/ . Embryol. exp. Morph. Vol. 50, pp. 155-161, 1979
Printed in Great Britain (g) Company of Biologists Limited 1979
155
Waves propagated during vertebrate development:
observations and comments
By ANTHONY ROBERTSON 1
From the Department of Biophysics and Theoretical Biology,
The University of Chicago
SUMMARY
The status and possible roles of propagated waves occurring during vertebrate embryogenesis are discussed. Some preliminary data for waves observed in time-lapse films of early
chick (Gallus domesticus) and Medaka (Oryzias latipes, a teleost) are given. The general
similarities between these phenomena and wave propagation during cellular slime mold
aggregation are pointed out and it is suggested that the control of development by propagated
waves in regulative embryos is not only widespread but also depends on a common cellular
mechanism.
INTRODUCTION
Two general mechanisms for supplying positional information (Wolpert,
1969) to cells in Metazoan embryos have been suggested. Both involve extracellular signalling, the first by a gradient in activity - e.g. in the concentration
of a molecule (Child, 1941; Crick, 1970) - the second by a propagated periodic
signal which might be by brief pulses of a small molecule, in analogy with
signal propagation, in the nervous system (Goodwin & Cohen, 1969). In both
cases the initial polarity of signalling (or sense of the gradient) is supposed to
arise from an inhomogeneity present in the fertilized egg. This might be represented by a gradient of cell size depending on differences in cleavage rate caused
by variations in yolk distribution, or, in general, a gradient of metabolic activity
with one end defined by a singularity related to the point of sperm entry. Thus,
both kinds of model depend on suitable initial conditions to, at least, define a
sense or polarity. Thereafter, the two mechanisms are quite different in kind.
If one allows the possibility of the control of development by a periodic signal
then it has also been argued (Cohen, 1971; Robertson & Cohen, 1972) that
morphogenetic movements should be observed, using time-lapse filming techniques, since movement is that cellular behavior occurring during development
whose time course and ease of observation are most likely to reveal the underlying dynamic of signals controlling development.
1
Author's address: Department of Biophysics and Theoretical Biology, The University of
Chicago, 920 E. 58th Street, Chicago, Illinois 60637, U.S.A.
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A. ROBERTSON
It is possible that the kind of signalling between cells in regulative metazoan
embryos represents an adaptation of a phylogenetically early mechanism
controlling cellular aggregation leading to the evolution of one branch of the
Metazoa. Although the Metazoa are polyphyletic in origin one way of producing a multicellular organism is clearly by aggregation of an initially homogeneous population of single cells, as is still true for the cellular slime molds.
There may, therefore, be more profound reasons for observing the dynamics of
cellular movement during early development in phylogenetically advanced
organisms. This point of view is reinforced by the identification of cAMP as
an aggregative attractant for some species of cellular slime mold (Konijn,
Barkley, Chang & Bonner, 1967; Konijn, van de Meene, Bonner & Barkley,
1968), by the growing evidence for an extracellular cyclic AMP signalling
system in the early chick embryo (Gingle, 1976; Robertson & Gingle, 1977;
Robertson, Grutsch & Gingle, 1978) and by the association with gastrulation
of acetylcholine in the Medaka (Fluck, 1978) and of acetylcholine and serotonin
in the sea urchin (Gustafson & Toneby, 1970). This suggests that small molecules, important for intercellular communication in adult Metazoa, may have
acquired similar roles early in eukaryotic evolution (Goodwin & Cohen, 1969;
Robertson & Cohen, 1972; Robertson, 1974).
The utility of observing cellular movements and analyzing their dynamics
has been amply justified by much experimental work on the cellular slime molds,
in particular Dictyostelium discoideum. Here observation of periodically propagated waves of cell movement during aggregation allowed strong inferences to
be drawn about the signalling mechanism controlling aggregation itself (Shaffer,
1962). Once the molecule propagated during aggregation was identified as
cAMP (Konijn et al. 1967, 1968) these inferences were quickly substantiated
and many details of the waveforms observed during aggregation could be
understood (Durston, 1973, 1974). Furthermore, it was shown, as had been
suspected (Bonner, 1949), that the signalling mechanism was retained throughout development and controlled multicellular morphogenesis as well as aggregation (Rubin & Robertson, 1975; Rubin, 1976). It is also interesting that both
gradients and propagated signals are used within the slime molds, the former
being associated with the most simple multicellular morphogenesis, as exemplified by D. minutum (Gerisch, 1968; Raman, 1976); and the latter with the
more complex morphogenesis of D. discoideum and of the Polysphondylium
species (Jones, 1976).
