiently from the midline of pattern that they represented
intermediate separations in the series between those dealt with in this paper and
the lateral halves described in the next (see Fig. 1 of this paper and paper II
discussion).
Early development after isolations, and the schedules of gastrulation in relation to
subsequent mesodermal patterns
The abnormal zone of naked new membrane, caused by blastomere separation,
appeared to be removed from the embryo surface by a bilaterally symmetrical
inward rotation of blastomeres around the site, so that only externally normalappearing cell junctions were apparent by around control 64-cell stages. This rearrangement was aided by a deviation from their presumptive positions of the
cleavage planes in cell cycles immediately following separation, such that more
cells occupying a single tier were characteristically produced before a horizontal
cleavage set in. The only major redistribution of material that was apparent,
however, was the final suturing together of what would have been two lateral
regions at a new (midline) position in each half embryo (Fig. IB). Only cases in
which this occurred promptly were studied further in detail. Individuals in which
an internal, blastocoel-like surface was exposed to the medium for much of the
blastula period sometimes eventually healed, but always gastrulated on altogether
late, extended time courses. If of dorsoanterior presumptive fate, such isolates
Pattern in Xenopus blastomere pairs
101
finally formed externally 'balanced'-looking miniature bodies, though with varying
degrees of spina bifida. These proved to have disproportionately small or absent
notochords and head parts, however. Presumptive posterior such isolates
invariably gave non-axial, 'bauchstucke' forms. This effect of early prolonged
healing may be important and interesting in relation to the slight delaying effect
upon gastrulation of the original method of separations, and to possible
interpretations of the discrepancy between the morphogenetic results in this paper
and in previous ones (see Discussion).
Dorsoanterior cell pairs, thus isolates, are typically smaller than their posterior
partners as mentioned earlier, though the tendency varies greatly between egg
batches. A study of absolute cell numbers in isolates remains to be done using
proper techniques, but counting around the diameters and marginal zones of
several reciprocal isolates at control stage 10 revealed a smaller surface cell
number in the dorsoanterior partners, which still look smaller as gastrulae. In
larval pattern analysis (see previous subsection) the mesodermal cell counts of
posterior but axial part-patterns slightly exceeded 50 % of the within-set controls,
in conformity with expectation from the normal contributions. Dorsoanterior
part-patterns, while appearing to contain less than 50% control cell numbers,
were too different geometrically from the control plan for safe comparison to be
made. There is thus a strong indication that the two types of isolate enter
gastrulation with different cell numbers, but no indication that this represents a
deviation from their role in normal development. The causes of variation in
numbers of cells produced, from eggs and early blastomeres of different sizes, will
be discussed in paper II of this series. Observations of cell numbers throughout the
present work, however, are in line with previous findings that surgical interruption
of tissues and changes of configuration do not of themselves affect the cell cycle
schedule before pattern establishment is complete in this experimental system
(Cooke, 1979, 1981).
Dorsoanterior isolates initiate the external signs of gastrulation within 10 or
15min of their entire controls, i.e synchronously with them. Observation of
isolates from egg batches in which the pigment gradation across the animal
hemisphere was pronounced enough to act as a marker have not indicated that the
dorsal lip forms on a meridian differing from its presumptive one in the egg of
origin. The subsequent lateral spreading and fusion of the lines of surface bottle
cell activity, to give a ring-shaped blastopore, completes itself precociously in
these isolates however. They thus show the ring blastopore characterizing normal
stage 11 or 11+ while controls are still in the spreading lip phase 10+ or 10!.
Surface cell counting suggests, on some occasions, that this precocity is simply
because of the smaller tissue extent around the marginal zone in isolates, with the
spread of new cellular activity occurring at control rates on a 'per cell' basis. This
would represent a mosaic partial time course of gastrulation, with all material
behaving according to its local presumptive schedule. In other instances, however,
the initial lip is broad and rapidly spreading in relation to the control configuration, so that progression to give a ring occurs on a more rapid time course per
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J. COOKE AND J. A. WEBBER
cell than is occurring in controls. This represents departure from presumptive
schedule to give true precocity of cellular activity in the more 'lateral' material.
