Immunocytochemical evidence for an NMDA1 receptor subunit in dissociated cells of<Emphasis Type="Italic"> Hydra vulgaris</Emphasis>

We have previously reported immunocytochemical, biochemical, behavioral, and electrophysiological evidence for glutamatergic transmission through (±)--amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)/kainate receptors in hydra. We now report specific localization of the N-Methyl-D-aspartic acid receptor subunit 1 (NMDAR1) in epithelial, nerve, nematocytes, and interstitial cells of hydra. Macerates of tentacle/hypostome pieces of Hydra vulgaris were prepared on agar-coated slides, fixed with buffered formaldehyde/glutaraldehyde, and fluorescently labeled with monoclonal antibodies against mammalian NMDAR1. Negative controls omitted primary antibody. Digital images were recorded and analyzed. Specific localized and intense labeling was found in ectodermal battery cells, other epithelial cells, nematocytes, interstitial cells, and sensory and ganglionic nerve cells, and in battery cells was associated with enclosed nematocytes and neurons. The labeling of myonemes was more diffuse and less intense. In nerve and sensory cells, punctate labeling was prominent on cell bodies. These results are consistent with our earlier evidence for glutamatergic neurotransmission and kainate/NMDA regulation of stenotele discharge. They support other behavioral and biochemical evidence for a D-serine-sensitive, strychnine-insensitive, glycine receptor in hydra and suggest that the glutamatergic AMPA/kainate-NMDA system is an early evolved, phylogenetically old, behavioral control mechanism.     

Role of the cellular environment in interstitial stem cell proliferation in Hydra

Summary The role of the cellular environment on hydra stem cell proliferation and differentiation was investigated by introduction of interstitial cells into host tissue of defined cellular composition. In epithelial tissue lacking all non-epithelial cells the interstitial cell population did not grow but differentiated into nerve cells and nematocytes. In host tissue with progressively increased numbers of nerve cells growth of the interstitial cell population was positively correlated to the nerve cell density. In agreement with previous observations (Bode et al. 1976), growth of the interstitial cell population was also found to be negatively correlated to the level of interstitial cells present. The strong correlation between the growth of the interstitial cell population and the presence of interstitial cells and nerve cells implies that interstitial cell proliferation is controlled by a feedback signal from interstitial cells and their derivatives. Our results suggest that the cellular environment of interstitial cells provides cues which are instrumental in stem cell decision making. Offprint requests to: T.C.G. Bosch     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Nematocyte development in Hydra attenuata is dependent on both the interstitial cells and the epithelial cells

The aberrant, a morphological mutant of Hydra attenuata, has altered patterns of the development and distribution of nematocytes. The number of nematoblasts and nematocytes is higher in the aberrant than in the normal. Stenotele differentiation is incomplete and the numbers of desmonemes and holotrichous isohrizas mounted on the body column are much higher than normal. Because nematocytes arise by differentiation from the interstitial cells, epithelial cell/interstitial cell chimeras between the aberrant and normal strains were made to determine whether the lesion giving rise to the alterations in the mutant was due to the epithelial cells or a cell type in the nematocyte lineage. Only the chimera in which both cell types were derived from the aberrant exhibited the altered nematocyte development. If the chimera contained a normal cell type, either epithelial cell or interstitial cell, nematocyte development was normal. Thus, both epithelial cells and cells of the nematocyte lineage are involved in the control of nematocyte development. A defect in one of the lineages can be compensated for by the other cell type.     

