The results are reported of a series of experiments, the exact geometry of which has been presented in a previous paper. Late blastulae and early stage-10 gastrulae are supplied with a second head organizer region at varying angular distances, in the marginal zone, from the presumptive site of their own organizer. The configuration of positional information existing in the mesodermal mantle of the late gastrula or earliest neurula, as a final result of such operations, was recorded by observing the pattern of axial organ differentiation obtained by tailbud stages (26–28).

The operational differences between various current theories as to the nature of embryonic differentiation fields are briefly discussed, as a framework within which to consider the results of experiments such as those reported here. It is suggested that in the future, and using the present results as a basis, experiments may be possible that are more critical in distinguishing between the various theoretical suppositions involved.

Evidence is presented that the final configuration of positional information, achieved as a result of the implantation of a second head organizer at or before the onset of host gastrulation, becomes stable some time before it is irreversibly expressed in terms of a pattern of cell commitment in the mesodermal/endodermal mantle. It is insensitive both to relative ages of host and graft at the time of operation, over the range employed and, probably, to the ambient temperature of development between operation and the time of cell differentiation, being dependent only on the angular distance originally existing between graft and presumptive host organizer sites.

Tn the discussion, a model is given for the visualization of positional information in partially double fields, produced in a two-dimensional sheet of cells where the normal endpoint of field formation is a bilateral symmetry of differentiation zones.

In a previous paper of this series (Cooke, 1972) it was shown that when a plug of cells consisting of the head organizer region of a stage-10 Xenopus embryo is implanted in normal orientation into the marginal zone of a host late blastula or early gastrula, the two organizers finally seen there are autonomous with respect to one another. They appear to divide up the territory of cells available to them into two fields, in terms of the initial cell adhesive/locomotory changes which characterize early organizer activity, but such competition is only apparent for pairs of organizers initially fairly close together on the surface, and then only for some short time after they have left it during the process of gastrulation. The detailed geometry of the operations is given in Paper 1 of this series.

By the close of gastrulation, no successfully operated embryos are detectably different from controls to external examination, the visible cell orientation tending to be normal to the blastopore lip, and neural induction not yet apparent. However, dissection of half-gastrulated embryos and the use of vitally stained grafts has shown that the graft normally heals into a full continuity with the cells of the host, and tends to create its own axis of elongation and cell stretching amongst the host cells behind it, comparable with that of the normal presumptive dorsal midline.

At some time during the late gastrula/early neurula stages, the process of cell determination within the mesoderm and endoderm has proceeded to completion for the primary axial organization, thus expressing in an irreversible manner the pattern of positional information obtaining there at this time. From classical work using the differentiation found in expiants cultured in isolation (see, for example, Holtfreter & Hamburger, 1955), a provisional estimate of the time of such a loss of lability would be the late gastrula/early neurula stages, reached in the Xenopus embryo some 5 h after the onset of gastrulation. The term ‘positional information’ is used in these papers essentially in the sense developed by Wolpert (1969), to mean that variable, whatever its nature, that is perceived by cells at each point in a regulative individuation field, constituting information as to their relative position within the field, and ultimately causing an appropriate pathway of differentiation to be pursued.

Now one of the expressions of the positional information and presumptive pattern of differentiation in the axial mesodermal field, around the time of cell determination there, is the pattern of inductive influence (see Nieuwkoop et al. 1952) that is transferred from it to the overlying neurectoderm, producing ultimately the form of the axial nervous system and the various accessory structures (cement-gland, external components of the otic structures, tailfin, etc.). Nieuwkoop (1967 a, b) is of the opinion, after much experimental work upon the process of induction, that although the neural plate must ultimately have regulatory properties of its own as an individuation field, its proportions nevertheless closely reflect those of the pattern of inductive influence from the underlying axial mesoderm. It seems justifiable then, to use tailbud stages (st. 26–28, Nieuwkoop & Faber, 1956) for examining the final result of regulation in the case of these operated embryos. By this time visual information is obtainable, by external examination, on the state of the pattern of positional information as it has been at the closing phases of primary embryonic induction.

The operated embryos fall into a series where a dual anterior axial structure tends to become a normal, single posterior structure, the two limiting cases being those of regulation to give a complete normal, single embryo, and on the other hand completely double organization, i.e. belly-joined twin axes. This paper reports these observations, including the results of experiments investigating the effects, upon the level of fusion of such partially paired individuation fields, of (a) relative age of host, i.e. time of appearance of its own organizer, relative to the time of implantation of the grafted one, and (b) the ambient temperature of development, between the operation and the scoring of results, over a range where normally proportioned development occurs in undisturbed, demembranated embryos.