So far it has not been possible to supply such strong evidence for the control
of development by a propagated signal in metazoan embryos, although there is
much circumstantial supporting evidence, particularly for vertebrate embryos.
Many morphogenetic movements do, indeed, show a clear periodic component
(listed by Robertson & Cohen, 1972) and there have been reports of propagated
waves in the chick embryo (Robertson & Gingle, 1977; Stern & Goodwin,
1977). Stern & Goodwin inferred the presence of propagated waves by making
Propagated waves during vertebrate development
157
measurements of markers moving on the ventral surface of the embryo during
primitive streak formation, and Robertson & Gingle observed clear propagated
waves of refractive index change, often associated with cell movements counter
to the direction of propagation, at all stages of development to stage 12
(Hamburger & Hamilton, 1951, hereafter referred to as H-H). Films at later
stages have not yet been examined systematically.
In this paper I therefore report some measurements of propagated waves in
the chick embryo and in the embryo of the Japanese Medaka (Oryzias latipes),
comment on their general similarity and suggest a mechanism which could
account for their presence, based on that discovered in D. discoideum. The work
began when early casual observations (with J. Cooke) of wave propagation led
me, with my colleagues (A. Gingle and J. Grutsch) to begin a detailed analysis
of some morphogenetic movements in the early chick embryo. This paper is a
preliminary statement of the thought behind the experimental work, intended
to give a conceptual framework within which the results can be comprehended.
RESULTS
1. Materials and methods
Embryos. Chick embryos were obtained and filmed as described previously
(Robertson & Gingle, 1977), the only difference being that a x 10 water immersion objective was used to obtain the clearest films of propagating waves.
The use of a water immersion objective avoids multiple image formation by
refraction and reflection at the several interfaces inevitable when a dry objective
is used. Although waves can be seen with both optical systems they are clearest
with an immersion objective; presumably multiple image formation can lead to
phase averaging and degradation of the appearance of the waves. Embryos of
the Medaka (Oryzias latipes) were obtained by removing fertilized eggs from
female fish, kept at 26 °C, 1 h after the beginning of a 12 h light period and
were filmed by transmitted light while bathed in Yamamoto's solution
(Yamamoto, 1967). A dry objective was used, since it was not necessary to
cover the culture medium with a layer of mineral oil as is required for the chick
embryo, and the waves could be seen clearly under these conditions. For
filming, the embryos were at room temperature (21-23 °C).
Film analysis. 16 mm films, taken at frame rates of 8 and 15 per minute, were
analyzed with a computer-based system in which the observer uses an acoustic
stylus to enter coordinates of the object followed for each frame of the film
(Potel & Sayre, 1976, 1977). Data for wave periods and velocities were obtained
in this way and plotted as functions of developmental age. Some of the films
on which this paper is based were shown publicly at the EMBO meeting on
Oscillatory Phenomena in Biological Systems, held in Dortmund during 3-6
October 1976 (Boiteux, Hess & Plesser, 1977).
II
EMB
50
158
A. ROBERTSON
2. The chick embryo
(a) General observations
Several hundred embryos have been filmed for periods of up to 36 h beginning
at H-H stages 2-12. Visual evidence for periodicities in gross movement and in
local movements of cells was found at all stages. Propagating waves of
refractive index change within sheets of cells were also observed in some films
of allj stages, but their properties varied, although some general features
emerged.
(1) Waves always propagate counter to cellular movements when these can
be seen; they never pass through each other.
(2) Wave propagation may be quite local, particularly in regions where there
are high cell densities surrounded by regions of sparser cell density, for example,
close to the somites and in the mesoderm from which the endothelial layers of
the extra-embryonic vascular plexus is forming, close to the margins of the
emphalo-mesentoric veins (H-H, stages 10-13). I have seen both spiral and
circular waveforms.
(3) Aperiodic twitching of the whole embryo, reflected particularly by movements of the margin, occurs frequently, particularly in early (H-H 2-3) and
later (H-H 10-13) stages, when many pairs of somites have formed. These are
reminiscent of movements noticed by De Haan (personal communication). I
have also observed at all stages occasional cellular movements in the form of
twitches, in the area opaca, as well as occasional propagated waves.
(4) In most films, especially those taken at low power, individual waves are
not clear; instead waves appear to propagate in at least two layers, but in
different directions, giving a strong impression of a moving optical interference
pattern.
