/ . Embryol. exp. Morph. Vol. 58, pp. 107-118, 1980
Printed in Great Britain © Company of Biologists Limited 1980
107
Somitogenesis in amphibian embryos
III. Effects of ambient temperature and of developmental stage
upon pattern abnormalities that follow short temperature shocks
By JONATHAN COOKE 1 AND TOM ELSDALE 2
From the Division of Developmental Biology, National Institute for
Medical Research, Mill Hill, London
SUMMARY
Temperature shocks of a few minutes duration at 37 °C to tail-bud embryos of Rana
induce zones of abnormal segmentation along the somite files subsequently produced. The
immediate result of a temperature shock is a temporary arrest of development as a whole,
following which the schedule of somite determination and formation is resumed at the normal
rate. It is during the period immediately following this that the zone of abnormal somite
pattern is determined. Thus the length of the abnormal zone reflects the total time taken by
the morphogenetic system to recover from the disturbance, and might depend upon variables
affecting both the duration of the initial arrest and the duration of the recovery period itself.
Observations are presented demonstrating how the lengths of abnormal zones, caused by
a temperature shock of any particular severity, are affected by three variables: (1) the ambient
temperature to which the embryos were adapted before shock, (2) the ambient temperature
of post-shock development, (3) the stage in somitogenesis, i.e. the number of somites already
formed at the time of shock.
The data (in this and previous papers of the series) support models postulating that the
spatial periodicity in cell behaviour, that is somite morphogenesis, reflects a normal interaction between two hidden aspects of development, one a wavefront of cellular activation
passing down the body axis, and the other having the character of a temporal periodicity
throughout the tissue. Temperature shock, as well as halting the wavefront (i.e. stopping
development) temporarily, leads to a subsequent period during which there is only gradual
recovery of normal co-ordination between the periodicity within cells of the tissue and the
wavefront progress. It is the relative rate of this recovery, alone, that is responsible for variation in the length of the abnormal zone.
INTRODUCTION
In recent papers (Elsdale, Pearson & Whitehead, 1976; Pearson & Elsdale,
1979; Cooke, 1978) the occurrence has been described of localized disruptions
in the normally regular pattern of somites and the fissures between them, following the exposure of anuran amphibian embryos to high temperature (37 °C)
shocks of a few minutes duration. The embryos as a whole show no signs of
1
Author's address; Division of Developmental Biology, National Institute for Medical
Research, Mill Hill, London NW7 1AA, U.K.
a
Author's address; M.R.C. Population and Cytogenetics Unit, Western General Hospital,
Crewe Road, Edinburgh, U.K.
8
EMB 58
108
J. COOKE AND T. ELSDALE
damage beyond a few hours initial delay in the developmental schedule and
somite cells in the disturbed region are able to complete normal histodifferentiation. The precise spatio-temporal sequence of cellular activities in forming
somites has, however, been disrupted over regions of paraxial tissue that would
normally have formed some three to ten segments. It should be emphasized
that whereas the high temperature is experienced for minutes only, the determination and then later execution of the cellular activities which are found to
have been disorganized extends over hours or days (depending upon species,
ambient temperature and the length of the abnormal region seen). It is this
feature which makes the results of interest. The cell movements of somite
formation in Xenopus, in their spatial and temporal aspects, have been described
by Hamilton (1969) and by Cooke & Zeeman (1976).
There are two separate developmental time periods during which local
abnormalities may be induced in the somite series by high temperature, in either
Rana temporaria or Xenopus laevis. Shocks to late blastulae or gastrulae induce
areas of disruption, on a random basis, approximately evenly distributed through
the tissue due to form about the anterior 25 somites of the body pattern, or
clustered towards the more posterior of these after shocks delivered near the end
of this period. Similar shocks to stages from early neurula onwards induce, with
great reliability, a single, bilateral zone of disrupted pattern precisely in that
tissue which was due to begin the visible sequence of somite morphogenesis at
a particular time after the shock (2-6 h at lab. ambient temp., dependent upon
species). Temperature sensitivity in tissue during this second sensitive period
thus moves back through the body pattern in a regular way, just ahead of the
steadily advancing wavefront of morphogenesis itself (Elsdale et al. 1976;
Pearson & Elsdale, 1979). These two developmental periods are separated by
a recognizable interval during which any temperature shock that allows development to continue at all will leave the somite pattern unaffected. One
possible meaning of this refractory period and of the two spatial patterns of
effect on either side of it has been discussed by Cooke (1978), but the early
sensitive period may be understandable in quite other ways than the one suggested in that paper (see Elsdale & Pearson, 1979).
