/. Embryol. exp. Morph. 76, 95-114 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
95
Evidence for specific feedback signals underlying
pattern control during vertebrate embryogenesis
By JONATHAN COOKE 1
From the Division of Developmental Biology, National Institute for Medical
Research, London
SUMMARY
Experiments are described in which sectors including dorsolateral mesoderm from earlyneurula-stage amphibian embryos are grafted to the mid-ventral region of gastrula-stage
hosts. The grafted tissue pursues an autonomous developmental sequence, though integrated
into the host mesodermal mantle, so that such embryos develop a ventral strip of ectopic
somite tissue, occasionally with a pronephric formation at one side. When the proportions in
which mesodermal tissue has been assigned to the four basic territories of the host's
mediolateral pattern are assayed, a significant deficit in somite is characteristically found,
though the phenomenon is variable in magnitude. It seems that the size of the host's pronephric territory may be diminished in a similar way, if an earlier differentiating ectopic
pronephros is already joined to the system. These phenomena are discussed in relation to
theories of biological pattern formation.
INTRODUCTION
Pattern formation during early development of vertebrates involves the
assignment of particular determined states to successive groups of cells within a
sheet of tissue. Cells' fates in this process are determined by their positions, in
relation to the amount of tissue available in each individual embryo and in
relation to a restricted 'organizer' region (Spemann, 1938 review; Cooke,
1972£, 1979a, 1981). Lineage is not involved in the early designation of cell fate
as it is in some other types of embryo (Laufer, Bazzicalupo & Wood, 1980). It
has been proposed that such development is controlled by a simply shaped
gradient in the level of some positional signal which is set up across the tissue,
with local levels of the signal being 'interpreted' directly by cells in effecting
developmental decisions (Wolpert, 1971). On this view, no specific interactions
are believed to occur between emerging pattern parts to control their relative
proportions. This latter control would occur automatically by the independent
positioning of frontiers between the successive parts, according to threshold
levels of the positional signal in the normal gradient profile across the original
cell sheet.
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K.
96
J. COOKE
In amphibian embryos, the medial-to-lateral pattern that characterizes the
vertebrate mesoderm becomes established over a period when this layer is essentially a cylindrical jacket, one or a very few cells thick, migrating anteriorly
between neurectoderm and yolky endodermal mass. It consists of mid-dorsal
notochord and then paired somite, pronephric and lateral plate/blood island
territories respectively, and these territories become fixed in a mediolateral
sequence (see Fig. 1) across a period extending from early in gastrulation
(notochord), probably into later neurula stages. Frontiers within the mesodermal population are positioned with considerable accuracy in normal development, in the sense that quite constant proportions of the cells are used to found
each territory (Cooke, 1981, 1982). In this paper I present evidence that the
proportion of cells devoted to the somite territory is specifically reduced in the
host's pattern when gastrulae develop after ectopic (i.e. mid-ventral) implantation of additional determined somite tissue from neurulae. The effect is concentrated in the region of the long axis lying opposite the differentiated ectopic
LPI
Fig. 1. Determination of mediolateral pattern in amphibian embryos. Schematic
cross sections of (A) midgastrular and (B) early larval stages of development are
shown. The mesodermal cell layer and the four territories it forms in the developed
pattern are stippled with decreasing density from mid-dorsal (arrowhead) to lateral
or ventral. Stippling density indicates differentiation tendencies at the early stage
where the mesoderm is essentially a monolayered cylinder; see the later territories
notochord (NC), somite (Som), pronephros (PN) and lateral plate (L PI), after the
dorsal convergence movements and induction of the overlying nervous system
(CNS). Except for notochord in the dorsal midline, however, determination of the
precise positions of frontiers between the pattern parts in the cell sheet does not
occur until later in gastrulation and neurulation. Scale bar represents 1 mm approx.
Specific feedback signals during vertebrate embryogenesis
97
pattern part, but size of the host pronephric territory can be similarly reduced by
prior, ventral differentiation of pronephros in grafted material anywhere along
the axis.
Such phenomena are not to be expected if a purely positional information
mechanism controls the pattern, and I propose a model that departs from that
idea for this particular system. This model shares with earlier ones of Rose (1957,
1970) and Braverman (1961) the proposal that substances or signals, produced
specifically within each emerging pattern part, flood the system successively to
form a record of its history and ensure that remaining uncommitted tissue
elsewhere is diverted to give other parts. The idea was proposed in outline
elsewhere because of recent data (Cooke, 1981,1982) on the pattern proportions
in mesoderms developing at artificially small size, and in those shared between
two organizing regions after Spemann dorsal lip grafts. These data are very
difficult to match with the expected performances of plausible physicochemical
systems such as might set up and regulate gradients, on the positional information hypothesis that the absolute levels of the gradient profiles attained would be
directly 'interpreted' to give the observed pattern configurations. A patterncontrol system of the type here proposed, by contrast, would explain both these
and the present quantitative results. It is also consistent with a certain statistical
feature of the pattern proportions within the whole population of embryos so far
measured in my laboratory collection, which is presented for the first time.
