Properties of the foot inhibitor from hydra

Summary A substance was isolated from crude extracts of hydra that inhibits foot regeneration. This substance, the foot inhibitor, has a molecular weight of 500 daltons. It is a hydrophilic molecule, slightly basic in character and it has no peptide bonds. The pruified substance acts specifically and at concentrations lower than 10^–7 M. At this low concentration only foot and not head regeneration is inhibited. Hydra are sensitive to purified foot inhibitor between the second and eight hour after initiation of foot regeneration by cutting. In normal animals the foot inhibitor is most likely produced by nerve cells. A substance with similar biological and physico-chemical properties is found in other coelenterates.     

The head and the foot inhibitor from hydra are not dowex artefacts

Summary In a recent publication in this journal (Berking 1983) it was claimed (1) that the head inhibitor we isolated from hydra is a Dowex artefact, (2) that a separate foot inhibitor does not exist in hydra and (3) that the only inhibitor that has so far been isolated from hydra is one which inhibits head and foot regeneration equally well. These statements are incorrect and require a response. In the following, I would like to summarise our evidence that the inhibitors isolated from hydra, including Berking's inhibitor, have different specificities for head and foot regeneration. In addition, I would like to show that none of our substances are Dowex artefacts.     

A new biochemical marker for foot-specific cell differentiation in hydra

Summary Mucous cells in the basal disk of hydra contain a peroxidase-like enzyme allowing specific staining of these cells with substrates for peroxidases. The peroxidase activity provides an excellent marker for foot mucous cell, differentiation and was used to follow the reappearance of footspecific cells during foot regeneration after amputation. By choosing the appropriate either soluble or precipitable substrate the peroxidase reaction was used both for a qualitative and for a quantitative evaluation of foot-specific differentiation in hydra. For histological studies diaminobenzidien was found to be a suitable substrate which forms a dark brown precipitate within the cells containing the peroxidase activity. For a quantitative evaluation of foot regeneration the soluble substrate 2,2-azino-di(3-ethyl-benzthiazoline-sulfonic acid-6) ammonium salt was used which after reaction with the enzyme gives rise to a diffusible green reaction product the concentration of which can be measured by its specific absorption at 415 nm. Based on the diffusible enzyme product a new quantitative assay for foot regenration was developed and applied to confirm the effect and specificity of morphogenetic substances which either inhibit or activate foot or head regeneration in hydra.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata. III. The shaping of the body column

Excised pieces of hydra body tissue of varying size and shape regenerate into cylinders with a head and foot at opposite ends. The numbers of cells along the axial and circumferential dimensions were determined before, during, and after regeneration. The main process in shaping the excised tissue into a body column was found to be a rearrangement of the cells. When regenerates of different size were measured, the proportions of the body columns were found to vary, such that the smaller the animal the squatter the body column was. The presence of the head in regenerates was necessary for the formation or maintenance of the cylindrical shape, while the size of the head determined the proportions of the cylinder. The formation of a gradient of adhesivity induced by the developing head is suggested as the basis for the rearrangement of the cells into the cylindrical form.     

Ultraviolet irradiation initiates ectopic foot formation in regenerating hydra and promotes budding

We have studied the effects of ultraviolet-C (UVC) and Ultraviolet-B (UVB) on growth and pattern formation inPelmatohydra oligactis. UVC brings about a significant increase in budding in intact hydra while UVB does not exhibit such an effect. Excessive budding could be a response for survival at wavelengths that damage biological tissues. If the head or base piece of a bisected hydra is irradiated and recombined with the unirradiated missing part, regeneration proceeds normally indicating that exposure of a body part with either an intact head or foot to UVC does not influence pattern formation. Most significantly, in the middle piece, but not in the head or the base piece of a trisected hydra, UVC leads to initiation of ectopic feet formation in almost one third of the cases. Thus, UV irradiation interferes with pattern formation in regenerating hydra, possibly by changing positional values, and promotes budding in intact hydra. This is the first report on induction of ectopic feet formation by UV in regenerating hydra and opens up the possibility of using UV irradiation as a tool to understand pattern formation in the enigmatic hydra     

