Grafting, together with tissue identification by monoclonal antibodies, has been used to study the allocation of Xenopus animal cap cells to the ectodermal or mesodermal lineages. Animal cap cells become responsive at stage and lose responsiveness to mesodermal induction at, or just after, stage (depending on the batch of embryos). The ability of the vegetal yolky cells to induce mesoderm disappears between stages and 11. It is present at stage 6– and may exist before this. The emergence of competence to respond at stage , coupled with the fact that the endoderm is already capable of induction at this stage, suggests that mesodermal induction begins at this point in the intact embryo.

The early amphibian embryo can be considered to consist of two different types of cell, the pigmented animal cap cells which primarily give rise to the epidermis and nervous system (Keller, 1975; Cooke & Webber, 1985) and the vegetal pole cells which will mainly form the gut. In isolation the animal cap region forms only differentiated epidermis (Holtfreter & Hamburger, 1955; Asashima & Grunz, 1983; Slack, 1984; Jones & Woodland, 1986). However, fate-mapping studies using 16- and 32-cell embryos show that the animal region also forms substantial amounts of the mesoderm (Moody, 1987; Cooke & Webber, 1985; Dale & Slack, 1987). It is thought that this mesoderm is formed by the inducing action of cells in presumptive vegetal cells on competent animal cells, the latter being reported to be able to respond to this induction up to the start of gastrulation (Nieuwkoop, 1969, 1973; Dale, Smith & Slack, 1985). This interaction can also be demonstrated in experimental tissue combinations. Thus, if a competent animal cap is placed in association with an inductive vegetal plug, the animal portion is induced to form mesoderm, which is often dorsal in nature (notochord and somite). We call these Nieuwkoop combinations, after one of their originators. Similar interactions can be demonstrated when animal cap cells are grafted into endodermal regions of a whole embryo (Jones & Woodland, 1987).

In this paper, we seek to determine the time when animal cells gain or lose their responsiveness to the mesodermal inductive signal, and the period during which vegetal cells are capable of producing this signal. The results presented in this paper confirm, within a stage, those published by others describing the end points of inductiveness and competence (Nakamura, Takasaki & Ishihara, 1971; Dale et al. 1985; Gurdon, Fairman, Mohun & Brennan, 1985). It was necessary to make very accurate determinations of these somewhat controversial time points as part of our strategy for detecting the onset of competence to respond and induce. This has provided an indication that mesodermal induction begins very early in the whole embryo.

Embryos were cultured and explants made as described by Jones & Woodland (1986). Nieuwkoop grafts were made by isolating animal caps from blastulae and early gastrulae and combining them with isolated vegetal pole explants in MBS (88 mM-NaCl; 1 mM-KCl; 24 mM-NaHCO3; 15 mM-Tris-HCl; 0·33 mM-Ca(NO3)2; 1 mM-MgSO4; 1 mM-NaHCO3; 2 mM-sodium phosphate pH 7·4 and 0·l mM-Na2EDTA; Gurdon, 1977). Grafts healed within half an hour and were then allowed to develop until control embryos were stage 25–30. Animal and vegetal combinations were made between X. laevis and X. borealis; X. borealis cells were identified by the presence of intensely fluorescent chromatin granules after quinacrine staining (Thiébaud, 1983).

Fixation, embedding, sectioning and staining with antibodies were as described in Jones & Woodland (1986). The monoclonal antibody 5A3.B4, raised against adult Xenopus skeletal muscle, was used to identify induced muscle. This antibody reacts with striated muscle from stage 20 onwards and reacts with no other tissue type. Notochord was usually identified on morphological grounds, but confirmed in a few cases using a notochord marker (Smith & Watt, 1985). In no case did cells with apparent notochord morphology prove to be negative.

