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.     

Characterisation of two types of head activator receptor on hydra cells

A head activator (HA) analogue is described which even at high concentrations does not lose its biological activity. By cross-linking two HA molecules over a C8 spacer, the conformation was sufficiently altered, such that self-inactivation of HA by dimerisation was prevented. In addition, the introduction of a tyrosine instead of phenylalanine in one of the two HA molecules allowed radioactive labelling with iodine. This HA bipeptide was used to investigate the effect of HA at different concentrations and as ligand for HA receptor characterisation. We found that low concentrations (0.1-10 pM) sufficed to stimulate interstitial cell mitosis, and that higher concentrations (10-1000 pM) were required for the determination of interstitial cells to nerve cells. Binding of the radioactive HA ligand to living hydra and to purified membrane fractions was saturable and specific. Binding was compatible with HA analogues with a stable monomeric conformation, but less well with dimerising HA and HA analogues. Scatchard and kinetic analyses revealed the presence of at least two types of binding site in the membrane fraction, one with a 'lower' affinity (Kd = 10(-9) M) and one with a 100-fold higher affinity (Kd = 10(-11) M). Autoradiography showed that interstitial cells were differentially labelled, suggesting that the number or types of HA receptors may vary depending on cell cycle status. A mutant of hydra with a multiheaded morphology contained 6-20-times more HA receptors per mg protein than other hydra species or mutants.     

Action of the head activator on the determination of interstitial cells in hydra.

In addition to its role as a growth hormone (preceding paper), at the cellular level the head activator functions as one of the substances which control the determination of uncommitted stem cells in hydra. In the presence of head activator the determination of interstitial stem cells to nerve cells is stimulated, the determination of interstitial cells to nematocytes is inhibited. The determination of interstitial cells to nerves occurs shortly before or in the very early S period of interstitial cells.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.     

Head activator and head inhibitor are signals for nerve cell differentiation in hydra

Head activator and head inhibitor control nerve cell differentiation in hydra. Head activator acts as a stimulatory signal on nerve cell differentiation by forcing nerve cell precursors, which are arrested before final differentiation, to develop into mature nerve cells. Head inhibitor acts antagonistically by keeping the cells in their arrested state, before mitosis and terminal differentiation. This and other evidence suggest that the arrest of the nerve cell precursors occurs in the G₂-phase of their cell cycle. Nerve cell differentiation can also be induced by wounding the animal. This is a consequence of an initial disinhibition caused by diffusion of head inhibitor out of the tissue and the subsequent release of head activator which then stimulates nerve cell differentiation.     

Action of the head activator as a growth hormone in hydra.

At the cellular level the head activator from hydra acts as a mitogen or growth hormone. It shortens cell cycle times by stimulating cells arrested in the G2 period to go through mitosis. This is true for continuously proliferating cell types like epithelial cells, gland cells, and interstitial cells, and for differentiating interstitial cells including those undergoing a last mitosis before differentiating into nerves or nematocytes.     

Head activator does not qualitatively alter head morphology in regenerates ofHydra oligactis

Summary The characterization of head activator (HA) as a morphogen capable of increasing the number of tentacles regenerated by hydra was re-examined. Gastric tissue was excised from HA-treated whole animals and allowed to regenerate. At the cellular level the differentiation of head-specific ectodermal epithelial cells was monitored by quantifying monoclonal antibody, CP8, labeling. This labeling has been correlated with a rise in head activation potential and the determination of tissue to form head structures (Javois et al. 1986). At the morphological level tentacle number was monitored. HA-treated regenerates began the head patterning processes and evaginated tentacles sooner than controls but did not produce extra tentacles. The kinetics of CP8 labeling did not reveal major differences between treated and control regenerates after the initiation of head-specific epithelial cell differentiation. HA appeared to act more like a growth factor stimulating the differentiation of head-specific cell types rather than a morphogen which altered head morphology. An additional aspect of the study examined axial-specific effects of HA on the initiation and extent of head-specific epithelial cell differentiation. The cellular response of ectodermal epithelial cells to HA was dependent on their original axial location. More CP8⁺ tissue differentiated in regenerates of apical as opposed to mid-gastric origin.     

