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Morphogenetic Interactions before Gastrulation in the Amphibian, Xenopus laevis—Regulation in Blastulae by A. s. G. CURTIS 1 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 considerable degree at the grey-crescent region by the 8-cell stage, probably by means of interactions within the cortex (Curtis, 19626). But this determination is still easily disturbed because excision of the grey-crescent cortex from the 8-cell stage does not prevent the appearance of a dorsal lip. The embryo forms a new dorsal lip by a process of regulation. Similarly, Votquenne (1933) destroyed the blastomere containing the grey crescent at the 8-cell stage in Ranafusca and nevertheless obtained nearly normal embryos; which result indicated that regulation had happened. At a later stage, late blastula, excision of the cells of the presumptive dorsal Up region does not prevent a dorsal lip forming and subsequent neural induction occurring (Goerttler, 1926) although the embryos are of the abnormal 'ring' type. But though regulation in Goerttler's experiments can hardly have occurred before gastrulation, due to the late stage used, it remains an open question whether the regulation of the embryo after Votquenne's excision of a micromere or my excision of cortex from the 8-cell stage embryo, happens soon after the operation of excision or whether the necessary interactions amongst cells are delayed until some later stage such as gastrulation. The answer to this question will help to reveal the extent of developmental change before gastrulation. There is reason for thinking that a degree of morphogenesis of the various presumptive regions occurs by late blastula stages because Dalcq (1933) showed that considerable localization of organs had occurred by the late blastula stage of R. fusca. For these reasons experiments have been made on regulation of the presumptive dorsal lip region in blastulae. Early blastulae have been used since there is a sufficiently long time before they become gastrulae to determine whether regulation occurs soon after removal of tissue or at gastrulation. In addition, since the presumptive dorsal lip region plays a major part in the imagination process when it reaches the gastrula stage (Schechtman, 1942) such experiments may throw light 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 a simple chemical treatment blocks regulation if applied just after excision of cells from the blastula, but does not do so if applied slightly later. These experiments will be described at length. The general deduction from them was that one essential step in regulation occurs fairly soon after excision. In consequence a series of experiments designed to elucidate further the mechanism of this interaction will be described. A preceding paper (Curtis, 19626) showed that important morphogenetic factors are located in the cortex. These experiments provide some suggestions that these factors are still located in the cortex at blastula stages and interact between various parts of the embryo during regulation. METHODS Xenopus laevis morulae were decapsulated and stripped of their vitelline membranes by hand: they were cultured in Holtfreter saline buffered to pH 7-0 with 0 001 M 2-amino-2-hydroxymethyl-1, 3-propanediol-h ydrochloric acid (tris-HCl). In experiments on regulation after excision, the whole of the dorsal lip and much of the presumptive nervous system were the regions excised. The tissues were removed down to the blastocoel roof. Since the exact extent of these presumptive regions is uncertain, more tissue than contains these regions was cut out (see Text-fig. 1); in consequence it is unlikely that any tissue which would normally invaginate and carry out induction remained. The controls for TEXT-FIG. 1. Side view of the embryo at early blastula stage. The dashes indicate the limits of these experiments were unoperated emthe regions which were excised in these experi- bryos. Control and experimental embryos ments. They include presumptive dorsal lip and much of the presumptive nervous tissue. were treated with 0001 M tetra-sodium The position of the lower limit was carefully ethylene-diamine tetra-acetate buffered kept to in all excisions but the position of the animal and anterior limit was somewhat vari- at pH 8-2 with 0-001 M 2-amino-2-hydroable, in many cases rather less presumptive xymethyl-1,3 -propanediol-hydrochloric nervous tissue was excised. acid, made up in calcium-free Holtfreter saline. Embryos were treated with this medium for either 1 hour with mechanical agitation when complete disaggregation was required or for half an hour when 'collapsed' embryos were required. Collapsed embryos are those in which most cells are only slightly disturbed from their normal positions although the embryo has flattened down on the bottom of the culture dish and the blastocoel has collapsed. In the various experimental series this treatment was applied at varying times after the excisions were made. A. S. G. CURTIS—REGULATION IN BLASTULAE 453 Embryos were returned to the normal culture medium after this treatment. In three additional series grafts