In the present paper we describe the effects of ambient temperatures, prior
to shock and during development after the shock, and the precise stage of
morphogenesis at shock itself, as variables influencing the amount of pattern
made abnormal by a given number of minutes spent at 37-5 °C during the second
of the sensitive periods. For this, as for all our more recent work we have used
only Rana temporaria eggs collected from the wild in central Scotland, having
established that the effect of temperature shocks on morphogenesis are essentially identical, in relation to the developmental scale, with those seen in Xenopus.
The tight natural synchrony and very homogeneous developmental rate within
egg clutches, the low incidence of spontaneously abnormal morphogenesis, the
slower tempo of pattern formation and the wide temperature range allowing
Somitogenesis in amphibian embryos, III
109
normal development in this species, all permit a more precise temporal analysis
of events in somite formation.
We establish in this paper that, during the second sensitive period, only the
precise stage of morphogenesis at shock - operationally, the number of somites
then already formed - determines the position of the anterior edge of the
resulting patch of pattern disruption. Since somite boundaries are determined
and then form in a strict head-to-tail time sequence, the length of the abnormal
zone, i.e. 'amount of pattern made abnormal' in terms of somites-worth of
tissue, reflects the amount of post-shock development that occurs before normal
relationships are re-established between processes which must interact to
organize the pattern. Normal development has of course its temperature/rate
coefficient. This differs apparently from that of some of the processes of recovery
from acute periods of high temperature, because development at different
ambient temperatures (after a given shock) changes the amount of pattern
liable to be made abnormal. This and other findings are discussed in terms
of a class of models that is currently being considered for the mechanism
underlying development of repeated structures.
MATERIALS AND METHODS
Spawn of Rana temporaria (2- to 4-cell stage) was collected in season from
ponds in central Scotland. Embryos were allowed to develop at particular
temperatures between 6 and 20 °C in aerated pond-water after separation into
small groups within the jelly to ensure uniform conditions. Development is
normal over this temperature range and survival unimpaired.
Embryos are manually decapsulated (but vitelline membranes left intact)
before dropping in batches of 15-40 from the submerged mouth of a pipette
into a 150 ml bottle of 5 % Dulbecco saline in pond water at 37-5 °C. After the
required number of minutes (8-12) they were gently pipetted into an excess of
medium at 15 °C. Five minutes later they were set to develop in shallow dishes
of pond water at a chosen ambient temperature within the aforementioned range.
There was no death or detectable cell loss following such shocks in these
experiments.
Somites were examined by stripping the integument from larvae about 5 min
after the onset of fixation in 1 % glacial acetic acid in 0-5 % K2Cr2O7 solution.
Larvae were stripped at stages that postdated the return to normal segmentation
in the forming tail somites. Abnormally shaped or absent fissures were scored
bilaterally after lining up larvae beside controls. Selected embryos were examined in this way at earlier stages after shock, or fixed in half-strength Karnovsky's
fluid at pH 7-4 for 1 jam plastic sectioning in exact horizontal section and staining with toluidine blue.
8-2
110
J. COOKE AND T. ELSDALE
•S
Fig. 1. Four typical abnormal zones, caused by lOmin. at 37-5 °C experienced
around the 4-somite stage, drawn in camera lucida. Left-hand sides of embryos with
epidermis stripped. P = outline of pronephros. S = outline of somitic mesoderm.
Lines of normal intersomiticfissuresand equivalent separations between cell populations, drawn in. For further details of abnormalities see Pearson & Elsdale (1979).
Fissures are initially formed as lines of de-adhesion separating populations of cells,
each such population assuming a rosette configuration. Later, due to cell realignment, spindle-shaped cells span the distance between adjacent fissures.
RESULTS
Temperature shocks of the order of 100 sec often cause minute but definite
disturbances in the shape and position of one or two fissures, at a point in the
somite pattern which would have been the front edge of the abnormal zone had
the shock been somewhat longer (Cooke, 1978; Cooke & Elsdale, unpublished).