Growth regulation of the interstitial cell population in hydra : I. Evidence for global control by nerve cells in the head

The interstitial cells of hydra form a multipotent stem cell system, producing terminally differentiated nerve cells and nematocytes during asexual growth. Under well-fed conditions the interstitial cell population doubles in size every 4 days. We have investigated the possible role of nerve cells in regulating this behavior. Nerve cells are normally found in highest concentrations in the head region of hydra, while interstitial cells are primarily located in the body column. Our experimental approach was to construct, by grafting, animals in which the density of nerve cells varied in (1) the head region, or (2) the body column. The growth of the interstitial cell population was then measured in these hydra. The results indicate that differences in head nerve cell density are closely correlated with how fast the interstitial cell population increases in size. Variations in the level of either nerve cells or interstitial cells in the body column showed no such correlation. These findings suggest the existence of a signaling mechanism in the head region. This signal, which is a function of the density of head nerve cells, emanates from the head tissue and exerts global control on the growth of the interstitial cell population in the body column.     

Effect of head activator on proliferation, head-specific determination and differentiation of epithelial cells in hydra

In hydra the differentiation of head-specific ectodermal epithelial cells from multipotent stem cells is a multistep process in which cell cycle progression is regulated at three restriction points. Head activator acts as a positive signal at these restriction points. At the G2/mitosis boundary of epithelial stem cells head activator functions as a mitogen, being necessary for cell division. Subsequently, in or before S phase, head activator acts as determinant to ensure commitment of epithelial cells to head-specific determination. This effect of head activator requires hundredfold-higher concentrations, and may also require longer incubation times, than for cell proliferation. Epithelial cells thus committed to head-specific differentiation become arrested in G2 as a third and last restriction point in the cell cycle. They require disinhibition by decapitation and probably the presence of head activator for final differentiation, which then occurs in G2.     

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.     

Nerve commitment during head regeneration in hydra.

Most important event in head regeneration in hydra is a wave of conversion of many interstitial cells into nerve cells. Experimental evidence lends support to the idea that the commitment of interstitial cells into nerve cells is the first morphogenetic prerequisite for emergence of head structures, when the number of nerve cells increases. This increase in nerve cells is delayed when regeneration occurs at a site lower in the body column.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

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.     

Development of the two-part pattern during regeneration of the head in hydra

The head of a hydra is composed of two parts, a domed hypostome with a mouth at the top and a ring of tentacles below. When animals are decapitated a new head regenerates. During the process of regeneration the apical tip passes through a transient stage in which it exhibits tentacle-like characteristics before becoming a hypostome. This was determined from markers which appeared before morphogenesis took place. The first was a monoclonal antibody, TS-19, that specifically binds to the ectodermal epithelial cells of the tentacles. The second was an antiserum against the peptide Arg-Phe-amide (RFamide), which in the head of hydra is specific to the sensory cells of the hypostomal apex and the ganglion cells of the lower hypostome and tentacles. The TS-19 expression and the ganglion cells with RFamide-like immunoreactivity (RLI) arose first at the apex and spread radially. Once the tentacles began evaginating in a ring, both the TS-19 antigen and RLI+ ganglion cells gradually disappeared from the presumptive hypostome area and RLI+ sensory cells appeared at the apex. By tracking tissue movements during morphogenesis it became clear that the apical cap, in which these changes took place, did not undergo tissue turnover. The implications of this tentacle-like stage for patterning the two-part head are discussed.     

