J. Embryol. exp. Morph. Vol. 34, 3, pp. 559-574, 1975
Printed in Great Britain
559
Mechanical stresses and morphological patterns in
amphibian embryos
By L. V. BELOUSSOV 1 , J. G. DORFMAN 1 AND
V. G. CHERDANTZEV 1
From the Department of Embryology, Moscow State University
SUMMARY
1. Embryos of Rana temporaria have been dissected and shape alterations of different
parts of the embryo, taking place within 1 h of separation, have been studied. Two categories
of deformation have been revealed.
2. The first category comprises those deformations which take place immediately after
separation. They are insensitive to cooling, cyanide and Cytochalasin B treatment. These
deformations, which consist of a shortening of initially elongated cells, are considered to be
the passive relaxations of previously established elastic tensile stresses.
3. Deformations of the second category proceed more slowly. They are inhibited by
cooling, cyanide and Cytochalasin B treatment, are accompanied by elongation and migration of cells and occasionally lead to rather complex morphodifferentiations of isolated
fragments. These processes are considered to be the result of the active work of intracellular
contractile systems, either pre-existing or induced de novo.
4. By analysing the arrangement of the passive deformations we have constructed maps
of mechanical stresses in embryos from late blastula up to the early tail-bud stage. At several
embryonic stages drastic transformations of the stress pattern occur, these transformations being separated by periods during which the pattern of stress distribution remains
topologically constant.
5. A correlation between the arrangement of stress lines and the presumptive morphological
pattern of the embryo is pointed out.
6. Some possible relations between tensile tissue stresses and active mechanochemical
processes are discussed.
INTRODUCTION
The role of mechanical stresses in the orientation of cell movements in tissue
culture was established long ago (Weiss, 1929), but the application of similar
principles to morphogenetic processes in entire embryos has been delayed by
lack of data on the existence and localization of stresses in intact tissues.
This gap has recently been filled by the demonstration of intracellular contractile
systems in developing rudiments (Baker & Schroeder, 1967; Wessels et al.
1971;Burnside, 1971).
Similar contractile fibrils have been revealed in cells of the distal regions of
the actively growing hydroid polyps (Hale, 1960). Periodical contractions of
1
Authors' address: Department of Embryology, Moscow State University, Moscow
117234, USSR.
560
L. V. BELOUSSOV AND OTHERS
these fibrils gave rise to pressure stresses in cell layers. Growth, morphogenetic
shape alterations and some kinds of rudiment interactions in these species were
considered to be directly determined by these stresses (Beloussov & Dorfman,
1974).
These data emphasize the role which mechanical stresses may play in regular
geometrical alterations occurring during development. Therefore, it seems
desirable to study them in other developing systems. The simplest method of
revealing mechanical stresses in embryos consists of detecting the tissue deformations just after dissection. These deformations may be considered as direct
manifestations of pre-existing stresses. A number of scattered data on the existence of similar deformations have already been obtained. These are: contraction
of dissected blastomere furrows in Loligo eggs (Arnold, 1971); contraction of
dissected periblast in Fundulus eggs (Trinkaus, 1969); shape alterations of
isolated vegetal parts of sea-urchin blastula (Moore & Burt, 1939); and, what
pertains closely to this study, the intensive rolling of the layers of dissected eye
vesicle in amphibian embryos (Lopashov, 1963).
A similar dissection method, combined with the action of some physiological
agents and followed by a histological study of intact and separated tissues, has
been employed in this work. As a result, a fairly regular pattern of mechanical
stresses has been revealed in amphibian embryos from late blastula up to early
tail-bud stages. Maps of mechanical stresses have been constructed for successive
developmental stages, representing the lines of tensions and the points of branching of these lines. Besides the above mentioned 'immediate' deformations, whose
passive relaxatory characters have been confirmed by their non-sensitivity to
some inhibitory agents, another class of slower (although still complete within
1 h) and much more sensitive deformations has also been observed in separated
tissues. The latter processes were obviously active, leading in several minutes
to the creation of new tensile stresses and to rather complex morphodifferentiations of a fragment. It is proposed that they may be considered as simplified
models of normal morphogenesis.
