1. The regulation of hydra into hypostomal and non-hypostomal regions is considered as a model for the regulation of a linear pattern into two compartments. Regulation of such a system requires graded differences and a ‘principle of limited realization’.

  2. The hypostome is the dominant region in hydra and the time for its determination in isolated pieces depends upon the original position of the piece along the linear axis. The time increases in a disto-proximal direction from 4 h to more than 20 h.

  3. The determination of the hypostome is the first detectable event in distal regeneration and is probably the time-determining step in this process. Work on other hydroids is reinterpreted in these terms.

  4. The time for hypostome determination in proximal regions is significantly affected by the presence of more proximal regions, while in distal regions the time appears independent of the presence of more proximal regions.

Many systems having a linear pattern of spatial differentiation are capable of regulating this pattern during development or regeneration. This is very evident in the early development of the sea-urchin embryo, the development of cellular slime moulds, and in the regeneration of hydroids, planarians and vertebrate limbs. Hydra is a classic example of such a system in that removal or isolation of almost any region leads to the reconstitution of the original pattern. As a result of earlier studies on such systems attempts were made to provide a general explanation in terms of axial gradients, apical dominance, and polarity (Huxley & de Beer, 1934; Child, 1941). Work since this time has not invalidated in any striking manner these concepts, but has rather tended to extend them (e.g. Rose, 1952, 1957). In some ways the concepts have tended to remain descriptive rather than explanatory (Webster & Wolpert, 1966).

Spiegelman (1945) has attempted to specify some of the minimum requirements for regulation in such systems in terms of these concepts. He emphasizes that the crux of the problem of regulation lies in the fact that while the potentialities for forming a particular region exist throughout a large part of the system, the region is normally formed from only a small part. This means that the potentialities of most of the system are not realized. He also points out that this fact cannot be accounted for by a simple gradient in rate of differentiation, since a difference between two regions in rate of differentiation does not of itself imply a restriction of differentiation to the region of greater rate. What is needed in such a system is a ‘principle of limited realization’, that is, a mechanism for suppressing potentialities. Gradients, or differences, are, however, a necessary component of such systems, since the fact that two regions are capable of a particular differentiation, and yet only one succeeds in differentiating, means that there must be some difference between them. If such differences vary in a continuous fashion, then they are gradients. These ideas seem fundamental to any consideration of pattern formation but, as will be discussed elsewhere, may not be applicable without modification to regions other than the dominant region (Webster & Wolpert, 1966).

The properties of a dominant region have been characterized by Huxley & de Beer (1934): (1) it is the first region to be formed; (2) its formation, once initiated, is an autonomous process, independent of the formation of other regions; (3) once formed, it exerts an organizing influence on other regions; (4) it inhibits the formation of further dominant regions.

There is a considerable amount of evidence which suggests that the distal hypostome (Fig. 1A) is the dominant region in hydra. The organizing ability of the hypostome was revealed by Browne (1909), who showed that a small fragment of hypostome was capable of eliciting the formation of tentacles and a short axis when grafted to a host animal. All other regions of the animal (with the exception of the basal disc) were absorbed. These results were confirmed by Yao (1945). Browne also observed that during distal regeneration the properties of the hypostome were acquired by the distal region some time before the tentacles were produced.

Fig. 1.

A, Diagram of hydra showing the principal regions of the axis. B, Regions used in measurement of time for hypostome determination in isolated pieces and in other transplantation experiments: a, Subhypostomal region; b, proximal digestive zone; c, distal peduncle; d, proximal peduncle.

Fig. 1.

A, Diagram of hydra showing the principal regions of the axis. B, Regions used in measurement of time for hypostome determination in isolated pieces and in other transplantation experiments: a, Subhypostomal region; b, proximal digestive zone; c, distal peduncle; d, proximal peduncle.

Rand, Bovard & Minnich (1926) shed further light on the organizing properties of the hypostome when they demonstrated that a grafted hypostome and tentacles could suppress distal regeneration in the host animal. No other region possessed this property. These workers drew two important conclusions with regard to the role of the hypostome: (1) it ‘initiates or controls the development of structures appropriate in relation to itself’; (2) it ‘inhibits the operation of a developmental mechanism where such operation would result in the formation of structures inconsistent with the attainment of normal form’.

The organizing properties of the hypostome—one of the characteristics of the dominant region of a regulative system—are thus well established.

