ABSTRACT
Although the main visible forms of morphogenesis commence at gastrulation it is of great interest to discover whether they are preceded by a series of preparatory changes just as essential to development, although they have no immediately visible effects. When Dalcq & Pasteels (1937) proposed the hypothesis that the cortex and yolk respectively contained morphogenetic factors which determine the main structural features of the embryo, they did not suggest when or how these factors act. I have described a method (Curtis, 1960) for grafting portions of the cortex of the Xenopus egg from one egg to another, and by its use was able to confirm that parts at least of the cortex contain a morphogenetic factor. In the present work this technique has been used to make grafts between the cortex of embryonic cells of varying ages in order to discover whether the cortex alters its morphogenetic properties as development proceeds. A related question is whether the cortex brings its morphogenetic factors into action only when gastrulation begins or does it act at an earlier stage.
A very similar question is posed by the regulation of blastulae from which the presumptive nervous tissue has been removed for these embryos still produce a nervous system (Bruns, 1931). Do some of the morphogenetic changes required to establish a new nervous system occur immediately after the operation in the blastula or are they all deferred until gastrulation begins ? Experiments to resolve this question are described in a succeeding paper.
The main problem approached in this paper is whether or not morphogenetic interaction occurs before gastrulation. Are the factors of morphogenesis stored unused until gastrulation, when they start to act simultaneously or do they act long before gastrulation initiating a sequence of operations through which the developing embryo must pass?
In earlier work (Curtis, 1960) it was found that grafts of grey-crescent cortex from uncleaved fertile eggs placed in the ventral margin of a second egg of the same age would induce a whole second embryonic axis. I have repeated this type of experiment, making grafts between embryos of differing age or of the same age but at later stages, in order to discover whether this induction occurs, or occurs in a different fashion. If the induction does not occur it would suggest that the inducing system has altered since the single-cell stage of the embryo. In addition, closely related effects should be revealed by excision of the greycrescent cortex—a type of experiment closely parallel to the cytoplasmic excisions in the protozoon, Stentor, described by Tartar (1961). Since the subcortical cytoplasm may be of importance in this morphogenetic system excisions and grafts of it have also been made.
METHODS
Uncleaved fertile X. laevis eggs were decapsulated and stripped of their vitelline membranes by hand and stored in Holtfreter solution buffered to pH 6·3 with 0001 M 2-amino-2-hydroxymethyl-l,3-propanediol-hydrochloric acid. Cortical grafts were made by a technique largely the same as that described previously (Curtis, 1960), with a number of modifications, which result in a larger proportion of successful grafts. Chief amongst these is that many of the grafts have been placed in the recipient cortex just after, instead of before, calcium ions have been returned to the medium and its pH dropped back to pH 6·3. In consequence the graft was placed in the wound just after it began to heal instead of somewhile before: presumably this modification makes for success because it obviates the danger of the graft being damaged while the medium is changed. A second modification has been the abandonment of the use of the supplementary medium containing heavy metal ions. A third change has resulted from the finding that embryos stripped of their vitelline membranes are very easily damaged if they are moved by pipette from dish to dish. This damage is of a deceptive nature because it does not appear until some stages later. In consequence once the embryos have been stripped of their vitelline membranes they are not moved throughout all stages of the operation. Two examples of cortical grafts soon after grafting are shown in Plate, figs. A, B. Grafts were about 150 μ× 150μin area in all operations.
Portions of the cortex were excised from embryonic cells with fine tungsten needles. The excisions were made with the embryos in 0·001 M tetra-sodium ethylene-diamine tetra-acetate in calcium-free Holtfreter saline buffered at pH 8·2 with 0·001 M 2-amino-2-hydroxymethyl-l,3-propanediol-hydrochloric acid. The medium was replaced with normal Holtfreter saline buffered at pH 6·3 as soon as the excision was completed.