MATERIALS AND METHODS
The work utilized embryos oiXenopus laevis the S. African clawed frog, and
Ambystoma mexicanum the axolotl. Very different cell numbers are involved in
their primary mesoderms, their rates of development differ more than twofold,
and appreciable differences in morphogenetic movements of gastrulation may
place these amphibians almost in separate vertebrate classes. Their embryos
have nevertheless been found to be susceptible to closely comparable surgical
operations and to develop their primary patterns with indistinguishable proportions (Cooke, 1981,1982). Fertile eggs oiXenopus were obtained from spawnings induced by human chorionic gonadotrophin (Pregnyl-Organon Ltd.) injections, while those of Ambystoma were from both spontaneous and induced
ovulations in the laboratory. Routine procedures for amphibian care, dejellying
and demembranation of embryos, preparation for operations and subsequent
development have been described (Cooke, 1972a, 1979a). Operations were performed in 66% Niu-Twitty saline brought to pH7-3 with diluted HC1, and
antibiotic (Gentamycin 25 jUg/ml) was used in all solutions after demembranation. Control and experimental embryos of both species were placed at a reduced
temperature of 14 °C (normal ambient 20-21 °C) for a 24 h period beginning at
the close of gastrulation some 2-6 h after operations. The latter procedure, now
routine in the author's laboratory, has been found to optimize graft-host tissue
98
J. COOKE
integration and interaction in a variety of recombination operations on young
embryos. Operations were performed with electrolytically burnished tungsten
needles and a hair loop, with embryos held in depressions in dental wax under
the operating solution. Ionic strength was reduced to 15 % Niu-Twitty saline
once continuity of the neurectodermal layer had been restored to avoid exogastrulation. Matched sets of experimental and control (sham-operated) synchronous sibling embryos (see Results (a) section) were kept in the same dishes
and fixed and processed together.
Fixation for histological analysis was at the tailbud or early larval stage 30 in
Xenopus (Nieuwkoop & Faber, 1957) and 33/4 in Ambystoma (Schrekenberg &
Jacobson, 1975), attained 48 h and 6 days, respectively, after operations on
gastrula stages. Fixation, embedding, precisely transverse serial sectioning
(7 /im), staining with Feulgen/Light Green/Orange G, and the assay of size and
proportions of mesodermal pattern by counting of nuclei in the four pattern parts
in sample sections were all as in previous work. In the present study the rate of
sampling varied, between different sibling sets, from every fifth section in the
series to every twelfth (in the longer Ambystoma larva). Sampling always extended between the anterior limits of the pronephroi, and the posterior limits of
ectopic differentiations in experimentals (or equivalent position in controls)
which lay just anterior to the proctodaeum. The estimate of a pattern's proportions and its extent (mean total cells per transverse section) thus characteristically involved some 25 sections and 5-8000 nuclei.
The histological schedule employed allows ready assignment of cells to the
four structures that comprise the pattern. Notochord is highly characteristic with
its vacuolated differentiation, circular profile and sheath; somite shows a distinctive cell alignment with bluish nuclei and large nucleoli; pronephros shows a
tubular configuration with cuboidal epithelium. Lateral plate and blood-forming
tissue is evident from its primitive state of differentiation and position in the
lateral or ventral part of the mesodermal mantle (see Fig. 2, and the schematic
representation of structures in transverse sections of Fig. 3). In previous work
(Cooke, 1979a) estimating total cell numbers in mesoderms at earlier stages of
morphogenesis, the present sample-counting technique was coupled with use of
the Abercrombie factor for correction of errors in estimating absolute nuclear
population from sections. This was not relevant to the present work, since no
absolute estimate of cell numbers incorporated into each pattern part is made or
indeed required in order to answer the questions being posed. The requirement
is for a parameter, the percentage of the total nuclei observed to fall into each
part in a standard section series, which varies sensitively with actual proportions
in which tissue has been assigned to structures at initial pattern formation. At
tailbud stages, configurations and sizes of cells differ between the pattern parts
within embryos of each set, but care was taken only to compare, as sets, control
and experimental siblings of equal axial length, as work in the other, horizontal
plane of section has shown that mean A - P distance between nuclei in tissues is
Specific feedback signals during vertebrate embryogenes is
99
constant in such cases. The cell cycle in mesoderm between stages of pattern
determination and the tailbud larva is slow relative to development, effectively
zero in notochord and somite after determination, and not affected by operations
altering mesodermal size and polarity relations (Cooke, I979a,b). Repeat sampling (using different sections) of particular embryos in the present study
revealed the error involved, in percentages to each pattern part and mean cells
per section, to be smaller than the real differences encountered between successive sibling embryos analysed.
RESULTS
(a) The heterochronic grafting operation
Operations were designed to test the effect, upon the final proportions of
patterns still to be determined in young gastrulae, of extra tissue grafted to one
'edge' of the system from determined pattern parts of more advanced
mesoderms. The definition for the 'determined' state of embryonic material
PN
Ec Som
Fig. 2. Transverse sections of experimental Xenopus embryos with midventral
ectopic structures. Sections are from posterior trunk levels of stages 30-32. Symbols
for territories of the host pattern as for Fig. 1. Ectopic somite (Ec Som). A small
concentration of tissue with the appearance of lateral plate, but probably graftderived, often flanks the ectopic somite. Its inclusion or exclusion from the total cell
count for the 'host' pattern makes too little difference to affect the results in this
species (see text). In the examples, there has been no development of ectopic CNS
by graft ectoderm. Scale bar represents 1 mm approx.
