J.\Embryol. exp. Morph. Vol. 51, pp. 21-50, 1979
27
Printed in Great Britain @? Company of Biologists Limited 1979
Somitogenesis in amphibian embryos
I. Experimental evidence for an interaction between two
temporal factors in the specification of somite pattern
By MURRAY PEARSON 1 AND TOM ELSDALE 2
From the MRC Unit of Clinical and Population Cytogenetics,
Western General Hospital, Edinburgh
SUMMARY
Somitogenesis is described in two species of anuran amphibians, Xenopus laevis and Rana
temporaria, in which the cellular mechanics of somite formation are distinctly different. Heat
shocks are employed to demonstrate a wave of cellular change which precedes somite formation down the body axis. This prior wave is shown to be kinematic. It is not a propagated
wave. It is a consequence of the temporal activities of the cells laid out in space, but there is
no evidence that these activities depend upon an interpretation of their position.
Heat shocks cause characteristic segmental abnormalities over a zone of somites which is
formed several hours after the shock. Evidence from double heat shock experiments suggests
that the pattern of abnormality is the result of (i) a disturbance of co-ordination between
pre-somitic cells, and (ii) the time available to those cells for recovery before they are recruited
into a segmental pre-pattern at the time of passage of the prior wave. It is a temporal coordination that is disturbed and subsequently recovered following a heat shock. This temporal
co-ordination of pre-somitic cells does not depend upon position along the axis.
The evidence for two physiologically independent temporal patterns of cellular processes,
which interact to specify the segmental pattern of somites (their size, shape and number),
gives experimental support for the theoretical account of somitogenesis proposed by Cooke &
Zeeman (1976).
INTRODUCTION
Segmentation is characteristic of the development of many animal forms and
a basic principle of their morphogenesis. Somitogenesis in the vertebrate embryo
is a striking example. The various aspects of somitogenesis in amphibians have
been discussed by Deuchar & Burgess (1967) and Cooke & Zeeman (1976): the
craniocaudal sequence; the constancy of the pattern within a species; the coordination with the whole body pattern; the precision and versatility of the
control mechanisms that regulate the number and size of somites, not only
under normal conditions, but also abnormal regimes of ploidy (Hamilton, 1969)
and experimental manipulation (Cooke, 1975).
In our approach to the problem of somitogenesis we deal first with temporal
1
Author's address: 30 Albion Hill, Brighton, Sussex, U.K.
Author's address: MRC Unit of Clinical and Population Cytogenetics, Western General
Hospital, Crewe Road, Edinburgh, EH4 2XU, Scotland.
2
28
M. PEARSON AND T. ELSDALE
aspects of the process because only by doing so shall we gain insight into the
essential continuity of the whole process as well as the mechanism of discontinuity between somites. Segmentation begins just behind the head and
sweeps down the axis to the end of the tail; we refer to this as a wave of somite
formation. (When we refer to a wavefront we mean the movement of the
frontier, for instance in this case the frontier between segmented and not yet
segmented tissue.) In temporal terms, the problem of the specification of
segmental pattern is to understand how the period taken by the wave of somite
formation to traverse the axis is specified, and how it is partitioned into just so
many of the shorter intervals between the formation of one somite and the next.
We have previously demonstrated (Elsdale, Pearson & Whitehead, 1976) how
we can identify a crucial change in the properties of the pre-somitic tissue
occurring some hours before participation in somite formation. The basic
observation was that heat shocks given just before or during somitogenesis lead
to abnormalities in the segmental pattern. Heat shock does not disturb already
formed somites, and for a while afterwards new somites continue to form as if
nothing had happened. After a standard number of normal somites have been
made, however, abnormal somites begin to appear. Abnormal segmentation
continues for a longer or shorter time depending on the severity and duration
of the shock; eventually there is a return to normal segmentation. The anterior
part of the abnormal zone is the most disturbed; and the last normal somite is
immediately followed by maximally disturbed tissue to give a sharp anterior
boundary. Across this boundary, tissue immune to heat shock lies immediately
adjacent to tissue maximally disturbed. This means that tissue disturbable by
heat shock earlier in its maturation suddenly becomes insensitive in the run up
to somite formation. Abnormal zones commencing further and further caudally
are produced by shocks to progressively older embryos, from which it follows
that the frontier between immune and disturbable tissue moves as a wavefront
down the axis ahead of visible segmentation. The wave of somite formation is
preceded by a prior wave of change which confers immunity to heat shock.
It is not clear, however, whether the normal segmental pattern is specified at
this time of acquisition by cells of an immunity to heat shocks, or whether cells
then merely become insensitive to any disruption of a segmental prepattern
which has already been set. In this paper we present observations and experiments to show that the prior wave of cellular change is concomitant with segmental specification. Several observations support the inference that although
heat shock is used to demonstrate the existence of the prior wave, this wave
itself is not directly affected by heat shock. There is another component in the
specification of segmental pattern.
The demonstration of another component which is sensitive to heat shock
comes from analysis of the abnormal zone following a single or two successive
shocks, and leads to the conclusion that heat shock disturbs a co-ordination of
the paraxial mesoderm cells established before the arrival of the wave.
Somitogenesis in amphibian embryos. I
29
The results are interpreted according to a scheme for the specification of the
somite pattern which involves the coupling of both components, a wave of
cellular change by which cells are committed to subsequent somitogenesis, and
a co-ordinated periodicity, in the establishment of a segmental prepattern: there
is a wave of somite determination. Finally a model coupling suggested by Cooke
& Zeeman (1976) is discussed.