Simple excision of cytoplasm from beneath the cortex was carried out by lifting a small flap of the cortex at the site of excision with a needle: a micropipette working by capillary action was inserted into the subcortical cytoplasm to suck a small amount of cytoplasm out of the cell. These excisions were made with the embryo in Holtfreter saline containing twice the normal calcium concentration. A small pit remains for a short while after the excision, but it soon disappears and the cortex heals over the excised region.
In order to make grafts of subcortical cytoplasm a site for the graft in the recipient embryo was prepared by excising a portion of the existing cytoplasm. The graft was quickly cut out of a second cell with needles and pushed into the prepared site. These grafts were made in a Holtfreter saline containing four times the normal calcium concentration. Under these conditions the cytoplasm is sufficiently firm to be coherent during the brief process of grafting. The cortex soon healed over the grafted material.
The embryos and unoperated controls were cultured in normal Holtfreter saline pH 6·3 for several stages after operation: thereafter the tonicity of the medium was slowly dropped until the medium was completely replaced by 0·5 Holtfreter saline. Embryos were grown at 19–21° C. until they reached Nieuw-koop stage 21–22 (late neurula) and were then fixed in formol-bichromate solution, embedded in celloidin-paraffin, sectioned at 8–9 μ thickness, and stained with celestin-blue and eosin.
In the present work all grafts were made with material from the grey-crescent site placed in the ventral margin. Grafts were made between cells in these sites in embryos of varying ages according to the following plan.
Donor stage 4; Recipient stage 4 (i.e. 8-cell stage).
Donor stage 4; Recipient stage 1.
Donor stage 1; Recipient stage 4 (Nieuwkoop stages).
In addition subcortical cytoplasm was grafted from the grey crescent of stage 4 to the ventral margin of stage 4. Grey crescent cortex was excised from stage 1 and 4 embryos and subcortical cytoplasm from the grey crescent of stage 4.
RESULTS
Cortical grafts
Stage 4 to stage 4
Grafts of grey crescent from stage 4 were made to the ventral margin of embryos of the same stage. Of the ten grafts made all survived to fixation at stage 21–22. The graft could be recognized for some stages after the operation, see Plate, fig. A. During gastrulation slight loss of endoderm cells occurred from the yolk plug in four embryos. Neurulation proceeded normally. Late cleavage stages and blastula stages were entirely normal.
Examination of the sectioned material showed that a primary axis was established in the normal position in every embryo and there was no sign of the induction of a second axis. The head structures of two embryos were slightly distorted. In three embryos very small masses of pigmented material lying in the anterior belly epithelium could be found in one or two sections. I think it possible that this material represents the remains of the graft.
Stage 1 to stage 4
Ten grafts were made in this series. The results were like those of the preceding series in the main respects : there being no sign of induction by the grafts in embryos which showed normal development till fixation. In one embryo a slight thickening of the belly epithelium, so that it was 3–4 cell layers thick in an area about 30 μ by 40 μ, was observed, but otherwise there was no sign of the graft influencing the host.
Stage 4 to Stage 1
Thirteen embryos received this graft and developed well after the operation. One such embryo is shown in the Plate, fig. B. Eleven embryos showed the induction of a second axis complete with neural tube, notochord, and somite material. The neural tubes were rather abnormal in the arrangement of tissue around the central lumen; often being eccentric, see Plate, fig. C. In the remaining two embryos there was no proper induction but a large mass of rather disorganized epithelial material appeared on the ventral flank. Although only two embryos were observed at the beginning of gastrulation, when two separate dorsal lips formed in each embryo which had a common blastopore; it could be deduced from observation of the remainder at the end of gastrulation that separate dorsal lips had probably formed.
