Bud formation inHydra: Inhibition by an endogenous morphogen

Summary From crude extracts ofHydra tissue a substance has been purified which prevents or retards the asexual reproduction by budding. The molecular weight is in the range of 300 to 1000 daltons. Inhibition of bud formation can be observed with concentrations equivalent to the extract from one hydra per 4 ml, that is, to a more than 10,000-fold dilution of the initial crude extract of a hydra. The purified inhibitor is active at a concentration of less than 10^–8 M.Most of the inhibitor present inHydra is bound to cells. Within the cells the substance is mainly bound to particulate structures which sediment at 10,000 g. Its concentration is highest in the hypostomal region and decreases in the direction of the tentacles and peduncle. A second, lower, peak has been found in the basal disc. Treatment of the animals with a toxic agent (nitrogen mustard) which depletes the animal of interstitial cells, nematocytes and nematoblasts excludes the possibility that the inhibitor is present to any great extent in these cells. In conjunction with cell separation experiments by centrifugation of fixed cells in suspension, these results indicate that nerve cells are the most likely sites of storage of the inhibiting substance, although epithelial cells are not excluded as sources for the inhibitor.     

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.     

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.     

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.     

An IQGAP-related gene is activated during tentacle formation in the simple metazoan Hydra

Differentiation of body column epithelial cells into tentacle epithelial cells in Hydra is accompanied by changes in both cell shape and cell-cell contact. The molecular mechanism by which epithelial cells acquire tentacle cell characteristics is unknown. Here we report that expression of a Hydra homologue of the mammalian IQGAP1 protein is strongly upregulated during tentacle formation. Like mammalian IQGAP, Hydra IQGAP1 contains an N-terminal calponin-homology domain, IQ repeats and a conserved C terminus. In adult polyps a high level of Hydra IQGAP1 mRNA is detected at the basis of tentacles. Consistent with a role in tentacle formation, IQGAP1 expression is activated during head regeneration and budding at a time when tentacles are emerging. The observations support the previous hypothesis that IQGAP proteins are involved in cytoskeletal as well as cell-cell contact rearrangements. Received: 25 January 2000 / Accepted: 2 May 2000     

Head regeneration and polarity reversal inHydra attenuata can occur in the absence of DNA synthesis

Summary Regeneration in hydra is considered to be morphallactic because it can occur in the absence of cell division. Whether DNA synthesis is required for regeneration or other repatterning events is not known. The question was investigated by blocking DNA synthesis with hydroxyurea and examining several developmental processes. Head regeneration, reversal of regeneration polarity and battery cell differentiation all took place in the absence of DNA synthesis. Hence, morphallactic regulation in hydra is independent of both DNA synthesis and mitosis.     

Regulation of interstitial cell differentiation inHydra attenuata

Summary The axial position of interstitial-cell (i-cell) differentiation into nematocytes inHydra was studied. Nests of developing nematoblasts of three types of nematocytes were distributed in a non-uniform manner along the body column. Stenotele nematoblasts were distributed in a gradient with a maximum in the peduncle. Desmoneme and atrichous isorhiza nematoblasts were found predominantly in the upper half of the body region. These results suggest that the type of nematocyte differentiation an i-cell undergoes is influenced by the axial position of the i-cell. Because the assayed stage of nematocyte differentiation occurred 6–7 days after beginning of differentiation, the axial position of the anticedent i-cell at the time of commitment was determined by correcting for tissue displacement.     

Why polyps regenerate and we don't: towards a cellular and molecular framework for Hydra regeneration

The basis for Hydra's enormous regeneration capacity is the "stem cellness" of its epithelium which continuously undergoes self-renewing mitotic divisions and also has the option to follow differentiation pathways. Now, emerging molecular tools have shed light on the molecular processes controlling these pathways. In this review I discuss how the modular tissue architecture may allow continuous replacement of cells in Hydra. I also describe the discovery and regulation of factors controlling the transition from self-renewing epithelial stem cells to differentiated cells.     

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.     

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.     

Neural networks as a tool for control and management of a biological reactor for treating hydrogen sulphide

Based on an experimental database consisting of 194 daily cases, artificial neural networks were used to model the removal efficiency of a biofilter for treating hydrogen sulphide (H₂S). In this work, the removal efficiency of the reactor was considered as a function of the changes in the air flow and concentration of H₂S entering the biofilter. In order to obtain true representative values, the removal efficiencies (outputs) were measured 24 h after each input was changed. A MLP (multilayer perceptron 2-2-1) model with two input variables (unit flow and concentration of the contaminant fed into the biofilter) rendered good prediction values with a determination coefficient of 0.92 for the removal efficiency within the range studied. This means that the MLP model can explain 92% of the overall variability detected in the biofilter corresponding to a wide range of operating conditions.     