of either presumptive dorsal lip from blastulae or part of the margins of the wound left in blastulae after excision of the presumptive dorsal lip, were placed in the embryos after treatment with the EDTA solution. All embryos were cultured in the normal Holtfreter saline at 19-21° C. until they reached an age equivalent to stage 22 (Nieuwkoop & Faber, 1956). They were then fixed in formol-bichromate, embedded in celloidin-paraffin, sectioned at 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 repeated as a control whenever experiments on regulation were carried out. The embryos had their presumptive dorsal lip removed in the early blastula stage, and were then placed in the EDTA solution within 5 minutes. They were either treated with this solution for 30 minutes without mechanical agitation to produce the 'collapsed' type of embryo, or for 1 hour with agitation which produces complete disaggregation. Thirty-six 'collapsed' embryos were produced in six batches. At the end of the treatment with the EDTA solution the embryos had partially flattened down on to the bottom of the culture dish giving them a slumped appearance. Most of the cells in these embryos were still in roughly the correct relationship to one another. Their ectoderm was little damaged though the excision wounds had opened out. After their return to the Holtfreter saline the embryos healed rapidly and redeveloped their rigidity so that they rested on the bottom of the culture dish in a normal (unslumped) condition. Thirty-three of these embryos showed no definite sign of gastrulation, though three gastrulated normally. Unfortunately, it is hard to be certain whether or not slight invagination occurred in these thirty-three embryos, but no definite sign of the positioning of the tissues which would result from gastrulation was found. The ventral surfaces of these embryos were composed of endodermal cells; this fact suggests that invagination had failed. None of these thirty-three embryos showed any sign of neural differentiation, although all were composed of healthy cells. However, somite and/ or notochordal tissue lying dorsally in an unoriented mass appeared in fifteen embryos (see Plate, fig. A); in only one of them was the mesodermal differentiation solely notochordal. No elongation of these embryos occurred. The three embryos which had gastrulated developed nervous systems, somites, and notochord. In two embryos the nervous systems were neuroid in nature, but the other embryo had a fairly normal nervous system. When complete disaggregation was allowed to occur care was taken to mix the various cell types thoroughly by mechanical agitation. Twelve embryos were 454 A. S. G. CURTIS—REGULATION IN BLASTULAE disaggregated, and after their return to the normal Holtfreter saline all reaggregated successfully. They were grown for 20 hours, a period of time more than sufficient for complete cell sorting out to occur (Curtis, 1961). At the end of this time they were fixed. None of the twelve reaggregates showed any appreciable sign of the sorting out of the various cell types, though the outermost cells were rather flattened in an epithelial manner. Prospective ectodermal, mesodermal, and endodermal cells were mixed up randomly in these embryos, see Plate, fig. C. The next series forms 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 lips removed and were allowed to heal in normal Holtfreter saline for 3 hours. Then they were treated with the EDTA solution for 30 minutes to produce 'collapsed' embryos; thereafter they were grown in normal saline. Twelve of these embryos showed differentiation of all main cell types: they had well-organized neural tissue, notochord, and somites which formed axial structures. Of the remaining sixteen embryos seven differentiated mesoderm cells, but in nine embryos no sign of morphogenesis was found. Comparing these results with those of the previous series suggests that the time of treatment after excision affects the degree of regulation. It is necessary, however, to discover whether these effects of ageing would appear in embryos in which no excisions had been made. (c) Intact blastulae treated with EDTA Three groups of experiments were performed. Firstly, twenty-seven early blastulae were placed in the EDTA solution for 30 minutes which brought them to the collapsed state, returned to normal saline and cultured to fixation at stage 22. Of these embryos fourteen showed development of neural tissues. In eleven of them the nervous structures were either unclosed neural tubes or neuroid structures, or spina bifida. Four embryos were without any sign of tissue differentiation, but the remaining nine embryos produced somite and/or notochordal tissue. The experiment was performed in four batches. It was already known from earlier unpublished work that late blastulae could be disaggregated with EDTA solution without affecting the subsequent sorting out after reaggregation of the various cell types, so only a small confirmatory series of twelve experiments on early blastulae was undertaken. This second series forms a control to those disaggregates of excised embryos, see (a). These twelve disaggregates