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 a foot regeneration deficient strain of Hydra oligactis.

A foot regeneration deficient strain of Hydra oligactis with altered size regulation was investigated. Analysis of the concentration of four morphogenetically active substances in Hydra oligactis showed that the foot regeneration deficiency was mainly due to a drastically reduced foot activator concentration. Foot activator was distributed as a very steep gradient in Hydra oligactis leading to a more severe impairment of foot regeneration the closer to the head cutting was done. The concentration of foot inhibitor was comparable, that of head activator slightly increased and that of head inhibitor reduced compared to Hydra vulgaris. Studies at the cellular level implied that the enlarged gastric region was due to an elevated level of inhibition hinting at a shift in the ratio between bound and free head inhibitor in this animal.     

Morphogenetic substances in nerve-depletedhydra

Summary By a double colchicine treatment the nerve-cell population ofhydra was reduced to less than 1% of the normal complement. Such severely nerve-depletedhydra contained normal or higher than normal concentrations of head activator, head inhibitor, foot activator and foot inhibitor which in normal animals are produced by nerve cells. According to typical chromatographic properties all four morphogenetic substances were chemically identical to those found in normal animals. It is suggested that in nervedepletedhydra the epithelial cells, as the only remaining cell type, have taken over the morphogen-producing function of nerve cells.     

Hydra as a model organism for the study of morphogenesis

Pattern formation in hydra is controlled by two sets of morphogens: an activator and an inhibitor o f head and bud formation, and an activator and an inhibitor o f foot formation.     

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.     

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%.     

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.     

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.     

Isolation of a substance activating foot formation in hydra

We have developed an assay for a substance from hydra that accelerates foot regeneration in the animal. This substance is specific for the foot as evidenced by the following findings: (1) It is present in the animal as a steep gradient descending from foot to head, paralleling the foot-forming potential of the tissue (2) It does not accelerate head regeneration, nor do the head factors of hydra discovered by Schaller (1973) and Berking (1977) accelerate foot regeneration. We propose that the foot-activating substance is a morphogen responsible for foot formation in hydra. The foot activator can be extracted from hydra tissue with methanol and separated from other known morphogens of hydra by gel filtration and ion-exchange chromatography. A substance with similar biological and physicochemical properties can be isolated from sea anemones.     

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.     

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.     

Action of foot activator on growth and differentiation of cells in hydra

Foot activator is a small peptide found in hydra and specifically activates foot formation. I present a method for the further purification of foot activator by high-pressure liquid chromatography. The morphogenetically active fractions were assayed for their effect at the cellular level. Foot activator acts as a mitogen by pushing epithelial and interstitial cells, which are arrested in G2, into mitosis. In the presence of foot activator, epithelial stem cells are stimulated to differentiate into foot mucus cells, and interstitial nerve precursor cells differentiate into mature nerve cells. The interaction of foot activator with head activator in the development of hydra is discussed.     

Ectopic head and foot formation in Hydra: Diacylglycerol-induced increase in positional value and assistance of the head in foot formation

In wild type Hydra magnipapillata, daily application of the protein kinase C activator diacylglycerol (DAG) evokes sprouting of periodically spaced ectopic heads along the body column and leads to loss of the ability to regenerate proximal structures including the foot. The present transplantation studies show that the appearance of ectopic heads is preceded by an early increase in the 'positional value' (P-value) or 'head activation potential' of the gastric column. Long before ectopic head structures emerge, pieces of DAG-treated tissue transplanted into the corresponding positional level of untreated hosts induce head formation instead of being integrated, whereas pieces implanted from untreated donors into DAG-treated hosts form feet. Foot formation implies a decrease in the P-value. This down-regulation is promoted through long-range assistance by the head. Thus, after termination of the DAG treatment ectopic feet are intercalated midway between the periodically spaced heads; moreover, untreated polyps onto which additional distal heads have been grafted regenerate feet faster than do one-headed polyps and may form supernumerary feet. Multiheaded animals can also be produced using two substances (K-252a and xanthate D609) that interfere with signal transduction, but the mode by which secondary heads arise is different from DAG-induced ectopic head formation. Presumably because the assistance by the parental head is impaired, buds fail to form a foot and detach and instead give rise to stable secondary body axes. It is assumed that the P-value along the body varies according to the number of cellular receptors for factors serving as intercellular signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The head activator is released from regenerating Hydra bound to a carrier molecule