MATERIALS AND METHODS
Rearing
Xenopus embryos came from the Genetics Department, University of Edinburgh, or from Dr C. Ford, School of Biological Sciences, University of Sussex,
from spawnings induced by the injection of chorionic gonadotrophin. Embryos
were reared in dechlorinated tapwater, sorted into batches of the same stage
during blastula, and again at gastrula stages. Synchronous groups were reared
at room temperature or controlled temperatures. Observations on somites were
carried out after stripping the epidermis (Elsdale et al. 1976).
Clutches of Rana embryos were collected from natural habitats in Perthshire.
Synchronous batches were reared in pondwater.
Heat shocks
After removal of their outer jelly coats, embryos were pipetted into bottles
containing 150 ml of water and standing in a 37 °C water bath. After the
measured period, usually 15 min for Xenopus and 8 min for Rana, they were
pipetted back into an excess of water at room temperature or some other
controlled temperature.
Microscopy
Material fixed in Smith's or Bouin's fixative was dehydrated, embedded in
paraffin wax and sectioned at 5-6 ^m. The sections were lightly stained with
haemalum for histological examination.
For scanning electron microscopy, specimens were prepared by the method
described by Bard, Hay & Mellor (1975) and examined in a Cambridge Stereoscan SI 80.
Bisection o/Rana embryos
Embryos were cut in two with tungsten needles in agar dishes containing a
solution of 65 % Dulbecco's medium. After 1 h the fragments were cleaned of
dead cells and the medium drawn off to be replaced by a 5 % Dulbecco solution.
Anterior halves were stripped for scoring at a relatively early stage; the tail
halves were cultured longer, until segmentation in controls was complete, and
then fixed and stripped.
3
EMB 51
30
M. PEARSON AND T. ELSDALE
RESULTS
1. Normal somitogenesis
Somite formation in Xenopus is shown in Fig. 1. Paraxial mesoderm cells
first elongate perpendicular to the notochord; then a bundle of the most anterior
cells turns through 90° to give a somite, with myoblasts now lying parallel to
the notochord. After several hours these myoblasts differentiate as uninucleate
muscle fibres stretching from end to end of the somite. The process has been
fully described by Hamilton (1969).
In Rana the mechanics of somite formation are different. The somites form
before cell elongation, and in a manner comparable to many other vertebrates
where each somite forms initially as a rosette (Fig. 3). The adhesion between
cells of a segmenting somite confers a roughly radial organization on the group,
and de-adhesion from the non-segmenting cells posteriorly creates an intersomitic fissure (Fig. 2). Later, myoblasts elongate within the somite and fuse to
give multinucleate muscle fibres (Figs. 4 and 5).
Counts for the total number of somites formed in Rana vary from 39 to 45,
but it is unclear how great is the genuine variation between embryos because
boundaries between the small, last formed somites are very difficult to see once
myoblasts fuse, and yet before fusion one cannot be certain that the final somite
has formed. In Xenopus some 46 somites form to the end of the tail proper;
thereafter very small somites are added to the curious tail process which grows
throughout larval life.
F I G U R E S 1-5
Fig. 1. Somite formation at ca. 26-somite stage in Xenopus laevis. A horizontal
section showing each segmental group of cells turning through 90°. At this level, each
somite (23-26) comprises 4-5 myotomal cells in width, ps, Pre-somitic mesoderm;
n, notochord; nt, neural tube. Anterior is to the right.
Fig. 2. Somite formation in Rana temporaria. A horizontal section through 15somite embryo. Caudally (left) pre-somitic cells are unsegmented. Further forward,
somites have been formed by rounding up of the segmenting cells to present in
section a vague rosette profile. Somites at bottom show this better than at top, the
latter are cut slightly oblique, nt, Neural tube; ps, pre-somitic mesoderm; 12-15,
ordinal somite number, counted antero-posteriorly from first post-otic somite.
Fig. 3. Scanning electron micrograph of Rana neurula showing a partially formed
segmental furrow. Unlike Xenopus cells at the segmenting stage are not elongated.
Segmentation begins with a furrow dorsally (arrow).
Fig. 4. Rana. A horizontal section through a 15-somite embryo showing the
anterior first three somites. This is the same embryo as in Fig. 2, but anteriorly at
this stage the myoblasts have elongated and fused to give multinucleate cells which
differentiate as muscle fibres, n, Notochord; v, otic vesicle.
Fig. 5. Scanning electron micrograph of Rana embryo showing elongation and
fusion of myoblasts. Elongation begins six or seven somites anterior to the most
recently formed, and fusion in somites more than ten anterior to the most recent.
Such fusion is evident in the most anterior somites {ant) shown here.
31
Somitogenesis in amphibian embryos. I
Figs. 1-5. For legends see facing page.
3-2
32
M. PEARSON AND T. ELSDALE
Xenopus: rate of somite formation
44 -|
(a)
40 •
36
32
28
I 20
.16
12
10
20
30
40
50
60
Time (h)
70
80
90
100
Fig. 6a. For legend see facing page.
In spite of the different methods of forming somites, in both species the
effects of heat shocks on the pattern of somitogenesis are very similar.
2. Characterization of the prior wave
(a) The course of the prior wave in relation to the wave of somite formation
A graph of somite formation against time is simply constructed from counts
of the number of somites in embryos sampled successively from a synchronous
batch (Figs. 6 and 7). In both species the first several somites form in rapid
succession, thereafter a slower rate of formation is constant to the end of the
measuring period, to the 37-somite stage in Rana, the 46-somite stage in Xenopus.
Hamilton (1969) computed just such a linear relation (r = 0-995) for Xenopus
from the data in Nieuwkoop & Faber's (1956) Normal Table.
Comparable data on the prior wave of cellular change derive from counts of
the total number of normal somites anterior to the abnormal zone developed in
Somitogenesis in amphibian embryos. I
33
Xenopus
32 -
Stages N/F 12
13
14 15 17 18 19 20
10
15
20
25
30
40
50
60
70
80
Hours at 15 °C
Fig. 6. (a) The rate of somite formation in Xenopus laevis at different temperatures.
