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Preferential expression of a pp60c-src related protein tyrosine kinase activity in nerve cells of the early metazoan Hydra (Coelenterates).

It has been suggested that the proto-oncogene c-src plays a functional role in developing neurons, and in the mature nerve cells of higher vertebrates. The coelenterate Hydra represents the most primitive known organism possessing nerve cells. With Southern blot hybridizations we have demonstrated src-related sequences in Hydra. Antisera specific for the c-src gene product (pp60c-src) of birds and mammals precipitate a protein from Hydra cell extracts with a tyrosine-specific protein kinase activity. Studies of tissues and cells fractionated from a temperature sensitive mutant of Hydra which is depleted of interstitial (including nerve) cells at the non-permissive temperature, have indicated the src-like kinase of Hydra to be preferentially expressed in nerve cells. The high conservation of structural features and of the expression pattern indicates a basic function for pp60c-src in neurons.

Animals↗

Effects of ultraviolet light (2535 A) and dactinomycin upon hydra (Pelmatohydra oligactis).

Hydras exposed to ultraviolet light (2535 A, 12 erg/mm-2/s-2) for 15 minutes suffer a depression immediately, individuals that survived recover quickly and lastingly. Hydras treated with dactinomycin of specific concentrations do not suffer a depression until several days later, do not recover and die eventually. The results are also comparable when hydras are exposed to light and treated with cytostatic of such concentration. In even milder solution of dactinomycin all unexpected hydras recover and survive. But, almost all of the hydras exposed to ultraviolet light and treated with this solution die eventually. The effect of ultraviolet light upon them is then distinctly adverse. Buds are much less damaged and they do not die in this concentration. They grow and reproduce normally. This paper deals with the above mentioned phenomena.

Animals↗

Viruses of symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis.

We previously reported that isolation of symbiotic Chlorella-like algae from the Florida strain of Hydra viridis induced replication of a virus (designated HVCV-1) in the algae. We now report that isolation of symbiotic Chlorella-like algae from four other sources of green hydra and one source of the protozoan Paramecium bursaria also induced virus synthesis. Algae from one of these hydra contained a virus identical to HVCV-1 (based on its rate of sedimentation, buoyant density, reaction to HVCV-1 antiserum, and DNA restriction fragments) whereas algae from the other three hydra contained another similar, but distinct, virus (designated HVCV-2). The virus from the paramecium algae (designated PBCV-1) was distinct from both HVCV-1 and HVCV-2. The symbiotic algae in the hydra could also be distinguished ultrastructurally. Chloroplasts of both algae that produced HVCV-1 lacked a pyrenoid whereas chloroplasts of the other three symbiotic algae contained pyrenoids. Since all symbiotic eukaryotic algae we have examined have had virus, a potential viral role in symbiosis is suggested.

Journal Article↗

Role of epithelial cells and programmed cell death in Hydra spermatogenesis.

Spermatogenesis in higher animals is a tightly regulated process, in which survival and death of sperm precursor cells depends on the presence of somatic cells in gonads. In the basal metazoan Hydra spermatogenesis takes place in anatomically simple testes and in the absence of accessory structures. Hydra sperm precursors are derived from interstitial stem cells. Here we show that large numbers of sperm precursors in testes of Hydra vulgaris undergo programmed cell death (apoptosis) and that ectodermal epithelial cells phagocytose the apoptotic sperm precursors. This is surprising since so far no evidence has been reported that epithelial cells are directly involved in germ cell differentiation in Hydra. We propose that, similar to Sertoli cells in mammals, in Hydra epithelial cells support and perhaps even control spermatogenesis.

Journal Article↗

Self/nonself recognition in Cnidaria: contact to allogeneic tissue does not result in elimination of nonself cells in Hydra vulgaris.

