Morphogenetic Interactions before Gastrulation in the Xenopus … · Morphogenetic Interactions before Gastrulation in the Amphibian, Xenopus laevis—Regulation in Blastulae by A

Morphogenetic Interactions before Gastrulation in theAmphibian, Xenopus laevis—Regulation in Blastulae

by A. s. G. CURTIS1

From the Department of Anatomy and Embryology, University College, London

WITH ONE PLATE

T H E site of the future dorsal lip of the gastrula is already determined to a con-siderable degree at the grey-crescent region by the 8-cell stage, probably by meansof interactions within the cortex (Curtis, 19626). But this determination is stilleasily disturbed because excision of the grey-crescent cortex from the 8-cell stagedoes not prevent the appearance of a dorsal lip. The embryo forms a new dorsallip by a process of regulation. Similarly, Votquenne (1933) destroyed the blasto-mere containing the grey crescent at the 8-cell stage in Ranafusca and neverthe-less obtained nearly normal embryos; which result indicated that regulation hadhappened. At a later stage, late blastula, excision of the cells of the presumptivedorsal Up region does not prevent a dorsal lip forming and subsequent neuralinduction occurring (Goerttler, 1926) although the embryos are of the abnormal'ring' type. But though regulation in Goerttler's experiments can hardly haveoccurred before gastrulation, due to the late stage used, it remains an openquestion whether the regulation of the embryo after Votquenne's excision of amicromere or my excision of cortex from the 8-cell stage embryo, happens soonafter the operation of excision or whether the necessary interactions amongstcells are delayed until some later stage such as gastrulation. The answer to thisquestion will help to reveal the extent of developmental change before gastrula-tion. There is reason for thinking that a degree of morphogenesis of the variouspresumptive regions occurs by late blastula stages because Dalcq (1933) showedthat considerable localization of organs had occurred by the late blastula stageof R. fusca.

For these reasons experiments have been made on regulation of the presump-tive dorsal lip region in blastulae. Early blastulae have been used since there is asufficiently long time before they become gastrulae to determine whether regula-tion occurs soon after removal of tissue or at gastrulation. In addition, since thepresumptive dorsal lip region plays a major part in the imagination process whenit reaches the gastrula stage (Schechtman, 1942) such experiments may throwlight on the question of how the morphogenetic movements of gastrulation

1 Author's address: Department of Anatomy and Embryology, University College, Gower Street,London, W.C. 1, U.K.[J. Embryol. exp. Morpb. Vol. 10, Part 3, pp. 451-63, September 1962]

452 A. S. G. CURTIS—REGULATION IN BLASTULAE

are determined during development. In preliminary work I found that asimple chemical treatment blocks regulation if applied just after excision of cellsfrom the blastula, but does not do so if applied slightly later. These experimentswill be described at length. The general deduction from them was that one essen-tial step in regulation occurs fairly soon after excision. In consequence a seriesof experiments designed to elucidate further the mechanism of this interactionwill be described.

A preceding paper (Curtis, 19626) showed that important morphogeneticfactors are located in the cortex. These experiments provide some suggestionsthat these factors are still located in the cortex at blastula stages and interactbetween various parts of the embryo during regulation.

METHODS

Xenopus laevis morulae were decapsulated and stripped of their vitellinemembranes by hand: they were cultured in Holtfreter saline buffered to pH 7-0

with 0 001 M 2-amino-2-hydroxy-methyl-1, 3-propanediol-h ydrochloricacid (tris-HCl). In experiments on regu-lation after excision, the whole of thedorsal lip and much of the presumptivenervous system were the regions excised.The tissues were removed down to theblastocoel roof. Since the exact extentof these presumptive regions is uncertain,more tissue than contains these regionswas cut out (see Text-fig. 1); in conse-quence it is unlikely that any tissue whichwould normally invaginate and carry outinduction remained. The controls forthese experiments were unoperated em-bryos. Control and experimental embryoswere treated with 0001 M tetra-sodiumethylene-diamine tetra-acetate bufferedat pH 8-2 with 0-001 M 2-amino-2-hydro-xymethyl-1,3 -propanediol-hydrochloricacid, made up in calcium-free Holtfretersaline. Embryos were treated with this

medium for either 1 hour with mechanical agitation when complete disaggrega-tion was required or for half an hour when 'collapsed' embryos were required.Collapsed embryos are those in which most cells are only slightly disturbed fromtheir normal positions although the embryo has flattened down on the bottomof the culture dish and the blastocoel has collapsed. In the various experimentalseries this treatment was applied at varying times after the excisions were made.