After the first five or six somites, all subsequent somites form at a constant rate at
a given temperature. For both 16 °C and 20 °C, different symbols indicate different
synchronous batches of embryos. For one batch at either temperature, the range of
each somite count is shown by the vertical bar. For each count, the mean was taken
of at least five embryos (ten files); the twofilesof any one embryo are not exactly
synchronous, (b) The rate of somite formation at 15 °C ( • ) is plotted together
with the segmental rate of progress of the prior wave (#). The position of the prior
wavefront at any time is given by the number of normal somites before the abnormal
zone caused by heat shock at the time concerned. For embryos shocked before
appearance of the first somite, stages according to Nieuwkoop & Faber (1956)
were calibrated to the same time scale for 15 CC. The segmental rate of progress of
this prior wave shown by heat shock roughly fits a straight line.
embryos following a heat shock at a known time or stage. When the course of
the prior wave is thus plotted as presumptive somites against time, this course is
found to be linear from the beginning, and runs parallel to the linear part of
the curve for somite formation. The segmental rate of progress of the prior wave
is constant with time (Figs. 6 and 7).
The separation between the two waves can be read from the graph. It is the
difference between the number of somites present at the time of shock and the
number subsequently counted anterior to the abnormal zone; that is, the
number of normal somites formed following shock. The separation is ca. five
somites in Xenopus and between three and four somites in Rana, except for the
34
M. PEARSON AND T. ELSDALE
Rana
40 T
36
15 °C
32 •
* 24 •
P
14°C
5
£ 20
IJ6
12 •
4A
L /
J
10
20
30
40
50
Time (h)
60
70
80
90
Fig. 7. The rate of somite formation in Rana temporaria is also constant at different
temperatures ( # , • , A) after the first four somites. Vertical bars indicate the
range of counts; for each count the mean was taken of at least five embryos (ten
files). The broken line (— O —) plots the segmental rate of progress of the prior
wave in the same batch of embryos at 18 °C in which somite formation is also
plotted. The rate is constant throughout. The graph shows the 'lag' following the
prior wave before any given somite is formed.
first few somites. The constancy of the relation between the two waves after
these first few somites suggests the prior wave may be fundamental to the pattern
of somites, and somite formation a secondary consequence. In such case, the
rapid appearance of the first few somites would reflect their delayed formation
after the passage of the prior wave.
(b) The rate of recruitment of pre-somitic cells into somite formation and the
prior wave
The first 10-15 somites are of roughly equal size, after which they get smaller
in both species. This is not solely due to a decrease in cell size; fewer cells are
recruited into more caudal somites. To measure this decreasing rate of recruit-
Somitogenesis in amphibian embryos. I
35
I!
i
2
i
4
i
6
i
i
i
i
i
I
i
i
i
i
i
i
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
Somite number (n)
Fig. 8. This figure plots the number of cells per somite width against ordinal
numbers of the somites in Xenopus. The average value for each ordinal somite (A)
is obtained from a number of counts from different embryos in horizontal section
at the level of the notochord. Only counts which were unambiguous are included.
The inset diagram shows how the count of x cells is related to the mode of somite
formation, and to the length of pre-somitic material included in a somite.
Three series of embryos are represented, (i) Normal embryos given by ( # ) with
average counts for somites 1-42. Vertical bars show the range of such counts for
each ordinal somite, (ii) Embryos given a heat shock at the 13-somite stage which
results in an abnormal zone whose average length extends from somite 16 to 20. Counts
for somite width are given for these embryos posterior to the abnormal zone (A)(iii) A similar series shocked at the 17-somite stage whose abnormal zones extend
an average length from somite 20 to 24. Counts for somite widths posterior to this
abnormality are given by (O)For normal embryos ( # ) each average count is the mean of from 4 to 15 different
counts. Up to somite 26,10 or more counts contribute to this mean, smaller numbers
of counts in more posterior somites. All counts were made before myoblast differentiation. For heat shocked embryos no ranges are given, since no count varied
more than in normal controls; each average is the mean of two to eight different
counts.
Since the segmental rate of progress of the prior wave is a constant, the ordinal
somite number is a measure of time, and the curve describes the decreasing rate of
recruitment of pre-somitic cells along the axis into this prior wave, the curve of
velocity against time. This velocity is no different behind an abnormal zone following heat shock compared with the same position in normal embryos, since
somite width is the same for any ordinal somite.
raent, advantage is taken of the peculiar mode of somite formation in Xenopus.
In this species, the number of cells counted across a somite in horizontal section
gives the number of pre-somitic cells along the craniocaudal axis recruited into
that somite (see Fig. 8). We thus obtain in Fig. 8 a quantitative estimate of the
length of pre-somitic material along the axis incorporated into each somite.
Because the segmental rate of passage of the prior wave (number of presumptive
somites per unit time) is constant, this same plot of cells per somite width
against ordinal sequence of somites can be translated as the rate of recruitment
of cells by this wavefront against time. The only assumption here is that cell
proliferation in the interval between the prior wave and somite formation is
36
M. PEARSON AND T. ELSDALE
280
240
200
jj 140
,O
80
40
10
14
20
24
Somite number
30
34
40 42
Fig. 9. The plot of total number of pre-somitic cells along the axis incorporated into
somites against ordinal somite number. The total number of pre-somitic cells along
the axis incorporated into somites up to somite n is simply the sum of the average
somite widths of somites 1 to n. As in Fig. 8, ordinal somite number is a measure of
developmental time for the progress of the prior wave. The curve for cell recruitment therefore gives the axial length versus time curve for the prior wave. The wave
begins with a constant velocity and slows gradually in the posterior trunk and
anterior tail region.
negligible: mitoses are not seen at this stage. Figure 8 is thus the second order
curve for the prior wave, whose shape in space-time is given in Fig. 9 by plotting
the total number of cells along the pre-somitic axial dimension incorporated
into somites against ordinal somite sequence
(c) The prior wave is kinematic
A frontier of change moving across a tissue may reflect one of two quite
different processes. In the one case, the moving frontier may depend upon the
propagation of a stimulus from cell to cell in the direction of the movement.
