/. Embryol. exp. Morph. Vol. 51, pp. 165-182, 1979
Printed in Great Britain © Company of Biologists Limited 1979
165
Cell number in relation to primary pattern
formation in the embryo of Xenopus laevis
I. The cell cycle during new pattern formation in response
to implanted organizers
By JONATHAN COOKE 1
From the Division of Developmental Biology, National Institute
for Medical Research, Mill Hill, London
SUMMARY
Results are presented which offer strong evidence that extensive alteration of the fates of
embryonic Xenopus cells occurs independently of the schedule of cell division, after operations which lead to a doubling of the axial pattern of mesodermal differentiation in the
gastrula. The experimental strategy was to make estimates of total mesodermal cell numbers
and mitotic index in closely matched sets, each of three synchronous sibling embryos, fixed
during the ten hours following the close of gastrulation. Within each set two embryos, an
unoperated control and a sham-operated embryo whose own dorsal-lip (organizer) cells had
been replaced with an equivalent graft, were developing normally. The third, experimental
embryo had received an organizer implant to replace an equivalent number of cells from its
ventral marginal zone, and was thus developing two axial mesodermal patterns of differentiation in relation to two dorsal midlines, the extra pattern embracing much host tissue. Mitotic
index was also determined, in specific regions and throughout the mesoderm, in similar sets
of embryos but at mid-gastrula stages.
The conclusions are justified by the results of a control investigation which show that there
is normally no difference in cell cycle time along the presumptive dorso-ventral mesodermal.
dimension, during the interval between time of operations and the determination of patterni
The lack of any enhancement of mesodermal cell number in late embryos with dual axia
patterns, or intervening enhancement of mitotic index in younger operated embryos, thus
suggests that new patterns may be determined in the Xenopus gastrula without generation of
extra cells.
The results are discussed in relation to recent ideas about pattern formation, and the
concepts of morphallaxis and epimorphosis.
INTRODUCTION
In the study of the control of pattern during animal development, a distinction
has come to be drawn in principle between two modes of regulative response
to disturbance, known as Morphallaxis and Epimorphosis (Morgan, 1901;
Wolpert, 1971). In Morphallaxis, restoration to wholeness of the final pattern
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
166
J. C O O K E
of differentiation or the generation of new such patterns, after removal or
grafting of embryonic cells, occurs without any requirement for special production of new cells or for special cell migration. The behaviour is more
reminiscent of the redistribution of some spatially graded information, by an
averaging or perhaps a diffusion process amongst the cells in the system, and
the term positional information was recently introduced by Wolpert (1969,
1971) to discuss the mechanism of such regulation. In Epimorphosis on the
other hand, pattern restoration or extra formation occurs by specific recruitment of a local cell population at the site of disharmony, or stimulation of the
cell cycle there, to produce the new pattern in tissue that would not have been
made but for the disturbance (e.g. Nothiger, 1972; Bryant 1977). Cells in such
systems might therefore be said to have positional values which, sometimes in
conjunction with the disparate values of cells that they find themselves contacting,
determine the values of the daughter cells produced. But there is no evidence
for overall intercellular positional signalling.
Although we can describe clearly the formal distinction between morphallaxis
and epimorphosis and the variations on the basic rules that distinguish the
latter (see discussions by Wolpert, 1971; Cooke, 1975a), it is currently unclear
how many developing patterns show real behaviour which partakes of both
characters, or is of one character at early stages and the other later on (Faber,
1971; Summerbell, Lewis & Wolpert, 1973; Kieny & Pautou, 1976). In a
recent paper proposing a universal formalism to describe behaviour of epimorphically controlled patterns, French, Bryant & Bryant (1976) suggest that
primary differentiation fields of embryos, i.e. those controlling the whole body
pattern at early stages, will be found to be morphallactic (independent of
growth), while secondary fields that control specific patterns such as limbs or
appendages at later times will be found to be epimorphic, using growth both for
normal pattern formation and for regeneration where this is possible. The
present paper reports the results of experiments designed to investigate this
question for the primary embryonic field of the vertebrate embryo. This field
exists, during gastrula stages, largely within the layer of cells known as the
mesoderm.