In the present material a shock length of 8-12 min was chosen (see Fig. 1), so
that the typical result was a region of disrupted pattern embracing the tissue
that would have formed some three to ten successive somites, beginning very
abruptly anteriorly, and returning much more gradually, posteriorly, to a
normal sequence of cell populations separated by regular fissures. It is this
appearance which has led to the postulate (Pearson & Elsdale, 1979) that the
initial, sudden disruption among pattern-co-ordinating processes, engendered
Somitogenesis in amphibian embryos. Ill
111
Table 1. Ultimate developmental retardation, in relation to initial delay or arrest of morphogenesis and normal rate of morphogenesis (h/somite) at each of two post-shock temperatures,
caused by 10 min at 37-5 °C in a synchronous population
Temperature
of post-shock
development
20 °C
No. of
embryos examined at
each sampling
timepoint
post-shock
Duration
post-shock
examined
(h)
6
8
10
6°C
24
48
72
Mean no. of
somites formed
since shock
.". Estimated no. of hours and
somites-worth of initial arrest
(A) and ultimate retardation
(i?) in relation to (C) control
development rate
Experimental 0
Control
5
Experimental 0-75
Control
6-5
Experimental 2-5
A and R = 7 h approx or 5-75
Control
8-25 somites-worth. C = l-2h/
somite
Experimental 0
Control
2-33
Experimental 0
Control
4-33
Experimental 2-17 A and R = 50 h approx or 4-5
Control
6-67 somites-worth. C = 11 h /
somite
This investigation was made on eggs from one clutch, adapted to a lab. ambient temp, of 15 °C before
shock, and forming somite 22 (+ or — £) at the time of shock. Somite formation in each freshly dissected
embryo was counted to the newest £ somite on the right-hand side, as each somite forms in rapid dorsoventral sequence. Ultimate retardation due to shock, in somites, was measured at control 35-somite stage.
by the shock, is subject to gradual recovery after the resumption of development as a whole. Shocks of this length result also in a developmental delay of
some hours relative to synchronous control siblings, representing a deficit of
some five formed somites when the populations are later compared at any stage
prior to completion of the normal body complement of somites (40+) in the
controls. This delay is incurred as an arrest immediately following shock, with
subsequent resumption of development at normal rate (see Elsdale et al. 1976;
Pearson & Elsdale, 1979). Visible morphogenesis recommences after a delay
similar to the delay observed in developmental schedule much later on (Table 1).
We conclude from this that, after the initial arrest at shock, the wavefront
representing morphogenesis recommences its passage down the body at the same
rate as in control embryos even though there is to occur subsequently a zone
in the body (thus, a time period) within which this wavefront fails to achieve
normal expression as somite pattern. Temperature shock thus results in a time
delay in the schedule of pattern determination that is occurring tailwards of
visible morphogenesis, and also causes an abnormality in the pattern region
112
J. COOKE AND T. ELSDALE
Table 2. The effect of ambient temperatures of development before and after the
shock, as variables affecting the amount of pattern made abnormal by 8 mins at
37-5 °C
Experiment
no.
N
1
15
2
15
Temperature of
pre-shock devel.
Mean length of
abnormal zone
Temperature of in somites-worth
post-shock devel.
of tissue
15 °C
(7th somite forming)
20 °C
(9th somite forming)
12 °C
(14th somite forming)
20 °C
(15th somite forming)
20 °C
6°C
20 °C
6°C
6-7
4-3
2-8
2-6
20 °C
6°C
20 °C
6°C
7-3
5-9
5-7
41
3
15
15 °C
(20th somite forming)
18 °C
6°C
10-5
60
4
20
6°C
(5th somite forming)
20 °C
(5th somite forming)
20 °C
9-5
20 °C
2-9
' ]V' refers to the number of synchronous sibling larvae in each sample within an experiment. The exact stage of pattern formation at each temperature of pre-shock development,
in each experiment, is given. By t testing, only the post-shock temperature comparison at
20 °C pre-shock development in Exp. 1 was not significant at the 0-05 level. Significance of
difference according to post-shock temperature at 15 °C pre-shock development in Exps. 1
and 3, and according to pre-shock temperature in Exps. 1 and 4, exceeded the P = 001 level.
which is first to be laid down when development, including pattern determination, recommences.