Any part of hydra can become any other part following transplantation (Browne, 1909), and the work of many early investigators showed that most regions (exceptions are the tentacles and basal disc) are capable of complete regulation and therefore of forming a hypostome.

Regeneration in hydra is always polarized, i.e. distal structures (hypostome and tentacles) are formed from distal ends and proximal structures (peduncle and basal disc) from proximal ends. Polarity is rigorously maintained in isolated pieces (Morgan, 1901; Tardent, 1960) but can sometimes be altered in graft combinations (Peebles, 1900; King, 1901; Browne, 1909; Goetsch, 1929); in such cases the new polarity is usually determined in relation to the position of a hypostome.

This series of papers will consider the factors controlling the formation and localization of the dominant region—that is, the hypostome—in hydra. Effectively we will be concerned with regulation of a simple linear pattern consisting of two regions, one of which is the hypostome. It should be noted that this whole formulation of the problem is quite different from that of Burnett (1961, 1962), who considers regeneration of hydra as being due to the growth of a new hypostome and tentacles from a growth zone. He is concerned primarily with the factors controlling growth within this zone. Apart from any theoretical objections that can be raised against such a formulation of the problem, Campbell (1965) has shown that growth is not localized in such a zone.

In this paper the experiments were designed to investigate regional differences in rate of hypostome formation. Earlier work on hydra has shown that tentacle regeneration in hydra occurs more rapidly from distal than from proximal regions (Peebles, 1897; Browne, 1909; Weimer, 1928; Rulon & Child, 1937; Spangenberg & Eakin, 1961). However, although tentacle morphogenesis is dependent on the presence of a hypostome, the time required for this process does not necessarily reflect the time required for hypostome differentiation. A more direct measurement of the rate of hypostome differentiation is required.

Browne’s observations suggest a test which will indicate the presence of a hypostomal organizer at an early stage of regeneration. A piece from a regenerating animal when transplanted to a host will elicit the formation of a secondary axis if a hypostome is present and will be absorbed if it is not. Although this test is ultimately dependent upon formation of tentacles, the primary factor involved is some change in properties which confers upon a region the power to resist absorption when transplanted. By analogy with similar changes occurring during embryonic development (Spemann, 1938; Weiss, 1939), when regions acquire independence of, or resistance to, environmental influences, it seems permissible to refer to this change as hypostome determination.

The majority of experimental work was carried out using Hydra littoralis grown from a clone supplied by Dr H. M. Lenhoff.

Animals were cultured by the methods of Loomis & Lenhoff (1956) in a medium (‘M’ solution) containing 0·001 M tris (hydroxy) methylaminomethane, 0·001 M-CaCl2,0·001 M-NaHCO3,0·0001 M-KCI and 0·0001 M-MgCl2 in deionized water; the pH was adjusted to 7·5·7*8 with N-HCI (Muscatine, 1961). Hydra were grown at room temperature (20–22 °C) and were fed daily with washed brine shrimp nauplii and washed after feeding. Under these conditions, and with weekly subculturing, they grew logarithmically with a doubling-time of about 212 days.

In the majority of experiments actively growing ‘adult’ animals—possessing one or two buds—were used 15–20 h after feeding.

Operative procedures

Simple cutting and more elaborate transplantation experiments were performed using techniques similar to those employed by earlier workers (Rand, 1899; Peebles, 1900; King, 1901; Browne, 1909). Operations were carried out on a plasticene surface under ‘M’ solution. Strict cleanliness was maintained but sterile conditions were not necessary and no antibiotics were used. Animals were kept in an incubator at 26 °C throughout the course of an experiment and were examined every 24 h.

Pieces to be transplanted from intact animals were obtained by isolating rings from selected body levels (Fig. IB), and splitting these up into smaller fragments; small pieces which had been isolated for some time and allowed to heal became spherical and were split open or cut in half before use as grafts. Transplantation was accomplished by inserting the graft into small incisions made in a host animal, care being taken that the endoderm of graft and host was in close contact to facilitate adhesion (Papenfuss, 1934). With persistence and practice about 90 % of the transplantation experiments were successful.

After transplantation, animals were left undisturbed in the operating dish for 1 h to allow grafts to heal in place and were then transferred to individual solid watch-glasses or Petri dishes containing ‘M’ solution, and placed in the incubator.