Blastopore closure (stages, Hi-13 equivalent appearances) continues on a
relatively precocious schedule in dorsoanterior isolates and is frequently followed
by an abnormal pear-shaped appearance, where the closed blastopore becomes
produced into a nipple-like structure that appears to give rise to much of the
slender trunk region that is later found. Those batches of isolates in which the truly
precocious spread of gastrulation followed by the nipple-like blastopore was most
pronounced, also gave rise to bodies that suggested the most restricted
anterodorsal part-pattern of mesoderm in relation to the fate map (see previous
subsection). They also included the smallest isolates, in some cases because of
relatively great eccentricity of the second cleavage, and in others because of small
original egg size.
Posterior isolates pursued more variable schedules of spreading gastrulation,
correlated with their more variable performance in pattern formation. Of 30
classified finally as showing pronounced axial elongation, somite segment
formation and neural induction, 28 had gastrulated on a spreading schedule
beginning 30-40min after control stage 10 (i.e. at control lateral lip stages), but
competing the blastoporal ring no earlier than controls. Of 18 classified as closest
to radially symmetrical, non-axial differentiation, 15 had shown essentially radially
symmetrical or patchy, rapidly fusing zones of onset of marginal cell 'bottling',
commencing 60-90 min after control stage 10 and at most 20 min before control
blastoporal ring completion. Such a gastrulation pattern is reminiscent of that seen
in those whole embryos which, having received u.v. irradiation to the vegetal
hemisphere at a precleavage stage, will produce similarly non-axial patterns
(Scharf & Gerhart, 1983; Cooke, 1985). On a crude binary classification,
therefore, only five examples of morphology would not have been predicted from
the time schedules of new cell activity at gastrulation that preceded them.
DISCUSSION
The results call into question various earlier assertions and ways of thinking
about anuran amphibian development, including some of the first author's. These
previous interpretations of data, on apparent pattern regulation, will be dealt with
first. We can then begin thinking anew about the primary spatial information
system in this development.
Previous interpretations
When large sectors surrounding the meridian of sperm entry are removed from
mid-blastulae, and new cell contacts finally result in normal-looking but sizereduced late blastulae, these often give rise to small bodies that look qualitatively
complete, and quantitatively balanced as to tissue types. (Cooke, 1981, 1982).
Such bodies also show a number of small somite segments that is correct for
developmental age (Cooke, 1975). This result remains obtainable in our
Pattern in Xenopus blastomere pairs
103
laboratory after blastula operations, even when it is most unlikely that
presumptive notochord territory has been removed. It is now clear that on simple
fate-map grounds (Fig. 2), there is no need to postulate regulative events to
explain constancy in the tissue proportion of somite to lateral plate, or in somite
number after these ablations. What had been believed to be a 'ventral' ablated
sector, normally contributing only to lateral plate mesoderm, in fact contributes to
a great majority of somites and substantially so to many of them. It contains, if
anything, proportionately more somite than lateral plate presumptive territory,
and probably contributes also to pro-nephros.
We can also now see that the dorsoanterior sector, left in blastulae after
ablations, is due to contribute to the entire somite series, though its normal
contribution is a partial one for all but the first few segments which are in any case
reduced in cross section. The present work tells us, moreover, that by the earliest
time the system can be partitioned in a plane that segregates out a contribution to
only part of the somite file - namely the posterior isolate at the 4-cell stage,
information leading to the development of appropriate (complete or incomplete)
numbers of segments in isolation is often present. This observation, though
startling from the standpoint of dynamic theories of development, simplifies the
interpretation of the small embryos previously constructed. They represent largely
a 'mosaic' development of part-patterns of mesoderm.