TEXT-FIG. 1. Side view of the embryo at earlyblastula stage. The dashes indicate the limits ofthe regions which were excised in these experi-ments. They include presumptive dorsal lipand much of the presumptive nervous tissue.The position of the lower limit was carefullykept to in all excisions but the position of theanimal and anterior limit was somewhat vari-able, in many cases rather less presumptive

nervous tissue was excised.

A. S. G. CURTIS—REGULATION IN BLASTULAE 453

Embryos were returned to the normal culture medium after this treatment. Inthree additional series grafts of either presumptive dorsal lip from blastulae orpart of the margins of the wound left in blastulae after excision of the pre-sumptive dorsal lip, were placed in the embryos after treatment with the EDTAsolution.

All embryos were cultured in the normal Holtfreter saline at 19-21° C. untilthey reached an age equivalent to stage 22 (Nieuwkoop & Faber, 1956). Theywere then fixed in formol-bichromate, embedded in celloidin-paraffin, sectionedat 8-9 fM, and stained with celestin-blue and eosin.

RESULTS

Regulation after excision(a) Treatment with EDTA immediately after excision

This experiment forms the main series of the present work, for it was repeatedas a control whenever experiments on regulation were carried out. The embryoshad their presumptive dorsal lip removed in the early blastula stage, and werethen placed in the EDTA solution within 5 minutes. They were either treatedwith this solution for 30 minutes without mechanical agitation to produce the'collapsed' type of embryo, or for 1 hour with agitation which produces com-plete disaggregation.

Thirty-six 'collapsed' embryos were produced in six batches. At the end ofthe treatment with the EDTA solution the embryos had partially flattened downon to the bottom of the culture dish giving them a slumped appearance. Most ofthe cells in these embryos were still in roughly the correct relationship to oneanother. Their ectoderm was little damaged though the excision wounds hadopened out. After their return to the Holtfreter saline the embryos healed rapidlyand redeveloped their rigidity so that they rested on the bottom of the culturedish in a normal (unslumped) condition. Thirty-three of these embryos showedno definite sign of gastrulation, though three gastrulated normally. Unfor-tunately, it is hard to be certain whether or not slight invagination occurred inthese thirty-three embryos, but no definite sign of the positioning of the tissueswhich would result from gastrulation was found. The ventral surfaces of theseembryos were composed of endodermal cells; this fact suggests that invaginationhad failed. None of these thirty-three embryos showed any sign of neural dif-ferentiation, although all were composed of healthy cells. However, somite and/or notochordal tissue lying dorsally in an unoriented mass appeared in fifteenembryos (see Plate, fig. A); in only one of them was the mesodermal differentia-tion solely notochordal. No elongation of these embryos occurred. The threeembryos which had gastrulated developed nervous systems, somites, and noto-chord. In two embryos the nervous systems were neuroid in nature, but theother embryo had a fairly normal nervous system.

When complete disaggregation was allowed to occur care was taken to mixthe various cell types thoroughly by mechanical agitation. Twelve embryos were


disaggregated, and after their return to the normal Holtfreter saline all reaggre-gated successfully. They were grown for 20 hours, a period of time more thansufficient for complete cell sorting out to occur (Curtis, 1961). At the end of thistime they were fixed.

None of the twelve reaggregates showed any appreciable sign of the sortingout of the various cell types, though the outermost cells were rather flattened inan epithelial manner. Prospective ectodermal, mesodermal, and endodermalcells were mixed up randomly in these embryos, see Plate, fig. C. The next seriesforms controls with which this series may be compared.

