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.     

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%.     

Analysis of morphogenetic mutants of hydra

The mutantreg-16 is deficient in head regeneration and abnormal in size regulation. The gastric region becomes twice as long as that of normal animals before the first bud is produced. Both mutant characteristics are due to changes in head-specific morphogen concentrations.Reg-16 contains twice as much head inhibitor and only half as much head activator in its head as normal animals. This leads to a higher level of free head inhibitor in the whole animal resulting on one hand in a greater distance of buds from the head, and on the other hand in a total blockage of release of head activator and head inhibitor which would be necessary to initiate head regeneration.     

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.     

Head regeneration inHydra is initiated release of head activator and inhibitor

Summary Hydra regenerating heads release at least two substances into the surrounding medium: one stimulates and one inhibits head formation. The inhibitor is released mainly during the first hour after cutting, the activator is released more slowly with a maximum in the second hour and with substantial release still during the following six hours. The release of both substances seems to be specific for head regeneration: it is not found in animals regenerating feet. The sequential release of these substances leads to the early changes observed at the cellular level during head regeneration inhydra: the inhibitor produces a decrease, the activator an increase in the mitotic activity of interstitial and epithelial cells, if assayed on intact animals. Head regeneration is blocked, if the release of the head activator is prevented. It is therefore suggested that these substances are necessary to initiate head regeneration inhydra.     

Size,shape and orientation of cells in budding hydra and regulation of regeneration in cell aggregates

Summary Changes in the number, shape, volume, orientation and vacuolization of cells involved in the budding of hydra were measured in histological sections. Before evagination, a group of about 800 epithelial cells are visibly recruited for the bud to be produced and this number increases to about 5,000 within a day. Thereafter, bud size increases mainly by proliferation of the cells within the bud. Upon recruitment for budding, the epithelial cells assume a columnar shape, with a smaller contact area facing the mesoglea, accompanied by a decrease in volume which is mostly accounted for by devacuolization. In later stages, cells progressively resume the form typical for non-budding areas of hydra. Evagination proceeds without reorientation of epithelio-muscular fibers, whereas elongation of the bud is accompanied by fiber reorientation.The process of sorting out and regeneration in aggregates of previously dissociated hydra cells was followed using various ratios of endodermal to ectodermal epithelial cells. From different initial compositions, the ratio in the regenerate rapidly approaches 11, the ratio found in normal hydra tissue.The experimental findings are discussed in the context of theoretical notions on pattern formation, evagination, elongation and stability of layered structures.     

Modulatory action of 12-O-tetradecanoylphorbol-13-acetate on bud production inHydra

Summary A low concentration of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.0 ng/ml) induced a transient inhibition of bud production in hydra which were fed daily. However, when hydra were starved following TPA-treatment, they produced further buds. Phorbol (1.0 ng/ml) and dimethyl sulfoxide (0.001%) did not influence bud production under either feeding or starvation conditions. These results indicate that TPA modulates asexual reproduction in hydra.     

A model for budding in hydra: pattern formation in concentric rings

Current models of pattern formation in Hydra propose head-and foot-specific morphogens to control the development of the body ends and along the body length axis. In addition, these morphogens are proposed to control a cellular parameter (positional value, source density) which changes gradually along the axis. This gradient determines the tissue polarity and the regional capacity to form a head and a foot, respectively, in transplantation experiments. The current models are very successful in explaining regeneration and transplantation experiments. However, some results obtained render problems, in particular budding, the asexual way of reproduction is not understood. Here an alternative model is presented to overcome these problems. A primary system of interactions controls the positional values. At certain positional values secondary systems become active which initiate the local formation of e.g. mouth, tentacles, and basal disc. (i) A system of autocatalysis and lateral inhibition is suggested to exist as proposed by Gierer and Meinhardt (Kybernetik 12 (1972) 30). (ii) The activator is neither a head nor a foot activator but rather causes an increase of the positional value. (iii) On the other hand, a generation of the activator leads to its loss from cells and therewith to a (local) decrease of the positional value. (iv) An inhibitor is proposed to exist which antagonizes an increase of the positional value. External conditions like the gradient of positional values in the surroundings and interactions with other sites of morphogen production decide whether at a certain site of activator generation the positional value will increase (head formation), decrease (foot formation) or increase in the centre and decrease in the periphery thereby forming concentric rings (bud formation). Computer-simulation experiments show basic features of budding, regeneration and transplantation.     

The fine structure of the hypostome and mouth of hydra

Summary The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed.     