Only the previously observed 'down-regulation' or reduction in size of the
notochord territory, not the normal result in the present work, needs explanation.
We have no definitive such explanation but clues may be offered by the following
observations. Early abnormal exposure of inner-type or new blastomere
membrane to the medium, as in the separation of blastomeres itself, delays
measurably the normally precise tempo of development, including the onset of
visibly new cell activities at gastrulation. Such delay becomes steeply more
exaggerated in relation to increasing amounts of time spent with blastocoelic
surface abnormally exposed or cell contacts relaxed, as in the unsuccessful
reconstructions of morulae after blastomere separations. Typical blastulae
resulting from previously published series of surgical excisions, where healing was
often prolonged but subsequent gastrulation not monitored, would be expected on
present evidence to have shown significantly retarded onsets of gastrulation. The
present isolations, performed at the outset of development, have shown clearly
that developmental delay beyond a certain rather strict threshold, occasioned by
some physiological trauma in certain cases, is the only circumstance that will cause
departure from fate in the patterns of mesoderm developed by reducing the
territory achieving notochord status (see results in this and in the following paper).
Such an effect is seen along with otherwise normal and healthy differentiation of
the tissue pattern that does develop, and tends to cause uninterrupted but slender
notochords rather than any deletion of particular regions of the rod-shaped
structure. It is thus not strictly linked to pattern but to notochord as a tissue type
(see later discussions and comparison with other types of notochord deficiency).
We suggest therefore that the small notochords of previous miniature Xenopus
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J. COOKE AND J. A. WEBBER
larvae have been fortuitous effects of slight physiological damage upon the
embryo's performance, though a systematic study of the morphogenetic effects of
prolonged blastocoel opening is needed. No good evidence remains for specific
regulative capacity as to whole body pattern in this embryo, from cleavage
onwards.
The range of results for frontal 4-cell separations in Xenopus, at least from
external appearance, approximates closely those reported by Kageura & Yamana
(1983). It is now hard to evaluate the classical claim for a different spectrum of
results, and for true regulation in 'dorsal' fragments, in Urodele development. The
two forms are possibly more deeply distinct, phylogenetically, than their postembryonic anatomies would suggest (Nieuwkoop & Sutasurya, 1976). The traditionally investigated species are also at the opposite end of a spectrum from
Xenopus as regards egg size and the rapidity of development. There is therefore
room for speculation that strategies for the spatial organization of early development are evolutionarily labile across even smaller phylogenetic gaps than that
between amphibians as a whole and amniotes (where specification of the body
plan is relatively delayed, in relation to an indefinite pattern of cleavage and early
cell mingling). The original urodele work may on the other hand have been
flawed, by inadequate numbers of examples and techniques for normal fate
mapping and for reconstruction of the internal mesodermal patterns in isolates.
The distribution of normal contributions to the mesodermal plan, around the
marginal zone in the urodele pregastrula, may be somewhat different from that in
anurans but is unlikely to be so divergent as to account for the different reported
results on fate map grounds alone.
The assertion that the Xenopus body plan lacks proportion-regulating
mechanisms, from cleavage stages, obviously demands re-examination of claims to
later regulative interactions continuing into gastrula stages (e.g. Cooke, 1982,
1983a,b). A future paper in this series following marked clones in embryos
where pattern twinning has been caused by dorsal lip grafting, will offer such a reexamination. It appears at the present time that certain interactions that tend to
harmonize proportions can operate within qualitatively 'complete' embryos as a
fine-tuning principle, whereas the present work concerns the inability of partembryos to re-establish missing boundaries to pattern.
The stability of a system specifying body position
The present results reveal that in many Xenopus eggs, very shortly after the
events of the precleavage interval, a configuration of stable information specifying
body position for mesodermal contributions is disposed around the system
approximately in register with the presumptive fate map. We can assume that this
information is in the vegetal-equatorial part of the egg (see later discussion). It is
laid out along a dimension at right angles to the animal-vegetal axis, in which cell
interactions will subsequently specify the definitive germ layers that contribute to
all the body regions in a co-ordinated way. We first explain why the results tell us
Pattern in Xenopus blastomere pairs
105
this, and then begin to ask what the nature of the information for body position
might be.