Regulation to restore wholeness of pattern after tissue removal, and production of pattern duplication in response to presence of an extra field boundary
or organizer, can occur extensively at pre-gastrula stages in amphibians, birds
and almost certainly mammals (Spemann & Mangold, 1924; Spemann, 1938;
Waddington, 1941; Waddington & Yeo, 1950; Waddington, 1952; Eyal-Giladi
& Spratt, 1965; Cooke, 1972a, b, 1973a). Strikingly, there has been no definitive
information as to whether such regulative responses involve alteration of the
schedule of cell division at their early stages. Amphibian embryos are highly
suitable for addressing this question, allowing performance of an operation
that causes extensive pattern duplication within a tissue consisting of a manageable number of cells. The geometry of normal mesoderm formation in anuran
Cell number during Xenopus pattern formation I
167
amphibians, such as Xenopus, has been investigated using various methods
(Nieuwkoop & Florschutz, 1950; Nakatsuji, 1974, 1975; Cooke, 19756; Keller,
1976), and grafting operations, producing a duplication of the mesodermal
pattern of differentiation within host tissue, have been described (Cooke,
1972a-c). During this latter work however, and in three further papers (Cooke,
1973a, b, 19756) on analysis of pattern regulation, the unjustified assumption
was made that the cellular responses involved were exclusively morphallactic.
The previous observation of appropriately small and few-celled structures,
arising in tailbud larvae after early size-reduction operations on blastulae
(Cooke, 1975 c), justifies the assertion that completion of spatial patterns does
not depend rigidly on production of particular cell numbers. Many complete
secondary axes, after early organizer grafts, are also obviously short and slender
compared with the host axis. The criterion for pure morphallaxis, however, is
more stringent than this; the production of new or replacement pattern parts
should take place without any effect upon the cell-cycle in the participating
tissues.
On theoretical and experimental grounds (Dettlaff, 1964; Graham & Morgan,
1966; Flickinger, Freedman & Stambrook, 1967; Chulitskaia, 1970; see also
Snow, 1977) it is very unlikely that real differences of cell cycle time, within one
'germ layer' of embryos at particular stages, could be concealed by a homogeneous mitotic index. Variation in the cell cycle occurs principally through
variation in phases other than M; thus the incidence of M is a sensitive index
of the relative length of the cycle within regions of an early cell layer. The
determinations of mitotic index in this study therefore serve to indicate, in the
event of a significant enhancement of ultimate cell number being observed,
whether such enhancement is caused by relatively rapid division among a small
population of cells near the graft-host boundary, or by only slightly elevated
division rate spread through much of the mesodermal field. In the latter event,
the small real differences in cycle time required could be expected to go unobserved, because of the low control mitotic index and the limitation upon the
numbers of cells that can be scanned.
The results offer strong evidence that the responses to disturbance in the
Xenopus primary embryonic field are truly morphallactic; that an endogenous
schedule of division frequency in mesodermal cells, nearly homogeneous
throughout the future body axis up until the time of determination, is unaffected by artificially provoked cell interactions that alter the pattern-forming
or positional values of large numbers of those cells.
The results are discussed in relation to the concept of 'quantal mitoses'
(Holtzer, 1964, 1970), to previous work showing independence of pattern development from mitosis (Cooke, 1973 a), and to possible secondary effects of
pattern size upon cell division (rather than, as here, the lack of any special role
of cell division in pattern creation).
168
J. COOKE
MATERIALS AND METHODS
Embryos were obtained by artificially induced spawning (Chorionic Gonadotrophin 'PregnyP-Organon, Ltd, 350 i.u. for females and 150 i.u. for males)
in Xenopus laevis kept on a diet of raw beef heart and liver. Pairs were not used
more frequently than every 5 weeks. Washing, handling and demembranation
of pre-gastrula stage embryos was as previously described (Cooke, 1972 a,
19756).
Operations were performed as previously described (Cooke, \912a, 1973a),
and as discussed in the results section, but using a solution of 48 % Holtfreter
saline, 16 % Niu Twitty saline and 36 % glass-distilled water, brought to pH 7-2
with 1 N-HC1. Following operations implanting stage-10 (Nieuwkoop & Faber,
1956) organizers into hosts during the 2 hours of their development preceding
stage 10, the healed embryos were stored on glass in 10% amphibian saline at
temperatures between 17 and 21 °C, from the onset of their gastrulation (stage
10). Unoperated, demembranated control embryos were passed through
exactly the same schedule as their synchronous operated siblings.
Sets of embryos were fixed at various stages, for 24 h, in a solution of 10%
40 (w/v) formaldehyde, 2 % glacial acetic acid, 50 % alcohol and 38 % amphibian
saline, followed by washing in 2 % formol saline for some days. They were
wax embedded and sectioned at 7 /*m in precise transverse section, care being
taken that the plane of section was very similar in embryos within one set for
comparison. Staining was by an adaptation of the Feulgen method, followed by
counterstaining with 0-2% alcoholic light green, and then orange G in 2 %
phosphotungstic acid. By this means, while nuclei and mitotic figures showed
up clearly, the different germ layers and the cellular structure within them were
visualized well enough for camera lucida drawings and, at later stages, identification of patterns of cell behaviour and differentiation.
