Developmental neurobiology of hydra, a model …Introduction to the nerve net of hydra: unique features of the diffuse nervous system The freshwater coelenterate hydra has a simple

REVIEW / SYNTHÈSE

Developmental neurobiology of hydra, a modelanimal of cnidarians1

Osamu Koizumi

Abstract: Hydra belongs to the class Hydrozoa in the phylum Cnidaria. Hydra is a model animal whose cellular anddevelopmental data are the most abundant among cnidarians. Hence, I discuss the developmental neurobiology of hy-dra. The hydra nerve net is a mosaic of neural subsets expressing a specific neural phenotype. The developmental dy-namics of the nerve cells are unique. Neurons are produced continuously by differentiation from interstitial multipotentstem cells. These neurons are continuously displaced outwards along with epithelial cells and are sloughed off at theextremities. However, the spatial distribution of each neural subset is maintained. Mechanisms related to these phenom-ena, i.e., the position-dependent changes in neural phenotypes, are proposed. Nerve-net formation in hydra can be ex-amined in various experimental systems. The conditions of nerve-net formation vary among the systems, so we canclarify the control factors at the cellular level by comparing nerve-net formation in different systems. By large-scalescreening of peptide signal molecules, peptide molecules related to nerve-cell differentiation have been identified. TheLPW family, composed of four members sharing common N-terminal L(or I)PW, inhibits nerve-cell differentiation inhydra. In contrast, Hym355 (FPQSFLPRG-NH3) activates nerve differentiation in hydra. LPWs are epitheliopeptides,whereas Hym355 is a neuropeptide. In the hypostome of hydra, a unique neuronal structure, the nerve ring, is ob-served. This structure shows the nerve association of neurites. Exceptionally, the tissue containing the nerve ring showsno tissue displacement during the tissue flow that involves the whole body. The neurons in the nerve ring show littleturnover, although nerve cells in all other regions turn over continuously. These associations and quiet dynamics leadme to think that the nerve ring has features similar to those of the central nervous system in higher animals.

Résume : Hydra fait partie des hydrozoaires dans le phylum des cnidaires. Hydra est un animal modèle pour lequel,parmi les cnidaires, il existe le plus de données sur les cellules et le développement. Je présente donc ici le développe-ment neurobiologique de l’hydre. Le réseau nerveux chez l’hydre est une mosaïque de sous-groupes neuraux qui com-pose un phénotype neural particulier. La dynamique du développement de ces cellules est inusitée. Des neurones sontproduits de façon continue par différenciation de cellules souches interstitielles multipotentes. Les neurones sont conti-nuellement repoussés vers l’extérieur en même temps que des cellules épithéliales et ils sont rejetés aux extrémités.Cependant, la répartition spatiale de tous les sous-groupes de nerfs est maintenue. Des mécanismes capables d’expliquerces phénomènes, i.e. les changements dépendants de la position des phénotypes neuraux, sont proposés. La formationdu réseau de nerfs chez Hydra peut être observée dans divers systèmes expérimentaux, mais les conditions peuvent va-rier d’un système à l’autre. Il est donc possible de comprendre les facteurs de contrôle à l’échelle des cellules en com-parant la formation des nerfs dans les différents systèmes. Les molécules de peptides reliées à la différenciation descellules ont été identifiées au cours d’un tri à grande échelle des molécules de peptides signalisateurs. La famille desLPW, composée de quatre unités qui ont en commun le N-terminal, L (ou I)PW, inhibe la différenciation des cellulesnerveuses chez l’hydre. En revanche, Hym355 (FPQSFLPRG-NH3) active la différenciation. Les LPW sont des épithé-liopeptides, alors que Hym355 est un neuropeptide. Il y a une structure neurale particulière, l’anneau nerveux, dansl‘hypostome de l’hydre. Cette structure a des associations nerveuses semblables à celles des neurites. Le tissu qui en-toure l’anneau nerveux ne subit pas de déplacement, malgré le flux des tissus dans le reste du corps. Les neurones del’anneau nerveux sont rarement remplacés, alors que les cellules nerveuses des autres régions sont continuellementchangées. Ces associations et cette dynamique lente permettent de penser que l‘anneau nerveux ressemble par certainsaspects au système nerveux central des animaux plus évolués.