MATERIALS AND METHODS
The majority of the experiments have been made on Rana temporaria embryos
from late blastula up to early tail-bud stages. Several operations were made also
on Rana esculenta and Xenopus laevis embryos of the same stages. The experiments consisted of complete isolation, three-side separation or dissection of
different parts of embryonic tissues. Under physiologically-normal conditions
embryos and separated fragments were kept at 18-20 °C in normal Holtfreter
solution twice diluted (several operations were performed in full-strength
Holtfreter solution and gave the same results). The influences of factors inhibiting
metabolism (cooling, moderate doses of KCN) and of Cytochalasin B upon
rapid deformations have also been studied. In cooling experiments fragments
Mechanical stresses in amphibian embryos
561
and donor embryos were kept in the same solution at 2-5 °C for not less than
10 min before operation, during the operation proper and throughout the whole
period of observation. KCN was used in a similar way in concentration 10 mg/
ml ( 1 0 ~ 4 M ) . In Cytochalasin experiments donor embryos and fragments were
placed in 0-2 mg/ml ( 0 - 4 X 1 0 ~ 6 M ) solutions of Cytochalasin B in dimethylsulphoxide (DMSO). The DMSO concentration was 0-4 % aqueous solution.
Control embryos were placed in DMSO solutions of the same concentration.
Deformations of tissue fragments have been studied both visually and by
means of time-lapse filming (35 mm film, exposure interval 1 sec). The rolling-up
of the isolated fragments with their external surface outside was designated as
positive rolling and that with external surface inside as negative rolling (or
bending, folding). A surface facing the gastrocoel cavity was considered as an
external one.
For histological purposes embryos or tissue fragments were fixed in Bouin's
fluid, totally stained with borax carmine, dehydrated in butyl-alcohols and embedded in paraffin-wax. Special attention was paid to the possible deformations
of separated fragments during fixation and subsequent treatment. In the great
majority of cases no significant post-vital deformations were observed.
The following well-known features of the histology of amphibian embryos
are relevant to this study. The ectoderm is bilayered, consisting of external
epiectodermal and internal hypoectodermal layers (terminology, from Detlaff,
1938). The hypoectoderm of the neural plate is several cell layers thick, whereas
in other regions it is a single cell layer. As a rule, the adjacent rows of epi- and
hypodermal cells are staggered, so that, in a section, each hypodermal cell is
opposite the junction between two epi-cells. This regular arrangement is disturbed in several narrow zones, most of which correspond to slight ectodermal
folds to be described in detail later.
RESULTS
Immediate deformations
External appearance of the immediate deformations
(1) Late blastula stage (Fig. 1: 1 A). An extirpated and dissected fragment of
the marginal zone immediately unfolded to an angle of 90° approximately. An
isolated superficial layer of the vegetal hemisphere bent slightly in the negative
direction (Fig. 1: 1B). No significant deformations took place in other regions.
(2) Middle gastrula stage (Fig. 1: 2 A). When any sector of the marginal zone
(lip of blastopore) was extirpated and separated in caudal direction (towards the
lip) the fragment walls immediately opened out to about 45-90° (Fig. 1: 2 A-B,
cr-caud). When the fragment was separated in a reverse, cranial direction
(away from the lip) the divergence angle did not exceed 10-20° (Fig. 1: 2 A-B,
caud-cr). When separated from the external layer, the already invaginated
material slightly expanded.
562
L. V. BELOUSSOV AND OTHERS
B
C
I
D
I
CSS®
Fig. 1. For legend see opposite page.