Alternatively there may be nothing actually spreading across the tissue to
prompt the change, the moving frontier reflecting the predetermination of the
cells to change one after another in a temporal sequence across the tissue. In
this case we have a kinematic wave. A simple experiment distinguishes between
the two sorts of wave: if the tissue is cut in two before the passage of the wave,
a propagated wave is stopped at the cut, whereas a kinematic wave is not stopped
and appears to jump across the cut.
Deuchar & Burgess (1967) proved that the wave of somite formation is
kinematic by cutting neurulae in two caudal to the last formed somite. We
describe a similar experiment proving that the prior wave is also kinematic.
Somitogenesis
37
in amphibian embryos. I
Table 1. Results of the bisection experiment
Rana embryos were cut in two at the stage of first somite formation, into anterior plus
posterior halves. In series 1 the bisected embryos were given no heat shock. Of 12 embryos only
2 failed to develop both anterior and posterior halves up to completion of segmentation.
Ten embryos were available for complete scoring. In series II embryos were heat shocked, then
immediately bisected. In 4 out of 12 embryos both halves did not survive to scoring, and in
another 2 embryos the tail half became so bent that it was only possible to score one side. There
were thus 14 out of an initially possible 24 files scored. In all but one of these, abnormal segmentation begins in the anterior half embryo.
In both series I and 11 the total number of somites formed by the two halves combined is
virtually identical to non-bisected control embryos in series III.
Mean
Abnormal
segmentation in
Total
no. of
A
oUI111ICo 111
37 °C
shock
files
recovered
I. 12
_
20/24
8-5
11. 12
+
14/24
11-6
+
12/12
—
No. of
embryos bisected
III. 6 controls
not bisected
(
"\
\Apan fircf*
IV! Cct 11 -111 o I
somites in ant. + post.
halves
ant. half
Ant.
half
Post.
half
abnormal
somite
39-43
(mean 40-5)
37-42
(mean 39-4)
39-42
13/14
13/14
6-9
—
—
7-4
Rana embryos at the neural fold stage, before the formation of the first
somites, were cut transversely in two immediately after an 8 min heat shock. At
this stage the prospective somitic material is confined to the posterior half of the
embryo; the cut therefore was made rather more than half way along the
embryos, nearer the posterior end. The two halves were reared separately. Not
only was the total number of somites formed by pairs of half embryos normal,
but also the sequence of somite formation was normal, and tail segmentation
was completed in posterior halves at the same time as in intact controls. The
anterior halves developed the beginning of an abnormal zone in all but one case
scored; the posterior halves showed a continuation of the abnormal zone
(Table 1).
Heat shock was used here to indicate, by the ordinal position of the first
abnormal somite, the position of the prior wavefront of cellular change at the
time of separation. The presence of abnormal somites in the anterior half
proves that the advancing wavefront had not yet reached the posterior half.
The demonstration of somitogenesis in the posterior half therefore proves the
kinematic nature of the prior wave.
(d) The wave of somite formation and the prior wave after heat shock
The rate of segmentation behind the abnormal zone is the same as in controls
in both species (Fig. 10). Heat shocked embryos are invariably retarded in their
development in comparison with controls. Figure 10 shows that the retardation
38
M. PEARSON AND T. ELSDALE
°36
-
(b)
(fl)
Xenopus laeris
1 32
0 24
.5 20
| 16
1 12
o
c
o
s
4
2
0
4
_l
12 16 20 24 28 32 36 40 0
4 8
Numbers of somites in controls
J
4
L
J
1
L
8 12 16 20 24 28 32 36 40
Number of somites in controls
Fig. 10. Retardation in somitogenesis caused by heat shocks at different stages in
(a) Rana and (n) Xenopus. In both cases the broken line represents the normal rate
of somitogenesis in controls. Arrows indicate the stage at which different groups
of embryos were heat shocked, (a) In Rana shocks given at three different stages
gave an identical result: a temporary arrest which leads to resumption of domitogenesis at the same rate as in controls, but now a constant 4 somites behind the
unshocked controls, (b) The same effect is seen in Xenopus. Heat shock does not
affect the rate of subsequent segmentation. The vertical bar shows the extent of the
abnormally segmenting zone; estimates of somites' worth of material segmented
in this abnormal zone are based on the most dorsal intersomitic furrows where
disturbance is least.
of somitogenesis is entirely due to a transient arrest immediately following
shock, an arrest which appears to affect the whole embryonic process and not
just segmentation.
If the overall rate of passage (i.e. rate of cell recruitment) of the wave of
somite formation were affected by shock, then, since the rate of subsequent
segmentation remains the same as controls, we should expect to find either
larger and concomitantly fewer somites, or smaller and more numerous somites,
formed as a result of a heat shock. We have gathered data on the normality
of the somite files as a whole in heat shocked embryos, and in particular on
whether the return to normal segmentation posterior to the abnormal zone
produces the correct number of somites, correctly sized according to their
position along the axis, in the correct time.
The following observations were made on batches of embryos receiving
shocks of intermediate duration (15 min in Xenopus, 10 min in Rana) resulting
in abnormal zones about 8 somites long.
