Combinations of tissues from Xenopus blastulae have been used to identify several mechanisms that limit the number of animal cells forming muscle after induction by vegetal cells. The results disagree with a model in which direct physical contact or very close proximity between animal and vegetal cells restricts the number of cells that receive the inductive signal. Rather it seems that a diffusible inducer is released by vegetal cells, and spreads through 4–8 animal cell diameters, equivalent to a distance of 80 μm, from the nearest vegetal cells. Several factors seem to cooperate to prevent the further spread of the mesoderm-forming induction. These include the slow diffusion and/or instability of the inducer, the time of loss of competence of animal cells to respond to induction, and the amount of vegetal tissue that releases inducer for a limited time. The combination of these, and perhaps other, processes seems to ensure that a consistent minority of animal cells are induced to form muscle, thereby leaving other animal cells available to form the nervous system and epidermis.
There are many instances in development and adult life where cell differentiation is influenced by products of other cells. In all these cases, it is important that only a limited number of cells in certain positions should respond to these signals. In long-range cell interactions, such as those mediated by hormones, the cells that respond to the hormone are selected by their possession of receptors. For very short-range interactions, such as ones involving neurotransmitters, only cells with membranes located very close to the point of release of transmitters respond; dilution and or instability of the transmitter causes its concentration to decrease strongly from the site of its release. Embryonic induction is intermediate in range, some of the cells that respond being up to 100 μm or 10 cell diameters away from the source of the inducer. It is not known what determines the number and position of cells that respond, though it is clear that more cells have the capacity to respond than normally do so. It is also of crucial importance that not too many cells respond to induction, since those that do not must contribute to different cell-types which would otherwise be deficient.
The experiments described here aim to identify mechanisms that localize the response to induction and this requires an experimental system in which inducing and responding cells can be varied in number and position. In most examples of embryonic induction, this cannot be achieved, because the response is lost as the number of cells is reduced, or because cell division and changes of cell position take place during a period of several days before a response is seen. The particular induction analysed here is the mesoderm-forming induction in amphibia. This takes place over a few hours, and can proceed in the absence of cell division and cell movement, making it possible to identify several processes that determine the number and location of cells that respond to induction.
Materials and methods
All experiments were carried out with embryos of Xenopus laevis. Embryo tissues and cells were cultured in modified Barth saline (MBS, Gurdon, 1977). Embryos were staged according to Nieuwkoop & Faber (1956).
Glass moulds were constructed from small pieces of 1 mm thick microscope slide attached by Araldite to each other and to a large piece which served as a floor. The top of each chamber was open. Any dead cells which emerged after placing embryo fragments into the moulds were removed at intervals. The moulds with embryo fragments were cultured in MBS until removed for fixation. Nuclepore filters were secured vertically in the middle of a glass mould, so that appropriately sized wells on each side of the filter provided a tight fit for vegetal or animal pieces of a blastula.
For some experiments, animal: vegetal conjugates were cultured in solid gelatin (BDH), made up in Ca2+ -free MBS. A 5 % gelatin solution was maintained at 25 °C until required. Then 2 μl of a 10 mg ml−1 stock solution of cytochalasin B (Sigma) in dimethylsulphoxide (kept at − 20 °C until required) was added to 6 ml of the 5 % gelatin solution (making a 3 μg ml−1 cytochalasin B solution). This was poured into a 35 mm plastic dish containing a thin layer of 2 % agarose (FMC, type LGT) in Ca2+-free MBS. Immediately after this, conjugates that had been formed in Ca2+-containing MBS were washed in Ca2+-free MBS and allowed to fall through the liquid gelatin solution, coming to rest on the solid agarose base. The whole dish was then placed in a 4 °C refrigerator for 15 min during which time the gelatin solidified, and was then transferred to an incubator for culture, usually at 19 °C.