Delay in development and pattern disruption are separable consequences of
temperature shock. After a 10 min, 35-5 °C shock, 2 °C lower than the normal
one, no abnormal pattern was seen in 20 larvae, despite a rather homogeneous
final delay in morphogenetic schedule of 4 | h (two formed somites).
Table 2 shows the results of experiments where two conditions were varied
between samples from a sibling population receiving the same shock at closely
comparable points in development (as monitored by visible somite formation).
These were: ambient temperature of development from early blastula stages
until shock, and ambient temperature of development in the period after shock.
Larvae were scored at an advanced but incomplete stage of pattern development,
so that the developmental delay incurred by shock could be computed in terms
of number of somites formed at the post-shock temperature of development,
and time taken to form each somite at that temperature. It can be seen that low
temperature of development after shock has a marked effect in decreasing the
Somitogenesis in amphibian embryos. Ill
113
Table 3
N
Temperature of
pre-shock and
post-shock
No. of somites
development formed at shock
Mean no. of somites'
Mean length of
delay in morphoabnormal zones
genesis, relative to
in somites-worth
sibling controls at
of tissue
15 °C, caused by shock
20
15 °C
4
2-9
70
20
13
41
7-5
By / testing, the comparison of abnormal zone lengths as between 4 and 13 formed somites
at shock was significant at the P = 001 level. The difference in mean final delay caused by
the shocks at the two stages did not approach significance.
amount of pattern made abnormal, provided that intrinsic sensitivity of the egg
clutch is such as to produce long zones of abnormality at normal laboratory
ambient temperatures. Ambient temperature up to the time of a particular
shock, however, is related in the opposite way to the amount of abnormal
pattern caused by that shock. The greater the difference between temperature
of prior development and that of the shock itself, the greater the pattern disruption effect tends to be.
A large batch of embryos was reared at 6 °C from the 2-cell stage and subjected to a 28 °C shock for 12 min (i.e. an equivalent temperature jump to a
37-5 °C shock experienced by embryos having developed at normal ambients in
these experiments). They showed no signs of abnormal somite pattern. Much
work on Xenopus and Rana indeed suggests a very steep relationship between
absolute temperature of a shock, around 36-40 °C, and the onset of developmental disruption (see, for example, the 35-5 °C shock with delay but no pattern
disruption, reported here). We cannot therefore suppose that the effect of preshock ambient temperatures upon abnormalities is due entirely to the difference
between the temperature to which embryos have become adapted and the high
one to which they are then acutely subjected.
In Table 3 and Fig. 2 results are presented of experiments where the critical
variable is the precise stage reached in pattern determination of the somite
series at the time of shock. For Table 3, samples were withdrawn from a
synchronous population, developing at constant ambient temperature, to
receive similar shocks (8J min) at two developmental stages 24 h apart. For
Fig. 2 a spread population of embryos was built up from one egg clutch by
releasing small groups of eggs from their very slow development through
cleavage stages at 6 °C, at a succession of times. They were then pooled to
develop together to tail-bud stages at 15 °C, when a single shock was experienced
by them all at a time corresponding to between 4 and 13 formed somites (or
48-72 h of adaptation to 15 °C). These procedures produced the expected range
of first abnormal somites, around 7-16, and in the experiment of Fig. 2 the
114
J. COOKE AND T. ELSDALE
O
11
0
o
o
O/
/
/
/
/
o
/°/ (9o
/
o
o
o
o
O
O
o
4 1st abnormal somite
16
Fig. 2. The correlations between developmental stage at shock and the length of
abnormal zones produced by 8 min at 37-5 °C, under two ambient temperatures of
post-shock development. Open circles, 18 °C post-shock ambient. Correlation
coefficient r = 0-88, significant at the P = 001 level. Filled circles, 6 °C post-shock
ambient. Correlation coefficient r = 0-60, significant at the P — 0-05 level.
effects of the shock were also examined after development at each of two postshock ambients.