The surface cell layer of an entire gastrula, when separated in the immediate
vicinity of the marginal zone, immediately bent as shown in Fig. 1: 2B, epi. In
other areas no significant deformations were observed.
(3) Late gastrula stage (blastopore almost closed) (Fig. 1: 3 A). The results of
caudal separation of the dorsal lip zone were the same as in the preceding stage,
but now the angle of the opening of the fragment walls was also the same during
Mechanical stresses in amphibian embryos
563
D
postbrf.
(5)
prebrf.
(5)
Fig. 1. Rapid deformations after dissections of amphibian embryos, (A) 1-12,
schemes of separations; (B) immediate deformations; (C) deformations taking
place 1-5 min; (D) 5-20 min; (E) 20-60 min after separation. Black wedges
indicate directions of cutting; numbers in parentheses denote how many operations
of each type were made. Dotted outlines in column A, lines 1-3, 5-8, indicate the
areas extirpated; 6-8, latent deformations of the same directions as the preceding
immediate deformations, caud.-cr., Caudo-cranial; cr.-caud., cranio-caudal;
chin., chordamesoderm; ent., entoderm; epi., epiectoderm; hyp., hypoectoderm;
npl., neural plate; postbrf., postbranchial fold; prebrf., prebranchial fold; snf.,
subneural fold. For other designations see text.
36
EMB 34
564
L. V. BELOUSSOV AND OTHERS
cranial separation, revealing a new tensile stress anterior to the lip. Dorsal
epiectoderm, when separated in the medial direction, now revealed a slight but
distinct negative bending (Fig. 1:3B, epi). A similar separation of the chordamesoderm led to its immediate negative rolling (Fig. 1:3B, chm). The same
was true of the ventral gastrocoel wall (Fig. 1:3B, ent).
(4) Early neurula stage. By this time some earlier tendencies have been
reinforced and several new areas of rapid deformation have arisen.
(4a) Anterior (hind-brain) area (Fig. 1:4A). Medial separation of neural
epiectoderm led to a sharp negative bending, localized somewhat laterally to
midline (Fig. 1: 4B, epi). Neural hypoectoderm bent during a similar operation
to a smaller extent.
At this stage the neural plate becomes limited laterally and anteriorly by a
shallow ectodermal fold which can be designated as the subneural fold (Fig.
1:4A, snf). This fold became much more pronounced immediately after
separation of its epiectoderm (Fig. 1:4B, snf). Medial separation of the
chordamesoderm also led to its extensive negative bending close to the midline
(Fig. 1: 4B, chm).
(Ab) Posterior (trunk) area (Fig. 1:5A). A similar separation of neural
epiectoderm also led to its negative bending, localized in this case just at the
midline (Fig. 1: 5B, epi). Neural hypoectoderm bent in the same direction,
but to a less extent (Fig. 1: 5B, hyp). The chordamesoderm behaved as in the
anterior area. So far no significant deformations were revealed in the subneural
fold region at that level.
(4 c) Deformations observed during longitudinal (cranial and caudal) separations at early-middle neurula stages (Fig. 1: 6 A). Cranial dissection of the hind
part of the dorsal embryo wall resulted in extensive divergence of neural plate
and chordamesoderm. In the head region a similar caudal separation led to an
extensive negative bending of the chordamesoderm (Fig. 1: 6B). After extirpation of the whole dorsal embryo wall the initial slight positive bending of the
neural plate increased to a certain extent (Fig. 1: 6B, npl). The completely
isolated chordamesodermal layer reproduced all the bendings observed during
partial separations.
(5) Middle-late neurula stage: deformations during transversal separations
(Fig. 1: 7A). As in the preceding stages, medial separation of the epiectoderm
of the invaginated neural plate led to its extensive negative bending, whereas
that of the neurohypoderm resulted in slight negative bending. Separation of
subneural fold tissues (including mesoderm) resulted in their negative bending
(Fig. 1: 7B, right side). A similar result was observed at early tail-bud stage.