Improved flax regeneration from hypocotyls using thidiazuron as a cytokinin source

Summary The effects of thidiazuron, benzyladenine and zeatin were tested with respect to bud regeneration of different flax explants from hypocotyls, cotyledons and apices of two fibre varieties (Ariane, Viking) and one linseed variety (Antarès). These three cytokinins were tested either alone or in combination with naphthalene acetic acid, indole acetic acid or 2,4-dichlorophenoxyacetic acid.Hypocotyls were the most responsive explants. Thidiazuron was significantly the most effective followed by benzyladenine, and then zeatin, in inducing organogenesis from hypocotyl segments. The optimal thidiazuron concentration for bud regeneration from hypocotyls was 0.1–0.3 M in combination with 0.01 M of naphthalene acetic acid. Six days after plating, shoot initials began to appear on hypocotyl sections compared with ten to fifteen days when using benzyladenine or zeatin.     

Macrotermes mounds as sites for tree regeneration in a Sudanian woodland (Burkina Faso)

The importance of mounds created by Macrotermes subhyalinus as safe site for tree regeneration was analysed in a savannah woodland of Burkina Faso. Plantlets (height <1.5 m) were sampled and followed over an year in 72 × 4 m² quadrats located on M. subhyalinus mounds and adjacent areas. The mechanisms of regeneration and plantlet mortality were also determined. We identified three regeneration mechanisms: seedlings regenerated by seed (abundant on mounds), sprouts (abundant on adjacent areas) and root suckers (a rare case on both sites). A total of 37 species representing 17 families and 30 genera were found on all quadrats, of which 29 species were found on termite mounds and 22 species on adjacent areas. Species richness and density of plantlets at the 4 m² scale were higher on mounds than in the adjacent area (P < 0.05). Among plantlet categories, seedling density was significantly different among microhabitats (P < 0.001) and across sampling periods (P < 0.01) and, the majority of plantlet individuals appeared within the 0–25 cm height class. The mortality of plantlets and particularly seedling mortality differed significantly between microhabitats (P < 0.01) and between periods (P < 0.01), whereas more than half the variation in the death of Acacia erythrocalyx seedlings (the most abundant species) were related to the density of the live seedlings of the same species (P < 0.001). The observed mortality rate was way below 50%; plantlet density remained higher on mound during sampling periods as compared to the adjacent area. It can thus be concluded that Macrotermes termite mounds are favourable sites for the recruitments of woody plants in savannah woodlands.     

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.     

应用水螅再生试验初探杜仲等中药的致畸作用

目的：探索三种安胎中药-杜仲、黄芩、苎麻根的发育毒性。方法：利用成体水螅与胃区体水螅形态学变化、中效中毒浓度与中效再生抑制浓度的比值(T50／I50)及Wilby评价标准,初步研究杜仲、黄芩、苎麻根的致畸作用。结果：致畸效用评价实验中,杜仲、黄苓、苎麻根的T50／I50比值分别为0．9、3．2、1．9,并呈剂量-反应关系;抗秋水仙碱抑制细胞分化实验中,杜仲还真有拮抗秋水仙碱对胃区体水螅分化再生的抑制作用,并呈较好的剂量-反应关系。结论：杜仲显示致畸作用阴性,有促进水螅分化及拮抗秋水仙碱抑制分化的作用;黄芩为致畸作用明显物质;苎麻根则显示致畸作用阳性。     

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 level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

 Several studies have provided strong, but indirect evidence that signalling through pathways involving protein kinase C (PKC) plays an important role in morphogenesis and patterning in Hydra. We have cloned a gene (HvPKC2) from Hydra vulgaris which encodes a member of the nPKC subfamily. In adult polyps, HvPKC2 is expressed at high levels in two locations, the endoderm of the foot and the endoderm of the hypostomal tip. Increased expression of HvPKC2 is an early event during head and foot regeneration, with the rise in expression being restricted to the endodermal cells underlying the regenerating ends. No upregulation is observed if regenerates are cut too close to the head to form a foot. Elevated expression of HvPKC2 is also observed in the endoderm underlying lithium-induced ectopic feet. A dynamic and complex pattern of expression is seen in developing buds. Regeneration of either head or foot is accompanied by an increase in the amount of PKC in both soluble and particulate fractions. An increase in the fraction of PKC activity which is membrane-bound is specifically associated with head regeneration. Taken together these data suggest that patterning of the head and foot in Hydra is controlled in part by the level of HvPKC2 expression, whilst head formation is accompanied by an in vivo activation of both calcium-dependent and independent PKC isoforms. Received: 10 July 1997 / Accepted: 8 November 1997     

N-arachidonoyl dopamine is a possible factor of the rate of tentacle formation in freshwater hydra regeneration

The effect of N-arachidonoyl dopamine, haloperidol, and their mixture on the rate of tentacle formation was studied during regeneration of the gastral and basal fragments of freshwater hydra. Some concentrations of haloperidol inhibited the tentacle formation, which was more pronounced in the basal fragment. N-arachidonoyl dopamine accelerated the tentacle formation in both fragments, particularly, in the basal one (an inversion of the natural difference in the rate of tentacle formation between the gastral and basal fragments). After the exposure to the mixture of these drugs, the effects of each of them were observed. Mass spectrometry assay has demonstrated endogenous N-arachidonoyl dopamine in the intact hydra homogenate. The possible involvement of this acyl-neurotransmitter in the regulation of the rate of tentacle formation in regenerating hydra is discussed.     

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.