Although Cnidaria have no specialised immune cells, some colonial forms possess a genetic system to discriminate between self and nonself. Allorecognition is thought to protect them from fusion with genetically different individuals and to prevent germ line parasitism. Surprisingly, when grafting tissue of two species of the solitary freshwater polyp Hydra, we found within the contact zone phagocytozing epithelial cells which selectively eliminated cells from the other species (Bosch and David, 1986). This led us to speculate that Hydra, which never undergoes "natural transplantation", can differentiate between self and nonself (Bosch and David, 1986). In a previous paper (Kuznetsov et al., 2002) we described that cells which accumulate in the contact region of these interspecies grafts are apoptotic and that apoptosis is induced by impaired cell matrix contact. Thus, observations in such interspecies grafts did not give hints concerning the presence of a discriminative allorecognition system. To clarify whether this fundamental aspect of immunity is present in these phylogenetically old animals, we examined epithelial interactions between different strains of Hydra vulgaris. Here, we show that contact to allogeneic tissue does not evoke any response in terms of phagocytosis and elimination of allogeneic cells. We, therefore, question Hydra's ability to discriminate between self and nonself and propose that, in contrast to colonial cnidarians, the solitary polyp Hydra has either lost or substantially reduced this ability.

Journal Article↗

Nerve ring of the hypostome in hydra. I. Its structure, development, and maintenance.

The anatomy and developmental dynamics of the nerve ring in the hypostome of Hydra oligactis were examined immunocytochemically with an antiserum against a neuropeptide and with neuron-specific monoclonal antibodies. The nerve ring is unique in the mesh-like nerve net of hydra. It is a distinct neuronal complex consisting of a thick nerve bundle running circumferentially at the border between the hypostome and tentacle zone. Immunostaining showed that the nerve ring was heterogeneous and contained at least four different subsets of neurons. During head regeneration and budding, the nerve ring appeared only after the nerve net of ganglion and sensory cells had formed. Every epithelial cell is continuously displaced with neurons toward either head or foot in an adult hydra. However, the ectoderm in the immediate vicinity of, and including, the nerve ring constitutes a stationary zone that is not displaced. Tissue immediately above this zone is displaced toward the tip of the hypostome, while tissue below is displaced along the tentacles. Correspondingly, the production of new neurons in the ring as measured by their differentiation kinetics is much slower than in surrounding areas. Thus, the nerve ring is static and stable in contrast to the dynamic features of the nerve net of hydra.

Animals↗

Migrating interstitial cells differentiate into neurons in hydra.

Interstitial cell migration has been observed numerous times in grafted hydra, but the extent to which graft injuries might stimulate the migration of otherwise nonmotile cells was unknown. The present study describes the migration and differentiation of vital dye-labeled interstitial cells in intact, normal hydra. Interstitial cells, stained with a fluorescent vital dye, migrated away from a labeled patch of ectodermal cells and subsequently were found throughout the body column. Shortly thereafter, labeled neurons began to appear among the migrating cells. The number of migrating interstitial cells remained constant over 5 days, whereas the number of labeled neurons increased. Labeled interstitial cells and neurons accumulated primarily in the head and peduncle, as observed in previous studies of short-term migration patterns in grafted hydra. The present study shows that migration of interstitial cells occurs in normal nongrafted hydra and that the accumulation patterns parallel results from graft experiments. The population of migrating cells appears to be limited to neuron precursors.

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Budhead, a fork head/HNF-3 homologue, is expressed during axis formation and head specification in hydra.

Accumulating evidence indicates that a common set of genes and mechanisms regulates the developmental processes of a variety of triploblastic organisms despite large variation in their body plans. To what extent these same genes and mechanisms are also conserved among diploblasts, which arose earlier in metazoan evolution, is unclear. We have characterized a hydra homologue of the fork head/HNF-3 class of winged-helix proteins, termed budhead, whose expression patterns suggest a role(s) similar to that found in vertebrates. The vertebrate HNF-3 beta homologues are expressed early in embryogenesis in regions that have organizer properties, and later they have several roles, among them an important role in rostral head formation. In the adult hydra, where axial patterning processes are continuously active, budhead is expressed in the upper part of the head, which has organizer properties. It is also expressed during the formation of a new axis as part of the development of a bud, hydra's asexual form of reproduction. Expression during later stages of budding, during head regeneration and the formation of ectopic heads, indicates a role in head formation. It is likely that budhead plays a critical role in head as well as axis formation in hydra.