For fixation, large syringe needles were used to cut out a solid cube of gelatin containing a conjugate, and the whole cube was immersed in a solution of 50 % methanol in MBS. After 8–24 h at room temperature, a 5 ml bottle containing the gelatin-conjugate in 50 % MBS was placed at 37 °C for 2 min, causing the gelatin to dissolve. Rotation of the bottle dispersed the gelatin, which was removed by careful washing with 50 % methanol:MBS, and which was then replaced by 100 % methanol. After a further fixation of a few hours or more at 4 °C, the conjugates could now be embedded in wax and sectioned by routine procedures. This particular fixative turned out to be best for the antigen recognized by the Kintner & Brockes (1984) antibody. The fixation by 50 % methanol:MBS was devised to avoid the gross distortion that results from immersing 5 % gelatin cubes in 100 % methanol.
Nuclease protection analysis and other procedures were the same as used before in this laboratory (Gurdon et al. 1985).
A localized response takes place in experimental conditions
The mesoderm-forming induction that takes place during the first few hours of amphibian development is too well known to need description (Nieuwkoop, 1969; Nakamura et al. 1970; review by Gurdon, 1987). We need to know, for the present discussion, that the cells that respond to this induction by forming muscle are located at and just above the equator of the blastula (Fig. 1). The cells that are near the animal pole of the embryo are not induced to form muscle or other mesodermal tissues, but form the whole nervous system and much of the epidermis. Therefore, the localization of response to mesodermal induction is of crucial importance in normal development.
Combinations of animal and vegetal regions of a blastula after removal of the equatorial one third have been widely used for the analysis of the mesodermforming induction; these constructions have the advantage that neither the animal nor vegetal pieces when cultured on their own will express muscle-specific genes, but the combination of the two always do so very strongly (Gurdon et al. 1985). Muscle cells are recognized in cultured animal: vegetal conjugates, in this and previous work, by the muscle-specific antibody of Kintner & Brockes (1984). Muscle cells are present in one (or sometimes two) coherent groups, and are located in an approximately central position (Fig. 2A). Therefore animal: vegetal conjugates provide a good experimental system in which to investigate mechanisms responsible for the location of muscle cells resulting from induction.
There is no predisposition among animal cells
The simplest explanation for the location of induced muscle cells would be a predisposition for muscle differentiation among the more equatorial cells of the animal hemisphere. This has been tested by placing cells from various regions of the animal hemisphere in conjugation with vegetal tissue. It is observed that about one third of all animal cells express muscle genes, whether they are taken as a piece of tissue from the animal pole or from the dorsal, ventral or lateral parts of the temperate zone. This is also true if cells from different positions are dissociated, mixed and reaggregated before being placed on vegetal tissue for induction. The outer strongly pigmented cells can be separated and tested independently of the inner animal cells, and are somewhat less strongly and less frequently induced to form muscle, but this is almost certainly attributable to the impermeability of the outer coat of their surface. Details of these experiments are not given here since they are in agreement with the experience of several laboratories (e.g. Jones & Woodland, 1987a,b). Even though there may be some small variation in the predisposition of animal cells from different regions to become muscle, it is clear that this cannot explain the equatorial location of muscle cells in conjugates and in normal development.
Muscle cells are near inducing cells, not in a midposition between two poles
Another hypothesis to account for the equatorial location of induced muscle cells is that they are in a midposition between the animal and vegetal poles of a blastula or conjugate. This may be contrasted with another possibility that induced muscle cells are always derived from the animal cells that are closest to vegetal tissue and hence closest to the source of inducer. These ideas have been distinguished by placing vegetal tissue and several pieces of animal tissue in the configuration shown in Fig. 3. It can be seen that the midposition between the two poles (fragment 4) does not coincide with the region where animal cells are closest to vegetal cells (fragment 2). Using RNase protection to recognize transcripts of a muscle-specific actin gene, it can be seen in Fig. 3 that muscle genes are not activated in the midposition of the conjugate, but are strongly and uniquely so where animal cells are in contact with vegetal cells. We, therefore, conclude that the central or equatorial position of muscle cells in normal embryos is attributable to their proximity to vegetal cells, and not to their distance from animal and vegetal poles.