100
J. COOKE
must always be operational, i.e. related to the precise treatment given to the cells
before they differentiate (grafting vs. explantation in vitro, disaggregation or
maceration vs. preservation of tissue structure, etc.). In the present work regions
of mesoderm are said to be determined when they differentiate according to
original fate, after heterotopic grafting as intact tissue from neurulae into
gastrulae (heterochronic grafting). In both species used, dorsolateral pieces
composed of invaginated presumptive somite from neurulae, but excluding the
visible notochord, gave rise to longitudinal strips of ectopically differentiated
somite after grafting to the midventral marginal zone of gastrulae. The operation
and its typical results are shown in Fig. 3A-D. Such autonomous behaviour was
reliably seen from donor pieces of Xenopus stages 13-14J (or Ambystoma
equivalent stages), grafted into host gastrulae of stages 101-12 with an average
host/graft difference of two stages. It is of interest that in other work (Forman
& Slack, 1980), comparable material grafted to the belly region appeared not to
express determination as somite, but the work in question involved smaller
grafts, and hosts of comparable age to the donors.
Certain cases in the present series of operations showed unambiguous
pronephric formations at one side of the midventral ectopic somite, in accord
with the location of the pronephric territory just lateral to that of somite in the
donor mesodermal mantle. Younger grafts, down to the host age, may be
assimilated to give a relatively undisturbed host pattern (no ectopic structures),
while any age disparity between tissues of more than two stages makes difficult
the matching up and fusing of graft and host marginal zones of mesodermal
recruitment (Keller, 1976; Cooke, 1979b). Such matching is required for successful integration of a strip of donor cells into the host mesodermal cylinder.
In Ambystoma, an enhancement of total mesodermal cell number in the host
pattern (seen entirely as addition to the lateral and ventral blood island territory),
as well as the presence of extra tissue as ectopic somite, was usual in successful
cases. In Xenopus the total host pattern cell numbers were not significantly less
than those of the controls. The slot cut in host gastrulae involved removal of
many fewer mesodermal cells than were then added as part of the graft with its
thickened (presomite) mesoderm, and indeed embryos cut as were the present
hosts, but then allowed to heal and develop without graft addition, show normal
cross-sectional cell numbers and pattern proportions (Cooke, in preparation).
The observation that the ectopic differentiations were additional to a normal or
even an unusually large cross sectional cell number in host patterns (Fig. 3C, D)
was therefore not unexpected.
Sham operations, performed on siblings of the experimental hosts, consisted
in removal of the midventral strip of marginal zone and its replacement with a
strip of similar origin from another gastrula (- homotopic, synchronic grafting).
This resulted in development of patterns indistinguishable from those of normal
embryos. Precisely matched synchronous sets of experimental and their shamoperated control siblings were fixed and sectioned as tailbud larvae (see
Specific feedback signals during vertebrate embryogenesis 101
CNS
CNS
NC
PN
EcSom
Ec Som
Fig. 3. The heterochronic grafting operation. (A) The operation whereby a dorsolateral sector of neurula, including neural fold (NF), presumptive somite mesoderm
(M) and the archenteron roof (AR) is grafted to a ventral site made by cutting a slot in
the ectoderm back to the marginal zone in a mid-gastrula host. Light arrow marks the
dorsal midline of the host, which is seen from the yolk-plug aspect. Heavy arrows show
how cell layers of graft and host, particularly in the blastoporal zones of mesodermal
recruitment (BP) where these layers join, are carefully matched up at the operation to
ensure development of an integrated cylindrical sheet of mesoderm. (B) Cross sectional appearance a few hours after operations such as in (A). The midventral graft has
integrated but is essentially pursuing its original, more advanced schedule of development as seen by the gathering of the mesoderm as in somite formation and, often ^ c o ordinated neural area in the ectoderm. The host is at early neural fold (Xenopus st. 13
I equivalent) stage. (C and D) Camera lucida outline cross sections of experimental
Xenopus and Ambystoma tailbud larvae at the time of pattern assay. The cellular textures of the four pattern parts, in which nuclei are sampled, are indicated schematically (cell numbers not to scale). Symbols as in Figs 1 and 2. Note the asymmetrical
development of ectopic structures (i.e. according to presumptive fate for the dorsolateral position of graft origin). The black arrowheads indicate limits of the
mesoderm which amounts to a normal cross-sectional cell population for the sibling
set, counting from the dorsal midline. It is seen that in Ambystoma there is characteristically an excess of lateral plate/blood tissue, presumably contributed by the graft.
Inset shows the (more anterior) region where the major proportion of the pronephric
tissue lies, illustrated for an Ambystoma larva. Scale bar = 1 mm approx.
102
J.
COOKE
Materials and Methods), and the proportions (% nuclei per pattern part) and
overall extents (mean nuclear number per T.S.) of their mesodermal body pat­
terns were recorded as already described. A few larvae showing ectopically
developing notochord at their graft positions (due to graft miscutting) were
excluded from the analysis since the aim was to study the effects, upon host
pattern, of tissues other than those developed from the organizer (Cooke, 1982).
In the remainder, presence or absence of ectopic differentiated somite and
pronephros was noted at each craniocaudal axial level throughout the region
examined. In Xenopus, subtotal results for anterior, middle and posterior thirds
of the total region (see Materials and Methods) were computed separately, while
in Ambystoma the region was treated as anterior and posterior halves (see Fig.
4). In both species, columns of midventral ectopic structures tended to occupy
about the posterior half of the axis between anterior host pronephros and host
proctodaeum. Ectopic somite nuclear numbers might rise to 30-50 % of typical
values for the host pattern at the levels of maximum ectopic developments.