The abnormal boundary of the fullest or most axial mesoderms made by
posterior isolates, i.e. their point of anterodorsal truncation, corresponds closely
with the partition made in the normal fate map by the cleavage plane. Frequently,
then, reciprocal isolates across this plane develop so as to suggest that further
communication between their descendant endomesodermal cell populations
would have been minimal and unnecessary during normal embryogenesis. The
dorsoanterior isolate always does something very close to this, so that the
impression remains that the dorsoanterior extreme or boundary of pattern is in
some sense a stable reference region in the egg. The deviations from mosaic
mesodermal part-patterns that occur in many posterior isolate bodies represent
losses to varying degrees of their full spread of information, or else failure to
acquire it, rather than attempts at regulative acquisition of more complete sets of
information. The system's behaviour can be illustrated graphically, as in Fig. 7, by
symbolizing the information as levels on a quantitative gradation of state in the
material. It formally resembles the behaviour of the ligatured insect blastoderm
described by Sander (1975). But the information levels here appear to be very
much more stable at early stages in relation to at least nuclear cleavage (the insect
blastoderm is multinucleate, but sometimes a syncytium, at time of ligature). Loss
of the correct gradation of information across material, when the boundary
regions are experimentally isolated from each other, can be slow or absent.
The morphology of at least two other types of experimental embryo bears out
the contention that all levels in the informational system specifying body position
can remain autonomously stable from early times. Precleavage u.v. irradiation
attenuates, in a dose-dependent fashion, the intracellular movements that somehow set up the information. The various low grades of this phenomenon result in
partial patterns bounded by normal tail formation but truncated anterodorsally at
a continuous series of levels in the normal fate map. Similarly, the minor group of
isolates, in which the egg has been transected obliquely in relation to the pattern
axis, show how detailed the adherence to specific coding for body position can be
around the egg substance. Axial but headless bodies, with partial notochords
confined to specifically posterior positions, are almost certainly the results of such
oblique partitioning. In all these cases, deprivation of an upper sector of the
normal complement of information levels occurred during or immediately after
the initial setting up of that information (Gerhart et al. 1981; Kirschner et al.
1980; Scharf & Gerhart, 1983).
Even posterior cell pairs isolated without any tipping, by sacrificing the
reciprocal pair, can develop the most fully mosaic part-patterns. It is unlikely that
such blastomeres have undergone a second, artificial activation of pattern
formation along the lines of that which can be induced in whole, u.v.-treated eggs
by tipping with respect to gravity. The rather striking co-incidence of the second
cleavage partition in the normal fate map, with the boundaries of the most
complete part-patterns, is further evidence that we are studying information
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J. COOKE AND J. A. WEBBER
Fig. 7. Some properties of the system specifying body position at cleavage stages. (A)
A spatial distribution of a graded property that specifies position value according to the
normal fates, in vegetal-marginal material, must exist in many individual eggs by the 4cell stage. This is because the developments implied by such a system are adhered to
quite precisely if the egg is partitioned (dotted line) at that time, and the boundary
between the reciprocal part-patterns produced corresponds with the line taken by the
cleavage plane through the normal fate map. (B) There may be a variable loss of
developmental information evident after isolated development, corresponding to
degrees of relaxation of the spatial gradation as shown. This is particularly seen in
posterior isolates. Regulation - a tendency to restore complete gradations of information in one or both isolated sectors - is never observed and thus not shown. (C)
Variation in the morphogenetic results of isolate development may be due to variation
in intensity of the initial, rapid mechanical events that set up asymmetry and a graded
profile of activation or position value. Thus in some eggs at the 4-cell stage the
information may be in a restricted distribution not corresponding to the normal pattern
contributions, so that a 'posterior' isolate will be permanently deprived, to a varying
extent, of its normal spread of information. In normal development of such eggs (and
in the later development of the dorsoanterior isolate), slow interactions corresponding
to a spread of activation might normalize the position value profile in material by the
time this is used to effect gastrulation and differentiation.
already disposed across the material at the time of separations. A further study of
isolates, from u.v.-treated eggs which will not otherwise form axes, is nevertheless
desirable.