In so far as they overlap, my observations agree with those of Stern &
Goodwin (1977) except that I have not seen waves propagating at velocities
greater than about 250 /rni/min. They inferrred much greater velocities (up to
2400 /*m/min) from measurements of the displacement of carbon particles. My
embryos were not marked; it is possible that more refined measurements would
show their fast waves, or that their measurements involved points separated by
several wavefronts, since their description implies that the fast waves were not
directly visible. Their slow wave (~200 /*m/min) seems to be comparable with
those I have observed, as do the small cellular displacements both of us have
observed, counter to the direction of wave propagation. They also observed
larger transient displacements of up to 50 /tm.
All these essentially qualitative features of wave propagation have yet to be
analyzed quantitatively and their natural history described. Analyses under way
include measurements of the morphogenetic movements involved in Hensen's
node regression and somite formation; collection of data for propagating
waves; and assays of the coupling between cellular signalling mechanisms and
100
Period number
200
300
Fig. 1. (A) Wave period plotted as a function of wave number for waves propagating anteriorly and parallel to the axis in the
most ventral layers of a chick embryo explanted at stage 12. (B) Wave period plotted as a function of wave number for
waves propagating lateral to the somites of an explanted stage-12 chick embryo.
Period number
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A. ROBERTSON
cell and tissue movements, contact formation, and differentiation (Gingle,
1976; Robertson & Gingle, 1977; Robertson et ah 1978).
(b) Detailed observations
Figures 1A and B show periods observed between waves propagating in two
situations both in embryos at H-H stage 12 in which the clearest waves have
been seen. The waves from the embryo of Fig. 1A are illustrated in Fig. 2. For
Fig. 1A the microscope was focused on the most superficial ventral layer of the
embryo; for 1 B on a deeper layer including the core of somite number 17, and
the depth of field was restricted by the use of differential interference optics.
In the first case wave propagation began at the posterior margin of the area
pellucida and the waves propagated anteriorly parallel with the embryonic axis
and counter to the regression of Hensen's node and the segmentation of the
somites. In the second case the waves propagated from the somite margins
laterally, grossly normal to the embryonic axis, thus also at right angles to the
waves seen in the superficial layer of the embryo.
In both situations the first periods are relatively long - 8-12 min - but decay
smoothly to a fairly constant value of 2-4 min. Wave velocities are quite
constant, showing no clear age dependence. For 15 waves parallel with the long
axis the mean velocity was 203 /an/min, with a maximum of 253 /^m/min and
a minimum of 115/*m/min. For each wave velocity remained constant for
distances of up to 1-2 mm. For 5 waves lateral to the somites the mean velocity
was x 69 jLcm/min, with arnaximum of 105 /*m/min and a minimum of 40 /im/min
3. The Medaka embryo
Films were made of four embryos. All developed successfully to at least
stage 46 (heartbeat); filming was begun about 2 h after fertilization at stages
5-6. The stages are identified in accordance with the normal table of Kirchen
& West (1975). Up to stage 14 (24 h under our filming conditions) epibolic
movements are continuous with no signs of any gross periodicity. At about
stage 14 periodic twitches of the embryo begin. These are correlated with waves
Fig. 2. Stage 12+ chick embryo from which data shown in Fig. 1A were taken.
The waves shown were moving anteriorly. The wavefronts were superimposed
optically on those observed with the film running (see Potel & Sayre, 1977). The
technique was to stop the film, draw in one wavefront using computer graphical
techniques, run the film again, adjusting the overlaid drawing projected by the
computer, until it matched the observed wave, then returning to the same frame
and repeating the process for the other two wavefronts. The fronts shown in 2B
are those in 2A, 2\ min later. Although Fig. 2 was produced by reconstruction
from the moving film, the resulting pictures give a surprisingly good impression
of what is seen. With practice the position of a wave can be found in a 'still', but
the net change in brightness is too small for convincing reproduction. The length
bars represent 500 /tm.
Propagated waves during vertebrate development
Fig. 2, For legend see opposite.
161
162
A. ROBERTSON
i
I
1
1
i
- A
-
6 -
_
o A
-
•
5 -
4 -
_
A
A
A
-
-
A
A
o
3 -
i
e
2 -
A\
t
A A
A
A
©
1 -
i
i
i
i
10
12
14
16
Period number
Fig. 3. Wave pericd plotted as a function of wave number for two Medaka embryos,
beginning at stage 14. Embryo 1, A; embryo 2, # .