Due to the time required for transplantation, most experiments were done on batches of 5–10 animals and the results presented in the tables are usually the total of at least two experiments. There was rarely any marked discrepancy between the results of such repeated experiments, but in cases where there was, the two batches are tabulated separately.

Nature and form of the secondary axis

The distal structures (tentacles and axis) formed as a result of transplanting a fragment of hypostome are derived primarily from the tissues of the host, the graft usually remaining as the hypostome of the new axis (Browne, 1909; Yao, 1945). Yao observed that in a few cases ‘self-differentiation’ occurred and the graft transformed partially or entirely into parts of the new axis in addition to becoming the new hypostome. Since in the present experiments grafts were not marked, no distinction could be made between induced and self-differentiated axes, but for the purpose of this investigation this was of little consequence since the primary consideration was the presence or absence of a hypostome and the subsequent formation of distal structures, irrespective of how or from what material they were formed. Therefore, throughout these papers the formation of secondary axes as a result of grafting will be referred to for convenience as induction.

The induced structures obtained in these experiments as a result of grafting were of four distinct types, and comparable to those described by Browne and Yao (Fig. 2). Type 1 : a short axis of diameter comparable to that of the distal regions of the host, a conical hypostome with mouth and two or more tentacles around the circumference of the hypostome—in other words, a more or less typical distal end. Type 2: a short axis of small diameter, a small hypostome and two or more tentacles—the whole structure being much smaller and of slightly different proportions to the host distal end. Type 3 : a short axis comparable with type 1 but with only one tentacle at its apex and no normal hypostome. Type 4: a single tentacle (rarely two) with no axis, obtained rarely.

Fig. 2.

Different forms of induced secondary axes.

Fig. 2.

Different forms of induced secondary axes.

All these types with the exception of type 4 were classified as positive inductions. Type 1 is of the typical form described by earlier workers (Browne and Yao), produced by grafting a hypostome fragment into distal regions. Type 2 is of the type described by Browne as being obtained when a hypostome is implanted in proximal regions; in the present experiments it was obtained under similar conditions. Type 3 is typical of the regeneration which is obtained at a distal end in an animal which has been treated with low concentrations of an inhibitory but not lethal substance, e.g. mercaptoethanol (unpublished observations). These induced axes are stable over a period of several days at least and, after the host animals have been fed, develop accessory tentacles and become identical with type 1. Type 4, although clearly a definite response on the part of the host, and therefore presumably indicating some degree of hypostomal influence, was not considered as a case of positive induction since in the majority of cases these tentacles disappeared (were absorbed) within 48 h, and were always absorbed after feeding. They are apparently rather unstable structures and are excluded from the results for this reason, bearing in mind that a primary criterion for hypostome determination is resistance to absorption. The comparative rarity of type 4 inductions means that their exclusion has a negligible effect on the results.

Experiment 1. Formation of hypostome from the subhypostomal region and from the distal peduncle

Animals were cut at the subhypostomal level (just proximal to the ring of tentacles) or at the distal end of the peduncle (just proximal to the youngest bud) to remove distal regions (see Fig. 1). At various times after cutting the distal tip of the regenerating piece was removed and transplanted to the middigestive zone of an intact host hydra. The transplanted piece was approximately the same size as a normal adult hypostome. Results are shown in Table 1.

Table 1.

Formation of hypostome from the subhypostomal region and from the distal peduncle

Formation of hypostome from the subhypostomal region and from the distal peduncle
Formation of hypostome from the subhypostomal region and from the distal peduncle

The results show a distinct difference between the two regions in the time at which a determined hypostome is formed. Pieces from the subhypostomal region induced 4 h after isolation while those from the distal peduncle produced only one positive induction after 12 h and required 18 h before a substantial number of pieces were determined. All the positive cases were type 1 or 3 inductions.

A direct, if crude, comparison of the rates of hypostome determination in the two regions is possible if an estimate is made of the time for 50 % of the pieces to become determined (T50). For the subhypostomal region T50 = 4 · 5 h; for the distal peduncle T50= 16 · 5 h.

In both pieces a determined hypostome was detected a considerable time before tentacle regeneration occurred. Animals cut subhypostomally developed tentacle buds about 18 – 20 h after cutting as compared with peduncles where tentacles did not appear until about 30 h after cutting. In both regions therefore the delay between hypostome determination and tentacle morphogenesis was about 13 – 15 h.