(b) Treatment with EDTA 3 hours after excision

Twenty-eight early blastulae in four batches had their presumptive dorsal lipsremoved and were allowed to heal in normal Holtfreter saline for 3 hours. Thenthey were treated with the EDTA solution for 30 minutes to produce 'collapsed'embryos; thereafter they were grown in normal saline. Twelve of these embryosshowed differentiation of all main cell types: they had well-organized neuraltissue, notochord, and somites which formed axial structures. Of the remainingsixteen embryos seven differentiated mesoderm cells, but in nine embryos nosign of morphogenesis was found.

Comparing these results with those of the previous series suggests that thetime of treatment after excision affects the degree of regulation. It is necessary,however, to discover whether these effects of ageing would appear in embryos inwhich no excisions had been made.

(c) Intact blastulae treated with EDTA

Three groups of experiments were performed. Firstly, twenty-seven earlyblastulae were placed in the EDTA solution for 30 minutes which brought themto the collapsed state, returned to normal saline and cultured to fixation at stage22. Of these embryos fourteen showed development of neural tissues. In elevenof them the nervous structures were either unclosed neural tubes or neuroidstructures, or spina bifida. Four embryos were without any sign of tissue differ-entiation, but the remaining nine embryos produced somite and/or notochordaltissue. The experiment was performed in four batches.

It was already known from earlier unpublished work that late blastulae couldbe disaggregated with EDTA solution without affecting the subsequent sortingout after reaggregation of the various cell types, so only a small confirmatoryseries of twelve experiments on early blastulae was undertaken. This second seriesforms a control to those disaggregates of excised embryos, see (a). These twelvedisaggregates reaggregated successfully, and sorting out of the presumptive celltypes occurred in them so that presumptive ectoderm lay at the outside, endo-derm innermost, and mesoderm in between. But no differentiation other thanthat pre-existing in yolk platelet size, cell size, and pigmentation was found inthese reaggregates, see Plate, fig. B.


In the third series a dozen intact blastulae were treated with EDTA solutionfor 30 minutes to produce 'collapsed' embryos, when they had reached an agecorresponding to that of the excised embryos given a 'delayed' treatment, see(b), namely at mid-blastula stage. Eleven of these embryos developed all themain tissue types, though bilaterally asymmetrical nervous systems and largenotochords were common. In one embryo somite tissue was the only cell typedifferentiated.

Although statistical evaluation of these results is deferred to the next mainsection it already appears that immediate treatment with EDTA probably hasmore effect in stopping regulation than delayed treatment. The prime stimulusto regulation may be the excision of cells. If so, the stimulus is transmitted overa wide area producing a reaction in many cells. The failure- of regulation afterimmediate EDTA treatment brings forward the question of whether its effectis upon the reacting system or upon the system that transmits the stimulus. Thefollowing experiments were designed to answer this question.

(d) Grafts placed in embryos treated with EDTA

The margins of the wound from which the excision has been made are pre-sumably the first regions to bear the stimulus to regulate. In consequence graftsof wound-margin made to embryos in which regulation has been blocked mightrestart regulation. If so this result would suggest that EDTA damages the trans-mission system and not the reaction system. Likewise grafts of presumptivedorsal lip might be able to induce fresh nervous systems in ectoderm if the EDTAhad not damaged its ability to react to a suitable morphogenetic stimulus.

Nineteen embryos had their presumptive dorsal lips removed as early blastulae.They were treated with EDTA solution for 30 minutes. Meanwhile, the sameexcision was carried out on other early blastulae to form wound-margins. Onegraft from the wound-margin of these embryos was placed in each of thetreated embryos within 5 minutes of the end of the EDTA treatment. The graftswere not treated with EDTA. They were placed in the wounded area of theembryos. Of the twenty-one grafted embryos only two showed regulation toform a nervous system; somite production alone occurred in three more embryos.No morphogenesis appeared in the remainder. Furthermore, the grafts showedno differentiation themselves, which suggests that the host embryos exert somekind of suppression on the grafts.