Hydra forced to regenerate a head releases head activator and head inhibitor during the first hours after cutting to induce head-specific growth and differentiation processes. Analysis of the size distribution demonstrated that the head-activator peptide is co-released with (a) large molecular weight carrier molecule(s) to which it is non-covalently bound. The carrier-bound head activator is fully active on Hydra indicating that a carrier does not hinder the interaction with receptors. In contrast to this the head inhibitor is released in its naked, low molecular mass form. The association or non-association with a carrier molecule results in marked differences in biological properties. The head activator has a short range of action, but a long half-life, the head inhibitor has a global range of action, but a short half-life. These results provide a plausible explanation why two antagonistically acting substances, although they are released from the same site and simultaneously nevertheless can give rise to a well-defined temporal and spatial pattern of differentiation as occurs, for example, during head regeneration in Hydra.     

The protein phosphatase inhibitor cantharidin induces head and foot formation in buds of Cassiopea andromeda (Rhizostomae, Scyphozoa)

The polyps of Cassiopea andromeda produce spindle shaped, freely swimming buds which do not develop a head (a mouth opening surrounded by tentacles) and a foot (a sticky plate at the opposite end) until settlement to a suited substrate. The buds, therewith, look very similar to the planula larvae produced in sexual reproduction. With respect to both, buds and planulae, several peptides and the phorbolester TPA have been found to induce the transformation into a polyp. Here it is shown that cantharidin, a serine/threonine protein phosphatase inhibitor, induces head and foot formation in buds very efficiently in a 30 min treatment, the shortest yet known efficient treatment. Some resultant polyps show malformations which indicate that a bud is ordinary polyp tissue in which preparatory steps of head and foot formation mutually block each other from proceeding. Various compounds related to the transfer of methyl groups have been shown to affect head and foot formation in larvae of the hydrozoon Hydractinia echinata. These compounds including methionine, homocysteine, trigonelline, nicotinic acid and cycloleucine are shown to also interfere with the initiation of the processes which finally lead to head and foot formation in buds of Cassiopea andromeda.     

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.     

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.     

Regulation with alpha-2-antiplasmin of Glu-plasminogen activation by tissue activator on fibrin

Interaction of tissue plasminogen activator with alpha-2-antiplasmin and its influence on tissue activator binding to fibrin was studied. Alpha-2-Antiplasmin decreases the binding of tissue activator to fibrin by 20%. The inhibitor formed a complex with tissue plasminogen activator (Kd 78.2 nM) and had no effect on amidolytic activity of the activator. The tissue activator binding to alpha-2-antiplasmin decreases by 20-35% in the presence of 6-aminohexanoic acid. It indicates that not only kringle 2 of the tissue activator molecule takes part in complex formation with alpha-2-antiplasmin, but also other activator domains. Two models were proposed to explain the alpha-2-antiplasmin effect on the Glu-plasminogen activation by tissue activator on fibrin. In the first place, the inhibitor binds to fibrin in the site where the activator complex is localized. It can create steric hindrances for the proenzyme interaction with its activator on fibrin. In the second place, alpha-2-antiplasmin in a complex with tissue plasminogen activator can bring to a change in the activator conformation and a decrease of its functional activity.     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Protein kinase modulators interfere with bud formation in Hydra vulgaris

The fresh water polyp Hydra can reproduce asexually by forming buds. These buds separate from the parent animal due to the development of foot tissue in a belt-like region and the formation of a constriction basal to that region. A single pulse treatment with activators of protein kinase C, including 1,2-dioctanoyl-rac-glycerol and 12-o-tetradecanoylphorbol-13-acetate, and inhibitors of various protein kinases, including staurosporine, H-7 and genistein, interfered with foot and constriction formation. The buds did not separate. Therewith, branched animals were formed, some of which bore a lateral foot patch. Simultaneous treatments with an activator and inhibitor led to a higher amount of branched animals than treatments with one of these agents alone. Based on the different specificities of the activators and inhibitors used we propose that activation of a protein kinase C and/or inhibition of a probably non-C-type protein kinase interfere with the decrease of positional value at the bud's base, a process necessary to initiate the pattern forming system leading to foot formation. Correspondence to: F. Perez