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Can. J. Zool. 80: 1678–1689 (2002) DOI: 10.1139/Z02-134 © 2002 NRC Canada

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Received 30 November 2001. Accepted 15 July 2002. Published on the NRC Research Press Web site at http://cjz.nrc.ca on15 November 2002.

O. Koizumi. Neuroscience Laboratory, Department of Environmental Science, Fukuoka Women’s University, Kasumiga-oka 1-1-1,Higashi-ku, Fukuoka 813-8529, Japan (e-mail: [email protected]).

1This review is one of a series dealing with aspects of the phylum Cnidaria. This series is one of several virtual symposia on thebiology of neglected groups that will be published in the Journal from time to time.

J:\cjz\cjz8010\Z02-134.vpWednesday, November 13, 2002 11:06:08 AM

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Introduction to the nerve net of hydra:unique features of the diffuse nervoussystem

The freshwater coelenterate hydra has a simple body plan.Like all coelenterates, it is a diploblastic animal: it is com-posed of two layers of epithelial cells, the endoderm and theectoderm. The apical end is the head and the basal end is thefoot. The head has a hypostome with the mouth opening atthe apex and several tentacles originating from the lowerpart of the hypostome.

Hydra has a simple nervous system consisting of a nervenet that extends through the body (Had�i 1909; Burnett andDiehl 1964; Lentz and Barrnett 1965; Lent�, 1968). Nervecells are interspersed among the epithelial cells of both layers(Fig. 1). No large concentrations of neurons such as gangliaare observed (Lentz and Barnett 1965; Lentz 1968; Bode etal. 1988b). The nerve net contains two types of nerve cells,ganglion cells and sensory cells (Davis et al. 1968; Koizumiand Bode 1991; Grimmelikhuijzen and Westfall 1995). Gan-glion cells lie close to the muscle processes at the basal endsof the epithelial cells. Sensory cells have elongated cell bod-ies that extend from the level of the muscle processes in anapical direction and an elaborate ciliary cone at the apicalend of the cell body (Fig. 1) (Westfall 1973; Westfall andKinammon 1978; Bode et al. 1988b; Koizumi and Bode1991; Grimmelikhuijzen and Westfall 1995).

The nervous system of hydra has several unique features.The most remarkable of these is the multifunction of neu-rons. Each neuron in hydra possesses the entire repertory ofnerve-cell functions (Westfall 1973; Westfall and Kinammon1978), i.e., the neurons are all sensory–motor-interneuronswith neurosecretory granules. For example, a sensory cellhas sensory cilia as a sensory neuron, synaptic connectionsto the muscle layer as a motor neuron, synaptic connectionsto neurites or the cell body of a ganglion cell as an interneuron,and aggregations of granules in non-synaptic regions of proxi-mal sites of the cell body as a neurosecretory cell (Fig. 2)(Westfall and Kinammon 1978). Ganglion cells have the samefeatures (Westfall 1973). Moreover, as is shown in Fig. 2,sometimes a single neuron innervates two different types ofeffectors: muscle fibers and a nematocyte (Westfall et al.1971; Grimmelikhuijzen and Westfall 1995).

Immunohistochemistry using neuropeptide antisera andmonoclonal antibodies specific to hydra neurons on wholemounts has made it feasible to study the nerve net of hydra(Grimmelikhuijzen 1985; Dunne et al. 1985). These studieshave shown that the hydra nerve net contains numerous sub-sets of neurons and that the spatial distributions are highlyposition-specific (Fig. 3). Numerous subsets of neuronscontaining different neuropeptides and several subsets ofneurons defined by monoclonal antibodies were noted(Grimmelikhuijzen et al. 1982, 1990, 1995; Grimmelikhuijzen1985; Dunne et al. 1985; Koizumi and Bode 1986, 1991;Koizumi et al. 1988; Yaross et al. 1986). The regional distri-bution of each subset tends to be constant (Koizumi andBode 1986; Koizumi et al. 1988; Bode et al. 1988b).