(b) Cell numbers in pattern territories, and the proportions
between them
A total of 20 experimental Xenopus embryos were analysed with 22 matched
sibling controls, in five sets. In this species the patterns of the experimental host
embryos, as a population, were indistinguishable in size (mean nuclear number
per T.S.) from those of their sham-operated siblings. The ectopic differentiations
could thus be treated as simple additions to one 'border' (the ventral border) of
the region wherein pattern was to form, and the results expressed either as
pattern proportions or as absolute nuclear numbers seen in pattern parts within
the normal medial-to-lateral sequence of four territories in the host. Table 1
displays these results for all Xenopus matched sets so far studied. Fig. 5 shows,
in histogram form with standard errors, the absolute nuclear numbers per section
per embryo for notochord, somite and lateral plate/blood island in anterior,
middle and posterior thirds of the total body region assayed. Only pooled results
for three matched sets whose 16 control members showed statistically
homogeneous proportions and cell numbers in T. S. have been used for the latter
figure, and pronephros has been omitted because its very unequal distribution
between the thirds makes its inclusion confusing and statistically invalid.
Fig. 4. External appearances of larvae. Control (sham operated) and experimental
Xenopus (A, B) and Amby stoma (C, D) larvae are shown from the left
lateral-ventral aspect. Ear vesicle (EV), pronephros and somite outlines (PN, SOM)
and proctodaeum (BP; the original blastopore) are indicated on (A). Heavy dashed
lines indicate cross sectional levels bounding the subregions of the total region
assayed for pattern proportion in the two species (see Fig. 5 and Table 3). Ectopic
structures are revealed as a ventral ridge (with or without a neural formation) run­
ning one third to one half way along the assayed region from the zone of original
mesodermal recruitment at the proctodaeum, and sometimes ending anteriorly at an
ear vesicle. Scale bar = 1 mm approx.
Specific feedback signals during vertebrate embryogenesis
Fig. 4
104
J. COOKE
Table 1. Proportions of nuclei encountered in each territory of the host pattern,
in control (sham-operated) Xenopus embryos and in their siblings with midventral
ectopic somite. Results over the total body region assayed (see text)
% Notochord
% Somite
% Pronephros
Control
C
C
C
C
C
Exp
E
E
3-6
4-1
3-7
3-7
3-3
3-2
3-2
4-0
4-1
42-3
44-0
43-8
45-2
43-7
42-9
39-5
38-4
41-0
10-1
(Ect PN)
E
3-8
Set 2
C
C
C
C
C
C
Setl
(Ect PN)
(Ect PN)
Set 3
(Ect PN)
Set 4
(Ect PN)
Set 5
% Lateral plate/
blood tissue
12-4
11-1
10-6
10-2
44-0
42-0
40-1
40-0
42-4
43-7
40-3
11-1
7-3*
11-0
6-9*
46-2
50-3
44-9
49-0
3-3
2-9
3-0
3-4
3-1
3-1
45-6
44-4
41-8
44-6
41-0
40-2
11-4
10-2
11-2
12-3
11-9
11-1
39-7
42-5
44-0
39-7
44-0
45-6
E
E
E
E
E
E
3-7
3-9
4-0
3-7
3-3
3-7
40-7
40-5
38-2
39-8
41-8
37-8
10-3
10-3
11-6
45-3
45-3
46-2
46-6
47-0
48-2
C
C
C
C
2-9
3-9
3-1
3-5
43-4
43-0
45-7
45-3
10-1
12-2
11-5
43-6
40-9
41-5
39-7
E
E
E
E
3-9
3-9
3-3
3-7
40-1
37-6
38-5
39-7
12-6
12-0
8-0*
10-7
43-4
46-5
50-2
45-9
C
C
C
C
3-0
4-0
3-2
3-8
38-4
40-0
39-1
39-7
12-0
10-7
11-1
46-6
45-3
46-6
46-7
E
E
E
E
3-7
4-0
3-7
3-9
34-2
33-8
37-2
33-4
9-5
7-0*
11-6
10-6
52-6
55-2
47-5
52-1
C
C
3-5
3-7
39-5
38-1
9-0
9-2
48-0
49-0
E
E
3-4
3-4
33-0
32-8
9-3
12-4
54-3
51-4
9-9
9-9
7-9*
10-3
9-7
9-8
Specific feedback signals during vertebrate embryogenesis 105
80
T
70
60
I x
x 5 0 13 4 0 - 30-
20 --
10--
nnn n n n
Cont
Exp
Notochord
Cont
Exp
Somite
Cont
Exp
Lat plate
Fig. 5. Mean cell numbers per pattern part per cross section, in control and experimental Xenopus embryos. Each group of three columns gives the mean and standard
error for cell number in a pattern part in anterior, middle, and posterior subregions
of the axis assayed, going from left to right. Data from three sibling sets where 16
control members showed statistically homogeneous cell numbers in somite.
* = different from the equivalent control value, P< 01. ** = different from the
equivalent control value, P< -001.
Though variable, the diminution in proportions and absolute cell numbers
devoted to somite, in host patterns flanked with ectopic somite, is highly statistically significant (x2 and Students T ' tests on null hypothesis that controls and
experimentals are random samples from a single population with the overall
mean value, within each set). The normal mesodermal cell numbers are incorporated as a reciprocal increase in the relative extent of the lateral plate/blood
territory. The phenomenon varies at the individual level from patterns with
somite proportions within the normal range (- though never as high as the
average control of their set) to those whose deficiency in somite cell numbers is
obvious even before counting, in the regions occupied also by ectopic structures.
It can be seen from Fig. 5 that the somite deficit in Xenopus patterns is not
apparent anteriorly, but is concentrated relatively posteriorly opposite the great
bulk of the ectopic somite. Lack of suppression of somite proportion in individual cases was associated with small and/or primitively developed ectopic
somite mass, whereas cross sections of normal total cell count opposite well-
106
J. COOKE
developed ectopic somite might contain only 50-60 % as many somite cells as
their controls.
