Mechanical stresse ans d morphological patterns in amphibian … · J. Embryol. exp. Morph. Vol. 34, 3, pp. 559-574, 1975 559 Printed in Great Britain Mechanical stresse ans d morphological

J. Embryol. exp. Morph. Vol. 34, 3, pp. 559-574, 1975 5 5 9

Printed in Great Britain

Mechanical stresses and morphological patterns inamphibian embryos

By L. V. BELOUSSOV1, J. G. DORFMAN1 ANDV. G. CHERDANTZEV1

From the Department of Embryology, Moscow State University

SUMMARY1. 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 categoriesof deformation have been revealed.

2. The first category comprises those deformations which take place immediately afterseparation. They are insensitive to cooling, cyanide and Cytochalasin B treatment. Thesedeformations, which consist of a shortening of initially elongated cells, are considered to bethe passive relaxations of previously established elastic tensile stresses.

3. Deformations of the second category proceed more slowly. They are inhibited bycooling, cyanide and Cytochalasin B treatment, are accompanied by elongation and migra-tion of cells and occasionally lead to rather complex morphodifferentiations of isolatedfragments. These processes are considered to be the result of the active work of intracellularcontractile systems, either pre-existing or induced de novo.

4. By analysing the arrangement of the passive deformations we have constructed mapsof mechanical stresses in embryos from late blastula up to the early tail-bud stage. At severalembryonic stages drastic transformations of the stress pattern occur, these transforma-tions being separated by periods during which the pattern of stress distribution remainstopologically constant.

5. A correlation between the arrangement of stress lines and the presumptive morphologicalpattern of the embryo is pointed out.

6. Some possible relations between tensile tissue stresses and active mechanochemicalprocesses are discussed.

INTRODUCTION

The role of mechanical stresses in the orientation of cell movements in tissueculture was established long ago (Weiss, 1929), but the application of similarprinciples to morphogenetic processes in entire embryos has been delayed bylack of data on the existence and localization of stresses in intact tissues.This gap has recently been filled by the demonstration of intracellular contractilesystems in developing rudiments (Baker & Schroeder, 1967; Wessels et al.1971;Burnside, 1971).

Similar contractile fibrils have been revealed in cells of the distal regions ofthe actively growing hydroid polyps (Hale, 1960). Periodical contractions of

1 Authors' address: Department of Embryology, Moscow State University, Moscow117234, USSR.

560 L. V. BELOUSSOV AND OTHERS

these fibrils gave rise to pressure stresses in cell layers. Growth, morphogeneticshape alterations and some kinds of rudiment interactions in these species wereconsidered to be directly determined by these stresses (Beloussov & Dorfman,1974).

These data emphasize the role which mechanical stresses may play in regulargeometrical alterations occurring during development. Therefore, it seemsdesirable to study them in other developing systems. The simplest method ofrevealing mechanical stresses in embryos consists of detecting the tissue deforma-tions just after dissection. These deformations may be considered as directmanifestations of pre-existing stresses. A number of scattered data on the exis-tence of similar deformations have already been obtained. These are: contractionof dissected blastomere furrows in Loligo eggs (Arnold, 1971); contraction ofdissected periblast in Fundulus eggs (Trinkaus, 1969); shape alterations ofisolated vegetal parts of sea-urchin blastula (Moore & Burt, 1939); and, whatpertains closely to this study, the intensive rolling of the layers of dissected eyevesicle in amphibian embryos (Lopashov, 1963).

A similar dissection method, combined with the action of some physiologicalagents and followed by a histological study of intact and separated tissues, hasbeen employed in this work. As a result, a fairly regular pattern of mechanicalstresses has been revealed in amphibian embryos from late blastula up to earlytail-bud stages. Maps of mechanical stresses have been constructed for successivedevelopmental stages, representing the lines of tensions and the points of branch-ing of these lines. Besides the above mentioned 'immediate' deformations, whosepassive relaxatory characters have been confirmed by their non-sensitivity tosome inhibitory agents, another class of slower (although still complete within1 h) and much more sensitive deformations has also been observed in separatedtissues. The latter processes were obviously active, leading in several minutesto the creation of new tensile stresses and to rather complex morphodifferentia-tions of a fragment. It is proposed that they may be considered as simplifiedmodels of normal morphogenesis.

