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Genetic analysis of developmental mechanisms in hydra. VI. Cellular composition of chimera hydra.

The homeostatic mechanisms that maintain constant cellular ratios in hydra tissue were studied using mutant and chimeric hydra strains. Mutants having abnormal cellular compositions are isolated through sexual inbreeding of wild hydra, as described in previous papers of this series. Chimeric hydra are produced by making use of a strain (nf-I) which lacks interstitial cells, nerve cells and nematocytes in its tissue. Reintroduction of interstitial cells from other strains (both normal and mutant) into nf-I leads to creation of chimeric strains having epithelial cell lineages from one strain (nf-I) and interstitial cell lineages from others. Analyses and comparisons of the cellular compositions of all these strains revealed that the numbers of nerve or interstitial cells in the chimeras were very similar to (statistically significantly correlated with) those in their interstitial cell donors. Since chimeras and their interstitial cell donors share the same interstitial cell lineages, this suggests that interstitial cells or their derivatives (nerves and nematocytes) play major roles in determining the nerve and interstitial cell levels in the hydra tissue. It is suggested that some form of homeostatic feedback mechanisms are probably involved in regulating the levels of these cell types.

Animals

Genetic analysis of developmental mechanisms in hydra. V. Cell lineage and development of chimera hydra.

Chimeric hydra were produced by making use of a strain (nf-1) which lacks interstitial cells, nerve cells and nematocytes. This strain arises by spontaneous loss of interstitial cells from its parental strain (sf-1) (Sugiyama & Fujisawa, 1978). Reintroduction of interstitial cells from other strains into nf-1 leads to the creation of chimeric strains that consisted of epithelial cells derived from strain sf-1 and interstitial cells and their derivatives (nerves and nematocytes) from other strains. In chimeras, interstitial or epithelial cells apparently maintain very stable cell lineages; no indication was obtained that suggested interstitial cell differentiation into epithelial cells or dedifferentiation in the opposite direction during the long courses of chimera cultures (up to one year). Developmental characters of chimeras were examined and compared to those of the epithelial cell (sf-1) and the interstitial cell donors. Almost all of the chimera's characters examined (growth rate, budding rate, tentacle numbers, polyp size, regenerative capacity, etc.) closely resembled those of the epithelial cell donor, but not of the interstitial cell donors. This suggests that epithelial cells, rather than interstitial or nerve cells, are the primary determinant of most, if not all, of hydra developmental characters.

Animals

Effect of insecticides (Dimiline WP 25, Torak EC 24 and Gamacide 20) on hydra (Hydra vulgaris Pallas).

Investigations showed that the three insecticides used had the most damaging effect upon hydra immediately after treatment. The tentacles and the hypostome are the parts most often damaged. Inse the affected cells, lesions appear in the intracellular membranes, the nucleus shell and the membranes of the mitochondria, Golgi complex and the endoplasmic reticulum, while the cell membrane is preserved. The damaged parts of the body regenerate within three days. Zymogen cells play a significant role in the course of regeneration. They dedifferentiate into gastrodermal interstitial cells and later into other types of cells of the ectoderm and the gastroderm. Apart from their intense participation in regeneration, these totipotent cells also invariably participate in the formation of new hydra buds. It was observed that Dimiline WP 25 and Torak EC 24 in the concentrations used stimulate asexual reproduction of this animal.

Animals

Development of Hydra lacking nerve and interstitial cells.

Hydra attenuata were rendered free of interstitial cells (I cells) and interstitial cell derivatives by colchicine treatment. These hydra were then cloned and cultivated for 18 months and their developmental capacities were studied. Some experimental hydra possessed a few (about 1% of the normal numbers) interstitial cells and retained this low level during prolonged culture and active growth without the differentiation of I-cells into specialized cells. Other hydra were completely freed of interstitial cells by the colchicine treatment. Maceration and histological analyses showed that once a hydra is freed of all interstitial cells it does not recover them, nor do its buds contain interstitial cells. I cell-free hydra also lack nerve cells, nematocytes, gametes and endodermal gland cells, and the tissue consists only of ectodermal and endodermal epithelial cells. Hydra completely lacking interstitial cells grow, bud, exhibit tissue renewal patterns, regenerate and preserve polarity generally typical of normal hydra. I cell-free hypostomal tissue has inductive capacity, as does normal hypostomal tissue, when implanted in I cell-free or normal gastric tissue. Regenerating I cell-free tissue undergoes precocious determination as does normal tissue. Only in some quantitative aspects do I cell-free hydra develop abnormally. We conclude that hydra consisting only of epithelial cells are capable of essentially normal development.