reaggregated successfully, and sorting out of the presumptive cell types occurred in them so that presumptive ectoderm lay at the outside, endoderm innermost, and mesoderm in between. But no differentiation other than that pre-existing in yolk platelet size, cell size, and pigmentation was found in these reaggregates, see Plate, fig. B. A. S. G. CURTIS—REGULATION IN BLASTULAE 455 In the third series a dozen intact blastulae were treated with EDTA solution for 30 minutes to produce 'collapsed' embryos, when they had reached an age corresponding to that of the excised embryos given a 'delayed' treatment, see (b), namely at mid-blastula stage. Eleven of these embryos developed all the main tissue types, though bilaterally asymmetrical nervous systems and large notochords were common. In one embryo somite tissue was the only cell type differentiated. Although statistical evaluation of these results is deferred to the next main section it already appears that immediate treatment with EDTA probably has more effect in stopping regulation than delayed treatment. The prime stimulus to regulation may be the excision of cells. If so, the stimulus is transmitted over a wide area producing a reaction in many cells. The failure- of regulation after immediate EDTA treatment brings forward the question of whether its effect is upon the reacting system or upon the system that transmits the stimulus. The following 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 presumably the first regions to bear the stimulus to regulate. In consequence grafts of wound-margin made to embryos in which regulation has been blocked might restart regulation. If so this result would suggest that EDTA damages the transmission system and not the reaction system. Likewise grafts of presumptive dorsal lip might be able to induce fresh nervous systems in ectoderm if the EDTA had 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 same excision was carried out on other early blastulae to form wound-margins. One graft from the wound-margin of these embryos was placed in each of the treated embryos within 5 minutes of the end of the EDTA treatment. The grafts were not treated with EDTA. They were placed in the wounded area of the embryos. Of the twenty-one grafted embryos only two showed regulation to form a nervous system; somite production alone occurred in three more embryos. No morphogenesis appeared in the remainder. Furthermore, the grafts showed no differentiation themselves, which suggests that the host embryos exert some kind of suppression on the grafts. In a subsidiary experiment the grafts were taken from the margins of excision wounds which had healed for 3 hours, and the host embryos had just been treated with EDTA solution. Of these nine embryos, only two showed signs of neural differentiation or other morphogenesis. Grafts of presumptive dorsal lip from early blastulae were placed in embryos which had been treated with EDTA after excision. Of the six embryos receiving this type of graft one alone showed development of neural structures; the remaining five were without signs of morphogenesis in either graft or host. 5584.10 Gg 456 A. S. G. CURTIS—REGULATION IN BLASTULAE Statistical testing of results The incidence of neural induction and mesoderm differentiation in the various experiments is displayed in Table 1. The effects of EDTA treatment applied to embryos immediately after regulation began or applied 3 hours later can be compared by the method of Cochran and Cox (see Snedecor, 1946) since the experiments were done in six and four groups respectively, with five to seven embryos in each group. There is significantly less neural induction in those treated with EDTA to produce 'collapse' immediately after regulation began, than in the other group, P < 0-05. Likewise there is significantly more mesoderm differentiation than neural differentiation in embryos treated with EDTA immediately 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 lip Immediate treatment with EDTA Collapsed embryos Disaggregates Delayed treatment Collapsed embryos No excision Immediate treatment with EDTA Collapsed embryos Disaggregates Delayed treatment Collapsed embryos Grafts into blocked embryos Fresh wound-margin 3-hour-old wound-margin Presumptive dorsal lip Mesoderm differentiation alone No. of embryos Neural induction 6 6 5 6 7 6 12 1 1 0 1 0 1 0 2 3 0 4 1 5 0 7 7 7 7 4 3 2 3 2 2 1 2 3 5 2 4 2 1 2 0 0 12 11 1 19 9 6 2 2 1 2 5 0 10 6 6 5 12 4 Neural differentiation is affected by EDTA treatment to a much smaller extent in unoperated embryos than in ones in which the presumptive dorsal lip has been excised (using method of Cochran and Cox, P < 0-05). Mesoderm differentiation is also more extensive in the unoperated embryos than in the excised ones, P < 005. A. S. G. CURTIS—REGULATION IN BLASTULAE 457 Comparisons with the remaining groups have been made by the 2 x 2 contingency test method. Despite the fact that there is more neural differentiation when EDTA treatment after excision is delayed there is still significantly less neural differentiation in the delayed group than in those unexcised embryos treated with EDTA at a