There are at present so many detailed theories as to the nature of the fields of positional information, utilized by sheets of cells in developing embryos, that a detailed discussion of them would be misplaced here. However, most of them embody some combination out of the pairs of opposed suppositions set out below, and the basic, although long-term goal of the present programme of experiments is to obtain evidence as to which of these suppositions are more nearly correct, for amphibian embryos.

(a) Throughout its existence, the field may be maintained by global properties of the cell sheet, whether as a diffusion landscape of substance or as a pattern of propagating temporal structure among the cells (e.g. Goodwin & Cohen, 1969). Each cell is then a passive recipient, or propagator of a certain value for the information at its location within the field. Alternatively, cells may become, before the actual process of cell determination, progressively more ‘homeostatic’ or organized towards the active maintenance within themselves of some ‘remembered’ value for the field information, as in the ‘sonk’ model of Lawrence (see Lawrence, 1971).

(b) Cells may develop, during the organization of the field, some intrinsically polar properties which constitute part of the mechanism for its maintenance, e.g. active transport of substance in one direction only, against a concentration gradient, or a polarity of migratory tendency which is autonomous within an isolated portion of the cell sheet. For a regulating model requiring that cells should show intrinsic polarity of transport, see Cohen (1971). Alternatively, fields may never require the acquisition of vector properties by individual cells or small groups of cells, for their maintenance, but exist simply as a landscape of some varying scalar quantity, such that cells from a particular presumptive region develop a particular ‘polarity potential’ with respect to others (see Wolpert, 1969).

(c) The actual field information may be the variation in absolute amounts, present in cells of different regions, of one or more substances whose titre progressively leads the cell towards the expression of a particular, more restricted developmental potency. Gradients of such substances might be built up and maintained by diffusion from constant, localized sources, with all cells constituting a ‘sink’, or in special cases with a localized sink, or else by polarized transport throughout the field (see, for example, Wilby & Webster, 1970). Alternatively, the information may consist in a range of cyclic, temporal metabolic structures available to cells, which have several underlying oscillatory metabolic processes going on in them, with the phasing of the separate processes partially intrinsically linked and partially modifiable due to propagation of more or less discrete events among the cells of a sheet. Such ‘phase-shift’ types of model, of which that of Goodwin & Cohen (1969) is only a primary and particularly well worked out example, do not postulate the continuous flux of appreciable amounts of any one substance through the field. Here, the ‘dominant’ region of the field, of which the head organizer region of the amphibian embryo is by classical criteria a good example, is conceived of as some type of propagation centre, for the underlying set of metabolic oscillations in cells of the field.

Experimental work has not as yet allowed a choice between any of these possibilities for the amphibian primary field. However, the experimental results reported here had in no sense been apparent or expected a priori, and they establish a phenomenology which is a necessary preliminary for future experiments which may thereby be enabled to be critical tests of some of the available models.

The embryos from which the data of this paper are taken consisted of the same sample dealt with in Paper I of this series, together with a large sample where the host was at stage 10 at the time of implantation of the second organizer. Several of the latter type of operation, selected for inter-organizer angles of near 90° or else near 65°, were used in the temperature experiments, being set to develop in the normal manner, but in incubators at 19 °C or at 25·5 °C, beginning immediately after the 35 min period of healing

For solutions used, the handling of embryos and the details of the operation procedure, see Paper I in the present series (Cooke, 1972).

Embryos resulting from the operations were examined at stages 26–28 (Nieuwkoop & Faber, 1956), the long tailbud stage, and the pattern of positional information that had obtained in their mesodermal/endodermal mantles at the close of gastrulation or during neural induction was inferred from the pattern of cement-gland differentiation and of induced axial nervous system, visible to external examination. Such of the underlying axial mesodermal system as could be seen seemed to correlate in structure with the central nervous system, and histology of selected examples confirmed this. That is, where a progressive fusion of two partially distinct axial nervous systems occurred as progressively more posterior sections were examined, the notochord and somite structures fused at a closely corresponding level, to a single bilateral structure.

A few transplanted organizer plugs were taken from whole embryos that had been stained from stage 9 onwards, in 0·05 % Nile blue sulphate in strength Holtfreter, buffered back to pH 7·4 with Tris. These organizers behaved in every way similarly to their unstained equivalents.