(6 a) Early tail-bud stage: dissection of a just closed neural tube (Fig. 1: 8 A).
Dissection of the neural tube along its midline led to the immediate divergence
of its walls. A similar result was obtained after transverse cutting of a tube
wall (Fig. 1:8B).
(6 b) Early tail-bud stage: separations in horizontal (frontal) directions
Mechanical stresses in amphibian embryos
565
(Fig. 1: 9 A). At this stage several new transversal ectodermal folds appear in
addition to the subneural folds, namely postbranchial, separating branchial and
trunk regions, prebranchial, separating branchial and head region, oral fold
(mouth rudiment) and two less-pronounced folds, separating the rudiments of
the branchial protuberances. When separated as shown at Fig. 1 (6 A), all these
folds immediately became much more pronounced (Fig. 1: 9B). This was also
true of the corresponding entodermal folds (Fig. 1: 9B, ent).
(7) Immediate deformations of non-folded areas of ventral and lateral ectoderm
and mesoderm (Fig. 1: 10-12A). The behaviour of these tissues did not alter
significantly during the whole developmental period under study. Ectodermal
fragments, excepting those taken anteriorly to the prebranchial fold (from the
presumptive oral field), contracted approximately to half their size and often
slightly rolled in a negative direction, thus forming a shallow cup (Fig. 1: 10B).
Considerable tension was shown to be present in the presumptive oral field
ectoderm, since its epilayer did not contract much after dissection whereas the
hypolayer did. Anterior to the prebranchial fold from the early tail-bud stage
onwards, and anterior to postbranchial fold from the middle tail-bud stage
onwards, immediate positive rolling of the epiectoderm was observed (Fig. 1:
9B, lower embryo wall).
In purely mesodermal and combined ectomesodermal fragments no significant
immediate contractions or other shape alterations were observed (Fig. 1:
11-12B).
Cooling by up to 2 °C did not influence the deformations listed in paragraphs
3-7 above in any way. Negative bendings of neuroectoderm occurred also in
KCN solutions of the concentrations employed. The opening angle of the dissected gastrula marginal areas slightly decreased after cooling. It is worth
mentioning that in Cytochalasin B solutions, deformations 4-7 above were
completely retained and were even more pronounced than normal. This result
contrasts sharply with those described below where slower deformations are
inhibited completely by the same agent.
During immediate deformations the previously elongated and/or oblique
cells contracted into rectangular and sometimes even spherical shapes. The
sharpening of pre-existing negative folds and similar bendings in other regions
seem to be a direct result of the mechanical pressure of these contracted cells
upon the adjacent ones.
Slower ('latent'') deformations (1-60 min after separation)
The immediate deformations described above were stable only at low temperatures and under the influence of Cytochalasin B. Under normal conditions
the shape of the fragments seen 0-5-1 min after separation changed again after
a further interval. These rather complicated morphological events of about
1 h duration are what we call latent deformations. They in turn fall into two
categories.
36-2
566
L. V. BELOUSSOV AND OTHERS
(A) Latent deformations in the same direction as the preceding immediate deformations (Fig. 1, lines 2, 6, 7, 8C-E, framed pictures)
(1) Prolongation of divergence of dissected marginal area walls. Under normal
physiological conditions the immediate opening of the dissected blastopore lip
was followed by a slower divergence of its walls, leading in 20-30 min to the
complete flattening of the fragment (Fig. 1: 2B-D). At the former tip of the
lip a new blastopore arose (Fig. 1: 2E).
(2) Foldings and closure of neural rudiments. The entire neural plate, extirpated in the early neurula stage, did not significantly alter its shape in 1 h
(Fig. 1: 5B-E, npl). On the other hand, the anterior part of the neural plate,
extirpated in the middle neurula stage, began to fold continuously along its
midline; and in 1 h it had almost closed (Fig. 1: 7B-E, npl). At the same time,
the neural plate bent transversely, thus increasing the sharpness of the immediate
bend already there (Fig. 1: 6 compare B and D).
