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The Cnidarian, hydra, is an appealing model system for studying the basic processes underlying pattern formation. Classical studies have elucidated much basic information regarding the role of development gradients, and theoretical models have been quite successful at describing experimental results. However, most experiments and computer simulations have dealt with isolated patterning events such as the dynamics of head regeneration. More global events such as interactions among the head, bud, and foot patterning systems have not been extensively addressed. The characterization of monoclonal antibodies with position-specific labeling patterns and the recent cloning and characterization of genes expressed in position-specific manners now provide the tools for investigating global interactions between patterning systems. In particular, changes in the axial positional value gradient may be monitored in response to experimental perturbation. Rather than studying isolated patterning events, this approach allows us to study patterning over the entire animal. The studies reported here focus on interactions between the foot and the head patterning systems in Hydra vulgaris following induction of a foot in close proximity to a head, axial grafting of a foot closer to the head, or doubling the amount of basal tissue by lateral grafting of an additional peduncle-foot onto host animals. Resulting positional value changes as monitored by antigen (TS19) and gene (ks1 and CnNK-2) expression were assessed in the foot, head, and intervening tissue. The results of the experiments indicate that positional values changed rapidly, in a matter of hours, and that there were reciprocal interactions between the foot and the head patterning systems. Theoretical interpretations of the results in the form of computer simulations based on the reaction-diffusion model are presented and predict many, but not all, of the experimental observations. Since the lateral grafting experiment cannot, at present, be simulated, it is discussed in light of what has been learned from the axial grafting experiments and their simulations.

Normal hydra head–body proportions were altered by axially grafting a second head in place of the lower body column. The resulting animals had double the head tissue and one-quarter the normal body column. Changes in the head activation potential of tissue subjacent to both heads were monitored by assaying the ability of these animals to regenerate heads. The host head, the grafted head, or both heads were removed at varying times following graft construction and the animals were scored for head regeneration and/or the ability to express a head-specific antigen recognized by monoclonal antibody, CP8. In the presence of the grafted head, tissue subjacent to the host head lost the ability to regenerate a head or express the head-specific antigen over a 48-hr period. In the presence of the host head, tissue subjacent to the grafted head regenerated heads at a very low frequency and lost the ability to express the head-specific antigen over the same 48-hr period. Following simultaneous removal of both heads, animals initially regenerated both heads for the first 48 hr after graft construction. Then, both head regeneration and expression of the head-specific antigen declined gradually over the next 3 days, though not to the very low levels observed when one head remained. These data, especially the loss of CP8 labeling, support the hypothesis that loss of regeneration ability was due to a loss of head activation potential in tissues subjacent to the heads. We propose that this reflected the attempt of grafted animals to compensate for the altered head–body proportions through reproportioning. In keeping with this hypothesis, feeding the grafted animals to stimulate growth of body column tissue and a shift toward more normal head–body proportions prior to decapitation resulted in animals which were capable of regenerating heads when decapitated. Several interpretations of the results based on the Gierer–Meinhardt reaction-diffusion model of pattern formation are discussed.

The de novo appearance of head organizing centers was monitored during regeneration of aggregates made from dissociated body column tissue of Hydra oligactis using the head-specific monoclonal antibody CP8 (L. C. Javois, R. D. Wood, and H. R. Bode, 1986, Dev. Biol. 117, 607-618). Newly differentiated CP8+ head organizing centers were present by 24 hr of regeneration and were restricted to one half of the aggregate. Depending on subsequent patterning events these CP8+ cells went on to develop into heads or body tentacles, or remained as CP8+ patches. A comparison of the number of initial CP8+ patches with the final number of CP8+ structures indicated that this number was fairly constant, suggesting that a limited number of head organizing centers were established early during regeneration. Examination of fully differentiated head structures revealed that they were segregated to one end of the aggregate with feet segregated away from the CP8+ structures. Increasing the surface area of the aggregates resulted in a more random initial spacing of CP8+ head organizing centers and ultimately a more random distribution of differentiated heads and feet. However, polarized polyps with one head and foot detached and the remaining structures were then segregated. The ability of existing pattern formation models to describe interactions between head- and foot-forming systems resulting in the observed spacing of heads and feet is discussed.