Fig. 1 represents diagrammatically the fate of an implanted organizer, following the basic operation, by the end gastrula stage. The distance along the front edge of the mesodermal mantle, between the host head organizer region and the graft, varies continuously (although not necessarily linearly) with the interorganizer angle in the marginal zone as measured at host stage 10, so that in the extreme case of an inter-organizer angle of 180°, the graft would be shown, in terms of Fig. 1, as split equally between the outer anterior corners of the flat-projected mesodermal mantle. Healed-in graft and the homologous host region have migrated in and travelled forward, relative to the expanding outer neurectoderm, approximately in parallel.

Fig. 1.

(a) Diagrammatic representation of the positions of typical grafted organizers in the mesodermal mantle at the close of gastrulation (hatched). Axis = middorsal line of neurula. Black-shaded area = host region homologous wth grafted cells. Small arrows = directions of apparent cell-stretching within mantle. (b) Configuration from mid-dorsal aspect within a whole gastrula/neurula

Fig. 1.

(a) Diagrammatic representation of the positions of typical grafted organizers in the mesodermal mantle at the close of gastrulation (hatched). Axis = middorsal line of neurula. Black-shaded area = host region homologous wth grafted cells. Small arrows = directions of apparent cell-stretching within mantle. (b) Configuration from mid-dorsal aspect within a whole gastrula/neurula

The shape given in the figure to the graft area is derived from the visible outlines of blue-stained grafts observed. Grafts closer than a certain angle to the host’s dorsal axis come to occupy instead a more compact position, even at the early neurula stage, whereas those whose shape is as shown, subsequently elongate further as an axis is organized under their influence, and often give evidence of a ‘trail’ of stained cells behind them in the midline of that axis. The great majority of the cells remain, however, as a relatively compact mass at the anterior end, and since ‘active’ grafted head organizers assume this compact though elongated shape, it is expected that the homologous host cells at this time occupy a similar shaped region. Fig. 2 shows typical instances, shown up by staining of the graft, of (a) the passive, compact appearance of a graft originally at a very small angle to the host organizer and (b) the active organization of a second axis by a graft at a reasonably wide angle to the host midline during gastrulation.

Fig. 2.

Diagram of 34 rear view of tailbud stages following: (a) operation to give inter-organizer angle less than 60°; no separate individuation due to graft. Densehatched area = position adopted by cellular mass of graft, as seen in vital staining experiments; (b) operation to give inter-organizer angle 90-100°, separate individuation due to graft.

Fig. 2.

Diagram of 34 rear view of tailbud stages following: (a) operation to give inter-organizer angle less than 60°; no separate individuation due to graft. Densehatched area = position adopted by cellular mass of graft, as seen in vital staining experiments; (b) operation to give inter-organizer angle 90-100°, separate individuation due to graft.

Table 1 shows the results of inspection of the total sample of operated embryos, at stages 26–28. They form a series, leading progressively from complete regulation to give a unitary normally proportioned axial structure, through dual bilateral structure anteriorly leading into single organization posteriorly, and finally to completely dual structure back to the tailbud level. Note that there are no examples of the converse series, namely, dual tailwards organization leading into single structure more anteriorly.

Table 1.

Relationship between cephalo-caudal level of fusion of axial structures in neurulae, and the angular distance observed between pairs of organizer apices at commencement of gastrulation, stage 10

Relationship between cephalo-caudal level of fusion of axial structures in neurulae, and the angular distance observed between pairs of organizer apices at commencement of gastrulation, stage 10
Relationship between cephalo-caudal level of fusion of axial structures in neurulae, and the angular distance observed between pairs of organizer apices at commencement of gastrulation, stage 10

Two further features of the results may be noted;

  1. Whenever axial nervous structures join to form a single organization more posteriorly, they do so at the same cephalo-caudal level of the total axial pattern in each. This is true even where the embryo’s own intrinsic axis remains clearly dominant, and the accessory structures are considerably smaller in absolute dimensions and eccentrically placed.

  2. With a frequency variable in different egg batches, implanted stage-10 organizers fail to promote individuation of a complete set of axial structures, the field being deficient apically. In such cases, the non-apical axis, e.g. a neural tube plus ear vesicles, nevertheless tends to join the main axis at a level appropriate for the initial inter-organizer angle observed at operation.