stage 3 hours later than the excision operation, P < 0-005. Likewise there is more neural differentiation in unoperated embryos treated 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 the effects of EDTA treatment through the blastula stage. Grafts of wound-margin do not unblock differentiation in embryos treated with EDTA immediately after excision, whether the wound-margin is fresh or 3 hours old, P > 0-1 in both cases, when compared with similar ungrafted embryos. Likewise grafts of presumptive dorsal lip are without effect in restarting differentiation in similar embryos; P > 0-1 when compared with the ungrafted embryos. If they are compared with unexcised embryos treated with EDTA to produce 'collapse' such grafted embryos show a significant reduction in neural differentiation, P < 0-005. It can be concluded that one essential step in regulation occurs soon after the excision of the tissue, long before gastrulation, and that thereafter EDTA has little effect on regulation in embryos in which the wound has healed. This step is blocked by the action of EDTA. However, this behaviour is probably superimposed on a general increase in the resistance of the unoperated embryo to the action of EDTA during the blastula stage. It appears that the ability of cell types such as mesoderm, endoderm, and ectoderm to sort out in reaggregation is destroyed by excision of the presumptive dorsal lip and surrounding regions if regulation cannot take place. But, although failure of regulation occurs with EDTA treatment and though the dorsal lip has been removed, morphogenesis of mesoderm is not extensively suppressed. Grafts of untreated wound-margin or presumptive dorsal lip will not restart regulation. DISCUSSION Goerttler (1926) had shown that excision of the presumptive dorsal lip from blastulae 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 a fairly ventral level in the future blastopore region could still form dorsal lip when the ventral half of the blastula was rotated under the dorsal half, and Brice (1958) observed that the isolated ventral half of a blastula might, 'develop a dorsal lip'. In the present experiments the same type of regulation of cells to form dorsal lip has been found many times. To a large degree the evidence for the formation of a dorsal lip has been the discovery of signs of invagination and induction. But, although this is not wholly conclusive evidence for regulation, 458 A. S. G. CURTIS—REGULATION IN BLASTULAE accepting any alternative effect would involve proposing new theories about the nature of gastrulation and neural induction. The results show that one step in the morphogenetic process which reforms the dorsal lip occurs soon after the excision of the cells. This step is susceptible to damage by the action of EDTA. In addition this part of the process appears to be related to the mechanism whereby cells acquire those properties which allow them to sort out in reaggregates. Sorting out of the various cell types does not occur in reaggregates from embryos in which regulation has been blocked. Although this might be explained on the hypothesis that since induction at gastrulation is inhibited by blocking regulation these properties of the cells are normally developed at gastrulation, there are several objections to such a hypothesis. First, disaggregates of normal blastulae do sort out correctly into the various cell layers, as observed in this work, and appreciable separation of the cell types occurs although none of the neural differentiation associated with gastrular induction happens. It is difficult to imagine how induction would occur in a disaggregate before sorting out when the various cell types are mixed up randomly. In a recent review (Curtis, 1962a) it was pointed out that the sorting out of cell types in reaggregates involves differences in both motile and adhesive properties between the various cell types. Gastrulation is a similar process in that considerable directed movement of the cells is involved. Thus it is reasonable to expect that such adhesive and motile properties of the cells would be differentiated before gastrulation, and the results of the present work suggest that this differentiation can occur anew soon after excision of cells from the early blastula. In the preceding paper (Curtis, 19626) it was suggested that important morphogenetic factors responsible in part for determining the site of the future dorsal lip were localized in the cortex during cleavage stages. These factors were organized in such a way that excisions were regulated for and additions by way of grafts were of no effect. It appears that during early blastula stages at least, the same type of system operates, since excisions of presumptive dorsal lip cells are regulated for. This correspondence suggests either that regulation for operations done on cleavage stages does not occur till the early blastula stage or that the same system operates at both stages. Since some of the properties affected by regulation appear to be cell surface properties it is possible that regulation is a phenomenon involving the cortex alone even during blastula stages. In this connexion it