Mechanoreception and synaptic transmission of hydrozoan nematocytes

Mechanoelectric transduction and its ultrastuctural basis were studied in the cnidocil apparatus of stenotele nematocytes of marine and freshwater Hydrozoa (Capitata and Hydra) as a paradigm for invertebrate hair cells with concentric hair bundles. The nematocytes respond to selective deflection of their cnidocil with phasic-tonic receptor currents and potentials, similar to vertebrate hair cells but without directional dependence of sensitivity. Ultrastructural studies and the use of monoclonal antibodies allowed correlating the mechanoelectric transduction with structural components of the hair bundle. Two other types of depolarising current and voltage changes in nematocytes are postsynaptic, as concluded from their ionic and pharmacological characteristics. One of these types is induced by mechanical stimulation of distant nematocytes and sensory hair cells. It is graded in amplitude and duration, but different from the presynaptic receptor potential. Adequate chemical stimulation of the stenoteles strongly increases the probability of discharge of their cnidocyst, if the chemical stimulus precedes the mechanical one. Simultaneously, the probability of synaptic signalling induced by mechanical stimulation is increased, reaching nearly 100%. The chemoreception of the phospholipids used could be localized in the shaft of the cnidocil, because of the water-insolubility of the stimulant. This chemical stimulation itself does not cause a receptor potential; its action is classified as a modulatory process. Electron microscopy of serial sections of the tentacular spheres of Coryne revealed synapses that are efferent to nematocytes and hair cells besides neurite–neurite synapses, each containing 3–10 clear and/or dense-core vesicles of 70–150 nm diameter. The only candidates to explain the graded afferent signal transmission of nematocytes and hair cells are regularly occurring cell contacts associated with 1(–4) clear vesicles of 160–1100 nm diameter. Transient fusion and partial depletion of stationary vesicles are discussed as mechanisms to reconcile functional and structural data of many cnidarian synapses. Review contributed to the Symposium on Neuro-Anatomy and -Physiology of Coelenterates; 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA; July 6–11, 2003.     

Characterization of interstitial stem cells in hydra by cloning.

A procedure has been developed for cloning interstitial stem cells from hydra. Clones are prepared by introducing small numbers of viable cells into aggregates of nitrogen mustard-inactivated host tissue. Clones derived from added stem cells are identified after 1–2 weeks of growth by staining with toluidine blue. The incidence of clones increases with increasing input of viable cells according to one-hit Poisson statistics, indicating that clones arise from single cells. After correction for cell losses in the procedure, about 1.2% of the input cells are found to form clones. This compares with estimates from in vivo experiments of about 4% stem cells in whole hydra [David, C. N., and Gierer, A. (1974). Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci.16, 359–375.]Differentiation of nematocytes and nerve cells in clones was analyzed by labeling precursors with [³H]thymidine and scoring labeled nerves and nematocytes 2 days later. Nine clones examined in this way contained both differentiated nerve cells and nematocytes, demonstrating that the interstitial stem cell is multipotent. This result suggests that the observed localization of nerve and nematocyte differentiation in whole hydra probably occurs at the level of stemcell determination. The observation that differentiated cells occur very early in clone development suggests that a stem cell's decision to proliferate or differentiate is regulated by shortrange feedback signals which are already saturated in young clones.     

Cell lineages in Hydra: isolation and characterization of an interstitial stem cell restricted to egg production in Hydra oligactis

In an attempt to isolate unipotent stem cells (progenitors to the nerve cells, nematocytes, gland cells, and gametes) from Hydra oligactis females, animals were treated with a drug (hydroxyurea, HU) that preferentially lowers or eliminates the interstitial stem cells, leaving the epithelial tissue intact. In this epithelial environment, interstitial cells remaining after treatment will proliferate and differentiate, permitting a long-term analysis of their developmental capabilities. Following treatment of females with HU, animals were isolated that contained interstitial cells that gave rise to eggs only. Two clones of animals containing these cells were propagated for several years and the growth and differentiation behavior of the interstitial cells examined in their asexually produced offspring. During this time, the cells displayed an extensive proliferative capacity (classifying them as stem cells) and remained restricted to egg differentiation. It is proposed that both the sperm- and the egg-restricted stem cells arise from a multipotent stem cell, which also gives rise to the somatic cells (see above), and that, in hydra, sex is ultimately determined by interactions between cells of the two germ cell lineages.     

Putative intermediates in the nerve cell differentiation pathway in hydra have properties of multipotent stem cells

We have investigated the properties of nerve cell precursors in hydra by analyzing the differentiation and proliferation capacity of interstitial cells in the peduncle of Hydra oligactis, which is a region of active nerve cell differentiation. Our results indicate that about 50% of the interstitial cells in the peduncle can grow rapidly and also give rise to nematocyte precursors when transplanted into a gastric environment. If these cells were committed nerve cell precursors, one would not expect them to differentiate into nematocytes nor to proliferate apparently without limit. Therefore we conclude that cycling interstitial cells in peduncles are not intermediates in the nerve cell differentiation pathway but are stem cells. The remaining interstitial cells in the peduncle are in G1 and have the properties of committed nerve cell precursors (Holstein and David, 1986). Thus, the interstitial cell population in the peduncle contains both stem cells and noncycling nerve precursors. The presence of stem cells in this region makes it likely that these cells are the immediate targets of signals which give rise to nerve cells.     

Localization of dopamine in the freshwater hydrozoanHydra attenuata

Summary High performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.