Figure 4 is a diagram showing all types of ectodermalneurons. Ganglion cells, sensory cells, and unique nerve cells,including those of the nerve ring, are all localized together

on the muscle sheet of one layer of ectodermal epithelia, asshown in Fig. 1 (Mackie and Passano 1968).

Developmental dynamics of neurons: otherunique features of the diffuse nervoussystem

Hydra has three types of cell lineages: an ectodermal epi-thelial cell lineage, an endodermal epithelial cell lineage,and a interstitial cell lineage. The interstitial cell lineage iscomposed of interstitial cells, nerve cells, nematocytes, glandcells, and gametes (Fig. 5). Interstitial cells are multipotentstem cells, committed precursors, and differentiating inter-mediates (Campbell and David 1974; David and Gierer 1974;David and Murphy 1977; Bode and David 1978; Bode 1996).

In an adult hydra, nerve cells are produced continuouslyby constant differentiation from interstitial cells (Bode et al.1988b; David and Hager 1994). Nerve-cell production in thenerve net is balanced by a loss of neurons at the extremitiesand by the supply of neurons to young buds. Therefore, neu-rons are continuously changing their axial location by mov-ing with epithelial cells either towards the apical end (theapex of the hypostome or the tip of a tentacle) or towards thebasal end (the basal disk) (Campbell 1967a, 1976b, 1973;Bode et al. 1986, 1988b; Bode 1992). However, the distribu-tion of each subset of neurons expressing a certain neuralphenotype is maintained (Bode et al. 1986, 1988b; Bode1992).

How is the constant nerve net maintained in spite of theactive growth dynamics in hydra described above? In experi-ments related to this question, it was demonstrated that neu-rons can change the expression of FMRFamide-like peptideand vasopessin-like peptide depending upon their position inhydra (Koizumi and Bode 1986, 1991). Moreover, it wasdemonstrated that ganglion cells were converted to sensorycells when the the neurons were moved from the body col-umn to the hypostome (Koizumi et al. 1988).

These dynamic features of neurons in the adult hydra cor-respond to properties of developing nerve cells in embryosof higher animals.

Mechanisms controlling nerve-netformation at the cellular level

Hydra possesses several advantages for the study of nerve-net formation, which can be examined in various unique

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Fig. 1. Organization of a cnidarian epithelium and two types ofnerve cells, a sensory cell and a ganglion cell (modified fromMackie and Passano 1968).



experimental systems: regenerating, repopulating, budding, andnormal. Nerve-net formation progresses under different con-ditions depending on the system, therefore we can clarifyimportant factors at the cellular level by comparing nerve-net formation (Koizumi et al. 1990; Koizumi and Minobe1992; Minobe et al. 1995).

Regeneration systemHydra has a high capacity to regenerate, and a completely

new nerve net appears when the new tissue regenerates. Inthis case, nerve-net formation progresses with morphogenesisof the tissue, which brings about changes in the epithelial en-vironment of nerve precursor cells and nerve cells. In thissystem, the importance of epithelial cells for the formationof the nerve net was demonstrated (Bode et al. 1988a;Koizumi et al. 1990; Koizumi and Minobe 1992).