The technique we have used to achieve mesodermal induction is to combine animal cap cells with vegetal pole plugs; individually neither forms mesoderm when incubated to an appropriate stage but, in combination, the animal cap is induced to form mesoderm. This technique was pioneered by Nieuwkoop (1969, 1973) and has recently been used extensively by Gurdon and his colleagues (1985) and Dale et al. (1985), among others. In all grafted combinations, mesodermal structures were identified with specific monoclonal antibodies and the animal cap origin of the induced cells confirmed using Xenopus borealis/Xenopus laevis graft combinations. Fig. 1 shows muscle staining and notochord structure in a typical inducing Nieuwkoop combination. In all cases, except where noted in the tables, blocks of muscle and patches of notochord were of a similar, and substantial, size and were not isolated cells, indicating that these combinations either responded, or did not respond, in an all- or-nothing fashion.

Fig. 1.

Nieuwkoop grafts sectioned to show the origin, extent and variety of mesoderm formed. (A) Graft of stage-9 X. borealis animal cap on to the vegetal pole from stage-9 X. laevis, showing muscle, identified by B4 staining (arrow), in cells derived from the grafted ectoderm. Inset shows the extent of the induced mesoderm in the whole graft. (B) Graft of stage-7 X. borealis animal cap on to stage-9 X. laevis vegetal pole, showing induced muscle, identified by B4 staining (arrow) and notochord. (C) Shows the quinacrine staining of the same section as B, confirming the animal cap origin of the mesodermal tissues. The X. borealis animal cap cells show typical punctate fluorescence (arrow), an, animal cap cells; veg, vegetal pole cells; nt, notochord: Bar, 35 μm.

Fig. 1.

Nieuwkoop grafts sectioned to show the origin, extent and variety of mesoderm formed. (A) Graft of stage-9 X. borealis animal cap on to the vegetal pole from stage-9 X. laevis, showing muscle, identified by B4 staining (arrow), in cells derived from the grafted ectoderm. Inset shows the extent of the induced mesoderm in the whole graft. (B) Graft of stage-7 X. borealis animal cap on to stage-9 X. laevis vegetal pole, showing induced muscle, identified by B4 staining (arrow) and notochord. (C) Shows the quinacrine staining of the same section as B, confirming the animal cap origin of the mesodermal tissues. The X. borealis animal cap cells show typical punctate fluorescence (arrow), an, animal cap cells; veg, vegetal pole cells; nt, notochord: Bar, 35 μm.

We have used these grafts to attempt to answer two very simple questions: when is ectoderm first competent to respond to the inductive stimulus from vegetal cells, and when can this stimulus first be detected? One way in which one can ask questions about the timing of these events is by grafting animal and vegetal regions of early and late embryonic ages together. If one of the tissues in these heterochronous combinations is close to the end of its period of giving or receiving the inductive signal, then the ability of the other tissues to respond or induce can be measured by the presence of induced mesoderm in the grafted combination. If the combination fails to generate mesoderm because of the early state of origin of one of the tissues, the time when tissues become responsive or inductive can be assigned to a particular stage of development (see below). Importantly, two assumptions are not made in this approach, whereas they are central to the detection of mesodermal induction by explanting the prospective mesodermal tissue. First, it is not necessary to assume that pure tissues are isolated. Second, it is not necessary to assume that the early steps in mesoderm formation are irreversible. In order to carry out these experiments, the end of both the competent and inductive periods must be established with high accuracy. We felt that it was important to do this since there are minor disagreements in the literature that would be of significance in our kind of experiment. Having confirmed the end points of both competence and inductiveness, we have used animal tissue very near the end of its period of competence in the assay concerned and combined it with progressively earlier inductive tissue. If inductiveness is absent at early stages, mesoderm should not be seen, because the animal cells will have lost competence by the time it appears. This assumes that the endoderm cells cannot reset the timing of the animal cell programme. This has been tested, with respect to a cardiac actin mRNA appearance by Gurdon et al. (1985), who found that this time was intrinsic to the animal cells. We have also described it with respect to the appearance of the epidermal marker recognized by 2F7.C7 and observed no changes in timing (unpublished data). The timing of expression of the antigens recognized by 2F7.C7 and B4 are identical in X. laevis and X. borealis embryos. Furthermore, the grafted combinations have been carried out using both species for the source of each tissue in the grafts. These combinations give exactly comparable results irrespective of the source of inducing or responding tissue. For simplicity results have been pooled in Tables 13.

Table 1.