Mitotic index was scored within particular regions of the mesodermal mantle
of embryos by counting metaphase and anaphase figures, every fourth section,
for 2000 cells. Where both future nuclei in an anaphase figure were seen, one
mitosis was scored.
Standard estimates of cell numbers were made by counting nuclei visible in
every sixth section of the mesodermal mantle throughout embryos, including
the thickening around the closed blastopore in neurula and later stages (see
Keller, 1976). The total count was multiplied by a constant which was the
appropriate Abercrombie correction factor (Abercrombie, 1946) multiplied by
6 to give a number which is referred to a s ' the number of cells in the mesoderm',
but which must be an estimate, variably biased at different developmental
stages. In fact neither nuclear diameter nor mean internuclear distance, used to
compute the correction factor, change consistently between stages 13 and 17,
but after this time elongation and flattening of mesodermal cells in the transverse plane will systematically bias such counts as absolute estimates of cell
Cell number during Xenopus pattern formation I
169
number. Their use as sensitive indices of relative cell number is justified by
comparison only of equivalently shaped, synchronous sibling embryos, sectioned
in identical planes.
RESULTS
Table 1 shows the data from an overall control investigation, on mitotic
index in dorsal and ventral mesoderm within sibling sets of unoperated embryos
during gastrula stages. These stages, about halfway between the times of
operations and of earliest scoring of cell numbers in the experimental series,
are of the order of one cell-cycle time or less after such operations, and probably
precede the time at which the configuration of positional values within the
mesodermal mantle is made permanent by the onset of determinations for the
basic pattern (Holtfreter & Hamburger, 1955). There is no evidence for overall
dorso-ventral difference in mesodermal cell cycle times during these stages
(although a very localized region of higher mitotic index in the presumptive prechordal mesoderm and anteriormost endoderm, seen at all later stages examined, is already present). Thus, replacement of ventral with dorsal mesoderm,
per se, would have no effect on future total cell number that might be mistaken
for a stimulation of the cell cycle caused by positional interaction. There is no
cell death intrinsic to early amphibian development, so that total cell number,
at stages some two average cycle-times subsequent to the experimental rearrangement of cells, will be a sensitive detector of any effect upon the division
cycle during the intervening period of pattern determination.
Beginning anteriorly at stage 13, the notochord is rapidly segregated physically from the remaining mesoderm. Some 1-| h later, and spreading more slowly
posteriorly than notochord segregation, cells destined as somite tissue segregate
by dorsal convergence from all the remainder of the mesoderm (presumptive
nephros and lateral plate). As will be discussed in a later paper, mitosis within
these two most dorsal parts of the axial pattern effectively ceases for a long time,
the possibility of mitosis seeming to decrease sharply a set time before the
visible change in cell behaviour that segregates the tissues off, at each successive
level of the axis. Thus apart from the prechordal centre, there is hardly any
mitosis in all but the most posterior dorsal midline by stage 12|, and almost
none in anterior presumptive somite by stage 14. However, this morphogenesis,
and progressive loss of mitosis in the dorsal pattern areas, itself takes place
rapidly compared with the average cell cycle (estimated in excess of 10 h, see
Discussion). It is therefore considered that such a relatively late effect of pattern
upon cell division, following a longer period of homogenous mitosis, cannot by
itself have a significant effect upon the total cell count when comparing patterns
in neurulae which are developing two dorsal midlines with those which are not.
Each set of observations derives from three carefully matched, synchronous
sibling embryos, one experimental, one sham-operated and one unoperated
control. First, estimates have been made of total mesodermal cell number in
170
J. COOKE
Table 1. Dorsal, lateral and ventral mitotic indices in unoperated gastrulae of
two egg-batches (18 °C)
Mesodermal M.I.% (1000 cells sampled
throughout future A-I' axis)
r
Stage
10£ (early gast.)
1 (batch 1)
2 (batch 1)
3 (batch 2)
4 (batch 2)
H i (mid-late gast.)