As soon as it became apparent that there was some relationship between interorganizer angle and the subsequent level of fusion of axes, experiments were done to determine whether the host’s age, at time of implantation of the stage-10 organizer, had any influence upon this relationship. Table 2 shows the results of two such experiments, each of which necessitated the finding, in the inevitably random sample of operations originally made into stage-8 hosts, of some examples suitable for comparison with the prepared stage-10 operations. They give no indication that the time elapsing after implantation of an organizer, before the host’s own organizer becomes visibly active, has any influence upon the final pattern of positional information obtained with given inter-organizer angle.

Table 2.

Comparison of results of operations made at host stage 10, with those of similar inter-organizer angle but made at host stage 8

Comparison of results of operations made at host stage 10, with those of similar inter-organizer angle but made at host stage 8
Comparison of results of operations made at host stage 10, with those of similar inter-organizer angle but made at host stage 8

Thus the data in Table 1 represent pooled results for hosts of all stages between 8 and 10 at operation. The best estimate of the threshold angle, below which regulation to give a unitary field tends to occur, is 60°. It will be noted that this is a distinctly larger angle, representing many more intervening cells, than the 30° given in Paper I as an upper limit for fusion of organizer activities with respect to initial cell behaviour. However, in view of the relatively large dimensions of the apical zones (e.g. cement-gland inducing area, gill endoderm, prechordal plate) of the primary axial field, expressed as proportions of the whole early neurula devoted to them, it is concluded that this does not imply a lesser autonomy, or in any sense a greater regulative capacity, of the field with respect to its later expression in cell differentiation, as compared with its early expression in cell behaviour. Thus even though, by the probable time of determination of the zones of differentiation in the mesodermal mantle, the field has had 3 or 4 times as long in which to regulate as was the case for the early organizer interactions discussed in Paper I, embryos with inter-organizer angles of near threshold, 60°, often have large and laterally extended, although morphologically single, apical parts as neurulae.

Demembranated and otherwise undisturbed embryos of Xenopus develop normally, to give neurulae with similar proportions, at both 19 °C and 25·5 °C. At the former temperature, however, up to stage 20, development takes approximately 65 % longer to pass through each of the stages of Nieuwkoop & Faber 1956, than is the case at 25·5 °C. Tables 3 and 4 show the results of comparison, for operations on stage-10 hosts, of the levels of fusion of axes obtained following development at each of the two temperatures. In such experiments, the angle 80–90° is used because, in these cases, the expected level of fusion of axial structures lies in a region (mid-brain-hindbrain) where maximum resolution is obtainable by visual inspection, due to the rapid succession of structures. Eye-size, in the case of ‘inner’ eyes, also seems to be affected.

Table 3.

The effect of ambient temperature of development, between healing in of a grafted organizer at host stage 10 and the tailbud stage, upon the level of axial fusion resulting from particular inter-organizer angles

The effect of ambient temperature of development, between healing in of a grafted organizer at host stage 10 and the tailbud stage, upon the level of axial fusion resulting from particular inter-organizer angles
The effect of ambient temperature of development, between healing in of a grafted organizer at host stage 10 and the tailbud stage, upon the level of axial fusion resulting from particular inter-organizer angles
Table 4.

A precise comparison of three operations, on hosts of stage 10, having inter-organizer angles of 80°, developing at three ambient temperatures

A precise comparison of three operations, on hosts of stage 10, having inter-organizer angles of 80°, developing at three ambient temperatures
A precise comparison of three operations, on hosts of stage 10, having inter-organizer angles of 80°, developing at three ambient temperatures

There is slight but inconclusive evidence for a tendency towards relative discreteness of fields at the higher temperature, when inter-organizer angle is near 90°. If real, the effect is small. There is no evidence, on the other hand, for any decisive effect of temperature near the threshold angle for fusion or discreteness of field apices.

Fig. 3 shows diagrams, from the dorsal surface, of the axial structures of typical embryos having had inter-organizer angles of (a) 70°, (b) 90°, (c) about 130°.

Fig. 3.

Schematic drawings, from dorsal aspect, of late tailbud stages following typical cases of operations: (a) inter-organizer angle ca. 70°, double ventral cementgland, and forebrain structure only; (b) inter-organizer angle ca. 90°, axial structures double back to just behind ear-rudiment level; (c) inter-organizer angle ca. 140°, axes separate back to region of anus.

Fig. 3.