Variance for pronephric proportion is greater, in embryos generally, than that
for somite (see also Cooke, 1981). Even so, a marginally significant deficit in
pronephros is seen in the experimental Xenopus population as a whole. This
modest and variable diminution may be related to the appearance, in many
Xenopus ectopic formations, of small intermittent cell groups of pronephros-like
morphology at one junction between somite and the host pattern's lateral/
ventral edge. In five cases however (Table 1), host pronephric size was
diminished altogether below the normal range, and this was associated with the
only well-defined and anatomically coherent ectopic pronephric developments
found in the series. Host notochord size appears unaffected by the operation.
In the Ambystoma material the situation is more complex, since the integrated
host patterns in experimental embryos tend strongly to contain more cells in toto
than do the control patterns. It is particularly in this species that the donor's
dorsolateral mesodermal strip contains more cells than are removed from the
host's mesoderm to accommodate it (see Results (a)). In the absence of a cell
marker, and in view of earlier work that makes intercalary growth an unlikely
response to these operations (Cooke, 1979a), it is reasonable to assume that the
extra tissue flanking the ectopically differentiated structures is of donor origin
and has been diverted from its original fate as somite or pronephros to become
lateral plate/blood island integrated into the host pattern. We are left with the
decision as to whether to assess the effects of ectopic differentiations in terms of
the proportions (%) devoted to structures within the total cross-sectional population of the host pattern, or in terms of the actual nuclear numbers in these
structures.
Proportions are known to be regulated towards constancy against overall size
variation in normally patterned embryos (Cooke, 1981). Thus % somite might
seem an appropriate statistic since we can deduce an expected % (on the
hypothesis of no effect), as being that seen in the normal-sized control patterns
in each matched set. This has been done for all Ambystoma sets so far studied,
in Table 2. The results appear more dramatic than those for Xenopus in that no
experimental embryos achieve somite proportions quite as high as any of their
control siblings, while in many the somite territory is under-represented quite
below the normal range of variation.
A more conservative and reliable indication of specific inhibitory effects of
ectopic structures, however, is the observation of actual reductions of cell numbers in the appropriate parts of host patterns. Mean nuclear numbers per cross
section in the pattern parts are given, for anterior and posterior halves of the
region scored, in Table 3. Absolute reduction in somite is seen in several embryos but there are also embryos where somite cell numbers are indistinguishable
from normal, though embedded in a pattern whose overall size has been enhanced as described. It is impossible to be sure whether such embryos are evidence
Specific feedback signals during vertebrate embryogenesis 107
Table 2. Proportions of nuclei encountered in each territory of the host pattern,
in control (sham-operated) Ambystoma embryos and in their siblings with midventral ectopic somite. Results over the total body region (see text)
% Notochord
% Somite
% Pronephros
% Lateral plate/
blood tissue
Control
C
C
3-6
3-2
4-1
56-5
54-0
53-1
8-9
6-9
8-3
31-0
35-9
34-4
Exp.
E
3-6
4-2
42-2
45-2
9-8
7-7
44-4
42-9
Set 2
C
C
C
6-1
4-2
5-6
53-6
49-1
51-3
5-9
5-6
7-6
34-4
41-1
35-5
(Ect PN)
(Ect PN)
E
E
E
6-7
4-9
4-0
38-6
38-6
43-0
5-6
5-3
7-3
42-3
51-2
45-7
Set 3
C
C
3-0
3-2
57-2
55-1
5-1
5-0
34-7
36-2
E
E
3-5
3-0
40-4
34-6
5-1
3-3*
51-0
59-1
Set 4
C
C
C
C
5-4
6-4
6-2
6-7
51-6
52-2
52-3
50-5
7-0
5-7
7-3
6-7
36-0
35-7
34-2
36-1
(Ect PN)
E
5-5
41-0
4-0*
49-5
Setl
(Ect PN)
for inhibitory effects or not, since we do not really know what their 'expected'
somite cell numbers would be. As with the Xenopus series of material, however,
the most striking cases of somite under-representation are correlated with the
greatest masses of ectopically differentiating somite, while marginal cases are
associated with the appearance that much of the implant was unable to maintain
its developmental tendency and was instead assimilated into the host pattern as
lateral plate/blood forming tissue.
In both species, the ectopic midventral somite formations of experimental
embryos are only sometimes accompanied by neural differentiation of the overlying ectoderm and its formation into a tube. Several examples of pronounced
host somite suppression were associated with absence of any ectopic neural
formations, but such suppression was always accompanied by a well-formed
ectopic somite mass. Fig. 2 shows examples of such experimental embryos for
Xenopus.
The observation of a well-formed ectopic pronephros in Ambystoma is
108
J. COOKE
--+0-3
•t
•
#• O
O
tO
0
O
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Fig. 6. The independence of variations in proportion between somite and pronephros. Within each subset of matched synchronous siblings (i.e. all controls or all
those with patterns experimentally influenced by heterochronic grafting), the means
of % nuclei scored in somite and in pronephros were computed, and the individuals
inserted in the scatter plot according to the relative deviations from these means
(expressed as decimal fractions of the mean % value) seen in their own patterns.
Black spots; control (sham-operated or unoperated) embryos. Open circles; host
patterns of embryos which also have ectopic structures. The resulting scatter shows
no significant correlation, positive or negative, between embryos deviations from
their sample mean for these two proportions. Ordinate; relative deviation in
pronephros proportion. Abscissa; relative deviation in somite proportion.