1 (batch 1)
2 (batch 1)
3 (batch 2)
4 (batch 2)
Dorsal
Lateral
Ventral
3-4
2-7
5-2
5-8
2-8
3-6
4-9
6-7
3-7
30
60
5-4
40
4-5
6-4
51
3-7
4-7
6-8
5-4
4-8
3-9
5-9
61
There are no significant dorso-ventral differences within each embryo. Mid-endodermal
M.I. in batch 1 over this period was 3-5%, in batch 2, 5-6%. (2000 cells, pooled from four
embryos in each batch.) M.I. among a small group of anterior (presumptive pre-chordal)
cells was 7-2 and 8 4 % in batches 1 and 2 respectively (1000 cells, pooled from four
embryos each), representing an enhancement above general mesoderm significant at P = 001
level over the whole series.
embryo sets fixed during the ten hours following the close of gastrulation, at
which time the basic spatial pattern of mesodermal determinations is considered
to have been fixed, and the first external signs of the existence of the secondary
pattern are to be seen. Determinations of mitotic index, in mesodermal pattern
parts where cell division is still to be expected (see Results and Discussion),
have also been made on this material. Secondly, mitotic index determinations
have also been made in various mesodermal regions of similar sets of embryos,
but at much shorter times after the operations; that is, while the secondary
pattern formation is actually occurring.
Figure \a shows the standard operation and its sham control version. The
graft, cut from a very early gastrula donor, is a group of a few hundred cells at
most, extending from mid-dorsal marginal surface to the blastocoele. ]t
embraces the bottle cells of Holtfreter (1943), a small sector of the presumptive
anterior dorsal mesoderm internally, and of the endodermal surface (future
archenteron of the head region) externally (Nieuwkoop & Florschutz, 1950).
It is implanted in harmonious orientation (see Cooke, 1972#, c) into the ventral
marginal zone of a host blastula within \-2 h of onset of the latter's
gastrulation, after excision of an equivalent group of marginal cells to accommodate it. In the sham operation, where a blastula is simply given a new organizer
to replace its own presumptive one, development is to a normal, single pattern.
After healing in, a ventral graft sinks inward while maintaining surface contact,
to produce an invagination which is usually met smoothly by the advancing
Cell number during Xenopus pattern formation I
111
Fig. 1. id) The operation (left) and its sham-control version (right). The core-shaped
stage-10 organizer graft is shown in surface aspect. The two recipient sites left by
excision of groups of cells from the ventral and dorsal (presumptive organizer)
marginal zones of host blastulae are shown in vegetal view. In these relatively late
hosts, stage 9+, and about to commence their own gastrulation, the internal and
external cell-size gradient is indicated, and the incipient dorsal lip activity in the
experimental host. The more substantial secondary axes tend to be formed (see
Fig. 2 c) when rather earlier hosts are used. The necessary re-orientation of the graft
relative to the experimental host is indicated, (b) A set of three siblings at stage 13,
in surface view from the rear, showing the outlines and flattenings of the early
neural plates, and the slanting blastoporal groove and smaller appearance (due to
the double 'gatherings' of the neural folds) in the experimental embryo. In this case,
host and secondary neural plates are of synchronous development, (c) A set of
siblings at stage 17, from the front, showing deeply indented cephalic neural folds
and the incipient induction of the ectodermal cement gland beneath them. In this
case the experimental secondary neural induction, while of considerable size, is
delayed in its morphogenesis. In such cases, the underlying mesodermal pattern also
tends to be of primitive development (as in Fig. 2d).
172
J. COOKE
lines of external gastrulation activity that extend ventrally from the host's
organizer to complete a blastoporal ring. ]n the best cases, this blastopore when
closed thus overlies two archenterons, but this is not necessary for substantial
second mesodermal patterns to be formed internally, correlated with second
neural plates. At the end of gastrulation the internal mesodermal mantle,
essentially a cylindrical sleeve of cells, has advanced further than usual ventrally,
having there a second axis of bilateral symmetry around which an extra pattern
of differentiations develops in opposition to those due to the host's field.
In this work, only cases where the second axis consists of an appreciable part
of the total mesodermal material are considered, the size of the second field
being revealed only at early neurula stages by alterations in shape, because of
dorsal elongation and convergence due to each axis, and by the flattening
defining each area of neuralized ectoderm (Fig. 1 b). The secondarily induced
ectodermal derivatives finally seen (e.g. Fig. 1 c) are relatively passive reflections
of the patterns of mesodermal determination beneath them (Nieuwkoop, 1970;
Cooke, 1912b) that the presentwork is concerned with. Previouswork has shown
that donor cells essentially form some anterior dorsal mesoderm, and anteriormost endodermal structures only. Much of the new pattern is within host tissue.
