Neuropeptides and their functions in Hydra.

In order to identify novel peptide signaling molecules involved in the regulation of developmental and physiological processes in the freshwater cnidarian, Hydra magnipapillata, we initiated a systematic peptide screening project, the Hydra Peptide Project. In the project, twelve neuropeptides were identified so far. The LWamide family is composed of seven members, which share a GLWamide motif at their C-termini. All the peptides have an ability to induce metamorphosis of Hydractinia serrata planula larvae into polyps. In Hydra, LWamides induce detachment of the bud from a parental polyp. A neuropeptide, Hym-355, enhances neuronal differentiation by inducing the multipotent interstitial stem cells to enter the neuron differentiation pathway. A myoactive neutopeptide, Hym-176, specifically and reversibly induces contraction of the ectodermal muscle of the body column, in particularly in the peduncle region of epithelial Hydra that totally lack nerve cells. Two members of a novel neuropeptide family (FRamides) were contained in the same precursor. However, they have opposite myoactive functions in epithelial hydra. From these results, it seems fair to say reasonable to conclude that the so-called 'primitive' nervous system of Hydra is in reality more complex than generally believed.

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.

A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra.

During the course of a systematic screening of peptide signaling molecules in Hydra a novel peptide, Hym-355 (FPQSFLPRG-NH(2)), was identified. A cDNA encoding the peptide was isolated and characterized. Using both in situ hybridization and immunohistochemistry, Hym-355 was shown to be expressed in neurons and hence is a neuropeptide. The peptide was shown to specifically enhance neuron differentiation throughout the animal by inducing interstitial cells to enter the neuron pathway. Further, co-treatment with a PW peptide, which inhibits neuron differentiation, nullified the effects of both peptides, suggesting that they act in an antagonistic manner. This effect is discussed in terms of a feedback mechanism for maintaining the steady state neuron population in Hydra.

A novel neuropeptide, Hym-176, induces contraction of the ectodermal muscle in Hydra.

During the course of a systematic non-targeting screening of peptide signal molecules in Hydra, we identified a novel myoactive neuropeptide called Hym-176. The primary structure of Hym-176 was determined to be APFIFPGPKVamide. It specifically and reversibly induced contraction of the ectodermal muscle of the hydra body column in vivo. However, it had no effect on the ectodermal muscle of the tentacles. The structure-activity relationship analysis showed that the sequence of FIFPGPKVamide is a minimal requirement for the myoactivity. Removal of an amide group from the C-terminus completely abolished the activity. By using the antibody specific to Hym-176, the tissue localization of the peptide in hydra was determined immunohistochemically. The intense immunoreactivity was found in the peduncle nerve cells, indicating that Hym-176 is a neuropeptide.

Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families.

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of Hydra magnipapillata were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display-PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone, Anthopleura fuscoviridis, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species, Hydractinia serrata. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of Hydractinia serrata. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.

Nerve cell differentiation in nerve-free tissue of epithelial hydra from precursor cells introduced by grafting. I. Tentacles and hypostome.

The differentiation of hydra nerve cells in the nerve-free tissue of epithelial hydra was examined in Hydra magnipapillata. Nerve cell precursors, the interstitial cells, were introduced into the upper half of epithelial hydra by grafting it onto the lower half of normal hydra. In the tentacles of grafted epithelial hydra, a small number of RF+ ganglion cells first appeared in the proximal area at 1.5 days after grafting, followed by the appearance of NV1+ sensory cells in the same area about a day later. In the following days, both neuron types appeared more numerously in more distal positions. The front boundary for each type moved gradually from the base to the tip of the tentacles in about 7 days. In the hypostome, a small number of RF+ ganglion cells first appeared in the apex at 1.5 days. More nerve cells appeared in the following days, eventually forming a cluster of RF+ sensory cells at the apex surrounded by numerous RF+ ganglion cells in the adjacent tissue. These results show that nerve cells do not differentiate randomly in the epithelial hydra host. Instead, differentiation occurs in a strongly region-specific manner in the same way as in normal hydra, suggesting that epithelial cells in each region provide different cues or signals to produce region-specific nerve cell distribution in normal hydra tissue.

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.

Plasticity in the nervous system of adult hydra. III. Conversion of neurons to expression of a vasopressin-like immunoreactivity depends on axial location.