In a subsidiary experiment the grafts were taken from the margins of excisionwounds which had healed for 3 hours, and the host embryos had just been treatedwith EDTA solution. Of these nine embryos, only two showed signs of neuraldifferentiation or other morphogenesis.

Grafts of presumptive dorsal lip from early blastulae were placed in embryoswhich had been treated with EDTA after excision. Of the six embryos receivingthis type of graft one alone showed development of neural structures; the remain-ing five were without signs of morphogenesis in either graft or host.

5584.10 Gg


Statistical testing of resultsThe incidence of neural induction and mesoderm differentiation in the various

experiments is displayed in Table 1. The effects of EDTA treatment applied toembryos immediately after regulation began or applied 3 hours later can becompared by the method of Cochran and Cox (see Snedecor, 1946) since theexperiments were done in six and four groups respectively, with five to sevenembryos in each group. There is significantly less neural induction in thosetreated with EDTA to produce 'collapse' immediately after regulation began,than in the other group, P < 0-05. Likewise there is significantly more mesodermdifferentiation than neural differentiation in embryos treated with EDTA im-mediately after regulation began, using the/-test, t = 22-56, d.f. = 5,P < 0-001.

TABLE 1

Summary of experimental results

Type

Excision of presumptive dorsal lipImmediate treatment with EDTA

Collapsed embryos

DisaggregatesDelayed treatment

Collapsed embryos

No excisionImmediate treatment with EDTA

Collapsed embryos

DisaggregatesDelayed treatment

Collapsed embryos

Grafts into blocked embryosFresh wound-margin3-hour-old wound-marginPresumptive dorsal lip

No. of embryos

665676

12

7777

10665

12

12

1996

Neural induction

110101

0

4323

3452

0

11

221

Mesoderm differ-entiation alone

230415

0

2212

4212

0

1

250

Neural differentiation is affected by EDTA treatment to a much smaller extentin unoperated embryos than in ones in which the presumptive dorsal lip has beenexcised (using method of Cochran and Cox, P < 0-05). Mesoderm differentiationis also more extensive in the unoperated embryos than in the excised ones,P < 005.


Comparisons with the remaining groups have been made by the 2 x 2 con-tingency test method. Despite the fact that there is more neural differentiationwhen EDTA treatment after excision is delayed there is still significantly lessneural differentiation in the delayed group than in those unexcised embryostreated with EDTA at a stage 3 hours later than the excision operation,P < 0-005. Likewise there is more neural differentiation in unoperated embryostreated with EDTA at a later stage (3 hours later) than an earlier one, P < 0-025.These results suggest that the unoperated embryo becomes more resistant to theeffects of EDTA treatment through the blastula stage.

Grafts of wound-margin do not unblock differentiation in embryos treatedwith EDTA immediately after excision, whether the wound-margin is fresh or3 hours old, P > 0-1 in both cases, when compared with similar ungraftedembryos. Likewise grafts of presumptive dorsal lip are without effect in restart-ing differentiation in similar embryos; P > 0-1 when compared with the un-grafted embryos. If they are compared with unexcised embryos treated withEDTA to produce 'collapse' such grafted embryos show a significant reductionin neural differentiation, P < 0-005.

It can be concluded that one essential step in regulation occurs soon after theexcision of the tissue, long before gastrulation, and that thereafter EDTA haslittle effect on regulation in embryos in which the wound has healed. This stepis blocked by the action of EDTA. However, this behaviour is probably super-imposed on a general increase in the resistance of the unoperated embryo to theaction of EDTA during the blastula stage.

It appears that the ability of cell types such as mesoderm, endoderm, andectoderm to sort out in reaggregation is destroyed by excision of the presump-tive dorsal lip and surrounding regions if regulation cannot take place. But,although failure of regulation occurs with EDTA treatment and though the dor-sal lip has been removed, morphogenesis of mesoderm is not extensively sup-pressed. Grafts of untreated wound-margin or presumptive dorsal lip will notrestart regulation.