MATERIALS AND METHODS

The majority of the experiments have been made on Rana temporaria embryosfrom late blastula up to early tail-bud stages. Several operations were made alsoon Rana esculenta and Xenopus laevis embryos of the same stages. The experi-ments consisted of complete isolation, three-side separation or dissection ofdifferent parts of embryonic tissues. Under physiologically-normal conditionsembryos and separated fragments were kept at 18-20 °C in normal Holtfretersolution twice diluted (several operations were performed in full-strengthHoltfreter solution and gave the same results). The influences of factors inhibitingmetabolism (cooling, moderate doses of KCN) and of Cytochalasin B uponrapid 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 than10 min before operation, during the operation proper and throughout the wholeperiod of observation. KCN was used in a similar way in concentration 10 mg/ml (10~ 4 M). In Cytochalasin experiments donor embryos and fragments wereplaced in 0-2 mg/ml ( 0 - 4 X 1 0 ~ 6 M ) solutions of Cytochalasin B in dimethyl-sulphoxide (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 bymeans of time-lapse filming (35 mm film, exposure interval 1 sec). The rolling-upof the isolated fragments with their external surface outside was designated aspositive rolling and that with external surface inside as negative rolling (orbending, folding). A surface facing the gastrocoel cavity was considered as anexternal one.

For histological purposes embryos or tissue fragments were fixed in Bouin'sfluid, totally stained with borax carmine, dehydrated in butyl-alcohols and em-bedded in paraffin-wax. Special attention was paid to the possible deformationsof separated fragments during fixation and subsequent treatment. In the greatmajority of cases no significant post-vital deformations were observed.

The following well-known features of the histology of amphibian embryosare relevant to this study. The ectoderm is bilayered, consisting of externalepiectodermal and internal hypoectodermal layers (terminology, from Detlaff,1938). The hypoectoderm of the neural plate is several cell layers thick, whereasin other regions it is a single cell layer. As a rule, the adjacent rows of epi- andhypodermal cells are staggered, so that, in a section, each hypodermal cell isopposite the junction between two epi-cells. This regular arrangement is dis-turbed in several narrow zones, most of which correspond to slight ectodermalfolds 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 ofthe marginal zone immediately unfolded to an angle of 90° approximately. Anisolated superficial layer of the vegetal hemisphere bent slightly in the negativedirection (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 thelip) 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 invaginatedmaterial slightly expanded.


B C DI I

CSS®

Fig. 1. For legend see opposite page.

The surface cell layer of an entire gastrula, when separated in the immediatevicinity of the marginal zone, immediately bent as shown in Fig. 1: 2B, epi. Inother areas no significant deformations were observed.

(3) Late gastrula stage (blastopore almost closed) (Fig. 1: 3 A). The results ofcaudal 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


D

postbrf. prebrf.

(5) (5)

Fig. 1. Rapid deformations after dissections of amphibian embryos, (A) 1-12,schemes of separations; (B) immediate deformations; (C) deformations takingplace 1-5 min; (D) 5-20 min; (E) 20-60 min after separation. Black wedgesindicate directions of cutting; numbers in parentheses denote how many operationsof each type were made. Dotted outlines in column A, lines 1-3, 5-8, indicate theareas extirpated; 6-8, latent deformations of the same directions as the precedingimmediate 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


cranial separation, revealing a new tensile stress anterior to the lip. Dorsalepiectoderm, when separated in the medial direction, now revealed a slight butdistinct negative bending (Fig. 1:3B, epi). A similar separation of the chorda-mesoderm led to its immediate negative rolling (Fig. 1:3B, chm). The samewas true of the ventral gastrocoel wall (Fig. 1:3B, ent).

(4) Early neurula stage. By this time some earlier tendencies have beenreinforced and several new areas of rapid deformation have arisen.

(4a) Anterior (hind-brain) area (Fig. 1:4A). Medial separation of neuralepiectoderm led to a sharp negative bending, localized somewhat laterally tomidline (Fig. 1: 4B, epi). Neural hypoectoderm bent during a similar operationto a smaller extent.

At this stage the neural plate becomes limited laterally and anteriorly by ashallow ectodermal fold which can be designated as the subneural fold (Fig.1:4A, snf). This fold became much more pronounced immediately afterseparation of its epiectoderm (Fig. 1:4B, snf). Medial separation of thechordamesoderm 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 neuralepiectoderm also led to its negative bending, localized in this case just at themidline (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 theanterior area. So far no significant deformations were revealed in the subneuralfold region at that level.