(i) The number of somites segmented in files, including an abnormal zone,
is the same as in controls in both species. These counts, to the end of the tail
proper in Xenopus (46 somites) and to the end of the file in Rana, include
estimates of the number of somites, or somites' worth of material, in the
abnormal zone. In zones about 8 somites long the most dorsal intersomitic
Somitogenesis in amphibian embryos. I
39
boundaries are often minimally disturbed and estimates are unlikely to err by
more than ± 1 somite.
(ii) In Xenopus the width of the somites behind the abnormal zone, measured
by cell number, is normal for their ordinal positions along the axis (Fig. 8).
(iii) Since the rate of segmentation is the same as in controls following the
initial arrest, the correct number of normal sized somites are formed at the
correct time along the axis.
Heat shock therefore does not alter the rate of passage of the wave of somite
formation. Within the abnormal zone itself, shocked embryos show the same
antero-posterior sequence of (abnormal) segmentation as in normally segmenting
embryos. The separation between this wave of somite formation and the prior
wave can be demonstrated in heat shocked embryos just as in previously unshocked embryos (Figs. 6 and 7). Rana embryos were given a second shock at
different stages after a first shock at the 1-3 somite stage. In each case the
number of normal somites which formed in the interval between this second
shock and the beginning of a second abnormal zone did not differ significantly
from controls which had been given no previous shock, as in Fig. 7. The prior
wave proceeds as usual at the same rate as the wave of somite formation,
preceding by about three to four somites.
3. The hidden effect of a heat shock
The previous demonstration that visible segmentation is normal in all respects
behind an abnormal zone might suggest that the effect of a heat shock is
exhausted with the abnormal zone. Double heat shock experiments however
indicated a hidden effect of heat shock that is more enduring.
Rana embryos were given two shocks so arranged that a minimum of two
normal somites intervened between the end of the first abnormal zone and the
beginning of the second. Shocks of 8 min and 9 min were employed. The first
was given at the very beginning of somitogenesis, the second around the
9-somite stage. Controls were given only the latter shock. Results are set out
in Table 2.
The interesting result is that the abnormal zone resulting from a second shock
is shorter than expected, and the severity of the abnormality considerably
reduced (Fig. 11). A heat shock therefore partially protects against a subsequent
shock.
4. The abnormal zone
Apart from the abnormal zone, the passage of the wave of somite formation,
and the prior wave, is not affected by heat shock. Within the abnormal zone,
only segmental pattern is affected; the cells subsequently differentiate normally.
(i) The setting of the abnormal zone. Heat shock is followed by a 'lag'
during which several somites segment normally before the disturbance is
registered. This 'lag' is independent of the duration of the shock, and following
40
M. PEARSON AND T. ELSDALE
Table 2. The effect of a double heat shock on Rana embryos
Experimental embryos were given an 8 min shock at the stage of first formed
somites, and a second shock during formation of the resultant abnormal zone. This
resulted in a second abnormal zone, separated behind the first by at least two normal
somites. Controls were shocked at the same stage as the experimental embryos at the
time of their second shock; there is in fact a difference of a mean one somite between
these two groups at the time of this shock, shown by the first abnormal somite
(FAS) for each. The number of abnormal somites (NAS) is larger by three somites
in controls.
The same effect of protection against a second shock is seen following 9 min
shocks.
Not only the length of the abnormal zone, but also the severity of abnormality is
reduced by the first shock, as shown in Fig. 11 (S.D., standard deviation; n, number
of files scored).
Control
Experimental (second shock)
(double shock)
only
8 min shock
Mean FAS
S.D.
Mean NAS
S.D.
n
9 min shock
Mean FAS
S.D.
Mean NAS
S.D.
n
13-3
0-9
2-1
1-4
34
120
0-8
5-5
1-9
27
10-9
0-6
4-4
11
15
10-9
H
6-9
2-5
14
shocks given after the earliest stages of somitogenesis, the number of 'lag'
somites is roughly constant (Figs. 6 and 7).
(ii) The abnormal zone may extend from 2 to upward of 20 somites' worth of
tissue, the length being proportional to the duration of the heat shock (Table 3).
The longer the abnormal zone, the more severe is the visible disturbance of
segmentation.
(iii) Asymmetry. The abrupt transition from the last normal to the first
abnormal somite has been mentioned. The disturbance is greatest anteriorly and
then gradually returns to normal posteriorly.
(iv) The essential disorder within the abnormal zone. The effect in Xenopus
was described by Elsdale et al. (1976): the elongation, alignment, and rotation
of cells occurs just as in normal segmentation, but instead of regular segmental
blocks, small irregular associations of cells create a chaotic myotome in which
regular segmental boundaries are lost (Fig. 12). Because in Rana cellular elongation is deferred until some time after segmentation, the picture seen on stripped
Rana embryos is a more direct reflexion of the disturbance caused by heat
shocks.
Somitogenesis in amphibian embryos. I
Fig. 11. A comparison of the abnormality induced by a 9 min shock given to Rana
embryos of the same stage (a) after a previous shock, and (b) after no previous
shock. In (a) both the length of the abnormal zone and the severity of the disturbance
are considerably reduced by the effect of the earlier shock.
41
42
M. PEARSON AND T. ELSDALE
Table 3. The correlation between length of the abnormal zone, measured
by the number of abnormal somites (NAS) and the duration of the shock
Rana embryos from the same ovulation were given 6, 8, or 12 min heat shocks
(37 °C) at the 9-10 somite stage. Essentially identical results were obtained with
embryos at other stages during trunk segmentation; in later tail stages a 12 min shock
produced abnormalities to the end of the tail, and shorter shocks corresponsingly
produced longer abnormal zones than in the trunk region.
The same relation between duration of shock and length of the abnormal zone
was described in Xenopus by Elsdale et al. (1976).
Duration of heat shock
Mean NAS
S.D.