In hydra the differentiation of head-specific ectodermal epithelial cells from multipotent stem cells is a multistep process in which cell cycle progression is regulated at three restriction points. Head activator acts as a positive signal at these restriction points. At the G2/mitosis boundary of epithelial stem cells head activator functions as a mitogen, being necessary for cell division. Subsequently, in or before S phase, head activator acts as determinant to ensure commitment of epithelial cells to head-specific determination. This effect of head activator requires hundredfold-higher concentrations, and may also require longer incubation times, than for cell proliferation. Epithelial cells thus committed to head-specific differentiation become arrested in G2 as a third and last restriction point in the cell cycle. They require disinhibition by decapitation and probably the presence of head activator for final differentiation, which then occurs in G2.

Previous studies have demonstrated that head activator (HA), an 11 amino acid peptide, stimulates head-specific differentiation processes in hydra. Additionally, HA enhances the differentiation of interstitial cells into nerve cells. This study investigated the effects of exogenous synthetic HA on the dynamics of both apical and basal regeneration in a piece of tissue excised from the body column of treated animals which comprised one-eighth of the original animal. The dynamics of apical and basal regeneration were monitored using the monoclonal antibody TS19. This antibody binds to apical and basal ectodermal tissue very early in the process of regeneration, before morphological structures are evident. Labeling is ultimately localized to the tentacles of the head and a ring above the basal disc. Thus, TS19 is a useful tool for analyzing the dynamics of both apical and basal patterning processes in the same regenerate simultaneously. Quantification of TS19 positive areas on regenerates over a time course of 72 hr revealed that HA treatment accelerated and amplified the dynamics of both apical and basal TS19 labeling. The specific basal effect was novel and was demonstrated to occur in the absence of a determined head independently of new nerve cell differentiation. It is proposed that the basal effect was the result of growth factor-like activity of HA.

The characterization of head activator (HA) as a morphogen capable of increasing the number of tentacles regenerated by hydra was re-examined. Gastric tissue was excised from HA-treated whole animals and allowed to regenerate. At the cellular level the differentiation of head-specific ectodermal epithelial cells was monitored by quantifying monoclonal antibody, CP8, labeling. This labeling has been correlated with a rise in head activation potential and the determination of tissue to form head structures (Javois et al. 1986). At the morphological level tentacle number was monitored. HA-treated regenerates began the head patterning processes and evaginated tentacles sooner than controls but did not produce extra tentacles. The kinetics of CP8 labeling did not reveal major differences between treated and control regenerates after the initiation of head-specific epithelial cell differentiation. HA appeared to act more like a growth factor stimulating the differentiation of head-specific cell types rather than a morphogen which altered head morphology. An additional aspect of the study examined axial-specific effects of HA on the initiation and extent of head-specific epithelial cell differentiation. The cellular response of ectodermal epithelial cells to HA was dependent on their original axial location. More CP8+ tissue differentiated in regenerates of apical as opposed to mid-gastric origin.

The body column of hydra is polarized such that a new head will regenerate from the apical end when both extremities are removed. This is due to a graded property of the tissue termed the head activation gradient. The aim of the experiments presented here was to determine what events connect a two-dimensional segment of the activation gradient in an isolated piece of tissue with the formation of a head structure at a particular location. To this end, tissue pieces with three different shapes were excised and analyzed during and after regeneration. The most apical tissue of each piece was labeled with the DNA-intercalating dye, DAPI, and the area where developmental changes were occurring was monitored using the monoclonal antibody CP8 (Javois et al., 1986). First, it was shown that polarity of regeneration was maintained regardless of the fraction of body length included in the excised pieces. Second, while head structures usually formed from the original apical tissue, they could be located anywhere in the regenerate. This was an effect of the healing process which shaped the apical edge differently in different pieces. Third, early CP8 binding occurred in similarly shaped areas suggesting that patterning events were initiated in a contiguous manner wherever apical tissue was located. And finally, not all of the CP8-marked tissue successfully formed structures. Apparently some regions were favored to continue the patterning process, and these in turn extinguished the process in neighboring regions.