Insert represents (a) a particular 'ideal' or 'normal' gradient profile for a signal,
controlled from one end of a system, which might be interpreted to give pattern, and
(b), (c) different families of variant profiles (shown in extreme form for clarity) that
might result from imperfect replicability of the gradient mechanism among individuals. Curves of the family (b) differ from the 'ideal' in steepness, while those of
family (c) differ in degree of inflection, or non-linearity. The marks on the ordinate
represent signal levels which could be taken to establish frontiers of two successive
'internal' pattern parts such as somite and pronephros in the present case. Reference
to these marks and to the families of curves shows that on a positional gradient
hypothesis for overall pattern control, a component of correlation (positive or conceivably inverse) would be expected, between deviations from the mean in proportions devoted to these pattern elements in individual embryos.
Specific feedback signals during vertebrate embryogenesis 109
Table 3. Mean total cell numbers per section in the host pattern, and numbers in
somite and pronephros, in control and experimental Ambystoma embryos.
Results for anterior and posterior halves of the body region assayed
Anterior; Cell Nos./Sect
Total
Som.
PN
212
220
208
224
218
152
125
127
117
116
231
210
20
18
26
208
231
208
247
189
217
124
117
120
130
86
119
173
194
183
217
188
231
19
20
191
214
121
128
188
205
E
E
96
108
70
68
21
15
223
228
108
109
216
232
Set 4
UUUU
Setl
80
87
76
87
25
19
25
20
214
211
209
200
134
125
129
103
205
200
194
178
(Ect PN)
E
70
16
228
110
213
Control
Set 2
nnn
C
C
Exp
E
(Ect PN)
(Ect PN)
www
Set 3
c
c
(Ect PN)
96
103
93
32
24
25
77
85
39
23
85
94
83
82
61
76
18
19
24
Posterior;Cell Nos. /Sect
Som.
Total
220
196
200
associated with a reduction in size of the host's in situ pronephros below the
normal range of variation in two cases, but not in another two. Another feature
of this more restricted set of results, as compared with the series from Xenopus,
is that the diminution in somite cell numbers can be present throughout the axial
region examined, including anteriormost (earliest differentiated) regions, as well
as opposite the somite from implants.
From our current understanding of the normal fate map of Xenopus development (Keller, 1976) it appears that tail somites could derive from precursor cells
situated in the mid ventral marginal zone of the young gastrula. On this view, the
contention that the present phenomena are true effects on pattern formation is
subject to the conceivable criticism that the grafts have mechanically prevented
the migration of specific cell populations into the dorsal axis in the more posterior
regions assayed, and trapped these cells instead to contribute to ectopic ventral
somite. In matched sets of Xenopus and of Axolotl, where several experimental
members showed marked somite diminution, the total cell populations
110
J. COOKE
were compared in the tailbud mesoderms, posterior to the proctodaeum and thus
to the region occupied by ectopic structures. This population is in fact very
largely of somite and presomite cells, and was found to be indistinguishable in
experimentals and controls. Tail regions of experimental embryos show no tendency to be deficient to outer inspection. It is thus highly unlikely that the effect
of midventral fragments is caused by any specific blockade to gastrulation movements, or to any selective aggregation of host presumptive somite cells into the
graft masses.
DISCUSSION
The two lateral parts of the pattern being assayed, namely pronephros and
lateral plate, continue with cell division at (possibly specific) cycle times of some
10-15 h, after their initial histogenesis. Their slow growth between foundation
and assay of the pattern could therefore complicate the interpretation. But
notochord is without cell division and, up to the tailbud stages here assayed, the
myotomal bodies of somites which alone were included in the counts show a very
low mitotic rate in urodeles while in Xenopus a mitotic figure has never been
observed. Thus for somite, the principal pattern part being studied in the
presence of grafted homologous tissue elsewhere in the embryo, the nuclear
numbers scored reflect directly the size of the cell population originally set aside
in pattern formation. Furthermore in the Xenopus series the overall cell population of hosts' patterns is of normal size at the time of assay, with the somite deficit
balanced by an excess of lateral plate. The strong presumption is thus that there
has been an alteration in the initial proportions in which the mesodermal cell
population has been allocated to found the pattern parts.
It is hard to avoid concluding from these results that tissue which is relatively
advanced, in its determination as particular territories of the embryonic pattern,
emits signals that can alter the balance of determinations elsewhere in the embryo. Specifically, it appears that such signals from implanted tissue can cause
diversion of younger cells from the homologous pathways of determination into
those proper to more lateral regions of the embryo, since the positions of certain
frontiers between pattern parts in the original mesodermal mantle are shifted
medially. Pattern determination probably proceeds from medial to lateral across
some considerable time, whose precise limits in relation to the progress of gastrulation are unknown, while the specific inhibitory signals whose existence is indicated by these results may each have a short time course in relation to development. Thus the inconstancy and variability of the observed effect on somite is not
surprising, in view of the variability in precise graft and host ages and age differences, among the operations performed to date. Although few in number, the
examples of apparent diminution of pronephric size by prior ectopic pronephros
differentiation are striking, especially as the ectopic territory lies well posterior
to the site of the host organ as well as in a ventral position. We do not understand
Specific feedback signals during vertebrate embryogenesis 111
enough about differences between gastrulation and the timing of determination,
in Xenopus and Ambystoma, to speculate on why in the latter case even the
earliest differentiating somite can be diminished by ectopic differentiations
which end up posteriorly, whereas in the former, somite inhibitory effects seem
confined to particular axial levels.