Such stability on the one hand and lack of regulative dynamics on the other, in
an informational system, contrasts with the behaviour to be expected of a
positional gradient set up and maintained by interactions obeying diffusion-like
laws, i.e. the reaction-diffusion, source-sink or fixed morphogen source models
that have usually been linked with the idea of positional information (Wolpert,
1971; Gierer & Meinhardt, 1972; Gierer, 1981). The information used by the
present isolates must be set up across essentially non-cellularized material within
at most an hour, and two cell cycles, after the significant events of the mid
precleavage interval. It would be simplest to assume that such information is
purely structural and given at the time of symmetrization movements, though this
is not necessarily so (cp. certain ctenophore and coelenterate embryos, Freeman,
1983). But it is often stable enough to direct development of part-patterns, when
we know that such patterns will not be fixated as irrevocable cell commitments for
another 10 or 15 h (Smith & Slack, 1983; Heaysman, Wylie, Hausen & Smith,
1984).
How might we explain the sequence of results in posterior isolates that deviate,
by loss of information, from the mosaic part-pattern? There may be partial loss of
Pattern in Xenopus blastomere pairs
107
originally existing information, during blastula/gastrula stages (i.e. relaxation of
the gradient in state as in Fig. 7B) or there may be a variability in the configuration
of information achieved at the stage of isolation, with some eggs having a reliably
polarized but still inappropriately restricted distribution of the state we will call
'activation' (see Fig. 7C). Such eggs would achieve the fully distributed activation
profile, and thus the normally proportioned development that they undoubtedly
give when left intact, by some slower 'back-up' mode of signalling which perhaps
more resembles a traditional morphogen gradient system. Certainly, eggs vary
within and especially between batches as to the apparent vigour of the structural
movements after sperm entry. It would be of interest to carry out frontal isolations
on individuals with records of the apparent extent of their early movements of
symmetrization. But on the evidence of these experiments, more than 50% of
Xenopus eggs would not require very much interaction along the presumptive axis
after the 4-cell stage in normal development. This indicates that while one may
choose to call the unknown variable specifying this pattern positional information,
the means of acquiring it, and its initial molecular nature, can be different from
those most recent workers on pattern have been considering. According to those
models, large isolated parts from the system should show dichotomous behaviour,
tending either to achieve small-scale patterns more complete than their fate-map
contribution (i.e regulation) or to lose pattern information entirely. The earlier
they are isolated in relation to the time of overt expression or differentiation of
pattern, the less mosaic should their behaviour be.
Observation of the real time course of gastrulation is limited by a) the variable
smoothness of the surface progress of lip formation in different batches, even
though stage-10 lip formation is very well timed as stated, and b) the imperfect
relation between spread of bottle-cell formation and epibolic pigment gathering at
the marginal surface, and the precise stage of true mesoderm involution underlying it (Keller, 1976). With these provisos however, the observations strongly
support the idea that a variable intimately linked with the signal that codes for
position within the body is the specific programmed rate of local development.
Thus, exactly timed onsets for mechanical contribution to gastrulation are probably set within mesoderm cells induced at particular meridians of the egg, which
are to construct particular levels of the pattern (see Fig. 2A).