Animals

Distinct cellular expression pattern of annexins in Hydra vulgaris.

The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.

Amino Acid Sequence

Metabolic relationships between green hydra and its symbiotic algae.

1. Hydra that maintain algal symbionts evidenced three modes of metabolism depending upon the nutritional state of the host and the photoperiodic conditions under which the animals were maintained. Animals either fed or fasted but maintained in the dark exhibited an RQ of 0.725 indicating fat metabolism. When they were fed Artemia nauplii and maintained in the light green hydra exhibited a mixed metabolism of carbohydrate and fat which gave an RQ of 0.862. Fasting green hydra, when maintained in the light, showed a pronounced carbohydrate metabolism typified by an RQ of 0.970. 2. Aposymbiotic hydra, whether fed or fasted, exhibited an RQ of 0.725 indicating a high degree of fat metabolism. 3. Symbiotic hydra which were fed and maintained in the light demonstrated a respiration rate of 14.4 microliter/day per hydra, which was 2.5 times greater than animals (symbiotic or aposymbiotic) maintained under any other condition of fasting or photoperiod. It is hypothesized that the enhanced respiration observed in these animals is due to the metabolic activities of the symbiotic algae associated with reproduction and development. 4. Calculations based on respiratory measurements and indirect calorimetry suggest that hydra consume approximately 0.0320 calories per day and that symbiotic algae may supply up to 69% of the host caloric requirements via the translocation of approximately 5 microgram of carbohydrate when the host is fasting and light is provided.

Animals

Hydra head activator peptide has trophic activity for eukaryotic neurons.

The synthetic undecameric peptide, pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe, known as the hydra head activator peptide, present in high concentrations in mammalian hypothalamus and intestine, was tested for neurotrophic activity in a survival assay using cultured chick embryonic sympathetic and dorsal root ganglion cells, and for morphological differentiation activity on neuroblastoma cells. Hydra head activator peptide supported neuron survival. The optimal active concentration, 1 pM, was very similar to the concentration that causes bud and head formation in hydra. Maximal neuron survival obtained with hydra head activator peptide was close to that obtained with nerve growth factor: both substances enhanced survival up to 3 times that of control cultures. Bradykinin, which has some amino acid sequence homology with hydra head activator, was inactive as a neurotrophic factor. Hydra head activator induced rapid morphological differentiation of the mouse neuroblastoma cell line Neuro-2A. Neuro-2A responded to the peptide by process extension, 4 h after its addition to the culture medium. Neurotrophic factors isolated to date have been characterized by their ability to maintain cell viability and enhance neurite outgrowth. Hydra head activator peptide met these two criteria when tested in 3 different neuron culture systems. Our results suggest that the head activator peptide may act as a neurotrophic factor for neurons in other species, including mammals.

Amino Acid Sequence

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.

Amino Acids

Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.

A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.

Amino Acid Sequence

Genetic analysis of developmental mechanisms in Hydra. IV. Characterization of a nematocyst-deficient strain.

The authors have previously found that mutant hydra strains showing various types of developmental defects can be isolated through sexual inbreeding of wild hydra. One such defective strain, called nem-4, contains virtually no stenoteles, one of the four types of nematocysts present in hydra, in its tentacles. However, stenoteles are present at a normal level in the body column of this strain, and they are turned over also at a normal rate. Grafting experiments between the head region of nem-4 hydra and the body column of wild type hydra (and vice versa) showed that wild type stenotele nematocytes can move into nem-4 tentacles but that nem-4 stenotele nematocytes can not move into the wild type tentacles. These observations suggest that the stenotele nematocytes are produced normally by differentiation from the interstitial cells in the body column of nem-4 hydra, but that they are somehow prevented from migrating into the tentacles in this strain.