of constriction which propagate from the region of the blastopore out across
the whole blastoderm, but not over the egg itself. Wave propagation continued
throughout the next 24 h, up to stage 23 when filming ceased. The velocity of
the waves is quite constant, at 1500 /tm/min. The waves begin before involution
of the embryonic axis, which, once started, is very rapid. In Fig. 3,1 show plots
of period length versus period number of waves from two embryos, beginning
with the first contraction. As for the two examples from chick embryos, the
first periods are relatively long, but the period rapidly declines to about 2 min
at the 15th period and changes only slightly thereafter. Once involution is under
way it is difficult to tell whether or not wave propagation is initiated at the
anterior pole of the axis since it is masked by events occurring in the superficial
layers of the blastoderm and since the egg rotates within the chorion, which is
anchored to the substrate by the chorionic filaments. Nonetheless, good viewing
conditions occur occasionally in the films; it then appears that waves start
propagating along the involuting axis and emerge to propagate across the
remainder of the blastoderm. This point could only be settled unambiguously
Propagated waves during vertebrate development
163
by making films of dechorionated eggs using Nomarski optics to obtain a
shallow depth of field. The superficial waves on the embryos' surface are easy
to follow, particularly since both the involuting axis and the large oil globules
provide references.
DISCUSSION
The results I report, together with earlier observations of propagated waves
and periodic cell movements in many developing embryos (Gustafson &
Wolpert, 1967; Robertson & Cohen, 1972; Goodwin, 1976; Robertson &
Gingle, 1977; Stern & Goodwin, 1977) suggest that these phenomena must be
taken seriously as reflecting a developmental control mechanism and are not
merely adventitious. This view is strengthened by the similarities in the evolution
of the periodicities in both chick and Medaka embryos, which also compare
closely to those reported for the cellular slime mold, D. discoideum (Durston,
1974). Since we know that cells from the early chick embryo can release cAMP
signals on stimulation with cAMP it is likely that a common cellular mechanism
is responsible for the genesis and propagation of periodic waves (Goodwin,
1976; Robertson et al. 1978; Robertson & Gingle, 1979). This raises two
general questions: what might the function of propagated waves be and how
are they produced? If these can be answered, a third question may be asked:
how do these phenomena relate to what we know of the function of the vertebrate organizer (Spemann, 1938; Rubin & Robertson, 1975)? First, however,
I shall suggest reasons for failure to observe propagated waves and some of the
constraints which must be considered when we attempt to account for them in
detail.
2. Wave propagation
Wave propagation, at least in a developmental context, is best understood
for the D. discoideum aggregation signal. Here it is easy to measure periods,
wave velocities and signal amplitudes and thresholds. A detailed theory of
signalling during aggregation has been developed (Cohen & Robertson, 1971a,
b; Gingle, 1976). This has been tested in various ways (Gingle & Robertson
1976; MacKay, 1978; Grutsch & Robertson, 1978) and we can now be sure of
the variables important to the system, which may therefore be used as a
minimal model for wave propagation in embryos.
In D. discoideum wave period depends both on the autonomous period of
the cells which initiate signalling and the mean refractory period of the field
into which they signal (Durston, 1973; Robertson & Drage, 1975). Late in
aggregation the central portion (tip) of the aggregate releases a continuous
signal triggering relaying at the refractory period of thefield(Rubin & Robertson,
1975) which is by then about 2 min. Earlier, however, the period declines from
10 min or more and the periodic signals from centres may be gated by the
refractory period of the field (Durston, 1974; Robertson & Cohen, 1974). The
similarities between periods recorded from both types of embryo and from
164
A. ROBERTSON
D. discoideum suggest that similar mechanisms are responsible for their evolution.
These mechanisms, in turn, can result from the response functions of cAMP
release which are remarkably similar for slime mold amoebae (Grutsch &
Robertson, 1978) and chick embryonic cells (Robertson et at. 1978) and would
give rise, ultimately, to a continuous cAMP source with upper concentration
bounded-very like the regulating gradients of embryology (Wolpert, 1969;
Cohen, 1971; Crick, 1971).
Wave velocity in slime molds depends on the density of relaying competent
amoebae, the diffusion constant of cAMP, signal size, threshold cAMP concentration for inducing relaying and extracellular phosphodiesterase activities
which reduce signal range, as well as on the intracellular delay between stimulus
and response (Gingle, 1976). All these variables, or their equivalents, must be
considered in accounting for the details of chemical wave propagation in
embryos. The rate-limiting factor, in slime molds, is the delay time which was
estimated to be 15 sec (Cohen & Robertson, 1971a) and measured at between
6 and 16 sec, and found to be concentration dependent (Grutsch & Robertson,
1978).