The data on cell numbers come from ten sets, each of three precisely synchronously developing sibling embryos, matched as closely as possible for
shape, and plane of sectioning, and each involving one successful example of
secondary pattern formation from stage 13 onwards, one sham operation
developing normally, and one unoperated control. Mitotic indices were also
scored for dorsal, lateral and ventral mesoderm in transverse sections of further
such sets of synchronous sibling embryos, at gastrula stages midway between
the time of operation and that of earliest cell counting. Figure 1 b and c show
typical external appearance of sets of three, at neurula stages 13 and 17.
Figure 2a-dshow the mid-axis transverse sectional appearance of, respectively,
a stage-ll£ unoperated gastrula, a stage-15 sham-operated neurula developing
normally, and its two siblings with doubled axial patterns. In Figure 2a the
boundaries are marked according to which mesoderm was scored as dorsal,
lateral or ventral (by reference to the archenteron) for mitotic index
purposes.
Table 2 shows the cell number estimates, and mitotic indices for appropriate
mesodermal areas, within the sets of matched synchronous siblings, together
with information about the particular operations, ambient temperatures of
development and stage of fixation of the embryos. Variation in mitotic index
and cell number between experiments and ovulations is much greater than that
within them. Indeed, only the remarkable consistency of cell number between
normal siblings makes the overall strategy a sensitive one to detect cell interactions with respect to the division cycle. The variation between experiments
could reflect properties of eggs from particular toads, but there is evidence
(reviewed in Dettlaff, 1964; Chulitskaya, 1970) for a differential effect of
Cell number during Xenopus pattern formation I
173
Dorsal
(a)
(c)
Arclienteron
Lateral
Ventral
Neural plate
Arclienteron
Fig. 2. Camera lucida outline drawings, with nuclei of the mesodermal layer marked
in, of transverse sections of control and experimental embryos at mid-axial levels.
(a) Stage-11-J- unoperated, 'anterior' to the blastoporal ring, to show the limits relative
to the dorsal midline for scoring dorsal, lateral and ventral M.I. (b) A sham-operated,
normally developing stage 15. At this axial level, although the notochord is well developed, the future somitic cells are only partially aligned and gathered dorsally to
leave the lateral plate (and presumptive nephros). (c) An experimental stage 15,
where the secondary axis is synchronous with the host's pattern in differentiation,
and associated with its own archenteron cavity in the endoderm. (d) A sibling of (b)
and(c), experimental, but where the obvious secondary axial pattern in themesoderm
and its overlying ectoderm is of relatively delayed development. There is as yet no
notochord differentiation or somitic alignment, and no archenteric cavity. On shape
criteria, (d) was not paired with (6) for cell counting, whereas (c) was.
temperature on the cell cycle and on early morphogenesis during amphibian
development, which could produce just the sort of variability seen.
It can be seen that comparisons within sets give no indications of enhancement of cell number in mesoderms within which two patterns are developing,
up to earliest tail-bud (20 s) stages, and no trend or significant differences in
12
EMB 51
14250
11150
11600
13400
14100
11650
12950
12200
16900
19150
11450
14200
13250
11000
13650
12200
18250
16750
17100
17200
11800
14100
12100
11150
10050
9800
9950
6800
Unop.
control
6450
Sham
op.
7050
Op.
Sham
op.
Unop.
control
1-2 dorsal
1-7 dorsal
1-5 dorsal
3-4 ventral
3-6 ventral
3-6 ventral
2-7
2-6
2-9
(Lateral plate and presumptive somite, between axes in op.)
41
4-5
51
(Lateral plate and presumptive somite, between axes in op.)
50
4-8
4-3
(Lateral plate, between axes and in sec. axis midline in op.)
4-2
3-9
4-5
(Lateral plate, between axes in op.)
3-3
2-7
3-5
(Lateral plate, between axes and in sec. axis midline in op.)
2-7
3-2
30
(Lateral plate, between axes in op.)
2-4
2-7
2-5
(Lateral plate, between axes in op.)
2-2
20
2-7
(Lateral plate, between axes in op.)
2-4
2-8
20
(Lateral plate, very small area between axes in asymmetrical op.)
Op.
M.I. % (2000 cells)
The M.I. data show no significant differences within embryos, or sets of matched siblings. Cell numbers show no trend in favour of operated embryos with
double axes. The range of stages investigated covers the time of estimated fixation of pattern-forming fates of cells, which probably begins dorsally before
stage 12£ and spreads ventrally through neurulation.