The walls of the dissected neural tube, after their immediate divergence,
slowly converged in several cases, completely closing again in 15-20 min. In
most cases, however, the dissected tube walls continued to diverge (Fig. 1: 8D).
All the processes described are obviously similar not only to the preceding
immediate deformations but also to the corresponding normal morphogenetic
processes. We believe this to be true not only for the closure of the neural
rudiments but also for the opening of the blastopore lips, since the latter process
may play a significant role in normal gastrulation, promoting the invagination
of cell material. It can be deduced therefore that these latent deformations are
determined by certain pre-existing mechanisms rather than induced by the
operation itself.
(B) Latent deformations of opposite direction relative to the immediate deformations
(1) Deformations of ectodermal fragments. Under normal conditions 0-52 min after extirpation all the ectodermal fragments, including the neuroectoderm, began to roll intensively in the positive direction, starting from the free
edge (Fig. 1,10C-E). The rolling was unequal, with sharp curvatures alternating
with practically flat areas. In 20-40 min the rolled fragments were subdivided
by narrow furrows, oriented in most cases transversely to the rolling axis
(Fig. 1: 10D). After a time some of these furrows disappeared whereas others
stabilized (Fig. 1: 10E).
(2) Deformations of purely mesodermal and ectomesodermal fragments
(Fig. 1: 11-12C-E). One to 2 min after separation the naked surfaces of the
fragments began to contract, thus leading to the bending of sufficiently large
fragments (Fig. 1: 11C). In 10-20 min the smaller fragments transformed into
smooth spheres (Fig. 1: 12D, a), the somewhat larger ones formed a single
invagination, and the largest ones were subdivided by several furrows, oriented
Mechanical stresses in amphibian embryos
567
Table 1. Numbers of internally (int.) and externally (ext.) situated cells in mesodermal fragments 3 min and 30 min after fragment isolation (counts on several
median sections)
Number of
fragment
1
2
3
4
5
6
Time after
isolation (min)
3
3
3
30
30
30
Absolute numbers
int./ext.
(
A
f
int.
ext.
75
60
33
310
392
182
264
145
158
205
339
264
O/\
\ /o)
28
41
21
146
116
70
radially and localized in the marginal area of the fragment (Fig. 1: 11D, 12D,
c, 12 E). During these transformations the cells contacting the naked surfaces
elongated perpendicularly to them. Later on some of the cells obviously migrated inside the fragment, an indication of this being the number of cells situated
outside and inside at different times after separation (Table 1). Formation of
radial furrows began from the immigration of individual epiectodermal cells
into the hypoderm. Some of these cells later on were connected to the opposite
(naked) surface by elongated cells. In such a way the marginal area of the fragment separated into several parts which rapidly rounded and became almost
completely isolated from each other (Fig. 1: 12E).
If a fragment included a piece of an intact ectodermal fold (for example,
subneural), it separated along this fold much more rapidly than in its absence
(Fig. l : 7 C , f l ) .
The following simple experiment demonstrated the rise of new mechanical
stresses during latent deformations. When dissecting an ectomesodermal fragment parallel to its surface immediately after its extirpation, no additional
divergence of dissected edges, i.e. no relaxation movements, were observed
(Fig. 1: 12B). However, 10-15 min after separation, a similar dissection led to
the immediate extensive rolling of the mesodermal layer (Fig. 1: 12 C, a), as
well as of the marginal area of the ectodermal layer (Fig. 1: 12 C, b). Both
deformations seem to be determined by the contraction of the previously
stretched transverse cell walls. As with other immediate deformations, these
were not influenced in any way by cooling.