Nerve-net formation during head regeneration was exam-ined by means of immunohistochemistry, using an antiserumagainst the neuropeptide RFamide. In the head of an intacthydra, there are RFamide-like immunoreactive (RFamide+)sensory cells at the apex of the hypostome and RFamide+ganglion cells in the lower part of the hypostome and thetentacle. The formation of the nerve net specific to the headprogresses in two steps during head regeneration. The firststep is the early appearance of ganglion cells at the apex.The second step is the late appearance of sensory cells at theapex and the simultaneous disappearance of ganglion cellsfrom the apex (Fig. 6). This sequential patterning corre-sponds to the behavior of epithelial cells as defined by amonoclonal antibody, TS19, which binds only to ectodermalepithelial cells in the tentacle. The labeling pattern of TS19progresses in two steps during head regeneration. First, TS19+epithelial cells appear at the apex, and later TS19+ epithelialcells disappear from the apex and are present only in the ten-

tacles. The similar two-step pattern of nerve-net formationand the appearance of tentacle-specific epithelial cells duringhead regeneration suggests that they have common controlmechanisms (Bode et al. 1988a).

Nerve-net formation was examined during head regenerationin three morphogenetic mutants, head-regeneration-deficientmutants, budding-deficient mutants, and multiheaded mutants,isolated by Dr. Sugiyama’s group in Mishima, Japan (Sugi-yama and Fujisawa 1977). The results showed that the twosteps of nerve-net formation are distinct processes that canchange independently. Moreover, abnormalities in the behav-ior of tentacle-specific epithelial cells in the mutants cor-respond well to the formation of their nerve net (Koizumiet al. 1990). These data strongly suggest that a commonmechanism controls the pattern of nerve-net formation andtentacle-specific epithelial cells.

It is possible to make hydra chimeras in which all epithe-lial cells are from a wild type and all nerve cells are from amutant or vice versa (Marcum and Campbell 1978a, 1978b;Sugiyama and Fujisawa 1978b, 1979). Nerve-net formationduring head regeneration in these chimeras was examined todetermine whether epithelial cells or neurons are the primarycause of abnormal nerve-net formation in mutants. The resultsshow that the epithelial cells are responsible for abnormali-ties in nerve-net formation (Koizumi et al. 1990). Nerve-netformation in chimeric strains (produced between wild-typeand mutant strains) demonstrated that it is controlled by theenvironment provided by epithelial cells during head regen-eration.

All of the above experimental results suggest that interac-tions between epithelial cells and nerve precursor cells areimportant for nerve-cell differentiation and nerve-net formationin hydra (Bode et al. 1988a; Koizumi et al. 1990; Koizumiand Minobe 1992).

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Fig. 2. Multifunctional nerve cells in hydra. A single sensory cell has synaptic connections to the muscle sheet of an epitheliomuscularcell, a nematocyte, and a ganglion cell. Moreover, it has sensory cilia and neurosecretory granules (drawing based on Westfall et al.1971; Westfall 1973; Westfall and Kinnamon 1978). The arrows show synapses and their polarities.



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Repopulation systemWe can examine nerve-net formation in a unique system

called the repopulation system. Many of hydra’s highly spe-cific cell types (neurons, nematocytes, gland cells, and ga-metes) are part of a single lineage of cells that is continuallybeing renewed by proliferation and differentiation of stemcells called interstitial cells (Fig. 5). The entire interstitial-cell lineage can be removed from a hydra by various means(Campbell 1976; Sugiyama and Fujisawa 1978a). The result-ing animal is termed an epithelial hydra and is composed ofonly ectodermal and endodermal epithelial cells. This viableepithelial shell can then be repopulated by interstitial cells,since they migrate into the depleted animal from a small

temporary graft of normal tissue (Marcum and Campbell1978a, 1978b; Sugiyama and Fujisawa 1978b, 1979).

Using the repopulation system, therefore, neuronal differ-entiation and nerve-net formation can be observed in neuron-free tissue. Figure 7 shows the sequential appearance ofnerve cells in the head that was observed. Various behavioralresponses corresponding to the sequences were recovered se-quentially.