Schematic drawings, from dorsal aspect, of late tailbud stages following typical cases of operations: (a) inter-organizer angle ca. 70°, double ventral cementgland, and forebrain structure only; (b) inter-organizer angle ca. 90°, axial structures double back to just behind ear-rudiment level; (c) inter-organizer angle ca. 140°, axes separate back to region of anus.

Fig. 4 shows a selection of embryos at around stage 26, exhibiting either double cement-gland and forebrain structures only (outer examples), or discreteness of axes anterior to a fusion at hindbrain or anterior trunk nervous system levels (inner examples).

Fig. 4.

A selection of embryos at around stage 26, following typical operations, showing varying degrees of axial doubling anteriorly. Outer examples - angles near fusion threshold 60-70°.

Fig. 4.

A selection of embryos at around stage 26, following typical operations, showing varying degrees of axial doubling anteriorly. Outer examples - angles near fusion threshold 60-70°.

Fig. 5 shows the 19 °C and the 25·5 °C examples from the experiment reported in Table 4. The animal having developed at the lower temperature is still at a very early stage of eyecup pigmentation (the upper example) but the median, dark body between the forebrain fields, which subsequently showed up as a small single median eye, can be seen in the photograph, as well as the rather greater degree of separation between the paired forebrain pigment fields in the lower example.

Fig. 5.

19 °C (upper) and 25·5 °C (lower) examples reported from experiment in Table 4. Upper animal is at earlier developmental stage than lower, but difference in configuration of the joined pigment lines is apparent for the two cases.

Fig. 5.

19 °C (upper) and 25·5 °C (lower) examples reported from experiment in Table 4. Upper animal is at earlier developmental stage than lower, but difference in configuration of the joined pigment lines is apparent for the two cases.

At inter-organizer angles of more than about 130°, the percentage of completely double axes, i.e. belly-joined twins, increases markedly, but the situation remains unstable in this zone, up to 180°, in that there is frequent twisting of axes at the rear so that they run side by side in that region, rather than ventroventrally opposed. Also, subsequent fusion of tailbud structures, which had begun development separately, is often observed. The nature of this relative instability of bilaterality in the rear part of the axis has not been further explored.

However, the evidence suggests that between the thresholds of 60° and, say, 140°, the geometrical configuration of anterior doubleness leading to posterior singleness for the ‘map’ or ‘landscape’ of positional information existing at the time of cell commitment in the mesodermal mantle, depends solely on the original inter-organizer angle within the limits of experimental and biological variability. Using criteria of absolute dimensions along the axis at the stage of scoring the results, the elongated tailbud stage, this dependence would appear to be non-linear. That is, in the range of larger angles a given change of angle leads to a greater change in the absolute distance down the axis at which the point of fusion is to be expected. However, when it is considered that determination of zones of differentiation, and thus the expression of the configuration of positional information, probably occurs at the latest during the unelongated neurula stages 15 or 16, then this apparent extreme nonlinearity of relationship can be understood as due to the increasing relative elongation that occurs, caudally, between stages 21 and 28.

Spemann and co-workers (Spemann, 1938) had observed that, in urodeles, when secondary axes were induced with their apices taking up a cephalad position in the embryo, as in the present experiments, the size and positioning of the successive cephalo-caudal zones of axial differentiation were under the control of the host ‘field’ so that host and graft structures lay in parallel at each level. The graft was supposed by these authors simply to induce a new axis of bilaterality in the dorso-lateral dimension of differentiation. Such a conclusion seems difficult to reach without data on the cephalo-caudal proportions of axes induced, where apical differentiation is obtained, but where the grafted head organizer itself lies well down in a presumptively more caudal position in the host’s field. Such a situation can be obtained in slowly developing urodela, but in Xenopus where cell determination sets in so much sooner after grafting, the result is almost always the irreversible loss of apical properties on the part of a caudally situated head organizer graft. However, it was considered that the following experiment might clarify the interpretation of the main series of operations.

Fig. 6 shows the three types of operation done, to investigate, as far as is possible in Xenopus, the cephalo-caudal size regulative capacity of secondary induced axial fields. The results are shown diagrammatically beneath in each case, and tabulated in Table 5. Fig. 6 a represents of course the normal operation, in a stage-10 host, with angle near 180°. The results indicate that there is little size regulation in a cephalo-caudal direction in cases of secondary induced fields having only a fraction of the normal axial distance available to them, being bounded posteriorly by the supplementary blastopore, or site of secondary intucking in the host’s belly. The two possible interpretations are either that; (a) the host field is indeed supplying the cephalo-caudal information component for the secondary axis as well as for its own; or that (b) the secondary field is autonomous, supplying both its own components of information, but entirely lacks cephalo-caudal regulative capacity (i.e. information is measured only from the head organizer end, without reference to the rear boundary, or blastopore zone, which, if its absolute distance from the anterior boundary is much less than that normal for the species, merely acts as a passive ‘stop’).