(3) Action of inhibiting agents on latest deformations. All the latent deformations were inhibited by cooling, KCN-treatment and Cytochalasin B. Under
these influences the fragments either remained flat or retained the folds established immediately after separation. The action of cooling and Cytochalasin B
was completely reversible, whereas that of KCN was irreversible. Cytochalasin
B led also to a slow unrolling of already rolled ectomesodermal fragments.
568
L. V. BELOUSSOV AND OTHERS
Moderate cooling (7-10 °C) promoted the irregular furrowing and subdivision
of ectodermal and ectomesodermal fragments. DMSO in the concentrations
employed had no significant influence on the processes under study.
DISCUSSION
Passive and active deformations
It may be seen that all the rapid deformations described may be naturally
divided into two categories. The first one comprises those deformations which
take place immediately after separation, are at the same time insensitive to
the inhibiting influences employed, and consist in shortening of the previously
elongated and/or oblique cells. Deformations of the second category on the
contrary proceed more slowly, are highly sensitive to inhibiting agents and are
accompanied by elongation and even migration of cells. Thus the first category
may be considered to be passive elastic relaxations of pre-existing stresses
whereas the second are active, energy-requiring processes, obviously connected
with the contraction of microfilaments. On the other hand, some of these
latter processes are due to pre-existing active mechanisms (morphogenesisimitating latent deformations; see Fig. 1C-E, framed pictures), whereas the
others are non-specific and are obviously caused by contractile systems activated
by the operation itself (see also Burnside, 1972, 1973).
The active deformations may be of interest as simplified models of morphogenetic processes, since they demonstrate rather rapid and complex morphodifferentiation and provide useful information on the mechanism of activation
of intracellular contractile systems. In this paper, however, the first category
of deformations will be mainly discussed in so far as it may be of use in constructing maps of the mechanical stresses existing over successive periods in the
development of amphibians.
Maps of mechanical stresses
To validate the employment of immediate deformations for the construction
of stress maps the cellular basis of these deformations must be discussed in
greater detail.
The existence of immediate relaxatory movements indicates that the cells of
amphibian embryonic layers are considerably deformed by the adjacent cells.
The layers as a whole are also deformed by the surrounding tissues. The mechanically stable cell shape achieved after separation is approximately cuboidal
when the integrity of the cell layer is not disturbed and approximately spherical
when cell connexions are disturbed during the operation, or in the regions
without a regular layer structure.
Let us consider now several somewhat idealized cases of relaxatory movements
and their interpretation (Fig. 2).
(1) Extirpation of fragments leads to their considerable shortening without
Mechanical stresses in amphibian embryos
569
.QrP
V>}
* * * * *
fh
i
\ \ \ \ \ vvv\
r
Fig. 2. Main types of immediate relaxatory movements in cell layers. Dissections are
indicated by dotted lines, stretched surfaces by heavy lines, the surfaces elastically
relaxing after dissections by asterisks. In E the largest angle a corresponds to the
greatest elastic contraction at left from dissection point. For other designations
see text.
570
L. V. BELOUSSOV AND OTHERS
G
E
C
Fig. 3. Maps of mechanical stresses for several successive stages of Rana temporaria
development and for a typical ectomesodermal fragment. (A) Late blastula;
(B) mid-gastrula, sagittal section; (C) same stage, transversal section (along the line,
indicated on B); (D) transition from gastrula to neurula, posterior region; (E) anterior region of early neurula; (F) posterior region, same stage (D-F - transverse
sections). (G) Early-middle neurula, sagittal section; (H) middle-late neurula,
frontal section; (I) similar stage, transverse section. (J) A typical ectomesodermal
fragment, 10-15 min after its isolation. Heavy contours represent distinct stress
lines; dotted contours, dispersed stress-lines; fine lines, non-tense surfaces,
separating embryo layers; pre-fo\d, corresponding to plica rhomboencephalica,
pe\<-fo\d, corresponding to plica encephali ventralis.
any bending (Fig. 2 A). This indicates that both surfaces were elastically stretched
to the same extent.