Amino Acid Sequence↗

Silencing of developmental genes in Hydra.

Numerous developmental control genes have been isolated in a variety of organisms by either homology cloning or system-specific strategies. Functional genetic tests, however, are available for only a few model organisms and particularly are missing in a number of animals that occupy key positions for understanding the evolution of development and gene function. Double-stranded RNA-mediated interference (RNAi) opens a way to perform functional studies in such "nongenetic" organisms. Here we show that RNAi can be used to test the function of developmental genes in the cnidarian Hydra, a classical model for developmental studies. Introduction of double-stranded RNA corresponding to the head-specific gene ks1 caused strong depletion of ks1 transcripts. ks1 loss-of-function polyps exhibited severe defects in head formation, indicating an important role of ks1 in Hydra head development. Our results demonstrate for the first time efficient gene silencing in Hydra. RNAi provides an entry point for a variety of functional studies and a direct approach for analyzing the hierarchy of regulatory genes in Hydra, which until now has not been amenable to loss-of-function genetics.

Animals↗

Developmental signaling in Hydra: what does it take to build a "simple" animal?

Developmental processes in multicellular animals depend on an array of signal transduction pathways. Studies of model organisms have identified a number of such pathways and dissected them in detail. However, these model organisms are all bilaterians. Investigations of the roles of signal transduction pathways in the early-diverging metazoan Hydra have revealed that a number of the well-known developmental signaling pathways were already in place in the last common ancestor of Hydra and bilaterians. In addition to these shared pathways, it appears that developmental processes in Hydra make use of pathways involving a variety of peptides. Such pathways have not yet been identified as developmental regulators in more recently diverged animals. In this review I will summarize work to date on developmental signaling pathways in Hydra and discuss the future directions in which such work will need to proceed to realize the potential that lies in this simple animal.

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Neuro-epitheliomuscular cell and neuro-neuronal gap junctions in Hydra.

Gap junctions have been described ultrastructurally between neurons and epitheliomuscular cells and between neurons and their processes in the hypostome, peduncle and basal disc of Hydra. All gap junctions examined in Hydra exhibit two apposed plasma membranes having a 2-4 nm gap continuous with the extracellular space. The gap junctions are variable in length from 0.1-1.6 micrometers and appear linear or V-shaped in section. Neuronal gap junctions in Hydra occur infrequently as compared to chemical synapses. Electron microscopy of serial sections has demonstrated the presence of adjacent electrical and chemical synapses (neuromuscular junctions) formed by the same neuron. In addition, multiple gap junctions were present between two neurons. This is the first ultrastructural demonstration of electrical synapses in the nervous system of Hydra. Such synapses occur in neurons previously characterized as sensory-motor-interneurons on the basis of their chemical synapses; these neurons appear to represent a type of stem cell characterized by having both electrical and chemical synapses.

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Hydra constitutively expresses transcripts involved in vertebrate neural differentiation.

The diploblastic Hydra is among the most primitive multicellular organisms. Using cross-hybridization with Xenopus probes, noggin-like transcripts were detected in the hypostome and basal disc of adult Hydra (Pelmatohydra oligactis), regions with properties similar to that of the amphibian organizer. This points to the possibility of a close molecular similarity between the Xenopus and Hydra organizers. The constitutive expression of a noggin-like gene in Hydra may be responsible for its regenerative capacity.

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Three digestive movements in Hydra regulated by the diffuse nerve net in the body column.

The mammalian digestive tract undergoes various digestive movements such as peristalsis and segmentation movement. How those digestive movements and the underlying mechanisms appeared in evolution remains unraveled. A widely accepted view has been that, early in evolution, the digestive process was static based upon diffusion, and later it became dynamic involving digestive movements. Here, we report digestive movements which occur in Hydra, a member of the phylum Cnidaria. We find that the body column of Hydra undergoes a series of movements when fed with Artemia. Comparison of the movements to those in mammals showed similarities in appearance to esophageal reflex, segmentation movement, and defecation reflex. When nerve cells were eliminated, polyps showed only a weak segmentation movement, demonstrating that the diffuse nerve net in the body column of Hydra primarily regulates the movements just as the netlike enteric nervous system does in mammals. Elimination of both secretory gland cells and nerve cells resulted in the complete loss of movement, suggesting that the gland cells are involved in the weak movement. Overall, these observations suggest that the digestive process in Hydra is dynamic and that the diffuse nerve net regulates the digestive movements as a primitive form of enteric nervous system.