The most obvious difficulty in evaluating the above results is that in the case of the subhypostomal region the formation of a hypostome is taking place in a piece of considerably greater size than in the case of the distal peduncle region. An animal from which the hypostome and tentacles only have been removed has about 4 times the mass (protein content) and 3 · 5 times the number of cells (DNA content) as an isolated peduncle (Webster, 1964). There is thus a close correlation between the relative sizes of the two pieces and the relative times for hypostome formation, and the possibility that the size of the regenerating piece rather than the position of the hypostome-forming region on the linear axis is determining the time in this experiment must be considered. The next experiment was designed to investigate this possibility.

Experiment 2. Formation of hypostome by isolated pieces of similar size

Pieces of approximately the same size (volume) were isolated from four positions on the linear axis : the subhypostomal region (a), the proximal digestive zone (b), the distal peduncle (c), and the proximal peduncle (d) (Fig. 1B). The average volume of the isolated pieces—calculated from ocular micrometer measurements—was 1 · 6 × 10−2 mm3, ranging from 1 · 0× 10 − 2 to 2·8 × 10−2. At various times after isolation the pieces were transplanted to the mid-digestive zones of intact host animals as in Exp. 1. None of the pieces at the time of transplantation showed any signs of tentacle formation. Results are shown in Table 2.

Table 2.

Formation of hypostome by isolated pieces of similar size

Formation of hypostome by isolated pieces of similar size
Formation of hypostome by isolated pieces of similar size

The first point to emerge from an inspection of Table 2 is that isolation of the subhypostomal region from more proximal regions has no measurable effect on the time for hypostome determination, thus proving that the results obtained in Exp. 1 were not due to the size differences between the two pieces. The T50 of 4·5 h can therefore be taken as the value for the time for hypostome determination in this region.

In the case of the distal peduncle, however, isolation of this region from more proximal regions has a considerable effect on the time for hypostome determination. The T50 calculated from Table 2 is 28 h, an increase of 75 % in the time compared with that measured in Exp. 1 (T50 = 16·5 h).

The T50 for the isolated proximal digestive zone is 22 h. The rate in situ for this region was not measured since it is known (see Burnett, 1961) that regeneration of tentacles from this region is interfered with by developing buds in the budding zone, just proximal to the distal cut surface.

No T50 can be obtained for the isolated proximal peduncle since even after 50 h only a very small number of transplanted pieces induced. Fragments from this region do not survive well after isolation, tending to disintegrate after 24 h or so. The endoderm is very sparse when the pieces are isolated, and seems to disappear entirely in many pieces which have been isolated some time, thus making transplantation very difficult—hence the small number of successful grafts. This region, unlike the other three, was never observed to undergo total reconstitution but the positive inductions indicate that, in some animals at least, this region is capable of forming a hypostome, albeit very slowly. It is felt that more significance should be attached to the small number of positive cases than to the larger number of negative cases in this sort of experiment when considering the possible properties of the region in situ, since a new factor—survival in isolation—is introduced.

The pieces from the subhypostomal region and the proximal digestive zone were about a fifth by volume of the whole digestive zone; the pieces from the distal and proximal ends of the peduncle were about a third of the volume of the whole peduncle. Using the values for regional protein and DNA content (Webster, 1964), and assuming that these values are uniform throughout a region, i.e. the same at distal and proximal ends (almost certainly true for the digestive zone at least), it may be concluded that the size of the four regions in terms of mass of living material or number of cells is similar. It is probable therefore that the differences between the four regions in time taken for hypostome determination are not due to size differences.

This experiment shows therefore that an important factor governing the time for hypostome determination in an isolated region is the original position of the region on the linear axis of the animal. The time is short in regions from distal levels and long in those from proximal levels.

The times, rates and relative rates of hypostome determination for the four isolated regions are summarized in Table 3.

Table 3.