(2) A similar operation leads to negative bending without any shortening of
lateral cell walls (Fig. 2B): the elastic stretching of the external surface was
greater than that of the internal surface.
(3) A similar operation leads to sharp negative bending with considerable
shortening of the lateral cell walls, up to complete cell rounding (Fig. 2C):
both external and lateral cell walls are stretched.
(4) Extirpation of an almost flat fragment leads to its strictly localized negative folding (Fig. 2Dj), whereas dissection of an initially bent rudiment leads
to its unfolding (Fig. 2D 2 ); both processes are accompanied by extensive contraction and rounding of initially stretched cells underlying the folded zone.
This indicates that, along with the tension of external and internal surfaces,
there exist tension line(s) going down from the fold and crossing the cell sheet.
Mechanical stresses in amphibian embryos
571
(5) Three-sided separation leads to a sharp negative bending localized exactly
at the border between separated and non-separated areas but without any
definite prelocalization in the intact embryo (Fig. 2E). This demonstrates the
tension of the external surface of the separated zone and the existence of an
elastic contraction zone somewhere near the zone of separation.
(6) A similar operation leads to negative bending (Fig. 2F) or rolling (Fig.
2G) of the separated area accompanied by initially oblique cells becoming symmetrical. This indicates the uneven stretching of both external and internal
surfaces and the elastic stretching of lateral cell walls.
Hence all the above deformations point to the stretching of at least one of the
layer surfaces, whereas deformations (3), (4), (6) indicates the existence of tension
lines which cross the cell layer(s) (cross-lines). In other words, in the latter
cases, the bifurcation of tension lines takes place at the corresponding points.
The maps constructed by these methods are presented at Fig. 3A-J. The
distinct lines of tensile stresses are indicated by heavy lines, those gradually
dispersing throughout the tissues by dotted lines and non-tense surfaces by
fine lines. The following general properties of stress patterns are to be emphasized. At any developmental stage the tension lines, including cross-lines, are
concentrated near to the restricted number of closed surfaces subdividing the
embryo. This tensile pattern does not alter gradually in the course of development. Instead it remains topologically constant for a certain finite period of
development, and then drastically transforms. The transformations comprise
the appearance of new cross-lines as well as (more rarely) the disappearance of
some old ones. The periods of development between the two next topological
transformations may be designated similarly as topologically invariant periods.
Successive topological transformations for the given period of Rana temporaria development are as follows:
(1) The establishment of fairly wide strips of stretched cells between the
blastocoel corners and the vegetal surface of the late blastula (Fig. 3 A). Their
positions correspond to the marginal zone of the gastrula.
This stress pattern is not changed qualitatively during gastrulation, although
stretching of the dorsal ectoderm increases (Fig. 3B, C). In the regions removed
from the blastopore, the stress pattern is circular and is localized mainly in the
ectodermal layer (Fig. 3 C).
(2) The next and perhaps the most important topological transformation
is that the circular stress breaks either exactly along the dorsal midline (in the
posterior region) or parallel to it (in the anterior region). Now the external
circular stresses pass along these lines to the archenteron roof and spread
ventrally, joining stress lines coming from the gastrocoel angles and then gradually dispersing (Fig. 3D). This transformation may be considered as the
demarcation point between gastrulation and neurulation.
(3) Shortly after, in the early neurula stage, the stretched cells of the dorsal
epiectoderm and mesoderm establish new contacts with the lateral embryo
572
L. V. BELOUSSOV AND OTHERS
surface just ventrally to the neural plate. This contact line corresponds to the
subneural fold which encircles the embryo laterally and anteriorly. Now several
other obliquely situated cross-lines dissect the neural plate (Fig. 3E,F). Thus,
instead of extended gradually dispersing tension lines, a number of closed
tense contours appear.