This “epithelial hydra host” provides an excellent experi-mental system for examining the relative roles of the epithe-lial factors described in the previous section (Regenerationsystem). Nerve-net formation in this system, in contrast tothe regeneration system, occurs without morphogenesis of

Fig. 3. Nerve net in hydra, visualized immunohistochemically. The patterns of the nerve nets differ depending on their position.(A) RFamide-like immunoreactive (RFamide+) nerve net in the hypstome. View from above the hypostome, showing the dark mouthregion in the center surrounded by a large number of epidermal sensory cells. (B) RFamide+ nerve net of the upper body column.Scale bars = 50 µm.



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epithelial tissue because the epithelial hydra host maintainsthe established morphology (Marcum and Campbell 1978a;Sugiyama and Fujisawa 1978b).

If epithelial cells indeed play an important role in producingregion-dependent nerve-cell differentiation, we would expectnerve-cell differentiation in the epithelial hydra host to havesimilar regional specificity to that of a normal hydra. How-ever, such specificity would not be expected if neurons in-stead of epithelial cells are the major factor determining theregional distribution of neurons. Instead, a random or totallydifferent neuron-differentiation pattern would be expected.

It has been reported that nerve-cell differentiation in epithe-lial hydra hosts occurs in the same region-dependent manneras in a normal hydra, thus providing direct evidence for therole of epithelial cells in regulating nerve-cell differentiation(Minobe et al. 1995).

Normal (and adult) systemIn an adult hydra, neurons arise continuously by differen-

tiation from multipotent stem cells among the interstitialcells (David and Gierer 1974; Bode and David 1978; Bode1992). Interstitial cells committed to neuron differentiationdivide to form a pair of small interstitial cells, which in turndivide and subsequently form neurons (Bode et al. 1990; Da-vid and Hager 1994). To compare the neuron-productionrates, animals were pulse-labeled with BrdU, and the label-ing index of various subsets of neurons was measured peri-odically. A particular subset was identified by double-labeling cells with an antibody against BrdU and with anantibody against a particular type of neuron.

To analyze the kinetics of neuronal differentiation, ani-mals initially labeled with BrdU were subsequently labeledwith an antibody against BrdU (Plickert and Kroiher 1988)and either DB5 or RC9. BrdU (1 mM) dissolved in hydra

Fig. 4. All types of ectodermal nerve cells in hydra. Neurons localized in the same place are illustrated separately in A–C. (A) Gan-glion cells. (B) Nerve ring and other unique neurons. (C) Sensory cells.

Fig. 5. The three cell lineages in hydra. (A) The ectodermal epi-thelial cell lineage. (B) The endodermal epithelial cell lineage.(C) The interstitial cell lineage, containing interstitial cells andtheir differentiation products. Interstitial cells consist ofmultipotent stem cells (I stem), committed precursor cells, anddifferentiating intermediates. Nv, nerve cells; GC, ganglion cells;SC, sensory cells; Nc, nematocyte; GL, gland cells.



culture medium was injected into the gastric cavity of a hy-dra. To measure the labeling index of neurons, labeled nu-clei were visualized with a monoclonal antibody againstBrdU (mouse IgG) and the cytoplasm of neurons with themonoclonal antibody DB5 or RC9 (both mouse IgMs). Thedifference in isotypes as well as the sequence of steps re-duced cross-reactivity to background levels in the double-labeling procedure (Fig. 8) (Koizumi et al. 1992).

The epithelial hydra host is also an excellent system forexamining the relative roles of neuronal factors. Since an ep-ithelial hydra is completely devoid of nerve cells, neurondifferentiation occurs in the absence of any influence fromexisting neurons. In contrast, nerve-cell differentiation oc-curs in the presence of fully matured nerve cells in the nor-mal (and adult) system. If there are differences in nerve-celldifferentiation and nerve-net formation between the repopu-lation system and the normal system, we can assess the roleof mature nerve cells in nerve-cell differentiation.

Other systems that can be used in the study of nerve-netformation are the budding system and dissociation/reaggregationsystem. Budding is the mode of asexual reproduction usedby hydra. A second axis formed by simple protrusion fromthe body column produces the head, body column, and footsequentially during budding (Otto and Campbell 1977). Inthe dissociation/reaggregation system, a normal hydra canregenerate from a cell mass after being dissociated into singlecells and then reaggregated by centrifugation (Gierer et al.1972). In both cases a normal nerve net eventually develops.