(4 c) Deformations observed during longitudinal (cranial and caudal) separa-tions at early-middle neurula stages (Fig. 1: 6 A). Cranial dissection of the hindpart of the dorsal embryo wall resulted in extensive divergence of neural plateand chordamesoderm. In the head region a similar caudal separation led to anextensive negative bending of the chordamesoderm (Fig. 1: 6B). After extirpa-tion of the whole dorsal embryo wall the initial slight positive bending of theneural plate increased to a certain extent (Fig. 1: 6B, npl). The completelyisolated chordamesodermal layer reproduced all the bendings observed duringpartial separations.

(5) Middle-late neurula stage: deformations during transversal separations(Fig. 1: 7A). As in the preceding stages, medial separation of the epiectodermof the invaginated neural plate led to its extensive negative bending, whereasthat of the neurohypoderm resulted in slight negative bending. Separation ofsubneural 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 divergenceof its walls. A similar result was obtained after transverse cutting of a tubewall (Fig. 1:8B).

(6 b) Early tail-bud stage: separations in horizontal (frontal) directions


(Fig. 1: 9 A). At this stage several new transversal ectodermal folds appear inaddition to the subneural folds, namely postbranchial, separating branchial andtrunk regions, prebranchial, separating branchial and head region, oral fold(mouth rudiment) and two less-pronounced folds, separating the rudiments ofthe branchial protuberances. When separated as shown at Fig. 1 (6 A), all thesefolds immediately became much more pronounced (Fig. 1: 9B). This was alsotrue of the corresponding entodermal folds (Fig. 1: 9B, ent).

(7) Immediate deformations of non-folded areas of ventral and lateral ectodermand mesoderm (Fig. 1: 10-12A). The behaviour of these tissues did not altersignificantly during the whole developmental period under study. Ectodermalfragments, excepting those taken anteriorly to the prebranchial fold (from thepresumptive oral field), contracted approximately to half their size and oftenslightly rolled in a negative direction, thus forming a shallow cup (Fig. 1: 10B).Considerable tension was shown to be present in the presumptive oral fieldectoderm, since its epilayer did not contract much after dissection whereas thehypolayer did. Anterior to the prebranchial fold from the early tail-bud stageonwards, and anterior to postbranchial fold from the middle tail-bud stageonwards, immediate positive rolling of the epiectoderm was observed (Fig. 1:9B, lower embryo wall).

In purely mesodermal and combined ectomesodermal fragments no significantimmediate contractions or other shape alterations were observed (Fig. 1:11-12B).

Cooling by up to 2 °C did not influence the deformations listed in paragraphs3-7 above in any way. Negative bendings of neuroectoderm occurred also inKCN solutions of the concentrations employed. The opening angle of the dis-sected gastrula marginal areas slightly decreased after cooling. It is worthmentioning that in Cytochalasin B solutions, deformations 4-7 above werecompletely retained and were even more pronounced than normal. This resultcontrasts sharply with those described below where slower deformations areinhibited completely by the same agent.

During immediate deformations the previously elongated and/or obliquecells contracted into rectangular and sometimes even spherical shapes. Thesharpening of pre-existing negative folds and similar bendings in other regionsseem to be a direct result of the mechanical pressure of these contracted cellsupon the adjacent ones.

Slower ('latent'') deformations (1-60 min after separation)

The immediate deformations described above were stable only at low tem-peratures and under the influence of Cytochalasin B. Under normal conditionsthe shape of the fragments seen 0-5-1 min after separation changed again aftera further interval. These rather complicated morphological events of about1 h duration are what we call latent deformations. They in turn fall into twocategories.

36-2


(A) Latent deformations in the same direction as the preceding immediate de-formations (Fig. 1, lines 2, 6, 7, 8C-E, framed pictures)

(1) Prolongation of divergence of dissected marginal area walls. Under normalphysiological conditions the immediate opening of the dissected blastopore lipwas followed by a slower divergence of its walls, leading in 20-30 min to thecomplete flattening of the fragment (Fig. 1: 2B-D). At the former tip of thelip a new blastopore arose (Fig. 1: 2E).

(2) Foldings and closure of neural rudiments. The entire neural plate, extir-pated 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 itsmidline; 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 immediatebend 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. Inmost cases, however, the dissected tube walls continued to diverge (Fig. 1: 8D).