(1) Stage 12^ (implants into
stage 8^, develop, at 19 Q
(2) Stage 13 (implants into
stage 9 + , develop, at 18 °C
(3) Stage 13 (implants into
stage 9, develop, at 18 °C)
(4) Stage 13£ (implants into
stage 9, develop, at 18 °C)
(5) Stage 14 (implants into
stage 8, develop, at 19 °C)
(6) Stage 15 (implants into
stage 9 + , develop, at 19 C)
(7) Stage 15 (implants into
stage 8^, develop, at 18 C)
(8) Stage 17 (implants into
stage 9 + , develop, at 20 Q
(9) Stage 19 (implants into
stage 8£, develop, at 18 C)
(10) Stage 21 (implants into
stage 8, develop, at 21 °C)
Set number, stage of development
and details of operation
No. of mesodermal cells
Table 2. Total estimated mesodermal cells, and examples ofmitotic indices in still dividing regions, within
matched sets of three embryos after operations
m
o
o
o
Cell number during Xenopus pattern formation I
175
Table 3. Mitotic indices in dorsal and ventral mesoderm {graft-host border) of
gastrulae after organizer implantations, in comparison with sham operations and
unoperated siblings
Set number, stage of
' C U J J J l l l C l l l a LIU 11VJL115
M.I. % (2000 cells)
f
since operation
Dorsal
Ventral
5-8
61
60
6-8
6-7
(1) Stage 11 (5h. at 18 °C)
Op.
Sham Op.
Unop. control
(2) Stage 11 (5h. at 18 °C)
5-9
Op.
6-3
4-7
Sham. Op.
5-8
Unop. control
(3) StagelH(8h . at 16 °C)
5-6
51
5-3
1-7
1-8
2-3
2-4
1-8
1-7
Op.
Sham Op.
Unop. control
Indices show no significant dorso-ventral differences within embryos, or trend when
operated are compared with control embryos. Since set 3 came from the same egg-batch as
sets 1 and 2, the significantly reduced overall M.T. is almost certainly due to the low ambient
temperature used.
mitotic indices. Furthermore, operations as such seem to be without effect
upon the cell cycle at any of these stages, even in the immediate region of the
expected graft-host boundary. Particularly interesting is the lack of any difference from control values in mitotic index of the still dividing lateral mesoderm
between closely situated dorsal midlines, in the stage-21 embryo shown in
Figure 3.
Table 3 shows mitotic index data, at two stages a few hours after operations,
in operated and control gastrulae. Again, no evidence for enhancement or
depression of mitotic index is seen due to any operation.
DISCUSSION
Taken overall, these data make it very unlikely that any enhanced cell
division is involved in creation of new patterns through cell interaction in the
Xenopus primary embryonic field. Many of the secondary axial patterns in these
experiments embrace a substantial proportion (estimated as about | ) of the
total mesodermal cells at neurula stages. By this developmental stage, at least
in vivo, the fundamental fates of all mesodermal cells as to axial structures are
almost certainly determined (Holtfreter & Hamburger, 1955). Thus the assumption, that the spatial pattern of differentiations in amphibian embryos
until these stages is controlled by morphallactic interactions, is given strong
176
J. COOKE
support. Further circumstantial evidence for this type of interaction is seen in
the transverse sections of several experimental embryos where the initial operation has been particularly asymmetrical, so that fewer cells have been situated
initially between host and graft dorsal midlines, tracing one way around the
embryo as compared with the other (Fig. 3). In these cases, evidence from cellsize and yolk platelet density in cells, and the positions of notochord rudiments
which may fuse posteriorly (see also Cooke, 1912b), strongly suggest that the
fates of cells have been so altered by the new interactions that the dorsal midlines of pattern (apexes of the fields in positional information terms) have not
necessarily developed from the cells whose presumptive fates were to form them
in the original graft and host fields. This phenomenon will be addressed fully
in a later paper, where the use of 1 ^m Epon sections will clarify such shifting
of the fates of cells. Such lability of cell fate is characteristic of morphallactic
interactions, as earlier studied for instance in sea urchin (Horstadius, 1939,1973)
or Hydra morphogenesis (WiVoy 8c Webster, 1970; Wolpert, Hicklin &Hornbruch,
1971), whereas in epimorphosis only a minority of the cells, those induced to
special growth, produce descendents with altered fates. Figure Aa depicts, in a
highly schematic way, the interpretation of positional interactions in gastrula
mesoderm required by the present findings, and contrasts it with extreme epimorphosis (Fig. 4b).