//
6 min
8 min
12 min
2-5
1-3
19
4-5
1-5
39
9-4
2-2
26
The anterior abnormal zone, especially following severe shock, often presents
a solid block of apparently unsegmented tissue equivalent to two or more
somites' length. Detailed examination reveals that this superficial appearance is
not due to a failure of segmentation but, on the contrary, to an extreme fragmentation of the impulse to segment, giving rise to a stippled appearance, the
result of condensation of cells into small, irregular groups (Fig. 11 b). Posteriorly
this fine stippling resolves into a chaotic segmentation characterized by haphazard and numerous short furrows and indentations. This appearance in turn
simplifies posteriorly, and with the appearance of clear intersomitic boundaries
and the disappearance of supernumerary furrows the normal segmental pattern
is re-established (Fig. 13).
In normal somitogenesis there appears to be a change in intercellular adhesion
at the time of segmentation; looser cell contacts are succeeded by a rather
tighter adhesion pattern (Fig. 14). Bellairs, Curtis & Sanders (1978) have
demonstrated in the chick that a wave of increased cellular adhesiveness
leads to somite formation.) In Rana embryos following heat shock, cells undergo the same change within the abnormal zone as in normally segmenting
embryos, but instead of forming large, somitic groups, the pre-somitic continuum breaks up into small irregular groupings.
The essential disorder observed here is an excessive segmentation, extreme
anteriorly, declining posteriorly, which reflects a disorganization of the normal
pattern of cellular condensation. There is no evidence of cell death.
Somitogenesis in amphibian embryos. I
43
Fig. 12. A light micrograph, horizontal section through Xenopus embryos following
a 15 min heat shock and subsequent abnormal segmentation. In the abnormal
zones indicated (
) cells have rotated, but chaotically. Thus there is no proper
alignment of myoblast nuclei as in normal somites seen anteriorly in (a). In both
(a) and (b) the return to normal segmentation posteriorly is evident. In both,
however, one side is seen to re-establish normal segmentation before the other.
The two files do show independent variation in the extent of the abnormal zone,
but the whole series of sections would need to be read to establish the true extent of
an abnormal zone in any embryo. A single section may be misleading, n, Notochord; nt, neural tube.
DISCUSSION
Heat shocks have been used to demonstrate, firstly, that segmentation can be
disturbed, and secondly that there is a wave of change in the presomitic tissue
prior to somite formation. After the passage of this wave of change segmental
specification is not disturbed by heat shock. Two observations together suggest
that this prior wave is a determinant of the succeeding wave of somite formation : (i) the constancy of the developmental relation between the two waves,
and (ii) the nature of the change before somite formation, an abrupt change with
which future somite boundaries become insensitive to disturbance by shocks.
Our hypothesis is that the behavioural changes responsible for the association
of cells into segmental groups, for somite formation, are triggered by the
passage of the prior wave.
44
M. PEARSON AND T. ELSDALE
Fig. 13. Scanning electron micrograph of stripped Rana tail-bud embryo showing
the typical effect of an 8 min heat shock during the neurula stage. The abnormal
zone is localized, is most severe anteriorly, and then returns gradually to normal
segmentation posteriorly. The nature of the abnormality is discussed fully in the
text. Note that the dorsal embryo is toward the bottom of the figure, ventral to the
top. Anterior to the left.
Fig. 14. Newly formed somites in the same embryo as Fig. 13, enlarged to show
apparently tighter adhesion in somites than in the posterior, non-segmented
pre-somitic mesoderm cells.
Somitogenesis in amphibian embryos. I
45
This hypothesis immediately raises two questions: (i) what is the cause of the
prior wave? and (ii) is this wave itself part of the mechanism of segmental
patterning and its regulation, or is it merely a temporal reflexion of an underlying spatial pattern ? These two questions are interrelated, and we shall approach
them by considering the kinematic nature of the prior wave.
A kinematic wave does not depend upon the propagation of a stimulus. Each
cell, pre-determined to do something at a particular time, acts autonomously.
The smooth passage of a kinematic wavefront implies that the cells are laid out
along the axis in the order in which they are set to change; if the order were
changed, the passage of the wavefront would be correspondingly erratic. A
kinematic wave does not depend on cells knowing where they are along the axis
and interpreting their positions, but on cells knowing when independently of
their whereabouts.
We would expect the wave of somite formation to be kinematic, as Deuchar &
Burgess (1967) showed, if it were the consequence of the prior wave. The
demonstration that the prior wave is likewise kinematic indicates that it too is
the consequence of an earlier primary event. Our experiment tells us only that
the paraxial mesoderm is temporally determined as early as the neural fold stage
at which embryos were cut in two; in a following paper, however, we shall
present additional evidence suggesting temporal determination as early as the
beginning of gastrulation. Zeeman (1975) envisages a temporal determination
established with the passage of a primary wave across the mid blastula coincidental with primary mesodermal induction. On this view, the kinematic
waves we demonstrate in connexion with somitogenesis are the reflexion of a
larger field of temporal determination, established across the marginal zone of
the young embryo, responsible for the intrinsic developmental dynamics of the
primary organizer. Zeeman's theorem describes the shape of such a wave,
arising out of the dynamical organization of the intercellular biochemistry, by
a cusp catastrophe. The waves we describe do not fit a cusp catastrophe at their
beginning or end (compare Fig. 8 with Fig. 3, Pearson & McLaren, 1977), but
in any case the substrate deformations during invagination rule out the likelihood of correspondence between such secondary waves and a postulated primary
wave. Our results provide no evidence to bear crucially on the theorem.