All the processes described are obviously similar not only to the precedingimmediate deformations but also to the corresponding normal morphogeneticprocesses. We believe this to be true not only for the closure of the neuralrudiments but also for the opening of the blastopore lips, since the latter processmay play a significant role in normal gastrulation, promoting the invaginationof cell material. It can be deduced therefore that these latent deformations aredetermined by certain pre-existing mechanisms rather than induced by theoperation itself.

(B) Latent deformations of opposite direction relative to the immediate deforma-tions

(1) Deformations of ectodermal fragments. Under normal conditions 0-5-2 min after extirpation all the ectodermal fragments, including the neuro-ectoderm, began to roll intensively in the positive direction, starting from the freeedge (Fig. 1,10C-E). The rolling was unequal, with sharp curvatures alternatingwith practically flat areas. In 20-40 min the rolled fragments were subdividedby narrow furrows, oriented in most cases transversely to the rolling axis(Fig. 1: 10D). After a time some of these furrows disappeared whereas othersstabilized (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 thefragments began to contract, thus leading to the bending of sufficiently largefragments (Fig. 1: 11C). In 10-20 min the smaller fragments transformed intosmooth spheres (Fig. 1: 12D, a), the somewhat larger ones formed a singleinvagination, and the largest ones were subdivided by several furrows, oriented


Table 1. Numbers of internally (int.) and externally (ext.) situated cells in meso-dermal fragments 3 min and 30 min after fragment isolation (counts on severalmedian sections)

Number offragment

123456

Time afterisolation (min)

333

303030

Absolute numbersf

int.

756033

310392182

A

ext.

264145158205339264

int./ext.( O/\

\ /o)

284121

14611670

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 surfaceselongated perpendicularly to them. Later on some of the cells obviously migra-ted inside the fragment, an indication of this being the number of cells situatedoutside and inside at different times after separation (Table 1). Formation ofradial furrows began from the immigration of individual epiectodermal cellsinto 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 frag-ment separated into several parts which rapidly rounded and became almostcompletely 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 :7C, f l ) .

The following simple experiment demonstrated the rise of new mechanicalstresses during latent deformations. When dissecting an ectomesodermal frag-ment parallel to its surface immediately after its extirpation, no additionaldivergence of dissected edges, i.e. no relaxation movements, were observed(Fig. 1: 12B). However, 10-15 min after separation, a similar dissection led tothe immediate extensive rolling of the mesodermal layer (Fig. 1: 12 C, a), aswell as of the marginal area of the ectodermal layer (Fig. 1: 12 C, b). Bothdeformations seem to be determined by the contraction of the previouslystretched transverse cell walls. As with other immediate deformations, thesewere not influenced in any way by cooling.

(3) Action of inhibiting agents on latest deformations. All the latent deforma-tions were inhibited by cooling, KCN-treatment and Cytochalasin B. Underthese influences the fragments either remained flat or retained the folds estab-lished immediately after separation. The action of cooling and Cytochalasin Bwas completely reversible, whereas that of KCN was irreversible. CytochalasinB led also to a slow unrolling of already rolled ectomesodermal fragments.


Moderate cooling (7-10 °C) promoted the irregular furrowing and subdivisionof ectodermal and ectomesodermal fragments. DMSO in the concentrationsemployed 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 naturallydivided into two categories. The first one comprises those deformations whichtake place immediately after separation, are at the same time insensitive tothe inhibiting influences employed, and consist in shortening of the previouslyelongated and/or oblique cells. Deformations of the second category on thecontrary proceed more slowly, are highly sensitive to inhibiting agents and areaccompanied by elongation and even migration of cells. Thus the first categorymay be considered to be passive elastic relaxations of pre-existing stresseswhereas the second are active, energy-requiring processes, obviously connectedwith the contraction of microfilaments. On the other hand, some of theselatter processes are due to pre-existing active mechanisms (morphogenesis-imitating latent deformations; see Fig. 1C-E, framed pictures), whereas theothers are non-specific and are obviously caused by contractile systems activatedby the operation itself (see also Burnside, 1972, 1973).

The active deformations may be of interest as simplified models of morpho-genetic processes, since they demonstrate rather rapid and complex morpho-differentiation and provide useful information on the mechanism of activationof intracellular contractile systems. In this paper, however, the first categoryof deformations will be mainly discussed in so far as it may be of use in con-structing maps of the mechanical stresses existing over successive periods in thedevelopment of amphibians.