In view of these results, it seems unlikely that introduction of a competing
second organizer into the primary field at an earlier stage than was accomplished
in this work would lead to the type of cell cycle stimulation which characterizes
epimorphosis. The latter may be restricted to later, secondary fields as suggested
by French et al. (1976). It is known that still earlier organizer operations in
Xenopus, or a graft into an equivalent host to that used here, but in slower
developing urodele species where there is more time for interactions, can lead
to essentially equal partition of the mesodermal cell population into the two
axial patterns. If total mesodermal cell number were found to be increased in
such newly determined twin patterns, it is predicted that this would be by
abnormally extended, or radially symmetrical, cell recruitment from the animal
neurectodermal zone to create the initial mesoderm, and not by any enhancement of mitosis. Such cell recruitment, or determination as mesoderm, is a yet
earlier pattern-forming process whose normal course has been documented by
Nieuwkoop (1969), and Weyer, Nieuwkoop & Lindermeyer (1977), who call it
'induction' of the mesoderm within the animal cap by the endoderm.
The question of the independence of pattern regulation from the generation
of new cells is quite separate from that of the ultimate control of pattern size
and proportions (in this case, the size and proportions of the whole body) by
feedback control of growth. Adjustment of newly formed patterns to normal
size by cell division, over a period of time, has been documented in insects
(Bohn, 1970, 1971). There is also evidence that this occurs during later developmental stages, when whole vertebrate embryos are initially formed from
Cell number during Xenopus pattern formation I
111
Neural tube
Notochord
Lateral
plate
Two
archenterons
Notochord
Neural tube
Fig. 3. Camera lucida outline drawing, with mesodermal nuclei marked in, of the
stage-21 experimental embryo of set 10 (Table 2) at mid-axial level. Note the small
cell number involved in pattern formation between the two dorsal midlines on the
left of the section, as contrasted with the right. Lateral plate mitotic indices in the two
areas were nevertheless indistinguishable, and of control level.
abnormally few cells, as seen in operatively produced Xenopus (Cooke, unpublished observations) and in the widespread occurrence of normally sized
identical twins, human and other. A cumulative increment of, say, two-fold
in the number of cells in each part of a pattern, would be achieved over weeks
of larval life by an enhancement of mitotic index undetectable at any one time.
The normal M.I. in the latest of the embryos reported here, even in a region
where pattern has been determined across many fewer cells than normal
between host and graft centres (see Fig. 3, and set 10 of Table 2), therefore
indicates only that there is no tendency even at this stage to complete the
ventral parts of disturbed patterns by any local epimorphic growth process.
We are not yet in a position to say at what point in development feedback interactions between growth and pattern size set in, or to speculate on their mechanism (i.e. whether by systemic, humoral controls, or by local intercellular interactions of the same type that initially regulate pattern itself).
178
J. COOKE
Donor cells
added, 7,
(a)
No of cells in system
at Tx and T2 both
i
normal for age of embryo ,
(b)
i
Cell number and
positional value
ABC D E F
cells
added, 7",
Zone of
intercalation
Dorsal
Space, or cell number
Ventral
Dorsal
Cell number and positional value
Fig. 4. Diagrams to show extremes of a continuum of behaviour possible for cell
positional interaction in pattern formation, (a) Pure morphallaxis, suggested to be in
operation in the system described in this paper, (b) Extreme epimorphosis. Intermediate phenomenaare imaginable, where an essentially morphallactic redistribution
of cell position values after operations, as in (a), is followed by enhanced cell division
throughout the new pattern, stimulated in some way by the steeper new gradient
of position values. The present results offer no evidence that this occurs by the
Xenopus stages studied. Time 7\ is that of operations, where donor cells (stippled
territory) of mid-dorsal (A) pattern forming value are introduced among host
presumptive mid-ventral (F) tissue. A-F represent presumptive pattern-forming
values in the dorsal-ventral dimension, i.e. a labile positional gradient of cell state
in model (a), ora fixed distribution of cell-labels in model (b). Time T2,several hours
after Tx is that at which cells pattern-forming values become irrevocable commitments that they or their descendents will differentiate as particular pattern parts.
Generation of a secondary set of such pattern values, between 7\ and Tz, is by
cellular interactions provoking changes independent of division in (a), or by specific
production of new cells with intercalated values in (b).