Heterochronous Nieuwkoop grafts: assessment of the competence of animal caps to form mesoderm

Heterochronous Nieuwkoop grafts: assessment of the competence of animal caps to form mesoderm
Heterochronous Nieuwkoop grafts: assessment of the competence of animal caps to form mesoderm

The end of animal cap competence

We defined the time at which animal cap cells lost their ability to form mesoderm by combining progressively later animal caps with fully inductive vegetal plugs (Table 1). Stage-10 animal cap was fully induced by stage-7 to -9 endoderm whereas stage-11 ectoderm was not. Stage- ectoderm gave somewhat variable results. In some experiments, it was totally refractory to mesoderm induction and in others gave fairly low percentages of induction. This indicates that the end of animal cap competence lies at some time very close to stage , variability in results perhaps being due to the morphological criteria used for staging these embryos, or perhaps from a genuine variation in the disappearance of competence.

The start of endodermal inductiveness

Since competence disappears close to stage , we used stage-10 animal cap, which should have a maximum of 45 min responsiveness, to test when inductiveness appeared in vegetal pole cells (Table 2).

Table 2.

Heterochronous Nieuwkoop grafts: time of disappearance of the ability to induce mesoderm

Heterochronous Nieuwkoop grafts: time of disappearance of the ability to induce mesoderm
Heterochronous Nieuwkoop grafts: time of disappearance of the ability to induce mesoderm

Endoderm from the earliest stage tested, stage 5, was clearly inductive in stage-10 combinations, resulting in the formation of both notochord and muscle from the grafted animal cap. This means that the endoderm must be inductive within 45 min of stage 5, that is by stage , or 64 cells, and it is possible that the endoderm is inductive from much earlier stages. We do know, however, that the oocyte is not capable of inducing mesoderm when animal cells are grafted on to its vegetal pole (data not shown). Of course, induction might not be actually happening at these early stages, because there might be no responsive animal cap cells.

The end of endodermal inductiveness

The time at which the endoderm in vegetal pole plugs loses its capacity to induce mesoderm was tested in a similar way (Table 2). Vegetal pole cells from stages induced mesoderm when in combination with stage-8 to -10 animal cap, whereas vegetal pole cells from stage 11 or greater never induced similar ectodermal cells.

The start of ectodermal competence

In combinations with endoderm at the end of its inductive period, stage , it is clear that stage-4 and -5 animal caps are not competent to respond to the inductive stimulus. In contrast, animal cells dissected from stage 6 or later formed abundant dorsal mesoderm in such combinations (Table 3). The failure of these early animal caps to form mesoderm was not a consequence of total inability of these grafts to respond, because when they were placed in combination with earlier endoderm, stage 9 or 10, they formed mesoderm. Stage-4 ectoderm failed to respond in all except one case in combination with stage-10 endoderm, but stage-5 ectoderm responded in the majority of these combinations. In these combinations the endoderm has respectively a maximum of or h of inductiveness remaining (see Table 2), during which time the animal cap will have aged beyond stage 6. However, stage- endoderm can have only a maximum of 45 min inductiveness remaining, during which time stage-4 animal caps could advance to stage 6 and stage-5 animal cells to stage . This indicates that competence to respond to mesodermal induction appears between stage 6 and . Notochord was seen in many of the grafted combinations, particularly when the animal cells were taken from embryos earlier than stage 7.

Table 3.

Heterochronous Nieuwkoop grafts: time at which competence to respond to mesodermal induction appears in the animal cap

Heterochronous Nieuwkoop grafts: time at which competence to respond to mesodermal induction appears in the animal cap
Heterochronous Nieuwkoop grafts: time at which competence to respond to mesodermal induction appears in the animal cap

These experiments suggest that vegetal pole cells are already generating mesodermal inductive signals close to the earliest point that they can be separated from the animal cap cells, and that animal cells are competent to respond to this signal from comparatively early stages – stage . However, our results suggest that animal cells cannot respond at stage 5 to 6, and hence that mesodermal induction cannot be occurring at this point.

In this paper, we are concerned with the times when the animal cap is competent to respond to an inductive signal from the vegetal pole cells, and the times when this signal is given. This is essential background for understanding how the future mesoderm of the embryo appears in its appropriate position and amount.