is of interest that once the wound caused by the excision has healed the process of regulation can no longer be affected by treatment with EDTA. This result implies that very extensive areas of cells in contact may be a necessary condition of regulation. Grafting untreated wound-margin to blastulae in which regulation has been blocked did not restart regulation. This result suggests that it is not the system which transmits the stimulus to regulation which is damaged by EDTA, but that it is the reacting system. The reason for such a conclusion is that the graft A. S. G. CURTIS—REGULATION IN BLASTULAE 459 from an embryo which had just been wounded would be expected to contain or develop the stimulus when it was placed in the blocked embryo. However, it is possible that transmission and reaction are carried out by the same system. In addition not only are grafts to blocked embryos without effect on regulation but there appears to be an active suppression of the development of the graft itself: for instance, grafts of presumptive dorsal lip fail to undergo self-differentiation even though they contain presumptive mesodermal and neural elements. Furthermore, the treatment with EDTA only affects morphogenesis extensively in embryos which have had cells excised from them. EDTA has little effect on the development of whole embryos. This result implies that regulation in some way 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 of the excision. The grafting experiments mentioned above suggest that it is the reaction to this stimulus which is affected. The reaction is not merely temporarily prevented but appears to be in some way directed into the wrong channel so that active suppression of morphogenesis occurs. EDTA tends to separate cells even when the treatment is of short duration. Thus its effect has a considerable similarity to that of wounding, and therefore EDTA might be expected to produce a stimulus similar to that occurring in regulation. The fact that intact embryos show some degree of susceptibility to damage of their differentiation mechanisms by EDTA at least in the early blastula stage may reflect this possibility. Presumably the lesser degree of inhibition of development found in such embryos compared with that in regulating embryos treated with EDTA, may be explained as being due to the chemical acting simultaneously at many points to separate the cells. Thus any stimulus to regulate the cells might produce on partial loss of contact, can only spread amongst a few cells before it is barred by a gap between cells. In the embryo in which an excision has just been made by operation cell contact is good on either lateral side of the excision so that the stimulus to regulate can be easily transmitted to many cells before the embryo is placed in EDTA. I do not think one should conclude that since EDTA treatment blocks regulation, calcium ions must be involved causally in the regulatory system. Not only does this chemical have effects other than the chelation of calcium but it is also possible that the important effect is a physical one, such as a temporary loss of contact between cells or alteration of the mechanical properties of the cell surface. Shoger (1960) suggested that the mechanical interaction of cells may be of importance in the regulation of the chick primitive node region. He treated whole nodes isolated from the rest of the embryo with EDTA and observed considerable though not total suppression of morphogenesis; the experiment is not exactly comparable with the present ones, for the node most nearly represents 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 460 A. S. G. CURTIS—REGULATION IN BLASTULAE one based on a' field' organization of morphogenetic factors originally developed by Dalcq & Pasteels (1937). For the purpose of this paper this 'field'will be defined as that network of spatial and temporal interactions involved in producing the dorsal lip of the blastopore and the related invagination. The ideas advanced in the previous paper (Curtis, 19626) to explain the organization of such factors in the cortex of the 8-cell stage will be extended. In the intact embryo the main arrangement of the morphogenetic field has been determined by the 8-cell stage, and the field may be centered on the future site of the dorsal lip. One particular structure, the dorsal lip, appears at the 'highest' value of this field. If this region is cut out another nearby region becomes that of highest value and thus becomes the dorsal lip. But how does this new region have the information that it alone is the now 'highest' region? This problem occurs in considering both the regulation of cortical excisions and of blastular excisions. Since regulation involves a change in function for many cells in the embryo it seems that the information must be transmitted between them. In addition the apparent necessity for cell contact to complete regulation, the behaviour of grafts in blocked embryos, and the sensitivity of regulation to the action of EDTA, all argue that a transmission system is involved in readjusting the field after excisions of cells have been made. It seems that this system