Characterisation of two types of head activator receptor on hydra cells

A head activator (HA) analogue is described which even at high concentrations does not lose its biological activity. By cross-linking two HA molecules over a C8 spacer, the conformation was sufficiently altered, such that self-inactivation of HA by dimerisation was prevented. In addition, the introduction of a tyrosine instead of phenylalanine in one of the two HA molecules allowed radioactive labelling with iodine. This HA bipeptide was used to investigate the effect of HA at different concentrations and as ligand for HA receptor characterisation. We found that low concentrations (0.1-10 pM) sufficed to stimulate interstitial cell mitosis, and that higher concentrations (10-1000 pM) were required for the determination of interstitial cells to nerve cells. Binding of the radioactive HA ligand to living hydra and to purified membrane fractions was saturable and specific. Binding was compatible with HA analogues with a stable monomeric conformation, but less well with dimerising HA and HA analogues. Scatchard and kinetic analyses revealed the presence of at least two types of binding site in the membrane fraction, one with a 'lower' affinity (Kd = 10(-9) M) and one with a 100-fold higher affinity (Kd = 10(-11) M). Autoradiography showed that interstitial cells were differentially labelled, suggesting that the number or types of HA receptors may vary depending on cell cycle status. A mutant of hydra with a multiheaded morphology contained 6-20-times more HA receptors per mg protein than other hydra species or mutants.     

Biosynthetic events of hydrid regeneration

Summary The results of a combined morphological and biochemical study of the role of DNA synthesis during distal regeneration inHydra oligactis revealed that a burst of³H-thymidine incorporation into DNA preceded the elaboration of each of the initial three tentacles. In addition, the relative level of each burst of precursor incorporation relfected the number of tentacles formed at that time. Cytological localization of concentrated amounts of labeled material in nuclei of the hypostome and tentacle regions provided corroborative evidence for the biochemical findings.Evidence that the increased DNA specific activity levels described above are associated with tentacle initiation derived from studies in which regenerating hydra were cultured in hydroxyurea and studies in which hydra regenerated proximally rather than distally. Hydra regenerating in 8 mg/ml (0.105 M) hydroxyurea developed morphologically recognizable hypostomes but no tentacles, and incorporated³H-thymidine into DNA at a level distinctly below that exhibited by uncut, untreated animals. Similarly, hydra regenerated a normal, functional basal disc in the absence of any increased DNA specific activity. Therefore, it is suggested that tentacle initiation inH. oligactis requires concomitant DNA synthesis and, as such, represents an epimorphic phenomenon.     

Variation in green hydra. A description of three cloned strains of Hydra viridissima Pallas 1766 (Gnidaria: Hydrozoa) isolated from a single site

Three different strains of green hydra were isolated in successive years from a single site in the River Frome, Bristol, England. The strains resembled each other in important taxonomic characters, and were identified as Hydra viridissima by their possession of smooth-walled single-chambered embryothecas, but were smaller, possessed smaller nematocysts and produced fewer eggs per individual than the Western European holotype. It is possible that English green hydra represent a geographical subspecies.
The three strains could be distinguished from each other by differences in rates of bud production and in characters associated with their symbiotic algae. These differences are considered varietal, and it is suggested that H. viridissima is a polytypic species. The possible origins of these variations and their significance is discussed.     

Effects of water hardness and alkalinity on the toxicity of uranium to a tropical freshwater hydra (Hydra viridissima)

In tropical Australian freshwaters, uranium (U) is of potential ecotoxicological concern, largely as a consequence of mining activities. Although the toxicity of uranium to Australian freshwater biota is comprehensive, by world standards, few data are available on the effects of physicochemical variables, such as hardness, alkalinity, pH and organic matter, on uranium speciation and bioavailability. This study determined the individual effects of water hardness (6.6, 165 and 330 mg l^-1 as CaCO₃) and alkalinity (4.0 and 102 mg l^-1 as CaCO₃), at a constant pH (6.0), on the toxicity (96 h population growth) of uranium to Hydra viridissima (green hydra). A 50-fold increase in hardness (Ca and Mg concentration) resulted in a 92% (two-fold) decrease in the toxicity of uranium to H. viridissima [i.e. an increase in the EC₅₀ value and 95% confidence interval from 114 (107-121) to 219 (192-246) µg l^-1]. Conversely, at a constant hardness (165 mg l-1 as CaCO₃), the toxicity of uranium to H. viridissima was not significantly (P > 0.05) affected by a 25-fold increase in alkalinity (carbonate concentration) [i.e. EC₅₀ values of 177 (166-188) and 171 (150-192) µg l^-1 at 4.0 and 102 mg l^-1 as CaCO₃, respectively]. A knowledge of the relationship between water chemistry variables, including hardness and alkalinity, and uranium toxicity is useful for predicting the potential ecological detriment in aquatic systems, and can be used to relax national water quality guidelines on a site-specific basis.     