The stage at which the animal cap loses its competence to form muscle

As background to our study of the time at which competence to form muscle appears, heterochronous combinations of tissues were used to determine when the animal cap loses its ability to form muscle, using stage-7 to -9 tissue as an inducer. Dale et al. (1985) found that the ability to form dorsal (though not ventral) mesoderm was lost by stage 10, our results aré closer to Nakamura et al. (1971) and Gurdon et al. (1985) and suggest that it is lost in a very short time interval close to stage . This is also consistent with Asashima & Grunz’s (1983) observation that a chick embryo extract induced stage- animal cap to form mesoderm. In agreement with these latter authors’ study using a heterologous inducer, we have found that inner and outer animal cap layers can individually form mesoderm up till stage , when naturally induced with vegetal cells (data not shown).

Like Dale et al. (1985), we found that notochord was formed more often when early animal caps were used. However, this is not a fundamental change in the responsiveness of the tissue, since in unpublished experiments we find that animal cap as late as stage can form notochord when grafted superficially into the dorsal marginal zone of a stage-8 blastula. This difference from Nieuwkoop grafts may derive from the changing sensitivity of animal caps in development, coupled to the strength of the inductive signal produced by parts of the endoderm which may or may not be included in vegetal pole fragments.

In conclusion, we can confirm the results of some of the previous workers, that the animal cap loses mesodermal competence very close to stage , with some variability between batches of embryos.

Stage at which mesodermal inductiveness disappears in the vegetal region

Using Nieuwkoop combinations of vegetal cells and competent animal caps Nakamura et al. (1971) and Gurdon el al. (1985) found a loss of inductiveness at mid stage 9. The former identified ventral and dorsal mesodermal tissues morphologically, whereas the latter, like us, focused on muscle, using molecular markers. On the other hand, Dale et al. (1985) with Xenopus, Boterenbrood & Nieuwkoop (1973) with axolotls, and Asashima (1975) with Triturus, obtained mesoderm, including muscle, from stage-10 inducers. However, all noted a decrease in dorsal induction. Dale et al. (1985) felt that this might be because they failed to dissect out the dorsal vegetal material. In our experiments, we obtained strong muscle induction at stage 10 and , using genetically marked animal cap cells. It therefore seems that, judged by this assay, mesodermal inductiveness disappears at late stage 10, concomitantly with mesodermal competence in the animal cap. Since the muscle was clearly striated, it seems unlikely that our difference from the results of Gurdon et al. (1985) relates to the fact that they used a different marker, cardiac actin gene transcripts.

Stage at which mesodermal inductiveness and competence arises

Once mesodermal competence and inductiveness have arisen, induction should occur and mesoderm should be determined. An obvious way to estimate the onset of the two phenomena might therefore appear to be to isolate the region fated to form mesoderm from embryos of different stages and establish when mesoderm first appears. This approach was adopted by Nakamura & Takasaki (1970), who identified stage as the stage at which much mesoderm appeared. It could hardly be earlier, since at stage 6 (32-cell) the cells that form most of the mesoderm also form ectoderm (Cooke & Webber, 1985; Dale & Slack, 1987), so induction, as normally conceived, could not have occurred. Nevertheless, this experiment is certainly flawed, as pointed out by Nieuwkoop (1973), since the region excised should contain both future endoderm and ectoderm, and therefore must be capable of self-induction after isolation. Despite the severe criticisms of this work, this has been the only study attempting to define the actual start of mesodermal induction. [It has recently been claimed that stage- cells are competent to respond and induce because when grafted together at this stage they later form muscle (Brennan, 1987). Of course, this experiment does not bear on the point because the cell interaction could have happened at any subsequent time.]