for transmission of the first stimulus to regulation and the reaction to it only comes into existence after an excision has been made, because EDTA damages it then but is without great effect on the morphogenesis of embryos in which no excision has been made. In other words, there is no transmission of information between the cells of the undamaged blastula, but excision activates the morphogenetic field so that interaction takes place and a new field is established in the regulated embryo. The activation is not susceptible to the effects of EDTA treatment but the reaction, namely the interaction between parts of thefieldto establish a new field, is affected. In consequence it would be expected that EDTA would have no large effect on the subsequent morphogenesis of whole unoperated blastulae not allowed to disaggregate, which of course is so. If a new field is established, which apparently requires that extensive cell contact be present, the embryo again is not greatly damaged by EDTA treatment. So it appears that the morphogenetic field in the blastula is normally arranged in an inactive manner so that no information is exchanged between cells, and regulation is a process in which it becomes activated and its various parts interact with one another. Since the blocked embryo has apparently some suppressive action on grafts of normal tissue it is possible that this influence may be organized in a fashion like a morphogenetic field. It is usual to presume that the properties in a field system are graded evenly from one part of thefieldto another. In consequence it may seem a little illogical that it should apparently be involved in determining the cell properties responsible 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 A. S. G. CURTIS—REGULATION IN BLASTULAE 461 reflecting a graded property. However, if these cell types represent various phase states of the cell surface as I suggested (1962a) grading of the chemical properties of the surfaces of various cells in accordance with the field would produce only a few different phase states and thus types of cell. The incidence of mesoderm differentiation in embryos in which regulation and gastrulation have been blocked suggests that mesoderm is predetermined before gastrulation to a considerable extent. Evidence for such a possibility was found by Dalcq (1933). SUMMARY 1. After the presumptive dorsal lip is excised from early blastulae of Xenopus Iaevis the tissues surrounding regulate to reform the dorsal lip. This regulation can be prevented by treatment of the embryos with the chelating agent, EDTA, provided that treatment is carried out fairly soon after excision. If regulation is prevented there is no differentiation of nervous tissue, but, nevertheless, there is a considerable mesodermal differentiation. If embryos whose presumptive dorsal lips have been excised are completely disaggregated with EDTA, reaggregation occurs but no sorting out of the cell types is found. Disaggregates of whole embryos reaggregate and show sorting out of the cell types though no further differentiation occurs. 2. These effects appear to be superimposed on a slight sensitivity of the intact embryo to damage to its neural differentiation by EDTA in the early blastula stage. But this sensitivity is greatly increased if excision of presumptive dorsal lip is carried out before treatment with EDTA. 3. Grafts of wound-margin material from embryos whose presumptive dorsal lips have been excised will not restart regulation in embryos blocked by EDTA treatment, whether the grafts are from freshly wounded embryos or from embryos wounded some hours previously. Likewise grafts of presumptive dorsal lip 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 the excision of tissue, long before gastrulation. This step is sensitive to the action of EDTA. It appears to be connected with the determination of those cell properties responsible for the sorting out of cell types in reaggregation, and there is considerable evidence that these properties are determined in the blastula stage. It seems probable that EDTA acts on the system which reacts to the stimulus to regulate and not on the actual transmission of this stimulus. Blocked embryos appear to contain a system such that additions, e.g. grafts, have their morphogenetic potentialities suppressed. Regulation in intact blastulae acts to replace missing tissues. The regulative properties of blastulae are probably the same as those to be found in mid-cleavage stages, save that there is some evidence of partial determination of mesoderm by the blastula stage. These results are 462 A. S. G. CURTIS—REGULATION IN BLASTULAE discussed in terms of a field theory, and it is suggested that regulation requires an intercellular communication system. RESUME Interactions morphogenetiques pre-gastruleennes chez VAmphibien Xenopus laevis. Regulation chez les blastulas 1. Apres excision de la levre dorsale presomptive de jeunes blastulas de Xenope, les tissus environnants reforment cette levre dorsale par regulation. Celle-ci peut etre empechee en traitant les embryons par l'ethylene diamine tetra-acetate (EDTA), pourvu que ce traitement soit accompli assez tot apres l'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 a ete excisee, la reagregation se produit mais on n'observe pas de segregation des types cellulaires. Les desagregats d'embryons entiers se reagregent et montrent une segregation des types cellulaires bien qu'il n'y ait pas de differenciation ulterieure. 