A consideration of what is understood about the early re-organization of plasms
in the egg, which must go far to set up the organization revealed in these
experiments, is a necessary prelude to thought about its molecular nature (Gerhart
et al. 1981; Kirschner et al. 1980; Neff, Wakahara, Jurand & Malacinski, 1984;
Elinson, 1984). Such consideration will be postponed for the integrated discussion
of the present results and those of separations at the 2-cell stage, in Paper II.
The flow of information that sets up the germ layers
How can separations at such early stages result in mosaic behaviour even though
territories for endoderm, mesoderm and neurectoderm have not yet been
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J. COOKE AND J. A. WEBBER
established? Experiments, on the origin of the mesoderm have produced partially
conflicting results so that the sequence of information flow in normal development
is unclear (Nieuwkoop, 1973; Nakamura et al. 1970; Gimlich & Gerhart, 1984).
Mesoderm is probably induced as a zone in the marginal region of the animal cap,
by information from the vegetal yolky region that will become the endoderm. This
information is still available at late blastula stage, but its normal spread may occur
much earlier. The graded information for body position, with which this paper is
concerned, may be present in those parts of the egg that will become both
endoderm and mesoderm. But even if it is originally confined to the presumptive
endoderm, there is some evidence that what is induced on each meridian in the
blastular marginal zone is not just mesoderm, but mesoderm that is specified for
development in co-ordination with its subjacent endoderm as a general body
region (Nakamura, Takasaki & Mizohata, 1970; Holtfreter & Hamburger, 1955).
If development is viewed as a series of often transitory specified states linked to
territories (Slack, 1982, 1983), then the simple germ layer mesoderm, without
further specification, may never exist. Mesodermal territories may from their
inception share elements of positional coding with the endodermal territories that
induce them. Some such assumption makes it easier to understand the present
results, and also the observed preservation of spatial contiguity in the normal body
plan by the endodermal and the mesodermal derivitives of large (early) clones.
The other view, favoured by Nieuwkoop (1973), is that the endodermal inducing
influence merely establishes a polarity by positioning the mesodermal organizer or
presumptive dorsal lip at one particular meridian. Pattern specification within
mesoderm is then assumed to occur, with a prolonged time course, mainly by
interactions around the marginal zone. But in this case, 4-cell-stage isolates
separated from the organizer-inducing region across the frontal plane could have
no basis for observing mosaic development according to presumptive fate for
mesoderm. They should be without sufficiently detailed information so that they
either lose polarity altogether or regulate to give new pattern.
We assume that primary information for the body plan is present relatively early
in both endoderm and (probably by patterned induction) in its accompanying
mesoderm. We have assayed pattern by inspecting mesoderm simply because, of
these two germ layers, it makes the greatly superior display of that pattern. The
featureless endoderm, although primary in causal terms, displays its pattern only
by gross architecture in the tailbud larva. We have noted that in dorsoanterior
isolates particularly but also in axially developing posterior ones, endoderm takes
up a largely mosaic 'shape' according to the normal endodermal contributions of
the blastomeres in the whole body, rather than disposing itself along the
mesoderm according to simple mechanical constraints.
The final phase of the informational flow that co-ordinates the germ layers is the
induction of the neurectodermal pattern with information from mesoderm. This
probably occurs only during gastrulation, as the appropriate spatial relationships
between patterned mesoderm and the neurectodermal layer are only then
established. Unlike the earlier one from endodermal to mesodermal zone, this
Pattern in Xenopus blastomere pairs
109
passage of inductive information involves pronounced geometrical re-organization
through morphogenetic movement. This difference is revealed in the development
of the present frontal isolates. The neurectodermal patterns, of those posterior
isolates whose mes-endoderms have developed mosaically according to fate, are
found to have involved massive departure from their normal presumptive
contributions by the ectodermal descendants of the original blastomeres. It was
recorded earlier that well-developed CNS pattern consisting of spinal cord,
hindbrain and ear vesicle characterizes such isolates. Examination of whole larvae
whose posterior cell pairs have been filled with FLDX reveals, on the other hand,
that these founder cells contribute almost entirely to the epidermal ectoderm and
to only very restricted parts of. the normal nervous system, namely a little to
forebrain (precisely what the isolate patterns lack) and to posterior or very lateral
spinal neural plate. We presume that the alteration of the course of development,
in the neurectodermal layer alone, is brought about by the enforced new spatial
pattern of contact between the mesodermal part in isolates, which embodies
considerable axial inductive information according to fate, and their ectodermal
part which would normally have been largely excluded from such contact with
inducing mesoderm and so would have become epidermis. This observation
extends the recent experimental confirmations of Spemann's original view of the
inductive origin of the CNS, that have been possible using lineage tracing
techniques (Gimlich & Cooke, 1983; Jacobson, 1984).