Since propagated waves have been seen in several situations in embryos we
must consider why they have not been observed more frequently. There may be
several reasons. Firstly, their observation requires filming at the appropriate
frame rates and under the right optical conditions. Secondly, propagation may
occur in dense sheets of cells and result in small net movement, or rearrangements, as suggested by Fristrom (1976) during evagination in Drosophila
imaginal discs. Thus, the net change in refraction may be very small and
propagation only inferred by indirect measurement (cf. Stern & Goodwin,
1977). Thirdly, as my results show, it is possible for waves to propagate in
different directions and different cell layers of chick embryos of the same age.
Thus, waves may cancel each other out optically, or even produce interference
patterns. Again, proper optical techniques must be used to reveal wave propagation unambiguously in these situations.
The discrepancies between early (Stern & Goodwin, 1977) and the later wave
velocities reported here are probably real. If so, they might indicate two different
modes of propagation. A velocity of 100/«n/min implies, for a mean cell
diameter of 5 fim, a delay time of 3 sec. This is consistent with the evoked
release of cAMP for early chick embryonic cells which is completed within less
than 5 sec. A velocity of 2400/mi/min implies a very brief ( ^ 1 / 8 sec) delay.
This seems unreasonably short, suggesting that the effective range of the signal
is much greater than a cell diameter, perhaps 60 jLtm, which is consistent with
the displacements (~50/«n) observed by Stern & Goodwin. One explanation
might be that the early waves are propagated by an extracellular signal, while
the later waves are carried through intercellular junctions. This could account
for the presence of waves in different cell layers propagating without direct
interference.
Propagated waves during vertebrate development
165
3. The functions of propagated waves
Goodwin & Cohen (1969) were the first to suggest that propagated waves
might supply positional information to cells in embryos. While the detailed
model they proposed may not be relevant to the subject of this paper they did
show ways in which propagated waves could account for many properties of
embryonic fields. In some slime molds propagated waves undoubtedly control
cell movements, and probably some cellular differentiations (Darmon, Brachet
& Pereira da Silva, 1976; Gerisch, Fromm, Huesgen & Wick, 1975). Such
functions are likely for propagated waves in the early embryo where cell movement is particularly important. Two virtues of propagated waves, when compared with concentration gradients set up by diffusion, are the greater efficiency
of a pulsatile signal both in terms of the drain on metabolism and of the all-ornothing nature of the signal which makes it unambiguous, and the greater
distance which can be covered by a signal within a fixed period. While these
benefits are particularly important for the control of aggregation by initially
dispersed cells, they may also be important within the embryo.
A second function may arise in situations where there is an abrupt change
of the geometry of the embryo, as, for example, during the imagination of
gastrulation and the evagination of imaginal disc development. In these
situations sheets of cells must undergo a large and rapid change in their radius
of curvature at a point or along a margin. Here, the periodic twitches evoked
by a propagated signal would help to surmount the potential energy barrier
associated with a sharp change in sign of the radius of curvature. This may
account for the pulsatile movements seen at the region of the sea urchin embryo
where the archenteron tip is about to invaginate, as well as for those associated
with gastrulation in the chick (Stern & Goodwin, 1977) and in the Medaka.
This is not to suggest that propagated waves evolved with this function, but
that an existing system is reflected during its performance (see Robertson &
Cohen, 1972).
4. Wave propagation and organizers
The data I have given in this paper support the analogy between wave
propagation in slime molds and in vertebrate embryos. Robertson & Gingle
(1979) developed this analogy further to produce a working hypothesis for the
development and functioning of the organizer. The new results here which add
to the model are the general similarities between the period v. period number
functions, the propagated nature of the signals and the evidence for the initiation of wave propagation at developmental stages where gross geometrical
changes are about to occur. From this point of view the organizer is a pacemaker,
as suggested by Goodwin & Cohen (1969). The new feature is that, as for
D. discoideum, the pacemaker is a continuous source of a regulated signal which
can entrain sensitive cells within its range. Features of both gradient and signal
propagation models are therefore retained in this view of the nature of the
166
A. ROBERTSON
organizer. While the generation of periodic signals probably always involves
fluctuations in intracellular cAMP levels, the signal itself, while in some cases it
is definitely an extracellular pulse of cAMP, may be another molecular species,
such as a neural transmitter, acting through a membrane receptor coupled to
intracellular adenylate cyclase.
This work was supported by a Grant-in-Aid from the Alfred P. Sloan Foundation and by
NIH grant HD 04722. I am grateful to Sally Hoskins for filming the Medaka embryos, to
J. Irwin, L. King, S. A. MacKay and M. J. Potel for data analysis, and to A. Gingle and
J. Grutsch for comments.
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{Received 1 August 1978, revised 13 November 1978)