Animals

Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity.

We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.

Amino Acid Sequence

Positional information and pattern regulation in hydra: enzyme profiles.

Certain key enzymes of alternative pathways of glucose metabolism, of amino acid metabolism and of redox systems have been measured in hydra and this profile compared with mammalian differentiated tissues with a view to locating pathways of specific importance in hydra. There was a marked constant proportionality in the major part of the enzymes investigated, the profile suggested a metabolic pattern geared to utilization of amino acids as a carbon source for biosynthesis and energy production and to the production and conservation of pyruvate. The importance of conversion to ionized forms was noted. The most notable specific proportion changes were the exceptionally low lactate dehydrogenase, malic enzyme and the relatively high citrate synthase. The proximal-distal gradients in hydra were examined and these gradients suggested a switch to a more anaerobic type of metabolism and an elevation of the pentose phosphate pathway as the basal region was approached. Measurements of the formation of 14CO2 from specifically labelled glucose provided additional evidence for the functional activity and polarity of the pentose phosphate pathway in hydra. The effect of oligomycin, which can reverse polarity in hydra, had a significant effect on gradients of enzymes eliminating all except that observed for G6P dehydrogenase. The profile suggested a movement towards a more anaerobic type of metabolism, in keeping with the known biochemical action of this inhibitor. It is suggested that redox states and/or phosphorylation states may be featured in the positional information of cells in hydra.

Alanine Transaminase

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.

Animals

A 33 kDa protein with sequence homology to the 'laminin binding protein' is associated with the cytoskeleton in hydra and in mammalian cells.

In hydra and in mammalian cells the monoclonal antibody V recognises an epitope which colocalises with cytoskeletal structures. Using this antibody for expression screening, a cDNA clone (955 bp) was isolated from hydra, which covers an open reading frame for a protein of 294 amino acids with a calculated molecular mass of 32.8 kDa. Northern blot analysis of hydra RNA resulted in a single mRNA species of 1.2 kb, and primer extension experiments proved this to be the full length message. 218 residues at the amino terminus of the hydra protein show extensive homology (73.5%) to a human protein designated 'laminin binding protein'. The carboxyl-terminal 76 amino acids possess no significant similarity (20%). The monoclonal antibody V, which recognises an epitope in this carboxyl-terminal part, reacts in Western blots, both in hydra and in mammalian cells, with a protein of 33 kDa and not with the 45 kDa 'laminin binding protein'. The 33 kDa protein is not extracellular or transmembrane, but has a strictly intracellular location as indicated by its amino acid sequence and by immunocytochemical and cell fractionation studies. In non-dividing mammalian cells the 33 kDa protein colocalises with filamentous structures; in dividing cells it dissociates from it and concentrates centrally. Presence of the SPLR-sequence, which is the consensus phosphorylation motif for the p34cdc2 kinase, links this 33 kDa protein to events occurring during the cell cycle.

3T3 Cells

Tissue economics of hydra: regulation of cell cycle, animal size and development by controlled feeding rates.

Epithelial cell production and epithelial cell loss in 6 different size classes of Hydra attenuata were examined to understand the relationships between growth and morphogenesis. The sizes of adult hydra, the sizes of their buds, and their budding rates are all nearly proportional to the amount of food the hydra eat. Hydra fed at high rates (4-25 Artemia nauplii per day) all have the same epithelial cell cycle time (about 4 days). Budding accounts for most of their cell loss. Hydra fed 4-12 Artemia per day maintain a steady state condition in which tissue loss balances tissue growth. Animals fed 25 Artemia per day are not in a steady state growth condition and change in size. At the lowest feeding rates (0-1 Artemia per day), the epithelial cell cycle time is lengthened to about 16 days. Cell loss from the tentacles accounts for most of the cell loss, and this loss is not completely balanced by growth. As a consequence these animals cease budding and shrink in size.

Animals

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