Cortical excisions
Large portions of the cortex can be excised from early embryonic stages; the wound heals over within 10 minutes in normal Holtfreter saline. However, if more than about 10 per cent, of the cortex is removed, although healing occurs it is followed by the appearance of damage to the cells some few cleavages later. The time of appearance of this damage seems to depend upon the size of the excision to some extent, for large excisions result in rapid damage but smaller ones cause damage which may not appear until blastula stages. The effect of large excisions from fertile uncleaved eggs appears at about the 16-cell stage; from 8-cell stages at about the 32-cell stage. Large blebs of cytoplasm protrude from cells in the vegetal region: apparently these blebs have no normal cell surface over them because they are very fragile by comparison with the normal embryonic cells. Simultaneously, small deeply pigmented bodies about 50 /z in diameter are budded off the cells at the animal pole, one cell producing several such bodies. These bodies remove most of the pigmentation from the animal pole cells and it is conjectured that they consist of cortex. Cells which form either blebs or pigmented bodies soon cytolyse completely but adjacent cells may remain unaffected if the excision was not large. The damage at the vegetal pole resembles that described (Curtis, 1960) when animal pole cortex was grafted into the vegetal pole.
If an extensive piece of cortex was removed these processes develop to such an extent that the embryo is completely cytolysed. But if less cortex was excised the damage is less extensive and an embryo containing many cells survives. Such damage occurred in about one-half of all the excisions attempted, but the following results refer alone to those embryos in which no such damage appeared-
Ten excisions of grey-crescent cortex from uncleaved fertile embryos were made. Cleavage continued unimpaired in all these embryos. Two formed normal neurulae. The remaining eight embryos showed no sign of morphogenesis or gastrulation at stage 22. In these embryos mitoses were still happening at fixation and it appeared that mitosis and cleavage had continued unimpeded. The cells were of increasing size from animal to vegetal pole, see Plate, fig. D. It is difficult to excise all the grey crescent without producing the damage described above; this may mean that not all the crescent was excised in those operations which produced normal embryos.
Eight stage-4 embryos had their grey-crescent cortex excised. The cortex was removed from either cell on each side of the midline. The wounds healed well and development continued normally. All the embryos showed no abnormality at stage 21.
Excisions of sub-cortical cytoplasm
About 50–100 μ3 of subcortical cytoplasm was removed from beneath the grey-crescent cortex of each of ten stage-4 embryos (8 cell). The wounds healed very rapidly. Development continued normally through blastula and gastrula stages. All the embryos showed differentiation of the main cell types. Seven embryos were normal in all major respects at stage 22 though three of these showed slight signs of bilateral asymmetry in the brain region. The remaining three embryos had neuroid types of nervous systems.
Grafts of subcortical cytoplasm
Grafts of cytoplasm from beneath the grey crescent of stage-4 embryos were placed in the ventral margin region of embryos of the same stage. Unfortunately, a certain amount of cytoplasm tends to spill out of the grafting site during the operation, in consequence of which it is impossible to be certain how much material was grafted into the embryo. The cortex soon healed over the graft and development continued normally. Of the ten embryos so grafted none showed any sign of an induction by the graft. Four developed entirely normally; in one embryo the host nervous system only reached the neuroid stage of development. The remaining five embryos showed slight defects in their nervous systems : in three the nervous system was abnormal in the head region so that the neural lumen was split into several in the front of the head. In two embryos a slight splitting of the nervous tissues occurred posteriorly so that spina bifida developed. These results are rather similar to those of Pasteels (1932) who obtained defects in the head region from embryos which had been wounded at the uncleaved stage in the ventral margin region.
These results are summarized in Table 1; and some are shown in diagram in Text-fig. 1.
A diagrammatic summary of the main results, a, excision of the grey-crescent cortex from a stage-4 embryo results in a normal embryo being formed (compare with d). b, grafting greycrescent cortex from stage 1 to the ventral margin of stage 4 does not result in the induction of a second embryonic axis, c, grafts of grey-crescent cortex from stage-4 embryos to the ventral margins of stage-1 embryos induce secondary embryonic axes, d, excision of grey-crescent cortex from stage-1 embryos prevents morphogenesis though cleavage and mitosis continue.