Table 5.

Relationship between relative length of secondarily induced axes, obtained by implantation of stage-10 organizers at various levels, and the completeness of the axial pattern subsequently induced. For geometry of the operations, see Fig. 6a, b, c 

Relationship between relative length of secondarily induced axes, obtained by implantation of stage-10 organizers at various levels, and the completeness of the axial pattern subsequently induced. For geometry of the operations, see Fig. 6a, b, c
Relationship between relative length of secondarily induced axes, obtained by implantation of stage-10 organizers at various levels, and the completeness of the axial pattern subsequently induced. For geometry of the operations, see Fig. 6a, b, c
Fig. 6.

(a, b, c). Representation of the three types of operation done to test anteroposterior size-regulative capacity of secondarily induced axes, together with typical results. The site of the implant, on stage-10 hosts in side view, is marked in outline. (a) Is the normal operation, with inter-organizer angle near 180°, and in this case the secondary blastopore forms a true anus, level with host’s own anus.

Fig. 6.

(a, b, c). Representation of the three types of operation done to test anteroposterior size-regulative capacity of secondarily induced axes, together with typical results. The site of the implant, on stage-10 hosts in side view, is marked in outline. (a) Is the normal operation, with inter-organizer angle near 180°, and in this case the secondary blastopore forms a true anus, level with host’s own anus.

The latter interpretation is the less plausible, since under abnormal circumstances such as excision of large amounts of the blastula, followed by perfect healing, normally proportioned early neurulae of less than normal linear dimensions have been observed in this laboratory. But in either case, these results imply that in studying the partially fused and partially discrete axial fields that have been set up in the main series of experiments, one need consider only the regulatory behaviour of the component of positional information governing bilaterality, and the dorso-lateral zones of differentiation tendency. The antero-posterior differentiation of the axes is here providing only a visualization of the relative position, behind the organizer apices, where regulation to a single bilateral organization has occurred.

It is unlikely that the process of determination, or irreversible restriction of differentiation potency, occurs very suddenly amongst the cells of the prospective zones of any one individuation field. There is some evidence (see Holtfreter & Hamburger, 1955) that in the case of the amphibian primary embryonic field, such determination occurs at different rates for different regions. Two alternative conceptions of the nature of fields, between which we cannot distinguish experimentally for amphibian embryos at the present time, are as follows. On the one hand, the variables constituting the positional information (e.g. titre of substances, temporal organization of metabolism in cells) may themselves cause the onset of restriction in potency, the entry of cells into differentiation pathways. On the other hand, an ongoing process of potency restriction may be intrinsic to cells in embryos, occurring after particular elapsed times at particular temperatures. The field may then be set up by partially independent mechanisms, merely channelling appropriately the choices made by cells at each point in an intrinsically timed programme of potency restriction.

In the present experiments temperature, over a range in which the visible processes of cellular differentiation occur at rates varying by a factor approaching two, has little or no effect upon the final geometrical results of the implantation of a second apical organizer near the beginning of gastrulation. Now in view of the processes involved in (a) the erection and maintenance of fields, and (b), the onset of cell determination (the former probably partially metabolic and partially diffusional in character, the latter presumably the results of cumulative metabolic activity), we should only expect a final complete temperature compensation in the results of modifying the field during development, under either of the following two sets of conditions:

(a) The onset of cell determination, to produce a pattern of differentiation, is caused by the erection of the positional information field itself and is ratelimited by the latter process.

(b) The onset of cell determination, partially independent of the actual pro-cesses erecting and maintaining positional information, occurs at some point in development considerably after the attainment of the regulated field, following the initial disturbance (i.e. the organizer implantation). Since many experimental embryos have configurations intermediate between regulation to give a single axis, and complete doubling of the axis, the partially double fields that these imply would have to be stable end-points in the process of regulation in the presence of two organizers. They would also have to be themselves independent of the ambient temperature of development, and dependent only upon the initial angle between organizers.