Mechanisms controlling nerve-netformation at the molecular level

To examine the molecular mechanisms of nerve-net for-mation, a joint project, “Large-scale non-targeting screeningof peptide signal molecules in hydra”, has started (Takahashi

et al. 1997; Bosch and Fujisawa 2001). Takahashi et al.(1997) developed a novel procedure for systematically iso-lating peptide signal molecules from hydra. Peptides wereextracted from large numbers of hydra, purified to homogene-ity using high-performance liquid chromatography (HPLC)without any biological assays. The isolated peptides weresubjected to structural analysis using automated amino-acidanalysis and then synthesized chemically, and the identity ofsynthetic peptides with native peptides was confirmed usingHPLC. The synthetic peptides were then subjected to a se-ries of biological test to examine their functions in hydra.

Using this approach, a number of peptides have been iden-tified that regulate development in hydra in addition to neuro-peptides controlling synaptic transmission and muscle con-traction (Table 1) (Takahashi et al. 1997, 2000; Yum et al.1998; Grens et al. 1999; Bosch and Fujisawa 2001; Harafujiet al. 2001).

Among these peptides, some that control nerve differenti-ation in hydra were identified. Some belong to the LPWfamily, and another peptide is Hym355 (FPQSFLPRGamide).A group of 4 peptides belong to the LPW family, whichhave 5–8 amino-acid residues and share the common N-terminal structure of L(or I)PW. All LPW peptides inhibitnerve-cell differentiation in hydra (Takahashi et al. 1997). Incontrast, Hym355 activates nerve differentiation in hydra(Takahashi et al. 2000).

Immunohistochemical analysis using antibodies to thesepeptides shows that Hym355 is a neuropeptide localized innerve cells but LPW peptides are epitheliopeptides whichare localized in epithelial cells. Cotreatment with a LPWpeptide and Hym355 nullified the effect of both peptides,which suggests that they act in an antagonistic manner(Takahashi et al. 2000).

Figure 9 illustrates the sequence of nerve differentiationand the effects of peptides. Interstitial multipotent stem cells

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Fig. 6. Reappearance of the RFamide+ nerve net during head regeneration in hydra. The numbers indicate days after decapitation.



(I stem) are committed to nerve differentiation in the bodycolumn; the committed nerve precursor cells (I Nv) migrateinto the head and foot and then differentiate into nerve cells(Nv) (Bode et al. 1990; Teragawa and Bode 1990; David

and Hager 1994; Bode 1996). At the same time theydifferentiate into the type of neuron appropriate for the finallocation (Bode 1996). Hym355 released from nerve cellsactivates nerve-cell differentiation, but LPW peptides from

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Fig. 7. Reappearance of the nerve net in the repopulation system. (A) RFamide+ nerve net. (B) RC9+ nerve net. RC9 is a monoclonalantibody specific to nerve precursor cells (interstitial cells) and ganglion cells. The time (hours or days) elapsed after nerve precursorcells were grafted into the epithelial host is indicated. Both are views from above the hypostome.



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epithelial cells inhibit it. Both might interact and act antago-nistically (Takahashi et al. 2000; Bosch and Fujisawa 2001).The next step of the study is to clarify the exact site of ac-tion. Is it commitment, migration, or differentiation?