A diagrammatic summary of the main results, a, excision of the grey-crescent cortex from a stage-4 embryo results in a normal embryo being formed (compare with d). b, grafting greycrescent cortex from stage 1 to the ventral margin of stage 4 does not result in the induction of a second embryonic axis, c, grafts of grey-crescent cortex from stage-4 embryos to the ventral margins of stage-1 embryos induce secondary embryonic axes, d, excision of grey-crescent cortex from stage-1 embryos prevents morphogenesis though cleavage and mitosis continue.
Interpretation of the results
Grey-crescent cortex from stage-4 embryos (8 cell) grafted to the ventral margin of uncleaved fertile eggs induces a second main embryonic axis, but does not do so when grafted to the same site in stage-4 embryos. This difference is statistically significant (P < 0·01) when a 2 × 2 contingency test is applied to the data of the results. A similar result is that there is no induction when greycrescent material from uncleaved fertile embryos is grafted to stage-4 embryos, although it is known from earlier work (Curtis, 1960) that it contains this active inducing factor. Comparing the results of this series with stage-4 to stage-1 grafts the difference is again significant (P < 0·01).
It is known that the grey-crescent cortex in the uncleaved fertile egg possesses the potentiality to induce an embryonic axis and that its ventral margin can accept this induction. But the stage-4 cortex, although still containing the inducing influence in the grey-crescent region, is unable to accept an induction. In other words, by stage 4 there has been a change in the system leading to the induction so that no secondary axis is produced.
These conclusions are extended by those derived from the experiments on cortical excision. The excision of grey-crescent cortex from uncleaved eggs results in a significant effect (P < 0·05), the arrest of morphogenesis, when they are compared with the effects of excisions from stage-4 embryos, whose morphogenesis was unaffected. Again a change in cortical properties is indicated between stages 1 and 4. These results also suggest that by stage 4 those parts of the morphogenetically important factors which are actually going to act are either no longer in the cortex or that they can in some manner be regenerated or regulated for when part of the cortex is removed. It may be felt that the failure of excision of cortex from stage-4 embryos to affect morphogenesis is due to the presence of cortical material in the cleavage furrows. This is, however, very unlikely, for the furrows open wide during the operation and cortex can be and was removed far into the furrows. In addition no pigmented cortex is found in the furrows, whereas it is the pigmented cortex which contains the factor at earlier stages. Selman & Waddington (1955) suggest that the cell surface in the furrow is synthesized de novo and is not formed by stretching the existing cortex. In consequence if the cortex of the furrow contains a morphogenetic factor this has been formed by the action of some transfer system from existing cortex, and represents a morphogenetic interaction.
Are the factors then present in the subcortical cytoplasm? If so the inducing ability of the grey-crescent cortex of stage-4 cells would be viewed as a sort of leftover part of the inducing system. However, excision of the cytoplasm from the subcortex of the grey crescent of stage 4 is without effect on morphogenesis, the results being without significant difference (P > 0·1) from those of the cortical excisions from this stage. Although this negative result suffers from the defect that it is always possible that I have not yet removed the ‘right’ piece of cytoplasm which would affect morphogenesis, it indicates that the inducing system has not moved into the subcortical cytoplasm.
This conclusion is in agreement with the result that grafts of grey-crescent subcortical cytoplasm do not induce second embryonic axes from the ventral margins of stage-4 embryos (P > 0·1) when compared with the effects of similar grafts of cortex. Obviously the subcortical cytoplasm does not contain such factors at stage 1 for excision of the cortex stops morphogenesis. It seems unlikely that in the short space of time between stages 1 and 4 the subcortex acquires the factors from the cortex and also becomes able to block the inductive effects of addition of subcortex (such as a graft) bearing the factors and yet neighbouring subcortex is able to acquire the factors again if it is excised.
In consequence it appears that the cortex undergoes some change between stages 1 and 4 which makes it impossible for fresh inductions to be made by grafting whenever it is the receptor of a graft. Nevertheless, the morphogenetic system does not leave the cortex but alters so that considerable restoration of excised parts can be made.