Maps of mechanical stresses

To validate the employment of immediate deformations for the constructionof stress maps the cellular basis of these deformations must be discussed ingreater detail.

The existence of immediate relaxatory movements indicates that the cells ofamphibian embryonic layers are considerably deformed by the adjacent cells.The layers as a whole are also deformed by the surrounding tissues. The mech-anically stable cell shape achieved after separation is approximately cuboidalwhen the integrity of the cell layer is not disturbed and approximately sphericalwhen cell connexions are disturbed during the operation, or in the regionswithout a regular layer structure.

Let us consider now several somewhat idealized cases of relaxatory movementsand their interpretation (Fig. 2).

(1) Extirpation of fragments leads to their considerable shortening without


* * * * *fhi

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V>}

\ \ \ \ \ vvv\r

Fig. 2. Main types of immediate relaxatory movements in cell layers. Dissections areindicated by dotted lines, stretched surfaces by heavy lines, the surfaces elasticallyrelaxing after dissections by asterisks. In E the largest angle a corresponds to thegreatest elastic contraction at left from dissection point. For other designationssee text.


G

EC

Fig. 3. Maps of mechanical stresses for several successive stages of Rana temporariadevelopment 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) an-terior region of early neurula; (F) posterior region, same stage (D-F - transversesections). (G) Early-middle neurula, sagittal section; (H) middle-late neurula,frontal section; (I) similar stage, transverse section. (J) A typical ectomesodermalfragment, 10-15 min after its isolation. Heavy contours represent distinct stresslines; 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 stretchedto the same extent.

(2) A similar operation leads to negative bending without any shortening oflateral cell walls (Fig. 2B): the elastic stretching of the external surface wasgreater than that of the internal surface.

(3) A similar operation leads to sharp negative bending with considerableshortening 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 nega-tive folding (Fig. 2Dj), whereas dissection of an initially bent rudiment leadsto its unfolding (Fig. 2D2); both processes are accompanied by extensive con-traction 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.


(5) Three-sided separation leads to a sharp negative bending localized exactlyat the border between separated and non-separated areas but without anydefinite prelocalization in the intact embryo (Fig. 2E). This demonstrates thetension of the external surface of the separated zone and the existence of anelastic 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 sym-metrical. This indicates the uneven stretching of both external and internalsurfaces and the elastic stretching of lateral cell walls.

Hence all the above deformations point to the stretching of at least one of thelayer surfaces, whereas deformations (3), (4), (6) indicates the existence of tensionlines which cross the cell layer(s) (cross-lines). In other words, in the lattercases, the bifurcation of tension lines takes place at the corresponding points.

The maps constructed by these methods are presented at Fig. 3A-J. Thedistinct lines of tensile stresses are indicated by heavy lines, those graduallydispersing throughout the tissues by dotted lines and non-tense surfaces byfine lines. The following general properties of stress patterns are to be empha-sized. At any developmental stage the tension lines, including cross-lines, areconcentrated near to the restricted number of closed surfaces subdividing theembryo. This tensile pattern does not alter gradually in the course of develop-ment. Instead it remains topologically constant for a certain finite period ofdevelopment, and then drastically transforms. The transformations comprisethe appearance of new cross-lines as well as (more rarely) the disappearance ofsome old ones. The periods of development between the two next topologicaltransformations may be designated similarly as topologically invariant periods.

Successive topological transformations for the given period of Rana tem-poraria development are as follows:

(1) The establishment of fairly wide strips of stretched cells between theblastocoel corners and the vegetal surface of the late blastula (Fig. 3 A). Theirpositions correspond to the marginal zone of the gastrula.

This stress pattern is not changed qualitatively during gastrulation, althoughstretching of the dorsal ectoderm increases (Fig. 3B, C). In the regions removedfrom the blastopore, the stress pattern is circular and is localized mainly in theectodermal layer (Fig. 3 C).

(2) The next and perhaps the most important topological transformationis that the circular stress breaks either exactly along the dorsal midline (in theposterior region) or parallel to it (in the anterior region). Now the externalcircular stresses pass along these lines to the archenteron roof and spreadventrally, joining stress lines coming from the gastrocoel angles and then gradu-ally dispersing (Fig. 3D). This transformation may be considered as thedemarcation point between gastrulation and neurulation.