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.

The pattern of differentiated wing structures formed following 180° rotation of the undifferentiated wing bud tip on its base was examined in detail. These analyses were performed to determine the handedness and origin of the supernumerary structures which arise. In contrast to the variable classes of symmetric and/or asymmetric limb anatomies observed following the same operation with amphibian regeneration blastemas, wings of predictable handedness were observed. Both the graft and stump contributed cells to the supernumerary structures. These results are discussed in the light of two current models describing the developing chick limb and analysed diagrammatically within the framework of one of these models, the polar coordinate model.

The formation of duplicated wing skeletal elements and/or extra wing muscles was studied by juxtaposing normally nonadjacent embryonic chick wing bud cells. A wedge of right or left stage 21 wing bud ectoderm and mesoderm was inserted in a slit made in a host stage 20 to 22 right wing bud at the same anteroposterior position as its position of origin. The distal edge of the donor wedge and host wing bud were aligned with each other. Donor tissue was grafted into a host wing bud in one of the following four axial relationships: both the anteroposterior and dorsoventral axes corresponded with each other ( aadd ); only the anteroposterior axes were opposed ( apdd ); only the dorsoventral axes were opposed ( aadv ); both the anteroposterior and dorsoventral axes were opposed ( apdv ). Of the 63 wings resulting from the control aadd operation and the 45 wings from the apdd operation, only 12 wings had a duplicated skeletal element; of the 69 wings sectioned from these two groups of operations, only one had an extra muscle. However, of the wings resulting from the aadv and apdv operations (48 and 52 cases, respectively), 23 had a duplicated skeletal element; of the 54 wings sectioned from these operations, 43 wings had one to four extra muscles. Furthermore, when the aadv operation was performed with a wedge of donor quail wing bud ectoderm and mesoderm or mesoderm alone, supernumerary muscles formed in these chimeric wings and they were made up of donor quail and host chick cells or only donor quail cells.

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.

A monoclonal antibody, termed JD1, was generated that bound to a subset of the nerve cells in the hypostome and tentacles of Hydra oligactis. Using a whole-mount technique the spatial pattern of the subset of nerve cells and their processes could be clearly visualized using indirect immunofluorescence. The subset largely corresponds to the epidermal sensory cells. Using the same technique the development of the pattern during head regeneration and budding was examined. The appearance of the nerve cells coincides with the formation of both the tentacles and hypostome. When head regeneration does not occur, JD1+ cells do not appear suggesting the differentiation of JD1+ cells is an integral event in head formation dependent on antecedent patterning processes.

The relationship between the position transplanted in a host limb bud, the orientation of a graft in a host limb bud, and the extra limb structures formed was studied by juxtaposing normally nonadjacent embryonic chick wing bud tissue. In one series of transplantation operations, two different wedges (ectoderm and mesoderm) of stage 21 right donor posterior wing bud tissue were transplanted to the middle of a host stage 20 to 22 right wing bud such that the dorsal-ventral polarity of the graft and host were the same or reversed. The results of these transplantation operations show that the formation of supernumerary limb structures depends on the position of origin of the donor tissue, the anterior-posterior position transplanted in a host limb bud, and the orientation of the graft in the host limb bud. In a second series of transplantation operations, the relationship between the proximodistal position where posterior donor tissue is transplanted in an anterior host site and the extra structures formed was studied. A wedge of posterior stage 21 right wing bud tissue was transplanted to an anterior proximal or anterior distal site of a stage 22 to 24 host right wing bud. The results of these transplantation operations show that when the donor tissue is transplanted to an anterior proximal position in a host wing bud, then limbs with only a duplicated humerus result, whereas, when transplanted to an anterior distal position, then limbs with a duplicated forearm element and extra digits result.