DISCUSSION
The mechanism which suggests itself as an explanation of these results is one modelled on the development of a gradient system. There are reasons for thinking that the morphogenetic influences in the cortex are organized as a field as Dalcq & Pasteels suggested (1937). The behaviour of the cortex in the 8-cell stage is such that additions to the cortex are without effect but excisions are replaced. This can be pictured as happening in a system in which the main features of the cortical field have been laid down. In consequence removal of a fairly large part of the cortex does not damage the system seriously because enough of the gradient remains to control development. For example, if the gradient works so that some part with the highest concentration of a substance or structure forms one definite tissue and the other parts form tissues in relation to this, the excision of the part of highest value will leave surrounding regions as those of highest value on which development will now centre. There is one objection to this hypothesis, which is that it may not seem to explain how grafts of stage 1 grey crescent into stage-4 embryos fail to produce an induction. An answer to this objection will be given a little further on.
In contrast the cortex in the uncleaved fertile egg may be pictured as containing no established gradient system, but only a morphogenetic factor centred in one place, the grey crescent. At some time between the first and third cleavages this grey crescent acts as a centre which initiates and activates the establishment of a cortical field. In consequence a graft of grey-crescent cortex into the uncleaved egg acts as a centre for activation. Hence in an embryo with such a graft the cortical field will spread from the host grey crescent and from the graft. This embryo with two separate cortical fields will, of course, produce a double embryo.
A hypothesis to meet the objection suggested two paragraphs previously can now be given. A graft of grey-crescent cortex made into a stage-4 egg is made after activation occurs, in consequence this graft cannot establish its own cortical field and hence it cannot affect development.
This hypothesis also explains how it is that excision of the grey crescent from uncleaved eggs prevents morphogenesis, because no activating centre is left. Even in the absence of this hypothesis the experimental results form a direct parallel with those of Seidel (1929) on Platycnemis from which he claimed that one part of the egg acted as an activating centre for development, for isolation of this region from the rest of the Platycnemis egg prevented development as does excision of one region of the Xenopus cortex.
In this discussion I have rather ignored the influence of the yolk gradient which Dalcq & Pasteels (1937) showed to be another important factor in morphogenesis. However, all grafts have been to the same part of the yolk gradient (ventral margin) as the grey crescent so that it does not become a direct factor in these experiments. But the yolk gradient plays an important part in activation since Dalcq & Pasteels showed that it was the interaction of this with the cortical factors that helped determine the formation of two embryonic axes in certain experimental situations. It might seem at first sight that the present interpretation is difficult to reconcile with Dalcq & Pasteels’s hypothesis about the interaction of these two morphogenetic factors. Their inversion or centrifugation experiments resulted in new regions of the cortex (sometimes two simultaneously) taking part in the formation of the dorsal lip of the blastopore. However, this result does not imply that the grey crescent is no longer the activating centre in such embryos. Their interpretation comes into complete agreement with the present hypothesis if we take their suggestion that the yolk gradient has some value at the junction of light and heavy plasms allowing the interaction which determines the site of the blastopore and add the following condition: that this interaction fixes and establishes the cortical field. If this is so the activation in inverted eggs would spread from the original grey crescent forming a rather labile system; on reaching those regions with the right value of the yolk gradient it would become fixed and irreversibly determined. Thus it would spread in this fixed condition outwards from one or two new centres forming one or two new morphogenetic fields.