If the configuration of positional information were to change continuously in time, after the operation, along some trajectory of regulation towards complete doubleness or complete singleness, until ‘frozen’ or expressed by the supervention of cell determination, then we should only expect such temperature independence of the final results of operations in the unlikely event of compensation throughout, amongst the temperature coefficients of the processes involved in field maintenance on the one hand and in cellular differentiation on the other (unless condition (a), above, were the case).

This final configuration of the regulated field, i.e. the precise level of axial fusion obtained in the neurula, is also independent of the relative stages of development of grafted organizer and host field, over a range of some 2·5 h up to host stage 10. There are two classes of explanation for this fact. One may need to consider models for fields where any two apical or ‘dominant’ sites, provided both are allowed to develop independently (see Discussion in Paper I of this series) beyond a certain point, will divide the territory of cells available to them into two fields alongside some boundary passing between them, and where the final position of that boundary will regulate to be independent of the relative number of hours that each centre has been active. Models of the class of the phase-shift one of Goodwin & Cohen (see Introduction), or hybrid models where some propagating phase-shift mechanism is conceived as polarizing substance transport in the cell sheet (e.g. Goodwin & McLaren, in preparation) have such properties rather naturally, without necessity of special assumptions to obtain temperature compensation, etc.

Alternatively, it may be that whilst there is evidently a deep-seated gradient in the marginal zone of the blastula, with respect to the tendency for any region to develop into one of organizer activity, the presumptive organizer site nevertheless does not start to influence the behaviour or differentiation tendencies of the cells around it until the very onset of gastrulation. Such a situation would ensure the stage-independence observed for the final results of the operation of implanting an extra stage-10 organizer. This observation does not therefore, at present, place substance gradient models in difficulty. The results of Paper I where early organizer interactions were studied, are in accord with this, implanted ‘precocious’ organizers becoming quiescent until re-activated synchronously with that of their host at stage 10.

Fig. 7 (a) and (b) show diagrammatically how the region of the early organizer which is initially approximately circular and could be organized, from one origin, only into roughly concentric zones of positional information is nevertheless formally sufficient, taking into account its change of shape during the gastrulation movements, to act as an organization centre for a bilaterally symmetrical field of positional information on two co-ordinates, using as origins P1 and P2, spread out posteriorly to it in the mesodermal mantle. ‘P’ here symbolizes, in a general way, either source or maintained high point for a gradient substance (dependent on the postulated mechanism for erection of the gradient), or ‘propagating cellular event that can be phase shifted, providing information’. Experiments in progress to investigate whether in fact a further origin, nearer the animal pole of the blastula and early gastrula, is utilized in setting up the cephalo-caudal information (derived entirely from origin P1 on this model), may form the subject of a future paper, but the consideration is irrelevant here as we are considering only the bilaterally organized, dorsolateral component of information, with origin P2.

Fig. 7.

(a) and (b). Representation of the formal adequacy of an initially radiallyorganized zone, around the stage-10 organizer in the presumptive mesoderm, for the subsequent erection of a bilaterally symmetrical field of positional information along two co-ordinates. For further explanation, see text.

Fig. 7.

(a) and (b). Representation of the formal adequacy of an initially radiallyorganized zone, around the stage-10 organizer in the presumptive mesoderm, for the subsequent erection of a bilaterally symmetrical field of positional information along two co-ordinates. For further explanation, see text.

The bilaterally symmetrical hill of Fig. 7b, the exact form of whose slopes cannot of course be known, is perhaps the best way of representing the landscape (whether of substance or otherwise) responsible for dorsolateral information. Since in the limiting instance (an inter-organizer angle of near 180°) a local, grafted head organizer can cause twinning extending to the tailbud region, a hill of the bilateral form shown must be able to be built up with the origin P2, initially at least, confined to the anterior part of its spine as shown (according to the results from vital staining of grafts), in the region of the graft. The bilateral symmetry of slope of such a hill, even well caudad from the head region, could be explained functionally by recourse to extreme cell stretching that occurs during gastrulation along the midline and in other regions of the mesoderm towards the midline. The anisodiametrical shape then assumed by the cells would mean that, if positional information were assumed to change in terms of number of cell boundaries crossed rather than of absolute distance, a likely assumption for active propagation, active transport or restricted diffusion models, then lines of greatest rate of change would be up the sides of the elongate hill, and only then along its spine towards the P2 region.