Inductive response requires proximity not physical contact
A simple way of limiting the number of animal cells that respond to induction would exist if physical contact between vegetal and animal cells, or such close proximity as normally separates two cells (∼1–2 μm), were required for the inducer to be successfully transmitted. Under these conditions, only a monolayer of animal cells would be induced during the blastula stage, but the combination of cell movement, which is considerable at this stage of Xenopus development, and cell division could result in the formation of a coherent group of muscle cells by the postneurula stage (as explained in Fig. 4A).
The need for physical contact has been tested initially by use of Nuclepore filters. Grunz & Tacke (1986) have reported that vegetal tissue can transmit its inductive effect through a Nuclepore filter with 0·1 μm pores. Our own results agree with this conclusion to the extent that animal pieces of a blastula can respond to vegetal tissue on the other side of a 0·1 μm Nuclepore filter (Fig. 5A) and that cytoplasmic processes do not extend through these pores within the period of ∼4h, after which competence ends. We do, however, observe a major weakening of the response across a filter. Using nuclease resistance as a quantitative assay, we find that about half of the test animal pieces are not delectably induced across a filter (i.e. to less than one tenth of the normal extent); of the other half that are induced, the response is weakened by 3–10 times (Fig. 5B). This very substantial weakening of the transfilter response suggests that this induction involves a diffusible molecule which is unstable or which is ineffective at a lower than normal concentration, or both. The conclusion from transfilter experiments is that the localization of inductive effect does not depend on physical contact, but is more likely to involve a restricted spread of the inducer from its source. It is conceivable that the inductive effect of vegetal cells can only be transmitted over a few microns to adjacent animal cells, which can then pass on the effect to a few other animal cells, as in homoiogenetic induction (Mangold & Spemann, 1927).
The inductive effect can spread over a few cell diameters
To show directly that the inductive influence of vegetal tissue can spread to animal cells not in contact with it, conditions have been used which were previously found to inhibit cell division and cell movement in conjugates: 5 % gelatin in Ca2+-free MBS containing 3 μg ml−1 cytochalasin B (Gurdon, in preparation). Inner animal cells from six blastulae were dissociated, extensively mixed and then reaggregated into small groups which were placed individually on vegetal tissue. Subsequent fixation, serial sectioning and reaction with muscle antibody revealed the number and location of muscle cells. As seen from the representative transverse section in Fig. 2B, and as shown diagrammatically in Fig. 4B, cells not in contact with vegetal cells have nevertheless undergone muscle differentiation.
The cytochalasin gel experiments also allow an estimate of how far this inductive signal can spread. In conjugates that have been examined in serial section from one side of a conjugate to the other, there appears to be a maximum of 4 cell diameters that separate vegetal tissue from the furthest animal cell that is muscle-antibody positive. This is the result of examining over 900 serial sections in six conjugates. Since these are cytochalasin experiments in which cell division has been arrested, 4 cell diameters of stage-8 animal cells corresponds to a distance of ∼80 μm. If cell division is allowed to continue, there can be up to 8 cell diameters separating vegetal and animal cells, though the distance across eight of these smaller cells is similar to that of the four larger midblastula cells.
Changing competence limits the spread of inductive effect
It would be surprising if the normally precise amount of muscle in normal development is determined entirely by the distance of diffusion and instability of inducer substances. In fact, it seems that an altogether different mechanism contributes, and this depends on the fact that animal cells lose their competence, or ability to respond to induction, at about stage 10 1/2 (Dale et al. 1985; Gurdon et al. 1985). To test the effect of competence termination on the eventual number of muscle cells, animal tissue from stages 8 to 11 was placed on vegetal tissue at stage 9. Subsequent analysis of antibody-stained serial sections showed that the older, less competent, animal tissues or ectoderm make substantially fewer muscle cells than the same tissue at an earlier stage (Table 1). It is noticeable that all muscle cells are, as observed before, in a homogeneous compact group located right up against vegetal cells. There also seems to be a sharp demarkation between antibody-positive and -negative cells. The main conclusion is that the time of loss of competence can be a significant factor in limiting the spread of response to induction.