DISCUSSION

Goerttler (1926) had shown that excision of the presumptive dorsal lip fromblastulae did not prevent the formation of a regulated dorsal lip in gastrulae.But this result was regarded with some doubt until Dalcq (1933) found that afairly ventral level in the future blastopore region could still form dorsal lipwhen the ventral half of the blastula was rotated under the dorsal half, andBrice (1958) observed that the isolated ventral half of a blastula might, 'developa dorsal lip'. In the present experiments the same type of regulation of cells toform dorsal lip has been found many times. To a large degree the evidence for theformation of a dorsal lip has been the discovery of signs of invagination andinduction. But, although this is not wholly conclusive evidence for regulation,


accepting any alternative effect would involve proposing new theories about thenature of gastrulation and neural induction.

The results show that one step in the morphogenetic process which reformsthe dorsal lip occurs soon after the excision of the cells. This step is susceptibleto damage by the action of EDTA. In addition this part of the process appearsto be related to the mechanism whereby cells acquire those properties whichallow them to sort out in reaggregates. Sorting out of the various cell types doesnot occur in reaggregates from embryos in which regulation has been blocked.Although this might be explained on the hypothesis that since induction atgastrulation is inhibited by blocking regulation these properties of the cells arenormally developed at gastrulation, there are several objections to such ahypothesis. First, disaggregates of normal blastulae do sort out correctly intothe various cell layers, as observed in this work, and appreciable separation ofthe cell types occurs although none of the neural differentiation associated withgastrular induction happens. It is difficult to imagine how induction wouldoccur in a disaggregate before sorting out when the various cell types are mixedup randomly. In a recent review (Curtis, 1962a) it was pointed out that the sort-ing out of cell types in reaggregates involves differences in both motile andadhesive properties between the various cell types. Gastrulation is a similarprocess in that considerable directed movement of the cells is involved. Thus itis reasonable to expect that such adhesive and motile properties of the cellswould be differentiated before gastrulation, and the results of the present worksuggest that this differentiation can occur anew soon after excision of cells fromthe early blastula.

In the preceding paper (Curtis, 19626) it was suggested that important mor-phogenetic factors responsible in part for determining the site of the future dorsallip were localized in the cortex during cleavage stages. These factors were or-ganized in such a way that excisions were regulated for and additions by way ofgrafts were of no effect. It appears that during early blastula stages at least, thesame type of system operates, since excisions of presumptive dorsal lip cells areregulated for. This correspondence suggests either that regulation for operationsdone on cleavage stages does not occur till the early blastula stage or that thesame system operates at both stages. Since some of the properties affected byregulation appear to be cell surface properties it is possible that regulation is aphenomenon involving the cortex alone even during blastula stages. In thisconnexion it is of interest that once the wound caused by the excision hashealed the process of regulation can no longer be affected by treatment withEDTA. This result implies that very extensive areas of cells in contact may bea necessary condition of regulation.

Grafting untreated wound-margin to blastulae in which regulation has beenblocked did not restart regulation. This result suggests that it is not the systemwhich transmits the stimulus to regulation which is damaged by EDTA, butthat it is the reacting system. The reason for such a conclusion is that the graft


from an embryo which had just been wounded would be expected to contain ordevelop the stimulus when it was placed in the blocked embryo. However, it ispossible that transmission and reaction are carried out by the same system. Inaddition not only are grafts to blocked embryos without effect on regulation butthere appears to be an active suppression of the development of the graft itself:for instance, grafts of presumptive dorsal lip fail to undergo self-differentiationeven though they contain presumptive mesodermal and neural elements.