(3) Shortly after, in the early neurula stage, the stretched cells of the dorsalepiectoderm and mesoderm establish new contacts with the lateral embryo


surface just ventrally to the neural plate. This contact line corresponds to thesubneural fold which encircles the embryo laterally and anteriorly. Now severalother obliquely situated cross-lines dissect the neural plate (Fig. 3E,F). Thus,instead of extended gradually dispersing tension lines, a number of closedtense contours appear.

(4) In the early-mid neurula stage the neural plate together with archenteronroof becomes dissected by a number of new cross-lines (Fig. 3 G; compare withFig. 1, 6B). Several fairly irregular indistinct cross-lines are localized in thecaudal region and one especially pronounced cross-line is found at midbrainlevel (Fig. 3 G, pre-pev).

(5) In late neurula-early tail-bud stage several new transversely orientedcross-lines arise in the anterior body region, joining post-, prebranchial and oralfolds with the corresponding folds of the oral ectoderm (Fig. 3H). Later onsimilar cross-lines dividing the rudiments of the branchial arches appear. Onthe other hand, the cross-line connecting the subneural folds and neural groovealmost disappears during neurulation and is replaced by a line localized slightlyventral and going along the roof of the intestinal cavity (Fig. 31).

According to the dissection results described above, the tensions of thepresumptive oral field ectoderm are localized mainly in its hypo-layer, theepisurface bearing no considerable tension (Fig. 3H).

A similar map for a typical ectomesodermal fragment, 10-15 min after itsextirpation, is presented at Fig. 3 J.

The presumptive significance of stress cross-lines: possible relations betweenmechanical tensions and the active mechanochemical processes in cells

It is easy to see that almost all the cross-lines revealed in intact embryos areof clear morphological significance. Indeed, stress lines established in the lateblastula outline the marginal zone of the gastrula; those established during thesecond topological transformation correspond to the neural groove and separatethe archenteron roof into its chordal and mesodermal parts. The third topologicaltransformation leads to complete separation of the neural plate from the moreventral regions, whereas a set of transformations (4) results in the differentiationof the tail-bud (in the posterior body region) and in the appearance of highlyspecific bendings in the head region. Thus in Fig. 3H pre corresponds to theso-called plica rhomboencephalica between mid- and hindbrain, whereas pevcorresponds to the plica encephali ventralis, marking the anterior chordaextremity. The destiny of the post-, prebranchial and oral cross-lines is clearfrom their designations. Therefore, the cross-lines mark the borders betweenthe embryo rudiments, as a rule, much earlier than they become visibly dif-ferentiated. Moreover, in several cases - for example, in the neural plate and inthe large mesoderm-including fragments - the directions of the cross-linescoincide 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 573arose (compare the tense but as yet passive neural plate at Fig. 1 (5) with theactively folded neural plate at Fig. 1 (7), npl). The existence of such a correlationindicates that there may be a causal connexion between the mechanical stressesand 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 areof course required to prove this hypothesis.

On the other hand, reverse relations are conceivable. Indeed, according to theabove data, the cross-lines are usually established along the direction of maximallayer stretching, which in its turn is caused by the activity of cellular contractilesystems in the preceding period of development. Thus, during gastrulation thedorsal embryo surface stretches most often in a cranio-caudal direction, whichcoincides with the direction of the dorsomedial and subneural folds. Later onthe active medial rolling of the neural plate stretches more ventral ectodermalareas in a ventro-dorsal (transverse) direction, which corresponds to the direc-tion taken by the post-, prebranchial and oral folds. It is also conceivable thatthe creation of cross-line pre-pev (Fig. 3G) is promoted by the lateral stretchingof the anterior part of the neural rudiment due to the backward folding of theanterior neural fold. Ectodermal and ectomesodermal fragments behave in asimilar way: the folds created are situated along the directions of the maximalstretching of fragment surfaces (e.g. radially in isotropically rolled fragments).These considerations make it possible to hope that in the not-so-distant futurea closed system of causal relationships between morphogenetic processes will beconstructed including mechanical stresses as one of its important components.

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{Received 4 November 1974, revised 19 May 1975)

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Mechanical stresse ans d morphological patterns in amphibian … · J. Embryol. exp. Morph. Vol. 34, 3, pp. 559-574, 1975 559 Printed in Great Britain Mechanical stresse ans d morphological