Distal vegetal cells contribute inducer
The results described above suggest that the amount of inducing substance released by vegetal cells could influence the eventual number of muscle cells. What therefore regulates the amount of inducer released? A simple idea is that only those vegetal cells, in a subequatorial position, that directly contact animal cells can contribute inducer. The blastomere graft experiments of Gimlich (1986) have established that, at the 32-cell stage of Xenopus, vegetal pole blastomeres as well as third tier subequatorial vegetal cells possess inducing activity. This does not, however, prove that the vegetal pole cells actually contribute inducer in normal development. To test this, constructs have been prepared as shown in Fig. 6, where the total amount of vegetal tissue varies, but the contact area with animal cells remains about the same. Although it is hard to be sure that the surface contact area between animal and vegetal cells remains constant, it does not appear to change during the first few hours of contact when the inductive effect must take place before competence is lost. Since the number of muscle cells is decreased below the normal value with lesser amounts of vegetal tissue, it seems that deep vegetal cells may contribute active inducer. It is also known that vegetal cells lose their ability to induce at about stage 10 (Nakamura et al. 1970; Gurdon et al. 1985; Dale et al. 1985; Jones & Woodland, 1987), and this is an additional factor affecting the amount of vegetal inducer released.
The concept that emerges from these results and those of Gimlich is that eggs or early embryos have a certain amount of inducing activity in the sum of their vegetal cells, and that this may place some, perhaps rather approximate, limit on the total number of induced muscle cells.
The results reported here all relate the differentiation of muscle cells to the early, mesoderm-forming, induction in Xenopus blastulae. In fact, muscle is a major, but not the only, cell type that results from this induction. Other mesodermal cell types include the notochord, lateral and head mesenchyme, and blood; it is not known how these cells arise after an initial mesodermal induction, but this does not affect our conclusions which are based on the formation of muscle.
The simplest way of limiting the number of cells induced to form muscle would be to suppose that physical contact between animal and vegetal cells is required, but this is clearly inconsistent with the results of our cytochalasin experiments. All our results point to a process in which inducer substances are released by vegetal cells and received by adjacent animal cells, beyond which the effect spreads only a short distance by diffusion or by homoiogenetic induction (Mangold & Spemann, 1927). The concentration of inducer appears to become too weak to be effective beyond a distance of about 80 μm from the nearest vegetal cells; this corresponds to about 4 cell diameters of stage-8 blastula cells, or to 8 cell diameters of later cells. The rather coarse limitation of inductive effect achieved by this means appears to be supplemented by the loss of competence of animal cells and by a limited amount and time of emission of inducer from vegetal cells. Further refinement and a sharpening of the border demarkating muscle from non-muscle seems to be achieved by cell movement, which brings like cells together, or by a community effect in which like cells enhance each other’s differentiation (Gurdon, in preparation). It is also important to appreciate the significance of the blastocoel, which probably acts as a block to the diffusion of inducer substances (Fig. 1). Indeed if the blastocoel is eliminated by high salt or centrifugation, development is very abnormal, partly because animal pole cells, which now lie in contact with vegetal cells, are incorrectly induced. The overall effect is one in which no single developmental process is of overriding importance, but where a consistent and accurate result is obtained by the combination of several processes each of which may be individually imprecise. This principle may be of common occurrence in morphogenesis.
The mesodermor muscle-forming induction in amphibian development is one of many medium-range cell interactions that occur throughout early development and adult life. Other examples occur in embryos of Drosophila (Doe et al. 1985; Martinez-Arias et al. 1988; DiNardo et al. 1988) and nematodes (Priess & Thomson, 1987), and may well be involved in the function of haemopoietic factors (review by Metcalf, 1987).
I am indebted to Dr J. Brockes and Dr J. Anderton for antibodies, to Barbara Rodbard for help in the preparation of this and many other manuscripts, to W. Westley and Anne Pluck for help with histology, and to the Cancer Research Campaign for supporting this work.