Furthermore, the treatment with EDTA only affects morphogenesis extensivelyin embryos which have had cells excised from them. EDTA has little effect onthe development of whole embryos. This result implies that regulation in someway alters the cells so that they are much more sensitive to the action of EDTA,Such an action indicates the spread of a stimulus amongst the cells as a result ofthe excision. The grafting experiments mentioned above suggest that it is thereaction to this stimulus which is affected. The reaction is not merely temporarilyprevented but appears to be in some way directed into the wrong channel sothat active suppression of morphogenesis occurs. EDTA tends to separate cellseven when the treatment is of short duration. Thus its effect has a considerablesimilarity to that of wounding, and therefore EDTA might be expected toproduce a stimulus similar to that occurring in regulation. The fact that intactembryos show some degree of susceptibility to damage of their differentiationmechanisms by EDTA at least in the early blastula stage may reflect this pos-sibility. Presumably the lesser degree of inhibition of development found insuch embryos compared with that in regulating embryos treated with EDTA,may be explained as being due to the chemical acting simultaneously at manypoints to separate the cells. Thus any stimulus to regulate the cells might pro-duce on partial loss of contact, can only spread amongst a few cells before it isbarred by a gap between cells. In the embryo in which an excision has just beenmade by operation cell contact is good on either lateral side of the excision sothat the stimulus to regulate can be easily transmitted to many cells before theembryo is placed in EDTA.

I do not think one should conclude that since EDTA treatment blocks regula-tion, calcium ions must be involved causally in the regulatory system. Not onlydoes this chemical have effects other than the chelation of calcium but it is alsopossible that the important effect is a physical one, such as a temporary lossof contact between cells or alteration of the mechanical properties of the cellsurface. Shoger (1960) suggested that the mechanical interaction of cells maybe of importance in the regulation of the chick primitive node region. He treatedwhole nodes isolated from the rest of the embryo with EDTA and observedconsiderable though not total suppression of morphogenesis; the experiment isnot exactly comparable with the present ones, for the node most nearly repre-sents the dorsal lip region, which was the excised area in my experiments,whereas in his the node was the only remaining area.

A theoretical mechanism which appears to account for these observations is


one based on a' field' organization of morphogenetic factors originally developedby Dalcq & Pasteels (1937). For the purpose of this paper this 'field'will bedefined as that network of spatial and temporal interactions involved in produc-ing the dorsal lip of the blastopore and the related invagination. The ideasadvanced in the previous paper (Curtis, 19626) to explain the organization ofsuch factors in the cortex of the 8-cell stage will be extended. In the intactembryo the main arrangement of the morphogenetic field has been determinedby the 8-cell stage, and the field may be centered on the future site of the dorsallip. One particular structure, the dorsal lip, appears at the 'highest' value of thisfield. If this region is cut out another nearby region becomes that of highestvalue and thus becomes the dorsal lip. But how does this new region have theinformation that it alone is the now 'highest' region? This problem occurs inconsidering both the regulation of cortical excisions and of blastular excisions.Since regulation involves a change in function for many cells in the embryo itseems that the information must be transmitted between them. In addition theapparent necessity for cell contact to complete regulation, the behaviour ofgrafts in blocked embryos, and the sensitivity of regulation to the action ofEDTA, all argue that a transmission system is involved in readjusting the fieldafter excisions of cells have been made. It seems that this system for transmissionof the first stimulus to regulation and the reaction to it only comes into existenceafter an excision has been made, because EDTA damages it then but is withoutgreat effect on the morphogenesis of embryos in which no excision has beenmade. In other words, there is no transmission of information between thecells of the undamaged blastula, but excision activates the morphogenetic fieldso that interaction takes place and a new field is established in the regulatedembryo. The activation is not susceptible to the effects of EDTA treatment butthe reaction, namely the interaction between parts of the field to establish a newfield, is affected. In consequence it would be expected that EDTA would haveno large effect on the subsequent morphogenesis of whole unoperated blastulaenot allowed to disaggregate, which of course is so. If a new field is established,which apparently requires that extensive cell contact be present, the embryoagain is not greatly damaged by EDTA treatment. So it appears that the mor-phogenetic field in the blastula is normally arranged in an inactive manner sothat no information is exchanged between cells, and regulation is a process inwhich it becomes activated and its various parts interact with one another. Sincethe blocked embryo has apparently some suppressive action on grafts of normaltissue it is possible that this influence may be organized in a fashion like a mor-phogenetic field.