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First identification and localization of a visual pigment in Hydra (Cnidaria, Hydrozoa).

The cnidarian Hydra does not possess identified photoreceptive structures or specialized cells for light detection; nevertheless, it shows a marked photosensitivity. So far no evidence has been previously reported about the localization of the proteins involved in the photoresponse. We used polyclonal antibodies and immunofluorescence microscopy on whole-mount Hydra to identify a putative rhodopsin-like protein. Our results show an immunoreactivity in the ectodermal layer of Hydra, which corresponds in position to the nervous epidermal sensory cells. These data provide the first identification of a rhodopsin-like protein in a phylogenetically old invertebrate and give a new insight into the Hydra photoreceptive response.

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.

Amino Acid Sequence↗

Immunocytochemical evidence for an NMDA1 receptor subunit in dissociated cells of Hydra vulgaris.

We have previously reported immunocytochemical, biochemical, behavioral, and electrophysiological evidence for glutamatergic transmission through (+/-)-alpha-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.

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Quantitative analysis of epithelial cell aggregation in the simple metazoan Hydra reveals a switch from homotypic to heterotypic cell interactions.

Hydra, a member of the diploblastic phylum Cnidaria, exhibits the most basic type of organized metazoan tissues. Two unicellular sheets of polarized epithelial cells - ectoderm and endoderm - form a double layer throughout the body column. The double layer can be reestablished from single-cell suspensions by tissue-specific cell-sorting processes. However, the underlying pattern of interactions between ectodermal and endodermal epithelial cells responsible for double-layer formation is unclear. By analyzing cell interactions in a quantitative adhesion assay using mechanically dissociated Hydra epithelial cells, we show that aggregation proceeds in two steps. First, homotypic interactions within ectodermal epithelial cells (ecto-ecto) and within endodermal epithelial cells (endo-endo) form homotypic cell clusters. Second, at an aggregate size of about ten epithelial cells/cluster, ectodermal and endodermal clusters start to form heterotypic aggregates. Homotypic ecto-ecto interactions are inhibited by a polyclonal anti-Hydra membrane antiserum, and under these conditions homotypic endo-endo interactions do not proceed beyond a size of about ten epithelial cells/cluster. These data suggest that homotypic cell clusters reduce their initial homotypic affinity and acquire a new heterotypic affinity. A link between cell adhesion and cell signaling in early Hydra aggregates is discussed.

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Extracellular matrix (mesoglea) of Hydra vulgaris. II. Influence of collagen and proteoglycan components on head regeneration.

Hydra are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. As an extension of the previous study which indicated that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial extracellular matrix, the present study was undertaken to analyze the role of mesoglea components during head regeneration in Hydra vulgaris. Studies were conducted that utilized drugs that affect collagen processing or secondary collagen structure (beta-aminoproprionitrile; 2,2'-dipydridyl; and cis-4-hydroxy-L-proline) and a drug that inhibits addition of glycosaminoglycan chains to proteoglycan core proteins (p-nitrophenyl-beta-D-xylopyranoside). These studies indicated that alterations in the structure of collagens or proteoglycans caused blockage of head regeneration in Hydra as monitored over a 48-hr period. Blockage of head regeneration was reversible once the drugs were removed, indicating that the drugs were not having a general toxic effect on the organism. Radiotracer studies also indicated that blockage of head regeneration was not simply due to a general depression of protein synthesis by the drugs. Various controls indicated that each drug was affecting mesoglea components under the conditions utilized in these studies. These observations indicate that preservation of normal mesoglea structure is required for Hydra head regeneration to proceed.

2,2'-Dipyridyl↗