The temporal determination of the prior wave in somitogenesis, already set by
early gastrulation (Elsdale & Pearson, 1979), suggests how this wave may be
involved in the specification and regulation of the segmental pattern. Cooke
(1975) has demonstrated that embryos artificially reduced in size at the early
blastula stage subsequently develop a normal body pattern including the normal
number of somites appropriately reduced in size and containing fewer than
normal cells. The interval between the formation of one somite and the next is
not altered and somitogenesis is completed in the same time as in control
embryos. This means that the wave of somite formation takes the same time to
traverse the shorter axis in reduced embryos as it does to traverse the longer axis
4
EMB 51
46
M. PEARSON AND T. ELSDALE
in normal embryos, implying that regulation in the former is achieved by a
slower progress of the wave of somite formation. The rate of segmentation is
the same in both cases, so that the necessary regulation must result from the
timing of cellular activities which lead to their incorporation into the kinematic
wave of somite formation. The timing of such cellular activities must be set by
an earlier process. If the prior wave is itself determined by some earlier process,
it is nonetheless the prior wave which appears immediately to regulate somite
formation, since only after its passage is somite specification fixed. This is our
second hypothesis: the timing of the cells' commitment to somite formation,
which is manifest at a supracellular level as a prior wave along the mesodermal
axis, is fundamentally a part of the patterning process. It is the timing of cellular
mechanisms rather than an original positioning of cells which underlies segmental pattern.
We could envisage two ways by which the segmental pattern might arise with
the prior wave and be affected by heat shocks. The segmental period might be
inherent in the timing of the cellular mechanisms which lead to the commitment
of cells to somite formation, or the period might arise independently of the prior
wave to be imposed upon it at the moment of this commitment. The crucial
considerations are the following.
Firstly, the asymmetry of the abnormal zone: behind the sharp anterior
frontier there is a gradual return to normal posteriorly. Secondly, the length of
the abnormal zone is proportional to the duration of the shock, so that a shock
longer by minutes leads to a longer abnormal zone formed over several hours.
We can view the whole somite file, including an abnormal zone, as a record of
the developmental history of pre-somitic cells up to somite formation; that is,
we can translate from the linear dimension of the file into a time course which
has been stamped on the final somite pattern by heat shock. Posteriorly, normal
segmentation reflects cells in an early phase of maturation at the time of the
shock; further anteriorly the maturing cells register heat shock in a subsequently
disturbed segmentation which increases up to the anterior frontier of the abnormal zone. Cells then switch from maximal sensitivity to insensitivity, and the
segmental pattern is fixed. Before cells reach this point of commitment, therefore, they may be deranged by heat shock depending on (a) how close they are to
the wavefront at the time of shock, and (b) on the duration of the shock. The
closer cells are to commitment, the more readily they are induced to abnormal
segmentation.
There are two possible explanations for this behaviour recorded by abnormal
files.
(i) The first explanation assumes that the picture provided by the file with an
abnormal zone directly reflects the responsiveness - or non-responsiveness - of
the cells along the axis at the time of shock. Once disturbed, a cell remains so in
the absence of any means of recovery. This explanation however offers no
obvious reason for the asymmetry of the abnormal zone beyond ascribing it to
Somitogenesis in amphibian embryos. I
Al
increasing sensitivity. Nor does it predict significantly longer abnormalities for
slightly longer shocks unless we postulate a subliminal sensitivity posteriorly
which can be brought to the point of disturbance by a longer shock. On this
hypothesis we might expect the effects of two shocks to be additive, the subliminal
effect of a first shock sensitizing the tissue for a greater response to the second,
but this is the opposite of what we find. In fact there is a shorter and less severe
abnormality, in comparison with controls, in response to the second shock. This
result of the double heat shock experiment indicates a restorative mechanism,
such that the effect of a first shock which is not seen in any visible segmental
abnormality enables the tissue to recover from the effect of a second
shock.
(ii) The second alternative explanation depends upon the ability of cells to
recover from the effect of heat shock. The pattern of visible abnormality may
then be translated into an account of recovery by labile cells which have not yet
been fixed in the segmental pattern by the passage of the wave of commitment.
On this basis it is the state of the cells at the time of commitment which is alone
crucial for orderly somitogenesis. Cells immediately behind the anterior frontier
of the abnormality register the most severe disturbance, not because they are
most sensitive, but because there is less time for recovery before the final determination of segmental pattern. Because the disturbance is transient, only those
cells committed immediately following a shock will stabilize the full disturbance.
Cells committed later will have more time to recover. A longer shock causes
more severe disturbance; and so recovery takes longer. In this case the abnormal
zone extends posteriorly into a region where recovery would have been complete
following a shorter shock. When a second shock is delivered, cells which would
have recovered from the first shock now recover more rapidly from the second
disturbance. Recovery therefore is an active process.
The evidence for independent recovery from heat shocks before a final
determination of the somite pattern means that segmental specification does not
emerge as an inherent pattern in the cellular maturation processes that lead up
to the prior wave. Segmental specification is concomitant with the passage of the
prior wave, but does not simply arise from it. There is no evidence that the prior
wave itself is disturbed by heat shocks.
The effect of heat shocks. The visible pattern abnormality is an excessive
segmentation, reflecting a tendency of the cells to associate into smaller, more
irregular groups than normal. Heat shock has fragmented some principle of
intercellular co-ordination established before the passage of the wavefront. It is
proposed that heat shock undoes this co-ordination over the whole length of the
paraxial mesoderm not yet overtaken by the prior wave. The loss is transient,
however, and a slow recovery toward re-establishment of co-ordination ensues.
To account for the fixation of the disturbance, and the delayed appearance of
abnormality after a shock, we propose that with the passage of the prior wave a
segmental pre-pattern is set up on the basis of the degree of intercellular
4-2
48
M. PEARSON AND T. ELSDALE
co-ordination pertaining at the time. This scheme (Fig. 15) accounts for all
the characteristics of the abnormal zone.