Actions of head activator and head inhibitor during regeneration in hydra

Abstract. Head regeneration in hydra is initiated by an extensive release of head inhibitor and head activator from tissue close to the cut surface. Release of both substances is less extensive after removal of the foot. Incubation of regenerating animals in medium with head inhibitor blocks not only regeneration of a new head but also release of head activator and head inhibitor. No effect was found of the head activator on the release of both substances. Release of head-specific substances is thus controlled by the inhibitor alone. Cellular determination in a head-specific direction and the production of new sources for head factors requires head activator.     

Analysis of head and foot formation inHydra by means of an endogenous inhibitor

Summary In tissue regenerating the head, the ability to initiate head formation in a host increases with the time allowed for regeneration before grafting, while the foot-initiating ability decreases concomitantly. The reverse was found for tissue about to regenerate a foot. The early divergent changes thus indicated are counteracted in both head and foot regeneration by treatment with an inhibitor (Berking, 1977) in low concentrations.The inhibitor also interferes with processes which determine wether or not hypostome and tentacles are formed, and how many tentacles (if any) appear. The circumferential spacing of the tentacles was regular whether their number was normal or below normal.Secondary axes caused by implanted tissue either detach after having formed a head and a foot (i.e. behave like buds) or do not detach, having only formed a head. This alternative depends on the origin and amount of the implanted tissue and on the position of the implant within the host.The following model based on these findings is proposed: Head and foot formation start with pre-patterns which cause a continuously increasing change of the tissue's ability to initiate a head or a foot. Along the body axis this ability is determined by a graded distribution of sources. As development progresses, the high source density which accumulates in the head region causes the formation of a hypostome and tentacles; the angular spacing of tentacles is also dependent on source density. At a certain low source density foot-formation is initiated. The inhibitor counteracts the increase of source density in head-forming tissue as well as the decrease of source density in foot-forming tissue. It thus appears to be part of the mechanism which controls morphogenesis in hydra.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

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.     

Interactions between the Foot and the Head Patterning Systems in Hydra vulgaris

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.     

水螅异常极性体轴的形成及矫正进程的观察

目的:如何建立和维持体轴是一个基本的发育生物学问题,而淡水水螅是适合进行形态发生和个体发育调控机制研究的重要模式生物。本文观察了大乳头水螅异常极性体轴的形成及矫正进程,初步探讨水螅极性体轴的维持和调控机制。方法:先切取水螅的整个头部,再获得带二根触手的口区组织。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,判别水螅基盘组织(水螅足区的末端)是否形成。结果:从40块口区组织再生得到的水螅个体中有1例极性体轴发育异常的个体,其身体两端均发育成头区,且两端的头区均具有捕食能力。随后水螅其中一端头区的触手逐渐萎缩、退化,最终该端头区转化成具有吸附能力的基盘组织。结论:水螅组织的再生涉及极性体轴的重建,而一些特殊因素可能造成临时性的水螅极性体轴调控紊乱。本研究表明水螅具备自我矫正异常极性体轴的能力。另外,本研究结果显示水螅触手可以萎缩直至退化,该现象涉及的细胞学过程可能是非常复杂的,有可能涉及到触手细胞的凋亡转化过程,也可能是触手的高度分化细胞仍然具备去分化能力、去分化后再转移到身体其他地方,其具体机制值得进一步探究。     

HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra

Developmental gradients are known to play important roles in axial patterning in hydra. Current efforts are directed toward elucidating the molecular basis of these gradients. We report the isolation and characterization of HyAlx, an aristaless-related gene in hydra. The expression patterns of the gene in adult hydra, as well as during bud formation, head regeneration and the formation of ectopic head structures along the body column, indicate the gene plays a role in the specification of tissue for tentacle formation. The use of RNAi provides more direct evidence for this conclusion. The different patterns of HyAlx expression during head regeneration and bud formation also provide support for a recent version of a reaction-diffusion model for axial patterning in hydra.     