Within these experiments all embryos therefore received the same shock, and
had adapted over long periods to the same pre-shock ambient temperature. In
what way is the amount of pattern made abnormal related to the position in the
body pattern where abnormality commences? Does the number of pattern units
or somites-worth of tissue affected remain constant, for instance, meaning that
a shorter tract of tissue is made abnormal by shocks experienced later where
each somite is made of fewer cells? The results show clearly that this is not so,
but that a given shock experienced later in the genesis of the body pattern
incurs a longer tract of disrupted pattern in terms of somite numbers. Although
the wavefront of morphogenesis slows down in terms of tissue (cells) traversed
per time as it progresses down the body, it proceeds at a uniform rate in terms
of somites formed (Cooke & Zeeman, 1976). This applies also to the earlier,
hidden wavefront, namely somite determination which occurs a set time before
actual somite formation at each body level (Elsdale et al. 1976; Cooke, 1977;
Pearson & Elsdale, 1979). The last results therefore mean that when shock is
experienced later in development, relatively more subsequent morphogenesis is
Somitogenesis in amphibian embryos. Ill
115
able to occur before the co-ordination among dynamic processes underlying
pattern determination returns to normal, as reflected in the regularity of the
pattern determined.
DISCUSSION
Experiments employing temperature shock have demonstrated the existence
of a wavefront of somite determination, advancing down the axis a set time in
advance of a further, visible wavefront, that of somite formation. The spatial
separation between the two wavefronts is given by the difference between the
number of somites already visible at the time of shock, and the ordinal number
of the first somite of the abnormal zone resulting from that shock (Elsdale et al
1976). In Rana, the separation is normally three somites-worth of material,
meaning that during most of morphogenesis, the boundaries on three more
somites than those visible have been determined, and this value is constant
except at the very beginning of the somite series (Elsdale & Pearson, 1979).
Observations in the present work indicate that it is also constant for a wide
range of ambient temperatures, although the absolute rates of passage of the
wavefronts are steeply temperature-dependent.
The most economical way of understanding these two successive wavefronts,
of determination followed by morphogenesis, is as expressions of two time points
in one intracellular process of development, which itself occurs according to a
timing 'gradient' along the body pattern to give the appearance of waves, even
though no truly wave-like propagation is involved at the time of expression
(a 'kinematic' wave). The spatially periodic somite pattern, whereby successive
similar-sized groups of cells participate in a similar sequence of activities to form
each somite, might then be due to regular punctuation of the progress of this
(otherwise smooth) developmental wavefront by its interaction with some other,
synchronized cellular activity within the tissue, having the character of a periodicity in effect if not in underlying mechanism (Cooke & Zeeman, 1976; Pearson
& Elsdale, 1979).
Previous work on post-neurula temperature shocks (Elsdale & Pearson 1979)
argues that the lengths of abnormal zones are a function of these two processes
recovering their co-ordination slowly after shock. The present effects, of postshock ambient temperature and of age at shock upon the lengths of abnormal
zones, offer further evidence for two component processes underlying the
genesis of normal somite regularity. They are normally co-ordinated, but
experimentally separable because their recovery after a temperature shock
responds differentially to ambient temperature of development and to stage of
development at shock. During recovery from shock, the travelling wavefront
appears to be resumed normally when development as a whole is resumed
whereas the resumption and intercellular co-ordination of the periodicity responds somewhat independently of development as a whole. The detailed
argument is as follows.
116
J. COOKE AND T. ELSDALE
On the general model for determination of somite pattern outlined above, rate
of recovery of normal co-ordination between periodicity and wavefront after
the shocks is less temperature-dependent than rate of development itself. At
lower post-shock ambients therefore, relatively less development takes place
with imperfect co-ordination; hence the shorter zone. Recovery of co-ordination
also appears to be relatively slower, in relation to the constant rate of pattern
determination (Elsdale et al. 1976) after shocks to later stages. Data in Tables 1
and 3 show that, after shocks of a particular severity, resumption of development at widely different temperatures or at different stages of development
requires very similar amounts of developmental time in terms of the incurred
morphogenetic delay (i.e. somite number x laboratory time taken to form each
somite at the temperature and stage concerned). If we did not know this we
might postulate that, at low post-shock temperatures (Table 2, Figure 2), all
aspects of development (wavefront and co-ordination) have recovered fully
before so much new post-shock pattern has been set up as is the case in siblings
at higher post-shock ambients. In fact, wavefront recovery requires a similar
amount of developmental time (in terms of number of somites determined and
made) at low temperature, but co-ordination recovery has been relatively faster
(i.e. less temperature dependent) thus exposing the embryo to less actual morphogenesis while in an abnormal state. Similarly, we might have postulated that the
effect whereby more pattern is disrupted by shocks to older embryos, affecting
posterior somites, is due to actually shorter delay in progress of the wavefront
despite a similar period for re-coordination, to produce the converse effect of
low ambient temperature. In fact, it must be the recovery period for co-ordination of periodicity which is longer, since the delay in wavefront is essentially
unchanged.