In Hydra magnipapillata the activator of protein kinase C diC8 causes multiple head formation along the body axis only when accompanied by feeding, but heavy feeding alone is sufficient to cause multiple head formation

In Hydra magnipapillata additional head structures can be induced to form by daily feeding accompanied by a daily treatment with diC8, an activator of protein kinase C. Based on these results, it was proposed that the PKC- pathway plays a central role in head formation in hydra. The results described here show that ectopic structures, as well as the ectopic localization of nerve cells, can be induced by heavy feeding alone. Furthermore, diC8 treatment does not induce ectopic head structures in starved animals. DiC8 reduces the rate of budding, leading to an unusual lengthening of the body column in reasonably fed animals.     

Identification of a vasopressin-like immunoreactive substance in hydra

Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells.     

Plasticity in the nervous system of adult hydra. II. Conversion of ganglion cells of the body column into epidermal sensory cells of the hypostome

Due to the tissue dynamics of hydra, every neuron is constantly changing its location within the animal. At the same time specific subsets of neurons defined by morphological or immunological criteria maintain their particular spatial distributions, suggesting that neurons switch their phenotype as they change their location. A position-dependent switch in neuropeptide expression has been demonstrated. The possibility that ganglion cells of the body column are converted into epidermal sensory cells of the head was examined using a monoclonal antibody, TS33, whose binding is restricted to a subset of epidermal sensory cells of the hypostome, the apical end of the head. When animals devoid of interstitial cells, which are the nerve cell precursors, were decapitated and allowed to regenerate, they formed TS33+ epidermal sensory cells. As this latter cell type is not found in the body column, and the interstitial cell-free animals contained only epithelial cells and ganglion cells in the part of the ectoderm that formed the head during regeneration, the TS33+ epidermal sensory cells most likely arose from the TS33- ganglion cells. The observation of epidermal sensory cells labeled with both TS33 and TS26, a monoclonal antibody that binds to ganglion cells, in regenerating and normal heads provides further support. The double-labeled cells are probably in transition from a ganglion cell to an epidermal sensory cell. These results provide a second example of position-dependent changes in neuron phenotype, and suggest that the differentiated state of a neuron in hydra is only metastable with regard to phenotype.     

Characterization of the VPS10 domain of SorLA/LR11 as binding site for the neuropeptide HA

The single transmembrane receptor sorLA/LR11 contains binding domains typical for the low-density lipoprotein receptors and a VPS10 domain which, in the related receptor sortilin, binds the neuropeptide neurotensin. SorLA is synthesized as a proreceptor which is processed to the mature form by a furin-like propeptidase. Endogenous sorLA and its hydra homologue HAB bind the neuropeptide head activator (HA). Transiently expressed partial sorLA constructs were investigated for ligand binding. We found that HA binds with nanomolar affinity to the VPS10 domain. The sorLA propeptide also bound to the VPS10 domain, whereas the receptor-associated protein RAP interacted both with the class A repeats and the VPS10 domain. The sorLA propeptide inhibited binding of HA to full-length sorLA and to the VPS10 domain. It also interfered with binding of HA to hydra HAB, which is taken as evidence for a highly conserved tertiary structure of the VPS10 domains of this receptor in hydra and mammals. The propeptide inhibited HA-stimulated mitosis and proliferation in the human neuroendocrine cell line BON and the neuronal precursor cell line NT2. We conclude that sorLA is necessary for HA signaling and function.     

Chemical anatomy of hydra nervous system using antibodies against hydra neuropeptides: a review

Polyclonal antibodies against hydra neuropeptides allow us to visualize the hydra nerve net. Chemical anatomy of the hydra nerve net was archived by means of immunocytochemistry with various antibodies to hydra neuropeptides. Our goal is to describe the hydra nerve net both qualitatively and quantitatively. The present chemical anatomy results indicate (1) differences in peptide expression between ganglion cells and sensory cells, (2) large differences in neuropeptide expression between ectodermal nerve cells and endodermal nerve cells, and (3) the usefulness of quantitative chemical anatomy to analyze the entire nervous system of hydra.     

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.