Effects of water hardness and alkalinity on the toxicity of uranium to a tropical freshwater hydra (Hydra viridissima)

In tropical Australian freshwaters, uranium (U) is of potential ecotoxicological concern, largely as a consequence of mining activities. Although the toxicity of uranium to Australian freshwater biota is comprehensive, by world standards, few data are available on the effects of physicochemical variables, such as hardness, alkalinity, pH and organic matter, on uranium speciation and bioavailability. This study determined the individual effects of water hardness (6.6, 165 and 330 mg l^-1 as CaCO₃) and alkalinity (4.0 and 102 mg l^-1 as CaCO₃), at a constant pH (6.0), on the toxicity (96 h population growth) of uranium to Hydra viridissima (green hydra). A 50-fold increase in hardness (Ca and Mg concentration) resulted in a 92% (two-fold) decrease in the toxicity of uranium to H. viridissima [i.e . an increase in the EC₅₀ value and 95% confidence interval from 114 (107-121) to 219 (192-246) µg l^-1]. Conversely, at a constant hardness (165 mg l-1 as CaCO₃), the toxicity of uranium to H. viridissima was not significantly (P > 0.05) affected by a 25-fold increase in alkalinity (carbonate concentration) [i.e. EC₅₀ values of 177 (166-188) and 171 (150-192) µg l^-1 at 4.0 and 102 mg l^-1 as CaCO₃, respectively]. A knowledge of the relationship between water chemistry variables, including hardness and alkalinity, and uranium toxicity is useful for predicting the potential ecological detriment in aquatic systems, and can be used to relax national water quality guidelines on a site-specific basis.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

The extracellular matrix of hydra is a porous sheet and contains type IV collagen

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 μm in diameter and about six pores per 100 μm² in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell–cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.     

Evaluation of tentacle regeneration as a biological assay inHydra

Summary Tentacle number in non-buddingHydra attenuata, randomly selected from mass culture varies <0.5 tentacles over a 3 month period. Replicate samples of untreated regenerates (n=50–60), however, show some variability in mean tentacle number regenerated (S _x0.13–0.15). The variability is similar whether experiments are performed using randomly selected animals or animals with identical tentacle numbers. The variability is, further, not the result of profound differences in the time of tentacle initiation in individual animals.Addition of 10^–5 M glutamate or a methanol extract to the assay medium results in both an earlier appearance of tentacles and in more tentacles being regenerated during early time periods. The mean tentacle number of methanol extract-treated animals is significantly higher than the mean tentacle number of either control or glutamate-treated animals at all time periods examined.The distribution of tentacle number classes among regenerates is normal in control and glutamate-treated animals but nonparametric in methanol extract-treated animals, making statistical analysis of the data using Student'st-test in-appropriate. The usefulness of the Mann WhitneyU and Kruskal-Wallis tests is discussed, as is the appropriateness of tentacle regeneration as an assay forhydra morphogens.     

The role of nerve cell density in the regulation of bud production in hydra

Summary The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.     

Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes,epithelial cells,and basement membrane

Summary Nematocytes (stinging cells) of hydra tentacles are anchored to the basement membrane by peculiar complex junctions in which a flattened tongue of an epithelial cell is interposed between the nematocyte and the basement membrane. In this paper we describe the arrangement of these junctions with emphasis on how they are related to the architecture of the epithelial cell. Each epithelial cell, called a battery cell, harbors 10–20 nematocytes and bears muscle processes that extend along the basement membrane. The epithelial cell component of the complex junction is usually a lateral extension of a muscle process. All nematocytes within a battery cell make junctions with muscle processes of the same (resident) epithelial battery cell despite the presence of numerous muscle processes from adjacent (foreign) cells. Some nematocytes make junctions with several resident processes, spanning the foreign processes to do so. Most junctions reside near the proximal ends of the muscle processes. New findings are reported on the substructure of the junctions. They are composed of aggregates of smaller elements, and the cytoskeleton within the complexes has a pronounced longitudinal organization. These observations are consistent with a suggestion that the complex junctions develop by aggregation of smaller junctional units originating elsewhere on the cells.