Regional differences in time for hypostome determination

Regional differences in time for hypostome determination
Regional differences in time for hypostome determination

The technique for assessing hypostome determination requires some comment. The formation of a secondary axis as a result of transplantation is not only dependent upon the acquisition of inductive ability by the region, and upon a morphogenetic response by the host to this inductive influence, but also upon acquisition by the graft of resistance to absorption. Providing that the piece is always transplanted to the same region of the host, the morphogenetic response of the host can be ruled out as an important variable in this test. This indeed confers upon the method a distinct advantage over that involving simple observation of tentacle formation in regenerating pieces, where the responding region varies with the level of the axis. It is, however, conceivable that resistance to absorption and inductive ability are independent properties and that under certain conditions one may be present without the other. There is no simple means of resolving this difficulty and it can only be pointed out that, in the entire course of the experimental work, a transplanted piece either induced a new axis or was absorbed within 24–48 h. In some cases the piece transformed into a peduncle and basal disc, but was never observed to persist unchanged. In view of this difficulty, it is evident that the method is really a test of both acquisition of resistance to absorption and of ability to induce. When hypostome determination is referred to, these are the changes which will be implied.

The main conclusions to be drawn from the experiments are that an important factor governing the time for hypostome determination in an isolated region is the original position of the region on the linear axis of the animal. It seems reasonable to postulate the existence of an axial gradient in properties which is expressed when regions are isolated as a difference in time for hypostome determination. The time required for hypostome determination by the subhypostomal region is independent of any more proximal regions being present. In contrast the time required by the distal peduncle region is dependent on the presence of more proximal regions. Finally, the hypostome is determined some time before any visible signs of regeneration, such as tentacle formation, are evident.

There is a considerable amount of experimental evidence that tentacle regeneration in hydra occurs more rapidly from distal than from proximal levels of the axis (Peebles, 1897; Browne, 1909; Weimer, 1928; Rulon & Child, 1937; Spangenberg & Eakin, 1961) and this Was also noted in Exp. 1. The results of Exp. 2 suggest that the difference in time required for tentacle regeneration is a consequence of the difference in the time for hypostome determination and that the formation of the hypostome is the primary, if not the only, time-determining, step in distal regeneration. This conclusion is borne out by the observation in Exp. 1 that the interval between the formation of a hypostome and the appearance of tentacles is practically identical (13–15 h) in animals regenerating from two levels of the axis; if regional differences in time for tentacle formation do exist then they must be very small.

The time of about 4h for hypostome determination by a subhypostomal region fits in well with the studies of Ham & Eakin (1950), who concluded from studies on the effect of inhibitors that an important change in response to chemical agents occurs after about 4 h regeneration.

It has been known for many years that differences exist between distal and proximal levels of marine hydroids in the rate at which a hydranth is reconstituted (reviews by Child, 1941; Tardent, 1960, 1963). The most detailed investigation is that of Barth (1938), who showed that the ‘inherent’ rate of hydranth reconstitution in isolated pieces of the stem of Tubularia decreased rapidly as the pieces came from successively more proximal levels. Evidently there is present in the stem an axial gradient in properties which is expressed as graded differences in the rate at which hydranth reconstitution can occur.

There is some evidence which suggests that the observed axial differences in rate of reconstitution do not reflect the rate at which all the processes involved in hydranth formation occur. Lund (1923), working with Obelia, was able to show that the axial rate differences arose as a result of differences in the time at which visible regeneration (out-growth of the coenosarc) commenced at different levels; once the process had started it proceeded at similar rates in all pieces, irrespective of their original axial position. Similar results were obtained by Steinberg (1954,1955) with Tubularia. The process of hydranth reconstitution was found to be divisible into two phases, the first consisting of the formation of a distal thickening as a result of distal migration of the coenosarc, the second of actual hydranth differentiation. Steinberg observed that the axial difference in rate of reconstitution arose as a result of a difference in the time at which the distal thickening was formed, the process of hydranth differentiation occurring at the same rate in all pieces. These results suggest that in other hydroids, as in hydra, the primary time-determining step occurs at an early stage in the process of reconstitution.

During hydranth reconstitution, differentiation or organization seems to proceed in a disto-proximal direction (Child, 1941; Rose, 1955) and it has been demonstrated in Tubularia that distal parts of the hydranth become determined before proximal parts when tested by isolation (Davidson & Berrill, 1948). It is known that the most distal region of some hydroids possesses organizing ability, comparable with that of the hypostome of hydra, and will induce a secondary axis when transplanted (Corymorpha, Child, 1941; Cordylophora, Beadle & Booth, 1938; Moore, 1952).

These facts suggest that the time-determining step which occurs at an early stage of reconstitution is, in all hydroids, the formation of a distal organizer or dominant region.