We have adopted a different approach to define when mesodermal competence and inductiveness start, using the heterochronous Nieuwkoop grafts described earlier. To test when the inductive stimulus is present we combined stage-10 with stage-5, and later, vegetal cells. These grafts usually form muscle, indicating that the inductive stimulus is present within 45 min of stage 5; i.e. when the vegetal plug is stage . Thus, we could not identify a negative time, and inductiveness may exist from the start of development, though it is absent from the oocyte. Since significant transcription is not seen until stage 8 (Bachrarova, Davidson, Allfrey & Mirsky, 1968; Gurdon & Woodland, 1969; Newport & Kirschner, 1982), the production of the inducer most probably depends on stored mRNA or protein.

The onset of animal cap competence to be induced was judged by using stage-9 to - vegetal tissue to induce progressively earlier animal caps. Stage-5 caps produced muscle and notochord in combination with stage-9 and -10 vegetal tissue, but not with stage even though the latter can clearly induce later ectoderm. This suggests that stage-5 animal cap is not competent to respond to mesodermal induction, but becomes responsive at some later stage at least by 45 min after stage 5, i.e. by the latest at stage , as indicated by the successful induction of stage-5 caps and stage-10 vegetal combinations. Thus mesoderm induction in the normal embryo is likely to begin at, or just before, stage , this time being set solely by the onset of competence. If mesodermal induction starts at the 64-cell stage, it is relevant to ask if there are cells of a purely mesodermal state at this stage. Fate-mapping experiments at the 256-cell stage indicate that derivatives of equatorial cells enter all three germ layers (Jacobsen, 1983). This result can be reconciled with the appearance of mesodermal induction at 64 cells if mesodermal differentiation is initially reversible (irreversibility is not assumed in our experimental design). This is actually to be expected since grafts conducted with late blastula tissue indicate that mesodermal induction takes , i.e. any changes that occur are reversible within this period (Gurdon et al. 1985). Thus by extrapolation to natural, earlier embryos, if induction starts at stage , it would not be complete (i.e. irreversible) for any cells until stage 8, which happens to be the time of general genome activation.

Nakamura & Takasaki (1970) concluded that the mesoderm becomes determined between stages 6 and . This was based on the observation that isolated stage-6i equatorial zones, but not those of stage 6, showed subsequent mesodermal differentiation on culturing. As pointed out above, the experiment is flawed because it depends on the explant containing only mesodermal progenitors. If this was so, and the mesoderm was originally generated as a result of induction, then this induction must have started h earlier, at the 2-to 4-cell stage, when there were not even animal and vegetal cells. Furthermore, our results show that the vegetal pole cells separated at the 16-cell stage are inductive, but that the animal cells are not responsive at this stage. This lends weight to Nieuwkoop’s (1973) conclusion that Nakamura’s experiment was faulty because the isolated equatorial zone must have contained endodermal and ectodermal progenitors and therefore could be capable of continued induction after explantation. Our conclusion differs from Nakamura’s in that we argue that mesodermal induction starts in the 64-cell-stage embryo, but that ‘committed’ mesodermal cells would not be expected before stage 8 (these would be ‘committed’ as defined by differentiation in isolated fragments, as in Gurdon et al.’s (1985) separated Nieuwkoop grafts, which were used to determine the timing of mesodermal induction).

The biological significance of these observations

The early time of mesodermal induction might be relevant to how much mesoderm is formed in this early phase of mesoderm formation. A working hypothesis is that in this early phase the mesoderm is formed from an annulus of animal cells which make contact with the yolky vegetal cells. It is proposed that the mesoderm itself is noninductive and that the large cells involved therefore act as a barrier to propagation of the inductive stimulus. ‘

The reason for the disappearance of mesodermal competence and inductiveness in normal development seems clear. In the blastula, the animal cap cells that neighbour vegetal cells all form mesoderm. The rest is separated from it either by the future mesoderm itself, or by the blastocoel. After the tissue rearrangements of gastrulation, prospective ectoderm comes close to endoderm, for example ventrally. If a mutant arose in which the former were competent to be induced to form mesoderm, or the latter was inductive, then more mesoderm would be generated in inappropriate positions, probably with lethal results. Thus the loss of pluripotency may be a vitally important aspect of any particular differentiated phenotype, although there is as yet no reason to believe that it is always permanent.

This work was funded by the Medical Research Council. The authors acknowledge the clerical assistance of Mrs Len Schofield and the technical assistance of P. Day.

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