It is usual to presume that the properties in a field system are graded evenlyfrom one part of the field to another. In consequence it may seem a little illogicalthat it should apparently be involved in determining the cell properties respon-sible for sorting out in reaggregation, for there are three distinct types of cell,ecto-, meso-, and endoderm, and there appear to be no intermediate types of cell


reflecting a graded property. However, if these cell types represent various phasestates of the cell surface as I suggested (1962a) grading of the chemical propertiesof the surfaces of various cells in accordance with the field would produce onlya few different phase states and thus types of cell.

The incidence of mesoderm differentiation in embryos in which regulation andgastrulation have been blocked suggests that mesoderm is predetermined beforegastrulation to a considerable extent. Evidence for such a possibility was foundby Dalcq (1933).

SUMMARY

1. After the presumptive dorsal lip is excised from early blastulae of XenopusIaevis the tissues surrounding regulate to reform the dorsal lip. This regula-tion can be prevented by treatment of the embryos with the chelating agent,EDTA, provided that treatment is carried out fairly soon after excision. Ifregulation is prevented there is no differentiation of nervous tissue, but, never-theless, there is a considerable mesodermal differentiation. If embryos whosepresumptive dorsal lips have been excised are completely disaggregated withEDTA, reaggregation occurs but no sorting out of the cell types is found. Dis-aggregates of whole embryos reaggregate and show sorting out of the cell typesthough no further differentiation occurs.

2. These effects appear to be superimposed on a slight sensitivity of the intactembryo to damage to its neural differentiation by EDTA in the early blastulastage. But this sensitivity is greatly increased if excision of presumptive dorsallip is carried out before treatment with EDTA.

3. Grafts of wound-margin material from embryos whose presumptive dorsallips have been excised will not restart regulation in embryos blocked by EDTAtreatment, whether the grafts are from freshly wounded embryos or from em-bryos wounded some hours previously. Likewise grafts of presumptive dorsallip material will not restart regulation or differentiation in blocked embryos,nor do the grafts themselves differentiate.

4. The results suggest that one step at least in regulation occurs soon after theexcision of tissue, long before gastrulation. This step is sensitive to the action ofEDTA. It appears to be connected with the determination of those cell propertiesresponsible for the sorting out of cell types in reaggregation, and there is con-siderable evidence that these properties are determined in the blastula stage.It seems probable that EDTA acts on the system which reacts to the stimulus toregulate and not on the actual transmission of this stimulus. Blocked embryosappear to contain a system such that additions, e.g. grafts, have their morpho-genetic potentialities suppressed. Regulation in intact blastulae acts to replacemissing tissues. The regulative properties of blastulae are probably the same asthose to be found in mid-cleavage stages, save that there is some evidence ofpartial determination of mesoderm by the blastula stage. These results are


discussed in terms of a field theory, and it is suggested that regulation requires anintercellular communication system.

RESUME

Interactions morphogenetiques pre-gastruleennes chez VAmphibienXenopus laevis. Regulation chez les blastulas

1. Apres excision de la levre dorsale presomptive de jeunes blastulas deXenope, les tissus environnants reforment cette levre dorsale par regulation.Celle-ci peut etre empechee en traitant les embryons par l'ethylene diaminetetra-acetate (EDTA), pourvu que ce traitement soit accompli assez tot apresl'excision. Si on empeche la regulation, le tissu nerveux ne se differencie pas,mais il se produit neanmoins une differenciation mesodermique considerable.Si on desagrege completement au EDTA des embryons dont la levre dorsale aete excisee, la reagregation se produit mais on n'observe pas de segregation destypes cellulaires. Les desagregats d'embryons entiers se reagregent et montrentune segregation des types cellulaires bien qu'il n'y ait pas de differenciationulterieure.

2. Ces effets paraissent etre surimposes a une legere sensibilite de l'embryonintact aux effets nocifs de l'EDTA, au stade de la jeune blastula, envers sadifferenciation nerveuse. Mais cette sensibilite est tres augmentee si on pratiquel'excision de la levre dorsale presomptive avant le traitement a l'EDTA.