The theory that positional information underlies pattern control, though abstract, is precise and makes certain potentially testable postulates. Some of these
concern the behaviour of pattern proportions at abnormally small field sizes and
under conditions of artificial bipolarity, in relation to the expected performances
of plausible physicochemical systems that might underlie the signal profiles
which are interpreted to give the patterns. Work of this type, whose results were
problematical for almost all gradient models, has been presented elsewhere
(Cooke, 1981, 1982). A further, strong postulate of the positional information
idea, as stated in the introduction, is that no specific interactions between emerging pattern parts are to be expected in the control of their proportions. Proportioning is achieved instead from interpretation of local signal levels along the
simple but regulated gradient profile between boundary values, so that frontiers
between cell groups progressing to different determinations are independently
positioned. The ectopic addition of advanced tissue belonging to some element
of the normal pattern should be without effect on the positions of such frontiers
elsewhere in the embryo, and thus should not affect the relative sizes of elements
in the host pattern.
The material of the grafts used in the present work does not represent the
organizing boundary for the pattern, since no notochord differentiated, and the
visible territory for this structure was in fact left in the donors. The boundary is
in fact represented by the dorsal blastoporsal lip, essentially the presumptive
notochord territory, transplanted in previous work (Spemann & Mangold, see
Spemann, 1938; Cooke, 1912a,b, 1979). The developmental stage of the present
grafts is such that they differentiate relatively autonomously and do not involve
the surrounding host material in a new edition of pattern centred on themselves.
They do affect the proportions of homologous pattern parts elsewhere, however,
in contravention of the expectations from the positional information hypothesis.
Together with the previous work, these results make it unlikely that positional
information, sensu stricto, underlies control of the mediolateral dimension of the
vertebrate body plan. There follows the outline of a model mechanism which
would accommodate the data. It is related to previous ideas of Rose (1957,1970;
see also Braverman, 1961) and, recently, of Meinhardt & Gierer (1980), though
differing in particulars from these. It is also perhaps a reversion towards the
original conception by Child (1941) of physiological gradients and dominance
hierarchies controlling morphogenesis.
The region of the embryo that will become the dorsal blastoporal lip and the
anterior medial mesoderm and endoderm is certainly an organizing boundary,
which controls the orientation with which the mesodermal body plan develops.
112
J. COOKE
It is the only region to give any evidence of autonomous boundary properties
after grafting operations in these embryos, being the organizer of Spemann
(review, Spemann, 1938). In the present model the organizer is assumed to
control only the overall field polarity, expressed as graded rates of physiological
progress towards determination by mesodermal cells, smoothly ranked from
medial or dorsal (i.e. in contact with the organizer) to presumptive lateral or
ventral (i.e. far from the organizer) extremes of the tissue. Such control from a
local region could be via a morphogen signal gradient declining smoothly with
distance from the boundary source, and setting local rates of cell development
by its level. Alternatively it could be by a spreading wave-like process of activation that was necessary to begin some schedule of cellular maturation. Such an
'activation wave' would produce a subsequent wavefront with respect to cells'
arrival at particular states of maturity, passing coherently across the mesoderm.
In the model now to be proposed, neither sort of control process would have any
further exact informational function, such as would have been required of a
regulating gradient used as true positional information. Either a primitive and
plausible diffusion-controlled mechanism or one involving spreading autocatalytic activation would be adequate to ensure the direction and continuity of such
a wavefront of development, and thus the reliable temporal order in which onset
of determination occurs within the tissue space. Normal spatial ordering and
proportional extent of determined territories are then assumed to result from (a)
the logic governing access to various determined states by cells and (b) the
history of the system as development proceeds. All cells that can develop fast
enough, mature towards one particular state, i.e. notochord determination. This
continues until a diffusible signal, produced specifically by cells that have
entered that particular committed state, builds up to a systemic level that
prohibits subsequently developing cells (i.e. those 'slower' because more lateral
in position) from developing further along the same pathway. The logic is such
that these cells are diverted towards a next available state, i.e. somite determination. But somite as a committed cell state involves production of a further diffusible systemic signal, which finally diverts even less-advanced cells towards a third
available state (pronephros), and so on. Each specific signal is sufficiently 'diffusible' within the mesoderm for its level to act effectively as a sensor for the
proportion of the individual embryo that has become devoted to the cell type that
produces it. The mediolateral wavefront of determination is thus serially diverted
in character during its passage across the tissue when particular proportions have
been reached for each territory.
The scaling down of pattern in small mesoderms and in those being simultaneously invaded by wavefronts from two dorsal organizing boundaries is an
automatic consequence of the mechanism, because of diversion of the character
of the progressing determination whenever a particular overall proportion has
been achieved, in the whole mesoderm, of cells producing each successive inhibitor. The negative feedback on sizes of specific territories from prior ectopic
Specific feedback signals during vertebrate embryogenesis 113
differentiations, reported in this paper, would be due to a priming or preempting effect when the system encounters appreciable concentrations of a
specific signal already supplied from elsewhere. The creation of the frontier
between pattern parts, corresponding to diversion of determination from this
particular character to the one lateral to it, will occur precociously and thus be
shifted medially to diminish the relative size of the pattern part.