2. Ces effets paraissent etre surimposes a une legere sensibilite de l'embryon intact aux effets nocifs de l'EDTA, au stade de la jeune blastula, envers sa differenciation nerveuse. Mais cette sensibilite est tres augmentee si on pratique l'excision de la levre dorsale presomptive avant le traitement a l'EDTA. 3. Des greffons de materiel marginal de la blessure, pris sur des embryons dont la levre dorsale presomptive a ete excisee, ne reprendront pas leur regulation chez les embryons bloques par le traitement a l'EDTA, — que ces greffons proviennent d'embryons fraichement blesses ou d'embryons blesses quelques heures avant. De meme, des greffons de materiel de la levre dorsale presomptive ne retablissent pas la regulation ou la differenciation des embryons bloques, et les greffons eux-memes ne se differencient pas. 4. Les resultats suggerent qu'au moins une etape de la regulation se situe tot apres l'excision du tissu, longtemps avant la gastrulation. Cette etape est sensible a Faction de l'EDTA. Elle apparait en liaison avec la determination de celles des proprietes cellulaires responsables de la segregation des types cellulaires au cours de la reagregation et il existe de nombreux faits montrant que ces proprietes sont determinees au stade blastula. II semble probable que l'EDTA agisse sur le systeme qui reagit au stimulus en regulant, et non sur la transmission reelle de ce stimulus. Les embryons bloques paraissent renfermer un systeme tel que des additions, c'est-a-dire des greffons, voient leurs potentialites morphogenetiques supprimees. La regulation dans les blastulas intactes agit pour remplacer des tissus manquants. Les proprietes regulatives des blastulas sont probablement les memes que celles observees aux stades moyens de la segmentation, sauf qu'il existe des indices d'une determination partielle du mesoderme au 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 A. S. G. CURTIS—REGULATION IN BLASTULAE 463 et on suggere que la regulation requiert un systeme de communications intercellulaires. 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, for his able assistance. The work was carried out during the tenure of grant C-4847 from the National Cancer Institute, U.S.A. REFERENCES BRICE, M. C. (1958). A re-analysis of the consequences of frontal and sagittal constrictions of newt blastulae and gastrulae. Arch. Biol, Liege et Paris, 69, 371-440. CURTIS, A. S. G. (1961). Timing mechanisms in the specific adhesion of cells. Exp. Cell Res. Suppl. 8, 107-22. (1962a). Cell contact and adhesion. Biol. Rev. 37, 82-129. (19626). Morphogenetic interactions before gastrulation in the amphibian, Xenopus laevis—the cortical field. / . Embryol. exp. Morph. 10, 410-22. DALCQ, A. (1933). Experiences de transplantation, de translocation et d'ablation relatives a la determination du systeme nerveux primitif chez les amphibiens. C.R. Soc. Biol. Paris, 114, 159-63. & PASTEELS, J. (1937). Une conception nouvelle des bases physiologiques de la morphogenese. 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 Publishing Co. SCHECHTMAN, A. M. (1942). The mechanism of amphibian gastrulation. Univ. Calif. Publ. Zool. 51, 1-39. SHOGER, R. L. (1960). The regulative capacity of the node region. / . Exp. Zool. 143, 221-38. SNEDECOR, G. W. (1946). Statistical Methods, pp. 485. Iowa: State College Press. VOTQUENNE, J. (1933). La disposition generale des ebauches presomptives dans Fceuf de Grenouille divise en huit blastomeres et les consequences de la destruction d'un micromere dorsal. C.R. Soc. Biol. Paris, 113, 1531-3. E X P L A N A T I O N OF PLATE FIG. A. Section through an embryo near its anterior end from which the presumptive dorsal lip had been excised at early blastula stage. The embryo was then treated with EDTA. The section is mainly transverse but somewhat tilted so that the dorsal half of the embryo is anterior to the ventral half. 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 bottommost. The vacuolation of the mesoderm cells, indicative of some degree of differentiation, is just visible in the middle of this section. FIG. B. Section through the reaggregate from a whole blastula which had been disaggregated. The reaggregate is positioned in this figure with its 'ventral' side on the left-hand side. Sorting out of the presumptive cell types into three roughly concentric layers has occurred, though mesoderm fails to surround the endoderm completely. Ectoderm covers the whole surface of the reaggregate. No further differentiation 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 from which the presumptive dorsal lip had been excised. Ectoderm cells, recognizable by their numerous pigment granules, lie side by side with endoderm cells, which can be identified by their large yolk platelets. Stain, celestin-blue and eosin. {Manuscript received 9:1:62)