A unique neural structure: the nerve ring inthe perihypostomal region of hydra

The nerve ring in the hypostome of hydra was observed

immunocytochemically using an antiserum against neuro-peptides and neuron-specific monoclonal antibodies (Fig. 10).The nerve ring in the mesh-like nerve net of hydra is unique.It is a distinct neuronal complex consisting of a thick nervebundle running circumferentially at the border between thehypostome and the tentacle zone. Immunolabeling showedthat the nerve ring is heterogeneous and contains at leastfour different subsets of neurons. During head regenerationand budding, the nerve ring appeared only after the nerve

Fig. 8. Double-labeling of BrdU-labeled nuclei and nerve cells using the BrdU antibody and the neuron-specific monoclonal antibodyRC9. BurU-labeled nuclei were visualized by fluorescein (green fluorescence) and nerve cells were labeled by Texas red (red fluores-cence). BrdU-labeled nuclei of double-stained nerve cells appear yellow with a dual-band filter. (A) Hypostome. (B) Tentacles. Scalebar = 50 µm.



net of ganglion and sensory cells had formed (Koizimi et al.1992).

The ectoderm in the immediate vicinity of and includingthe nerve ring constitutes a stationary zone that is not dis-placed. Tissue immediately above this zone is displaced towardsthe tip of the hypostome, while tissue below is displacedalong the tentacles. Correspondingly, the production of newneurons in the ring, measured by their differentiation kinet-ics, is much slower than in surrounding areas. Thus, thenerve ring is static and stable in contrast to the dynamic fea-tures of the nerve net of hydra (Koizumi et al. 1992).

These associations and quiet dynamics lead us to think thenerve ring has features that closely resemble those of thecentral nervous system in higher animals.

Conclusion

To understand a certain nervous system, both interdisci-plinary and overall neurobiological study is essential. Studyof the formation of the nervous system (developmentalneurobiology) is essential in addition to studies of structure(neuroanatomy) and function (neurophysiology, neuro-ethology, and behavioral physiology). In addition, studies atvarious levels are desirable, from the molecular level to thewhole-animal level.

According to current overall neurobiological studies of thenervous system of hydra, unique features of the neurons inthis primitive nervous system have appeared. Each neuron inhydra has the general properties of a cell, while neurons inhigher animals are highly specialized. Each neuron in hydrahas a complete set of nerve functions. Each neuron hasneurites as nerve fibers, but the differences between den-drites and axons present in higher animals are not observedin hydra.

Neurons in hydra show constant birth and death and con-stant displacement. They show considerable changes in phe-notype under the influence of the environment. Hence, theyshow active developmental dynamics and plastic properties.

Because of these properties we can use unique experimen-tal systems, such as the regeneration, budding, repopulation,normal, and dissociation–reaggregation systems, for study-ing nerve-net formation. In the near future, descriptions ofnerve-net formation in hydra will be made possible by com-bining studies at the molecular, cellular, and system levels.

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Fig. 9. Effects of peptide signal molecules on nerve differentia-tion in hydra. A neuropeptide, Hym355, activates nerve differen-tiation, while epithelial peptides, the LPW family, inhibit it. Istem, interstitial stem cell; I Nv, committed nerve precursorcells; Nv, differentiated nerve cells.

Peptide Structure Function Reference

Hym323 KWVQGKPTGEVKQIKF Morphogenesis Harafuji et al. 2001Foot activation

Hym346 AGEDVSHELEEKEKALANHS Grens et al. 1999Hym301 KPPRRCYLNGYCSPa Morphogenesis

Tentacle formationLPW family Cell differentiation Takahashi et al. 1997

Hym33H AALPW Inhibition of nerve differentiationHym35 EPSAAIPWHym37 SPGLPWHym310 DPSALPWHym355 FPQSFLPRGa Activation of nerve differentiation Takahashi et al. 2000Hym176 APFIFPGPKVa Muscle contractions Yum et al. 1998

EctodermGLWamide family Takahashi et al. 1997

Hym53 NPYPGLWa Bud detachmentHym54 GPMTGLWaHym248 EPLPIGLWaHym249 KPIPGLWaHym331 GPPPGLWaHym338 GPPhPGLWaHym370 KPNAYKGKLPIGLWa

Table 1. Hydra peptides whose function was identified.



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Fig. 10. RFamide+ nerve ring observed in the hypostome of Hydra oligactis. Scale bar = 100 µm.



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