The spontaneous incidence of local abnormalities in control populations,
corresponding to minimal versions of the disruptions due to temperature shocks,
increases sharply with somite number posterior to a certain position in the body.
This suggests that the periodic component of pattern formation, or the cells'
response to it, is less well organized at later stages even in undisturbed development. This may ultimately help to explain the fact that following shock in later
development, after more repetitions of the periodicity have elapsed, the
developmental time taken to approach normal co-ordination is relatively
greater.
The effect of low post-shock temperatures in reducing the length of abnormal
zones can be understood by an example. Suppose that embryos are shocked when,
say, somite 10 is forming. Then when somite 17 has formed at the end of the
experiment in all embryos developed at low post-shock temperature, we note
that in the high post-shock temperature embryos, the tissue that would have
been incorporated into this somite is usually within the abnormal zone where
small groups of cells are isolated by chaotic fissures, or form a continuous tissue.
This is despite the fact that in the low-temperature embryos, there would have
Somitogenesis in amphibian embryos, III
117
been an equivalent amount of total developmental time (including arrest)
elapsed when the wavefront crossed the position of this somite. The return to
full function and intercellular co-ordination for the periodic process, leading to
spatially regular fissure formation, must therefore have a markedly flatter
temperature coefficient than does development as a whole.
The particular model presented elsewhere (Cooke & Zeeman, 1976; Cooke,
1975, 1977; Zeeman, 1975) proposes that this second, periodic component process in somite pattern formation may be a smooth biochemical oscillator,
phase-linked among cells of the embryo, and acting to advance and retard the
development of cell behaviour expressed in the wavefront. The present results,
and indeed all the results from heat shocks to date, are by no means proposed
as evidence for such a theory, but only for the more general model of interacting wavefront and periodicity.
The actual period length of any rhythmical process, measured in terms of the
progress of the determination wavefront, must remain highly constant throughout development. This requires a very similar temperature response by the two
processes. Only in this way could relative constancy of somite numbers across
developmental temperatures be achieved, meaning that a closely similar number
of cycles of the periodicity are incorporated into the passage-time for the
determination wavefront down the whole embryo whatever the temperature.
In this context, it is interesting to note that vertebrates such as Rana, adapted
to develop in small bodies of fresh water, show strong constancy of somite
numbers over wide developmental temperature ranges, whereas certain marine
fish on the one hand and birds on the other (Fowler, 1970; Lindsay & Moodie,
1967), adapted to more temperature-buffered development, show poor canalization of the total number of somites against artificial manipulation of temperature.
The number of hours disturbance to development in eggs of a given clutch,
after a given shock, can depend on the ambient temperature to which the embryos
were physiologically accustomed prior to shock (Table 2), as if some adaptation
of cellular machinery to low temperature rendered it more disruptible by brief
exposure to a high one. The literature on acute high temperature effects would
suggest that there tends to be a pronounced threshold for onset of damage
within a certain temperature range for each species, but with this proviso, the
dynamics of the rhythmic process itself must be more disruptible in some way
after pre-adaptation to the cold. There are observations (Fraenkel & Hopf,
1940; Hoar & Cottle, 1952) including some on developing systems, that suggest
that cellular adaptation to a range of temperatures occurs partly via systematic
alteration in the lipid composition, through altered balance in the metabolic
pathways that construct such lipids. If membranes are involved in rhythmical
aspects of cellular activity, as at least one general model for biological clocks
would suggest (Njus, Sulzman & Hastings, 1974), we might expect temperature
contrasts between ambient and shock to play a role.
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J. COOKE AND T. ELSDALE
The work is supported by the Medical Research Council.
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