These data are not relevant to the question of whether cells must pass through
some particular phase of the cell cycle in order to register and react to a change
of positional information, or to pass from a pluripotential to a more highly
determined compartment in development (e.g. Holtzer, 1964, 1970, - t h e idea
of a ' quantal mitosis'). Both such strictures could apply during the development
of these supernumerary patterns after operations, since the estimated cell-cycle
time in the mesoderm is some 10 h, and a similar period elapses between operations that rearrange the cells and the subsequent determination of the patterns
of differentiation among them by the close of gastrulation. Previous work
(Cooke, 19736, c) established that if such a critical phase of the cell cycle does
exist, it is not cytokinesis or mitosis itself. Xenopus embryos developed from
pluripotential blastula stages up to highly determined tailbud stages under
complete inhibition of the latter two aspects of the cell cycle. Subsequent work
has shown that mesoderm cells in such embryos nevertheless incorporate
thymidine into nuclear DNA at appreciable rates over this period of morphogenesis, thus presumably becoming endopolyploid (B. C. Goodwin, unpublished
data). Thus, neither those data nor the present observations challenge the idea
Cell number during Xenopus pattern formation I
179
that some nuclear reprogramming or cellular transition through a part of
the replication cycle is necessary during development (e.g. Gurdon, 1969).
There is, however, no positive evidence for this, known sometimes as the
'clean gene' hypothesis, as regards the cellular transition between pluripotency
and restriction in vertebrate pattern formation, but only for that between stem
cell status and overt differentiation (Holtzer, 1964, 1970).
Of considerable interest is the appearance of typical gastrular levels of mitosis
in the obvious presumptive somite-notochord region of several newly determined axial patterns, at host stages when the original dorsal pattern parts have
for some hours been entered on their long, non-dividing phase of morphogenesis.
In fact, in such cases no cells as advanced in histogenesis as those of the host's
own dorsal midline could be found, even though any foreign cells in them (from
the donor) were even older developmentally than the host cells. An observation
such as this calls into question the idea, often implicit in discussions of development, that the schedule of intracellular (genetic) events leading to progressive
restrictions of developmental potential proceeds independently of any interactions modifying the positional information among cells, the latter interactions
simply determining which restriction compartment thecellswill enterat particular
ages or choice-points.
In the present material for instance, by virtue of the new cellular positional
interactions occurring, donor mid-dorsal cells have been delayed in their
schedule of maturation, while host presumptive ventral cells are entering the
dorsal differentiation compartments but again with a delay in schedule relative
to the normal dorsal precursor cells. There is every evidence, from cell behaviour and the series of embryos as a whole, that in such cases of delayed
morphogenesis a quite typical example of primary pattern is nevertheless forming. Each pattern compartment is determined after the lapse of a set 'physiological time' only in normal, undisturbed development.
Data in this laboratory (Steedman and Cooke, unpublished) suggest that
penetration times for thymidine and for colcemid are variable in gastrulating
amphibians after injections, probably because of differential access to mesodermal cells during morphogenetic movements. For this reason, it has not been
considered practicable to attempt the already complex task of determining the
pattern of absolute times of cell cycle phases in the mesoderm in vivo over the
period of pattern determination and axis formation. The best estimate from
the literature (Deuchar, 1958; Graham & Morgan, 1966; Flickinger et al. 1967),
by comparing present mesodermal M.I.S with endodermal, and using the assumption of other authors that mitosis itself has a relatively constant duration within
early embryos, is that Xenopus mesodermal cells from early gastrula onwards
until differentiation divide once in about 10-15 h at 18-20 °C. The apparent
variation in cycle time between different experiments in this paper might be
explained by temperature variation (Dettlaff, 1964; Chulitskaia, 1970) as well
as by individual female toad variability.
180
J. COOKE
There appears to be a significant discrepancy within the data as reported so
far. Over the 9 or 10 h of development between stages 12| and 21, as seen in
Table 2, there is something like a doubling of total mesoderm cell population.
And yet, precisely over this period, the great majority of the cells are being
removed from the cycling pool in conjunction with differentiation as notochord
of somite, having only contributed on average a doubling every 10 or more
hours, in an asynchronous manner, whilst in the cycle. The pre-chordal area of
continuing and slightly elevated mitosis is far too small to account for this, and
the tissue expansion is in any case posterior to the spreading zone of axial
differentiation rather than in front of it. A subsequent paper, in reporting exactly
how the cell complement of the mesoderm continues to be built up after the
'official close' of gastrulation, will resolve this apparent discrepancy.
I thank June Colville for expert technical work and my colleagues Susan Udin, Dennis
Summerbell, Michael Gaze and Malcolm Maden for their critical discussion during preparation of the manuscript. The work is supported by the M.R.C.
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