Non-equivalence. The nature of the body pattern
A final feature of the morphological results to which attention must be drawn is
the way in which they support the notions of non-equivalence (Lewis & Wolpert,
1976) and of a 'second anatomy' underlying development (Slack, 1982, 1983).
A straightforward consideration of cellular differentiation as studied by the
cytologist, and of the mechanics of cell adhesion and locomotion, might suggest
that early development is a matter of arranging that a simple pattern of territories
of certain shapes and sizes be formed within a cell sheet, with the material in each
territory embarking on one of the courses of differentiation that characterize the
later body. If each such course of differentiation were to be accompanied by a
particular set of earlier mechanical properties in the constituent cells, then the
observed normal correlation of morphogenetic movements and the pattern of
differentiation in the whole embryo might be expected to follow as a sort of
mechanical resultant of the local forces. The two above-mentioned ideas contrast
with this in proposing that a series of early decisions establishes a spatial pattern of
regions, but that the molecular coding system whereby the regions are marked out
is prior to, and independent of decisions underlying the development of the
recognized cellular differentiations or even anatomical structures. Each region is
indeed set to contribute to the final pattern in a defined way, but defined only by
position within the plan of the body. This may include contribution to all or any of
the differentiations and structures listed by the anatomist. The corollary is that
each gross element of the body, as defined by histodifferentiation, is composed of
110
J. COOKE AND J. A. WEBBER
contributions from diverse territories which were primarily specified as to the body
region each was to be responsible for.
In the current Xenopus experiments this shows up in the behaviour of somite
and notochord. Their material (or the material that induces them) appears to be
regionalized around the meridians of the egg at early cleavage stages, so as to set
up quantitatively specific contributions to the different geographical parts of the
final segment series and of the simple elongate notochord. The quantitative
aspects of posterior part-patterns of somite segments in relation to the complete
pattern, shown in Fig. 6, should be compared with the fate map as revealed in
Figs 2 and 3. This shows how the preservation of partial information, leading to
formation of particular posterior subsets of segments in each isolate, tends to be
linked with patterns of quantitative contribution that follow the normal fate of
material with that information in the complete development. Similarly the
patterns formed by the minority of oblique isolates, transitional between the main
results of this and of the following paper (Cooke & Webber, 1985), show the nonequivalence of regions even within the relatively restricted early notochord
territory. In these isolates and also in minimally affected u.v.-irradiated embryos
described in Paper III (Cooke, 1985), the fate map has been partitioned or reduced
so as to include only a specific minor fragment (usually relatively lateral in the egg)
of that territory. Partial notochords in restricted, posterior axial positions are
produced, rather than the abnormally slender rods occupying the whole available
space in the axis which a purely mechanical model of morphogenesis would
predict. Such embryos, made by the earliest manipulations, are striking evidence
of mosaicism and stability for something which we must call body-position value.
The complete notochord anlage presumably contains an almost complete set of
contributions from the belt-like equatorial zones, in the early embryo's mesodermal map (see Fig. 2A), which become coded for successive anteroposterior
axial positions.
This paper is dedicated to the memory of June Colville, skilled histologist for much of the
authors' work, who died on 30th August 1984.
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(Accepted 20 February 1985)