(4) In the early-mid neurula stage the neural plate together with archenteron
roof becomes dissected by a number of new cross-lines (Fig. 3 G; compare with
Fig. 1, 6B). Several fairly irregular indistinct cross-lines are localized in the
caudal region and one especially pronounced cross-line is found at midbrain
level (Fig. 3 G, pre-pev).
(5) In late neurula-early tail-bud stage several new transversely oriented
cross-lines arise in the anterior body region, joining post-, prebranchial and oral
folds with the corresponding folds of the oral ectoderm (Fig. 3H). Later on
similar cross-lines dividing the rudiments of the branchial arches appear. On
the other hand, the cross-line connecting the subneural folds and neural groove
almost disappears during neurulation and is replaced by a line localized slightly
ventral and going along the roof of the intestinal cavity (Fig. 31).
According to the dissection results described above, the tensions of the
presumptive oral field ectoderm are localized mainly in its hypo-layer, the
episurface bearing no considerable tension (Fig. 3H).
A similar map for a typical ectomesodermal fragment, 10-15 min after its
extirpation, is presented at Fig. 3 J.
The presumptive significance of stress cross-lines: possible relations between
mechanical tensions and the active mechanochemical processes in cells
It is easy to see that almost all the cross-lines revealed in intact embryos are
of clear morphological significance. Indeed, stress lines established in the late
blastula outline the marginal zone of the gastrula; those established during the
second topological transformation correspond to the neural groove and separate
the archenteron roof into its chordal and mesodermal parts. The third topological
transformation leads to complete separation of the neural plate from the more
ventral regions, whereas a set of transformations (4) results in the differentiation
of the tail-bud (in the posterior body region) and in the appearance of highly
specific bendings in the head region. Thus in Fig. 3H pre corresponds to the
so-called plica rhomboencephalica between mid- and hindbrain, whereas pev
corresponds to the plica encephali ventralis, marking the anterior chorda
extremity. The destiny of the post-, prebranchial and oral cross-lines is clear
from their designations. Therefore, the cross-lines mark the borders between
the embryo rudiments, as a rule, much earlier than they become visibly differentiated. Moreover, in several cases - for example, in the neural plate and in
the large mesoderm-including fragments - the directions of the cross-lines
coincide with those of the active elongation and migration of cells; however,
the latter processes are initiated later than the corresponding tensile patterns
Mechanical stresses in amphibian embryos
573
arose (compare the tense but as yet passive neural plate at Fig. 1 (5) with the
actively folded neural plate at Fig. 1 (7), npl). The existence of such a correlation
indicates that there may be a causal connexion between the mechanical stresses
and subsequent activation of the intercellular mechanochemical machinery.
These causal connexions, if proved, could be interpreted in terms of the
'positional information' concept (Wolpert, 1969). Further investigations are
of course required to prove this hypothesis.
On the other hand, reverse relations are conceivable. Indeed, according to the
above data, the cross-lines are usually established along the direction of maximal
layer stretching, which in its turn is caused by the activity of cellular contractile
systems in the preceding period of development. Thus, during gastrulation the
dorsal embryo surface stretches most often in a cranio-caudal direction, which
coincides with the direction of the dorsomedial and subneural folds. Later on
the active medial rolling of the neural plate stretches more ventral ectodermal
areas in a ventro-dorsal (transverse) direction, which corresponds to the direction taken by the post-, prebranchial and oral folds. It is also conceivable that
the creation of cross-line pre-pev (Fig. 3G) is promoted by the lateral stretching
of the anterior part of the neural rudiment due to the backward folding of the
anterior neural fold. Ectodermal and ectomesodermal fragments behave in a
similar way: the folds created are situated along the directions of the maximal
stretching of fragment surfaces (e.g. radially in isotropically rolled fragments).
These considerations make it possible to hope that in the not-so-distant future
a closed system of causal relationships between morphogenetic processes will be
constructed including mechanical stresses as one of its important components.
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{Received 4 November 1974, revised 19 May 1975)