The most important conclusion that can be drawn from these results is that a morphogenetic interaction occurs within the cortex before the 8-cell stage. This interaction is one in which the cortical field is determined. Since the cortical field helps control the future site of the dorsal lip it can be regarded as determining the main axes (anterior-posterior, lateral, and dorso-ventral) of the embryo. Because the cortical field acts in determining where the dorsal lip of the blastopore forms, it can be said to determine the main geometric axes of the embryo. Once the field is determined these axes are fixed. Some pertinent suggestions can be made in relation to this conclusion. The morphogenetic processes which happen in the cortex during activation occur in a very thin membrane. Whether the activation is a chemical process or a physical one its spread across the cortex is considerably impeded by the geometry of this membrane. The thinness and great extent of the cortex in the uncleaved egg would tend to slow down the spread of any change in the cortex. But once cell-division has occurred the diffusion of any change in the cortex becomes much easier because interaction can occur across the small gap separating apposed cell surfaces. The process of interaction is vastly aided because the diffusion process can occur over the wide area of contact between cells and across the narrow gap between them. Once three cleavages have been completed enough cell surfaces exist for these interactions to be made across apposed cell surfaces in each of the three axes of symmetry of the embryo. In this very tentative hypothesis I have assumed that the cortex or at least active derivatives of it are present in the cleavage furrows. The attraction of this hypothesis is that it does suggest how it is that the establishment of the cortical field occurs when it does.
The general argument given in this discussion is in agreement with the results of Dollander (1950), who found that the cortical field can be easily altered by ligaturing operations at the 2-cell stage. His experiments suggest that the cortical field has not been fixed in position at this stage. Votquenne (1933) found that removal of a micromere containing the grey crescent at the 8-cell stage in Rana fusca did not affect development : this result is paralleled in the present work by the excision of grey-crescent cortex at the same stage. Both results agree with the hypothesis in that once the field is established parts which are removed can be regulated for.
The damage following extensive excision of cortex in the 8-cell stage occurs not only in those cells which are derived from the original cell operated upon but also in other cells. This result implies that there is interaction between cells even in these early stages.
The absence of any large effect when cytoplasm was excised may at first seem to be in contradiction with the results of Milaire (1961) who found that various abnormalities developed in the nervous system after excision of subcortical cytoplasm. However, it is probable that much more cytoplasm was removed in his experiments than in mine.
One other general conclusion can be drawn from these results. This is that the next series of problems to be attacked are the mechanisms which establish the grey crescent in the fertile uncleaved egg. Presumably some of these are connected with the formation of the egg and others perhaps with fertilization; the function of the latter process in this respect has, of course, been extensively studied by Ancel & Vintemberger (1948).
SUMMARY
Grafts of grey-crescent cortex were placed in the ventral margin of cleavage stages of X. laevis embryos. Grafts from 8-cell embryos placed in uncleaved fertile eggs induced secondary embryonic axes, but such grafts placed in 8-cell embryos had no effect on morphogenesis. Grafts of cortex from uncleaved fertile eggs were without effect on the morphogenesis of the 8-cell embryo when placed in it.
Excisions of cortex from the grey crescent of uncleaved fertile eggs prevented morphogenesis although cleavage and mitosis were unimpeded. Excisions of such cortex from 8-cell stages were without effect on the normal morphogenesis of the embryos.
Excisions of sub-cortical cytoplasm from the grey-crescent cortex of 8-cell embryos do not affect development.
Grafts of subcortical cytoplasm from the grey-crescent region of 8-cell embryos were placed in the subcortex of the ventral margin of embryos of the same age. These grafts were without effect on morphogenesis other than slightly damaging brain formation.
These results taken in conjunction with those of previous work indicate that though the cortex contains morphogenetic factors determining the embryonic axes and the site of the dorsal lip at gastrulation, the nature of these factors undergoes some change between the beginning of the first and the end of the third cleavages. The change is such that the embryo becomes able to regulate for excisions or additions of cortex. The grey-crescent cortex appears to act as an activating centre in producing this alteration in the morphogenetic system of the embryo. There is no evidence that the cytoplasm beneath the cortex is directly involved in this interaction.