The nervous system of hydra consists of a nerve net that extends throughout the animal. Because of the tissue dynamics of hydra, the nerve net is in a steady state of production and loss of neurons. Neurons are continuously produced in the body column and are constantly lost by sloughing at the extremities and into developing buds. Consequently, every neuron is continuously displaced towards an extremity. A subset of the neurons of the nerve net, termed vasopressin-like-immunoreactive (VLI+) neurons, has been identified with an antiserum against vasopressin. This subset has a specific regional distribution in that it is found in the head, peduncle, and foot of an adult hydra. The VLI+ neurons in the head and peduncle are ganglion cells, while those in the foot include a newly described sensory cell. How is the regional distribution of the subset maintained when every neuron is continually changing location? Removal of the neuron precursors indicates the VLI+ neurons can arise by conversion from VLI- neurons of the body column. In the normal animal they probably arise by conversion as well as by differentiation. Conversion of VLI- to VLI+ neurons is due to a change in axial position, or region, instead of a maturation process.

Nerve net formation in the primitive nervous system of Hydra--an overview.

Nerve net formation was examined during head-regeneration and budding of Hydra using indirect immunofluorescence on whole mounts. The nerve net was visualized with an antiserum to a neuropeptide, RFamide. The nerve net forms in two steps: the first is the appearance of ganglion cells at the regenerating tip, and the second is the appearance of sensory cells at the apex and the simultaneous disappearance of ganglion cells from the apex. The behavior of epithelial cells during head-regeneration, as defined by monoclonal antibody TS19, corresponded with nerve net formation. We examined nerve net formation in 3 morphogenetic mutants: a head-regeneration deficient mutant (reg-16), a budding deficient mutant (L4) and a multi-headed mutant (mh-1). In addition, we also examined chimeric strains which consist of the epithelial cells from the wild type and nerve cells from a mutant, and vice versa. We obtained clear evidence that nerve net formation is controlled by the environment provided by the epithelial cells.

Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra.

The presence of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the peduncle of Hydra was demonstrated by immunogold electron microscopy. Thin sections of Lowicryl-embedded tissue labeled with antisera to RFamide and 5-nm gold-conjugated, secondary antibody and of Epon-Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms/ml RFamide. These results are the first demonstration that the dense-cored vesicles of Hydra neurons contain a neuropeptide.

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.

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.

Plasticity in the nervous system of adult hydra. I. The position-dependent expression of FMRFamide-like immunoreactivity.

The plasticity of nerve cells expressing the neuropeptide FMRFamide was examined in adult hydra. Using a whole-mount technique with indirect immunofluorescence, the spatial pattern of neurons showing FMRFamide-like immunoreactivity (FLI) was visualized. These neurons were located in the tentacles, hypostome, and peduncle, but not in the body column or basal disc. Since every neuron in the nerve net is continuously displaced toward an extremity and eventually sloughed, the constant pattern of FLI+ neurons could arise in one of two ways. When displaced into the appropriate region, FLI- neurons are converted to FLI+ neurons, or FLI+ neurons arise by differentiation from interstitial cells. To distinguish between these two possibilities, interstitial cells, the multipotent precursors of the nerve cells, were eliminated by treatment with hydroxyurea or nitrogen mustard. Following head, or foot and peduncle, removal from these animals, the missing structures regenerated. The spatial pattern of FLI+ neurons reappeared in the newly regenerated head or peduncle. This shows FLI- neurons in the body column were converted to FLI+ when their position was changed to the head or the peduncle. When the peduncle was grafted into the body column, it was converted to basal disc or body column tissue, and FLI disappeared. The appearance and loss of FLI was always position dependent. These results indicate that the neurons in the mature nerve net can change their neuropeptide phenotype in response to changes in their position.

Specific S-methylglutathione incorporation into a nematocyst-rich fraction of hydra.

1. Specific S-[C]methylglutathione incorporation from Hydra japonica into a nematocyst-rich subcellular fraction was observed. 2. This specific incorporation is not rapid reversible binding, as shown by the lack of saturation of the reaction with time. 3. Saturating kinetics of specific incorporation rate with S-[14C]methylglutathione concentration suggests the existence of intermediate reversible complex between a macromolecular and S-[14C]methylglutathione. 4. Activity of specific incorporation could be solubilized by Triton X-100 treatment of the nematocyst fraction, showing that the incorporation is not due to transport processes. 5. The incorporation was markedly diminished by the addition of cold trichloroacetic acid or urea, or by heat-treatment after the incorporation, showing that the complex is not stabilized by chemical bonding. 6. No chemical changes of free S-[14C]methylglutathione were detected in the reaction mixture, showing that the macromolecule interacting with S-[14C]methylglutathione is not a catalyzing enzyme. 7. These results suggest that this is a new type of glutathione incorporation and could be explained by a type of receptor protein which accumulates glutathione molecules.