The term ‘rate of hypostome formation’ has been carefully avoided in the description of our results since what has been measured here is the time required for hypostome determination. Even the concept of rate of hypostome formation is an obscure one, implying as it does a continuous transformation of nonhypostomal region into hypostome from the time when the original hypostome is removed. There is no reason to believe that this is the case. Barth (1938), in his work on the reconstitution of the hydranth in Tubularia, uses rate of regeneration to mean the amount of stem transformed into hydranth per unit time. However, as already pointed out above, hydranth reconstitution is not a unitary process, and it is not meaningful to talk about a rate for such a complex process. There is no evidence to suggest that either hypostome formation or subsequent hydranth morphogenesis can be treated as continuous processes with respect to time, particularly if switch mechanisms are involved, as in the case of hypostome determination which seems to be an ‘all or none’ phenomenon.

This raises the whole problem of what determines the time required for hypostome determination. It seems that if the formation of a hypostome is viewed as a ‘developmental pathway’ then regional time-differences could arise in three main ways: (1) all regions start from the same point on the pathway and at the same time, but differ in their rate of development; (2) all regions start from the same point, develop at the same rate, but differ in the time at which development is initiated; (3) all regions start at the same time, develop at the same rate, but differ in the point on the pathway from which they start. It is of great importance to establish which of these is in fact operative and it should be noted that only case (1) requires a gradient in rate of development. As pointed out above, for hydranth reconstitution the difference in time required for morphogenesis seems to result from the operation of the second mechanism.

Some consideration must be given to the difference in time for hypostome determination from the distal peduncle in isolated pieces as compared with intact peduncles. The increase in time required resulting from isolation could be the result either of a reduction in the total size of the piece, or of the removal of more proximal regions, or of the presence of a second cut surface. No definite decision can be made between these alternatives with the evidence at present available, but the view favoured at the moment is that the result is a size effect. The experiments of Barth (1938) and Steinberg (1954) on Tubularia led these workers to conclude that the size of the piece (volume or number of cells) had a definite influence on the rate of hydranth reconstitution though a more specific effect due to the absence of proximal regions was not considered. The role of the second (exposed) cut surface was excluded in Barth’s work where ligatures were employed, and the cut surface has been minimized as an important factor in the reconstitution of hydra by the few workers who have considered the problem (see, for example, Morgan, 1901). The view of Child (1941) that cutting somehow ‘activates’ a region and initiates the formation of a new dominant region is probably no longer tenable (Rose & Rose, 1941).

In contrast to the distal peduncle, isolation of the subhypostomal region has no apparent effect on the time for hypostome determination, even though the total size of the regenerating piece is reduced by a very much greater amount. A 75 % increase in the time required for hypostome determination would change the T50 for this region from 4·5 to about 8 h; it is possible that such an increase does occur but that the relative crudity of the experimental technique, coupled with the variability of the material, makes a change of this magnitude undetectable.

We have shown in this paper that there is an axial gradient in time for hypostome determination and this is probably the time-determining step in distal regeneration. As pointed out in the introduction, graded differences in properties are a necessary but not sufficient factor for regulation to occur. In addition a ‘principle of limited realization’ is required. This will be investigated in the following paper.

Etudes sur la régulation chez l’hydre. I. Différences régionales dans le temps requis pour la détermination de l’hypostome

  1. La régulation de l’hydre en régions hypostomale et non-hypostomale est considérée comme un modèle de régulation d’un type linéaire en deux compartiments. La régulation d’un tel système requiert des différences graduées et un ‘principe de réalisation limitée’.

  2. L’hypostome est la région dominante de l’hydre et le temps requis pour sa détermination en fragments isolés dépend de la localisation primitive du frag-ment le long de l’axe linéaire. La durée augmente en direction disto-proximale, de 4 heures à plus de 50 heures.

  3. La détermination de l’hypostome est le premier événement décelable dans la régénération distale et est probablement l’étape déterminante de ce processus. Le travail fait sur d’autres hydroïdes est ré-interprété dans ce sens.

  4. Le temps nécessaire à la détermination de l’hypostome dans les régions proximales est significativement affecté par la présence de régions plus proximales, tandis que dans les régions distales ce temps apparaît indépendant de la présence de régions plus proximales.

One of us (G. W.) is indebted to the Agricultural Research Council for a Postgraduate Research Studentship.

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