The inset in Fig. 6 shows a typical gradient profile (a) such as could be produced
by reaction diffusion or source-diffusion controlled at one boundary in a tissue,
and its family of variants (dotted curves b and c) such as might result from chance
fluctuations or imperfections in its regulatory mechanisms. Signal levels are
shown that might correspond in a positional information model to interpretation
thresholds for two 'internal' territories, such as the somite and pronephros
studied here. It can be seen how such a model predicts that a significant component of the irreducible variance in pattern proportions, among individual embryos, should take the form of positive correlations (or conceivably, for curves c,
inverse correlations) between the proportions devoted to these internal territories, with lateral plate always occupying the 'left-over' tissue. The scatter
diagram of Fig. 6 represents the deviations from mean proportion for somite
plotted against deviations from mean proportion for pronephros, in the patterns
of all normal-sized Xenopus and Ambystoma mesoderms ever assayed in this
laboratory. Deviations are estimated on the means within all control embryos of
each species and within all embryos where pattern had been influenced by ectopic
differentiation. A complete lack of correlation is seen between the intrinsic variations in proportion of each element, suggesting that in each embryo proportion
assigned to somite is independent of, and has no influence upon, proportion
assigned to pronephros. Such an observation is consistent with the idea that, as
in the present serial diversion model, each pattern element is controlled by an
independent size-assessment event in the mesoderm {via a diffusible inhibitor
and threshold arrangement) that is subject to its own performance variation.
There is a tempting analogy between the concept of serial diversion signals
controlling initial pattern proportions, and that of 'chalones' (Bullough, 1967),
the systemic feedback signals known to regulate the functional size of various
differentiated tissue systems in the later body. But at the time of pattern control
the embryo has no vascular system. Furthermore, what have been referred to
here as territories of a primitive pattern are not states of differentiation in the
histologist's or physiologist's sense, but states of potency restriction as to the
parts of the body to which cells' descendants can contribute. The postulated
signals are thus more allied to traditional morphogens than to hormones or
chalones. One demanding feature of the present model is the rapidity with which
specific signals appear to be able to spread through tissue, in relation to past
calculations about diffusion-controlled changes (see Crick, 1970). Empirical
study of the effective diffusion rates of marked molecules of known charge and
size through embryonic cell sheets is urgently required.
114
J. COOKE
I thank Jonathan Slack and Malcolm Maden for batches of Ambystoma embryos, and am
indebted to John Webber, Malcolm Maden and Fiona Harvey for critical discussions of the
work, June Colville for skilled histology and Fay Morris for typing the manuscript.
REFERENCES
BULLOUGH, W. S. (1967). The Evolution of Differentiation.
BRAVERMAN, M. H. (1961). Regional specificity of inhibition
London: Academic Press.
within chick brain. /. Morphol.
108, 163-285.
C. M. (1941). Patterns and Problems of Development. Chicago: University of Chicago
Press.
COOKE, J. (1972a). Properties of the primary organisation field in the embryo of Xenopus
laevis. I. Autonomy of cell behaviour at the site of initial organiser formation. /. Embryol.
exp. Morph. 28, 13-26.
COOKE, J. {1912b). Properties of the primary organisation field in the embryo of Xenopus
laevis. II. Positional information for axial organisation in embryos with two head organisers.
J. Embryol. exp. Morph. 28, 27-46.
COOKE, J. (1975). Control of somite number during development of a vertebrate, Xenopus
laevis. Nature 254, 196-199.
COOKE, J. (1979a). 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
organisers. J. Embryol. exp. Morph. 51, 165-182.
COOKE, J. (1979b). Cell number in relation to primary pattern formation in the embryo of
Xenopus laevis. II. Sequential cell recruitment, and control of the cell cycle during
mesoderm formation. /. Embryol. exp. Morph. 53, 269-289.
COOKE, J. (1981). Scale of body pattern adjusts to available cell number in amphibian embryos. Nature 210, 775-778.
COOKE, J. (1982). The relation between scale and the completeness of pattern in vertebrate
embryogenesis; models and experiments. Amer. Zool. 22, 91-104.
CRICK, F. H. C. (1970). Diffusion in embryogenesis. Nature 225, 420-422.
FORMAN, L. & SLACK, J. M. W. (1980). Determination and cellular commitment in the embryonic amphibian mesoderm. Nature 286, 482-484.
KELLER, R. E. (1976). Dye mapping of the gastrula and neurula oiXenopus laevis. II. Prospective areas and morphogenetic movements of the deep layer. Devi Biol. 51, 118-137.
LAUFER, J., BAZZICALUPO, P. & WOOD, W. B. (1980). Segregation of developmental potential
in early embryos of Caenorhabditis elegans. Cell 18, 569-577.
MEINHARDT, H. & GIERER, A. (1980). Generation and regeneration of sequence of structures
during morphogenesis. J. theoret. Biol. 85, 429-450.
NIEUWKOOP, P. D. & FABER, J. (1957). Normal Table o/Xenopus laevis. (Daudin). Amsterdam: Elsevier, North Holland.
ROSE, S. M. (1957). Cellular interaction during differentiation. Biol. Rev. 32, 351-382.
ROSE, S. M. (1970). Differentiation during regeneration caused by migration of repressors in
bioelectric fields. Amer. Zool. 10, 91-99.
SCHREKENBERG, G. M. & JACOBSON, A. G. (1975). Normal stages of development of the
Axolotl, Ambystoma mexicanum. Devi Biol. 42, 391-400.
SPEMANN, H. (1938). Embryonic Development and Induction. Reprinted 1967, Hafner, N.Y.:
Yale University Press.
WOLPERT, L. (1971). Positional information and pattern formation. Current Topics in Devi
Biol. 6, 183-224.
CHILD,
(Accepted 17 March 1983)