3. Des greffons de materiel marginal de la blessure, pris sur des embryonsdont la levre dorsale presomptive a ete excisee, ne reprendront pas leur regula-tion chez les embryons bloques par le traitement a l'EDTA, — que ces greffonsproviennent d'embryons fraichement blesses ou d'embryons blesses quelquesheures avant. De meme, des greffons de materiel de la levre dorsale presomptivene retablissent pas la regulation ou la differenciation des embryons bloques, etles greffons eux-memes ne se differencient pas.

4. Les resultats suggerent qu'au moins une etape de la regulation se situe totapres l'excision du tissu, longtemps avant la gastrulation. Cette etape est sensiblea Faction de l'EDTA. Elle apparait en liaison avec la determination de celles desproprietes cellulaires responsables de la segregation des types cellulaires aucours de la reagregation et il existe de nombreux faits montrant que ces pro-prietes sont determinees au stade blastula. II semble probable que l'EDTAagisse sur le systeme qui reagit au stimulus en regulant, et non sur la transmissionreelle de ce stimulus. Les embryons bloques paraissent renfermer un systemetel que des additions, c'est-a-dire des greffons, voient leurs potentialites morpho-genetiques supprimees. La regulation dans les blastulas intactes agit pourremplacer des tissus manquants. Les proprietes regulatives des blastulas sontprobablement les memes que celles observees aux stades moyens de la segmen-tation, sauf qu'il existe des indices d'une determination partielle du mesodermeau stade blastula. Ces resultats sont discutes sous forme d'une theorie de champ,

J. Embryol. exp. Morph. Vol. 10, Part 3

A. S. G. CURTIS


et on suggere que la regulation requiert un systeme de communications inter-cellulaires.

ACKNOWLEDGEMENTS

I thank Professor M. Abercrombie, F.R.S., and Professor J. Z. Young,F.R.S., for their advice and encouragement, and Mr. J. M. Pettitt, B.Sc, forhis able assistance. The work was carried out during the tenure of grantC-4847 from the National Cancer Institute, U.S.A.

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cortical field. / . Embryol. exp. Morph. 10, 410-22.DALCQ, A. (1933). Experiences de transplantation, de translocation et d'ablation relatives a la deter-

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Arch. Biol., Liege et Paris, 48, 669-710.GOERTTLER, K. (1926). Experimentell erzeugte 'spina bifida' und 'ringembryobildungen' und ihre

Bedeutung fur die Entwicklungs-physiologie der Urodeleneier. Z.f. gesamte Anat. 80, 283-343.NIEUWKOOP, P. D., & FABER, J. (1956). Normal table o/Xenopus laevis. Amsterdam: North-Holland

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EXPLANATION OF PLATE

FIG. A. Section through an embryo near its anterior end from which the presumptive dorsal liphad been excised at early blastula stage. The embryo was then treated with EDTA. The section ismainly transverse but somewhat tilted so that the dorsal half of the embryo is anterior to the ventralhalf. Note the absence of neural differentiation and the failure of any definite structures to form.Ectoderm cells lie towards the dorsal side of the embryo, mesoderm beneath, and endoderm bottom-most. The vacuolation of the mesoderm cells, indicative of some degree of differentiation, is just visiblein the middle of this section.

FIG. B. Section through the reaggregate from a whole blastula which had been disaggregated. Thereaggregate is positioned in this figure with its 'ventral' side on the left-hand side. Sorting out of thepresumptive cell types into three roughly concentric layers has occurred, though mesoderm fails tosurround the endoderm completely. Ectoderm covers the whole surface of the reaggregate. No furtherdifferentiation of the cells has occurred, e., ectoderm; m., mesoderm; end., endoderm.

FIG. C. High-power view of intermingled cell types in the reaggregate obtained from an embryo fromwhich the presumptive dorsal lip had been excised. Ectoderm cells, recognizable by their numerouspigment granules, lie side by side with endoderm cells, which can be identified by their large yolkplatelets. Stain, celestin-blue and eosin.

{Manuscript received 9:1:62)

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