Studies on pattern regulation in hydra: I. Regional differences in time required for hypostome determination

0 From the Zoology Department, King's College, University of London By G E R A L D - 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 1 Author's address: Strangeways Research Laboratory, Wort's Causeway, Cambridge, England. 2 Author's address: Department of Biology as applied to Medicine, Middlesex Hospital Medical School, London, W. 1, England. G. WEBSTER & L. WOLPERT 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. 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. 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. 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 hypostomeone of the characteristics of the dominant region of a regulative systemare 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 regionthat is, the hypostomein 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 transpl (...truncated)