Positional Information and Positional Signalling in Hydra

A model is proposed for regulation and regeneration of the headend of hydra in terms of positional information, which involvestwo gradients. One is a diffusible substance made at the headend, which may be regarded as a positional signal. The otheris a more stable cellular parameter which is the positionalvalue. The rule for head end formation is that the concentrationof the diffusible substance falls a threshold amount below thepositional value. This model, for which some computer simulationis provided, can account for head end formation in a wide varietyof grafts. Evidence for a diffusable signal is provided by experimentsin which the time/distance relationships for head inhibitionby a grafted head are determined. Changes in positional valueduring regulation have been assayed and are much slower awayfrom the boundary. Polarity is interpreted in terms of the interactionbetween the two gradients. The biochemical basis of the gradientsis not known, but an approach to the problem has been made bytreating hydra with a variety of chemical agents.     

Separation and specificity of action of four morphogens from hydra

Summary A procedure is presented by which four previously described morphogenetic substances can be purified from hydra: an activator and an inhibitor of head formation and an activator and an inhibitor of foot formation. We show that all four substances act specifically. At low concentrations, the head factors only influence head and not foot formation, and the foot factors only influence foot and not head formation.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Pattern formation in hydra tissue without developmental gradients

Ring-shaped pieces of hydra tissue were excised from a specified position on a body column of 20-30 polyps and grafted together in tandem like a chain of beads. A "tandem graft" prepared in this way has the same basic tissue organization and same tube-like morphology as a normal hydra body column, but lacks the head, foot, and developmental gradients ordinarily present. Three major types of structures were formed along the length of the tandem graft: heads, buds, and feet. The relative number of these structures produced was strongly affected by the origin of the tissue used to prepare the tandem graft. Evidence was obtained which suggests that tissue originally located outside of the budding zone in intact hydra has a strong latent capacity to form a bud, and that the level of this capacity forms a gradient from the budding zone toward the hypostome. Evidence was also obtained which is consistent with the view that the head and foot forming mechanisms cross-react positively, increasing the chances for these two structures to be formed next to each other on a tandem graft.     

A model of head regeneration in hydra

Abstract. Studies of the changes of the head inhibitor and the head activator during hydra head regeneration have shown that free head inhibitor blocks its own release from sources and that of head activator as well. On the basis of this feedback mechanism a system of differential equations was formulated which describes the changes of the free and bound substances during regeneration in a computer simulation. Removal of the head is assumed to cause a loss of free inhibitor by leakage. The resulting decrease in the concentration of free inhibitor allows extensive release of both substances from sources close to the cut surface. The fast diffusing inhibitor spreads out over the whole tissue. Due to its smaller diffusion rate the activator accumulates near the cut surface. This distribution is stable during the first hours of regeneration, and we propose that it is the necessary prerequisite for head formation. Using the stimulating property of the head activator on the production of nerve cells, which are in turn activator- and inhibitor-producing cells, restoration of the gradient of the sources in the model is assured. Budding, which is another important morphogenetic event of hydra, can also be described in terms of the model.     

Analysis of morphogenetic mutants of hydra

Summary A mutant ofHydra attenuata is analysed, theaberrant, which is distinct from the wild type in having a smaller head with fewer tentacles and only half the number of head-specific cells. The rate of head and foot regeneration and the doubling time are slower inaberrants than in normal hydra.The lower head-forming potential is paralleled by a reduced concentration of head-specific morphogens: compared to the wild type, in theaberrant the concentration of head activator is reduced to 70% in the head and to 50% in the body, the concentration of head inhibitor is reduced to 50% in the head and to 80% in the body. Theaberrant is more sensitive (3 times) to added head activator and less sensitive (>5 times) to added head inhibitor than the wild type.The slower rate of foot regeneration is paralleled by a lower content of foot-specific morphogens: compared to the wild type, in theaberrant the foot activator is reduced to 40% and the foot inhibitor to 70%.     

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.