It can be seen from inspection of Fig. 7 (b) that as one takes progressively more posterior transects through the field, at least at some point during its development, the bilateral profile of the gradient becomes progressively less steep. In terms of cellular responses to this gradient, one way of expressing this is to postulate that, at regions progressively further behind the level of a head organizer that is erecting an axial field under its own influence, bilaterality might take progressively longer to be achieved and to stabilize. Further, we postulate that when two such bilateral hills are being erected with their spines in parallel, a certain distance apart, which is the equivalent of the situation following on the operation described in this paper, then at a certain distance behind the line joining the pair of organizers, the flatness and shallowness of the slopes of the double transect will be beneath the threshold of discrimination for the cells in the middle region. From this level posteriorly, in the dual field, regulation will tend towards a stable single bilaterality of profile.

This situation is depicted in the block diagram of Fig. 8. It would seem applicable to all the types of theory under consideration; for the nature of positional information, including regulating double diffusion gradients, pumping/back-diffusion gradients, and dynamic propagating event models (since cells must have thresholds for perception of differences in phase-state as well as substance-concentration). If the assumption is made, as suggested earlier, that the processes of erection and maintenance of fields are to some extent independent of those leading individual cells towards differentiation as such, then the stability over time of these partially double positional information configurations, indicated by the temperature experiments, should be a property of the model. It is intuitively apparent that nowhere, even around the singularity marking the point of progression from double to single organization, is there a slope downwards in the direction of the head regions, i.e. a slope the converse of those in each field as a whole. Thus on all substance gradient models, in which diffusion plays a part either as mechanism or as antagonist in the setting up of the gradient, there need be no progressive tendency towards fusion due to diffusion’s acting in a sense opposite from that normal during the formation of a single field. Furthermore, any substance gradient model capable of regulation and self-maintenance over the time required (at least 5 h at 21 °C), in the single field situation, must incorporate mechanisms to prevent concentration rising, due to diffusion, above the level normal for any zone a given distance from the source of the principal substance. On the other hand, once the fusion of axes has occurred at a particular point due to the interpretation threshold and then regulatory activities of the cells, then the relative degree of separation of the hills of such a landscape can never increase, no matter how elevated or steep they subsequently become, in terms of absolute concentrations.

Fig. 8.

Representation, as a landscape, of a partially double field of positional information, considered to exist in the mesodermal mantle of the neurula following implantation of a second organizer at or before stage 10. Heavy arrows = local directions of maximal rate of change of positional information, along which diffusion and/or active transport, or phase shifting of cellular events, will occur. For further explanation see text.

Fig. 8.

Representation, as a landscape, of a partially double field of positional information, considered to exist in the mesodermal mantle of the neurula following implantation of a second organizer at or before stage 10. Heavy arrows = local directions of maximal rate of change of positional information, along which diffusion and/or active transport, or phase shifting of cellular events, will occur. For further explanation see text.

Diffusion on the one hand and substance production by metabolism on the other, which would be expected in general to act in opposition during the maintenance of a substance gradient, are normally characterized by different temperature coefficients. Thus ultimately, any substance gradient model requires the assumption of evolved, special compensation mechanisms in order that it shall produce and maintain the whole range of absolute information, in zones of similar proportion, during development at all temperatures within the range tolerated by the species. Since, as seen above, diffusion would not be expected to act in a counter-normal sense in the partially dual fields under consideration, the apparent constancy of the point of field fusion for particular inter-organizer angles, over a range of ambient temperatures, would not seem to require special explanation. The slight separating effect of high temperature, in the case of well-separated organizers, if a real phenomenon, might be interpretable as a result of some imperfection of temperature homeostasis, during its own development, on the part of a mechanism producing final constancy of proportion against ambient temperature in normal, singly organized embryos. That is to say, in normal embryos, the configuration of the field as developing at different temperatures, might be transiently slightly different, although finally identical.

Experiments are in progress, changing the temperature of development relatively suddenly at various times between operation and the onset of cell determination, in embryos similar to those discussed here. The detection of great differences in final level of field fusion, caused by temperature transients as opposed to variations in ambient temperature, may allow an approach to distinguishing between various substance gradient and active transport models. Such work will form the subject of a future paper.

At present, it is perhaps worth noting that models such as the phase-shift one of Goodwin & Cohen (1969), or hybrid models where such a mechanism is considered to form a field in which the values for cells are set, so that they then act as homeostats for substance concentrations as in the ‘sonk’ model of Lawrence (1971), would provide automatically for the observed constancies in the levels of field fusion in cases such as the present ones.

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