RÉSUMÉ
Interactions morphogénétiques avant la gastrulation chez l’Amphibien Xenopus laevis — le champ cortical
Des fragments de cortex du croissant gris ont été greffes dans la région ventrale de germes de Xénope en cours de segmentation. Des fragments prélevés au stade des 8 blastomères et greffés sur des œufs fécondés insegmentés y ont induit des axes embryonnaires secondaires, mais de tels greffons placés sur des germes au stade 8 bl. n’ont pas eu d’effets sur leur morphogenèse. Des fragments de cortex d’œufs fécondés insegmentés sont restés sans effet sur la morphogenèse de germes pris au stade 8 bl., quand on les y a greffés.
L’excision du cortex du croissant gris d’œufs fécondés insegmentés a empêché la morphogenèse, bien que le clivage et la mitose n’aient pas été inhibés. Mais l’excision de ce cortex pratiquée sur des germes au stade 8 bl. est restée sans effet sur la morphogenèse normale des embryons.
L’exérèse de cytoplasme sous-jacent au cortex du croissant gris n’a pas affecté le développement.
Du cytoplasme subcortical de la région du croissant gris de germes au stade 8 bl. a été greffé en position subcorticale dans la région ventrale d’embryons du même age. Ces greffes n’ont eu d’autre effet sur la morphogenèse qu’une légère altération de la formation de l’encéphale.
Ces résultats, groupés avec ceux d’un travail antérieur, indiquent que, bien que le cortex contienne des facteurs morphogénétiques déterminant les axes embryonnaires et l’emplacement de la lèvre blastoporale à la gastrulation, la nature de ces facteurs subit des modifications entre le début du premier clivage et la fin du troisième. La transformation est telle que l’embryon devient capable de régulation après excision ou adjonction de cortex. Le cortex du croissant gris paraît agir comme un centre activant en produisant cette altération dans le système morphogénétique de l’embryon. Rien ne permet de conclure que le cytoplasme subcortical soit impliqué directement dans cette interaction.
ACKNOWLEDGEMENTS
I thank Professor M. Abercrombie, F.R.S., and Professor J. Z. Young, F.R.S., for their advice and encouragement. Mr. D. West and Mr. J. M. Pettitt, B.Sc., have given able technical assistance. The work was carried out during the tenure of grant C-4847 from the National Cancer Institute, U.S.A.
REFERENCES
EXPLANATION OF PLATE
Fig. A. Graft of grey-crescent cortex (gr) placed in the ventral margin of an 8-cell embryo seen at the 16-cell stage. Graft clearly visible.
Fig. B. Graft of grey-crescent cortex (gr) placed in an uncleaved embryo at the lateral ventral margin seen at 32-cell stage. Graft beginning to merge into surrounding cortex.
Fig. C. Embryo, which had received graft of grey-crescent cortex at stage 1 from stage-4 embryo, seen in section after fixation at stage 21. The graft, which was placed in the ventral margin, has induced a second axis on right-hand side of section.
Fig. D. Section through an embryo which had its grey-crescent cortex excised at stage 1. Cleavage and mitosis have continued but there is no sign of morphogenesis in this embryo which was fixed at an age corresponding to stage 22. A.P. and V.P. refer to animal and vegetal pole positions respectively.
Fig. A. Graft of grey-crescent cortex (gr) placed in the ventral margin of an 8-cell embryo seen at the 16-cell stage. Graft clearly visible.
Fig. B. Graft of grey-crescent cortex (gr) placed in an uncleaved embryo at the lateral ventral margin seen at 32-cell stage. Graft beginning to merge into surrounding cortex.
Fig. C. Embryo, which had received graft of grey-crescent cortex at stage 1 from stage-4 embryo, seen in section after fixation at stage 21. The graft, which was placed in the ventral margin, has induced a second axis on right-hand side of section.
Fig. D. Section through an embryo which had its grey-crescent cortex excised at stage 1. Cleavage and mitosis have continued but there is no sign of morphogenesis in this embryo which was fixed at an age corresponding to stage 22. A.P. and V.P. refer to animal and vegetal pole positions respectively.