Turning to the nature of the second component in pattern specification we
consider the basis for intercellular co-ordination. This could in principle be
either a temporal or a spatial co-ordination. The latter suggestion however
would amount to the conceptual indulgence of a second pre-pattern. Furthermore, since the segmental rate of progress of the prior wave is constant throughout (Figs. 6 and 7), a segmental specification at the time of the prior wave must
be a constantly periodic process. It is most reasonable therefore to assume a
periodicity in the responsible mechanism and to implicate this periodic component in the partitioning of the longer time interval taken by the prior wave to
traverse the axis.
We propose therefore that each cell of the paraxial mesoderm carries two
independent pieces of equipment: first, a pre-set specification of the timing of
abrupt change, or a count-down apparatus, impervious to outside influence by
heat shocks, and second, a component uniquely susceptible to heat shock and
on the basis of which the intercellular co-ordination is established. The hypothesis that this component is a clock synchronizable with similar clocks in other
cells could be experimentally tested. While there is evidence that the first
component is present already by the beginning of gastrulation, co-ordination is
not established until the late gastrula (Elsdale & Pearson, 1979). There is no
evidence to suggest that cells are precipitated by heat shock into any abnormal
state other than to disturb their temporal co-ordination. It is the synchronization of a population of cells which is affected, upon which normal segmental
patterning depends. Similarly, in connexion with recovery from heat shock, it is
a resynchronization.
Coupling of the two components: the clock and wavefront model. Our results
indicate that crucial for the specification of the somite pattern is a process
involving the momentary coupling of both components in the setting up of a
segmental pre-pattern. The pre-pattern is established as a wave of somite determination which is, in practice, inseparable from the prior wave.
The components we describe are essentially the same as those proposed in a
theoretical model published by Cooke & Zeeman (1976), who suggest how the
coupling might work. Suppose a cyclic process in each cell having a period
equal to the interval between the formation of one somite and the next. The
cycles are phase-linked. The cells cycle in unison. Suppose further that there are
two parts of the cycle, an ON part and an OFF part, and that only during the
former can the cells undergo abrupt change. Cells that complete their 'countdown ' to the abrupt change which commits them to somite formation during the
OFF part of the cycle can do nothing and must wait until they re-enter the ON part
of the cycle. Hence, as the cells pass into the OFF part of their cycles, the wavefront
is halted, and on re-entering the ON part, a batch of waiting cells undergo their
abrupt changes synchronously, and are thus committed to define a somite.
49
Somitogenesis in amphibian embryos. I
The formation of an abnormal zone following lieat shock
A
Somites
B
1 2
3 4
D
C
5 6 7 8 9 10 1112 13 14 15 16 17 18 1 9 2 0 2 1 2 2 23 2 4 2 5
T4
T3
T2
*
*
*
*
Tl
Axis
Anterior
Posterior
Fig. .15. This is a board on which the reader is invited to perform two mobile
demonstrations, by placing a ruler along the base of the board and moving it
slowly upwards to exit from the top of the figure.
(1) The wavefronts of somite determination and somite formation. As the ruler is
raised the points of intersection with the lines labelled somite determination and
somite formation move along the ruler from left to right simulating the passage of
the two wavefronts along the embryonic axis as development proceeds.
(2) The creation of an abnormal zone following heat shock. The four superimposed
horizontal lines T1-T4 represent four sequential stages in the development of an
embryo following heat shock. At Tl six somites have been determined (A-B) and
the first somite is about to form. A heat shock at this time disturbs (asterisks) all of
the undetermined pre-somitic mesoderm, but determined tissue anterior to B is
immune. The first six somites form sequentially as the ruler is moved towards T2.
Concomitantly the wavefront of determination advances from B to C across tissue
disturbed by heat shock. Immediately behind B the tissue has no time to recover
before determination and the maximal disturbance is fixed; by the time the wavefront has reached C however, recovery is pictured as complete. Moving the ruler
beyond T2 towards T3 somites 7-12 form; these somites are to a greater (anterior)
or lesser (posterior) extent abnormal reflecting the degree of disturbance fixed at the
time of determination. Concomitantly the wavefront of determination continues
to advance across tissue now fully recovered from the heat shock. Moving upwards
from T3 to T4, somite formation returns to normal posterior to the abnormal zone.
50
M. PEARSON AND T. ELSDALE
The overall result is that a somite's worth of cells undergo abrupt change
together, followed by an interval of time before the next batch changes and so on
in discontinuous steps along the body axis. As Cooke & Zeeman see it the cells
become ready to change in smooth sequence down the axis and it is due to
coupling with the cyclic component that the observed wavefront of somite
determination is presumed to move in spurts and pauses, thus setting up the
segmental pre-pattern. The coupling is formally analogous to an escapement
mechanism in a clock by which a smooth energetic input from the mainspring
is transformed into ticks.
This model eschews reliance on any form of qualitative differentiation,
including positional interpretations, and thereby assumes that somitogenesis is
essentially the sequential division of a homogeneous loaf. In the absence of
evidence for any qualitative difference among pre-somitic cells, this seems a
realistic foundation for any account of somitogenesis.
Any explanation of somitogenesis does not principally concern the particular
changes of cell surface property nor the mode of cell behaviour, but rather the
spatio-temporal pattern of such changes within the morphogenetic cell population. Thus in Xenopus laevis and Rana temporaria the cellular mechanics of
somite formation are very different, yet the pattern of segmentation, and the
effects of heat shocks, are very similar. The same account suffices for both.
We thank Jonathan Bard for scanning electron micrographs and for criticising the manuscript; also Alison Abbott and Sandy Bruce for assistance. MJP thanks Professor Evans for
hospitality at the Western General, MRC for paying train fares and other expenses, Brian
Goodwin and Christ Ford for providing facilities at the University of Sussex.
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(Received 19 June 1978, revised 9 January 1979)