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AMER. ZOOL., 28:1053-1063 (1988) Maintenance and Regeneration of the Nerve Net in Hydra1 H. R. BODE, S. HEIMFELD, O. KOIZUMI, C. L . LlTTLEFIELD, AND M. S. YAROSS Developmental Biology Center, University of California, Irvine, California 92717 SYNOPSIS. Due to the tissue dynamics, the entire nervous system of a hydra is in a steady state of production and loss of neurons. Neurons arise by differentiation from the interstitial cells in the ectoderm. Nerve cell intermediates migrate along the body column, settle and complete differentiation. The type of neuron formed is a function of axial or regional location in which the differentiation intermediate settles. Another result of the tissue dynamics is that all neurons are constantly changing their location. As a consequence, many neurons switch from one type of nerve cell to another in accord with positional changes. spersed among the epithelial cells of both layers throughout the animal. They are connected with one another both within a layer and between the two layers by neuronal processes (Fig. 1), thereby forming a net of neurons (Hadzi, 1909; Burnett and Diehl, 1964; Lentz and Barrnett, 1965). These processes lie near the basal end of each layer just above the mat of muscle fibers, which are extensions of the epithelial cells. The neuronal processes run among the epithelial cells forming synapses with epithelial cells and nematocytes as well as with other neurons (Westfall et aL, 1971; Westfall, 1973). The organization is simple; only two types of nerve cells can be distinguished on a morphological basis: ganglion and sensory cells. They are very similar as Westfall (1973) has demonstrated that essentially all nerve cells in hydra exhibit properties of sensory, motor, and neurosecretory neurons. Yet, they differ in two respects. One MORPHOLOGY OF THE NERVE NET The body plan of a hydra is quite simple, is their location (Fig. 1). The ganglion cell consisting of a head (hypostome and ten- bodies lie near the base of either cell layer, tacles), body column, and basal disk (Fig. while the sensory cells are located in a more 3). The structure of the body is a hollow peripheral position (e.g., Diehl and Burcylinder whose wall is comprised of two nett, 1964; Lentz and Barrnett, 1965). Secepithelia, the ectoderm and the endoderm, ond, they differ morphologically in that the separated by a basement membrane, the cell body of a sensory cell is more elongate mesoglea (Fig. 1). Nerve cells are inter- (Westfall, 1973). Also, the cilium of a sensory cell extends from the apical surface of the nerve cell body towards the external 1 From the Symposium on Nervous System Regenerenvironment, and is surrounded at its base ation in the Invertebrates presented at the Annual Meet- by a stereociliary complex. In comparison, ing of the American Society of Zoologists, 27-30 the cilium of a ganglion cell projects from December 1986, at Nashville, Tennessee. INTRODUCTION The nervous system of the freshwater coelenterate, hydra, is one of the most primitive in the animal kingdom, consisting simply of a loose net of nerve cells. Due to the continuous expansion and turnover of the tissue of the animal, it is also highly unusual in two other respects. The entire nerve net is in a steady state of production and loss of neurons, and each neuron is constantly changing its location in the animal. As a consequence of these tissue dynamics, some of the properties of nerve cell differentiation are also unusual. In the following, these properties and the dynamics of the nerve net will be described. How these properties are involved in the maintenance of the nerve net in the context of the tissue dynamics in the adult, as well as in the development of the nerve net during head regeneration, will be discussed. 1053 1054 H. R. BODE ET AL. FIG. 1. Longitudinal section of a hydra showing the two tissue layers and the location of the nerve net. The two enlargements of the regions indicated by the respective boxes illustrate the location of the nerve cell bodies and their connecting processes within the tissue layers. All cell types except nerve cells and epithelial cells have been omitted. The mesoglea is the basement membrane separating the two layers. the lateral side and does not have a ciliary complex (Westfall, 1973). The nerve net is non-uniform in several respects. First, 70% of all neurons are in the ectoderm (Epp and Tardent, 1978). Of more importance, there is considerable variation both in the type and density of neurons found in different regions. Ganglion cells are found throughout the body, but their density varies considerably between regions. It is fairly constant throughout the body column, but increases rapidly at the apical end reaching on average a 6-fold higher density in the head (Bode et al., 1973). In the areas where the tentacles join the hypostome, the density is still higher forming clusters which approximate primitive ganglia (Kinnamon and Westfall, 1981). At the basal end the density rises gradually down the peduncle resulting in a 2-4-fold increase in the foot (Bodega/., 1973; Epp and Tardent, 1978). There are two types of sensory cells, each restricted to a specific region of the animal. The epidermal sensory cells are found only in the ectoderm of the head. Each sensory cell body is completely enveloped by an epithelial cell, and extends from the base to the apex of that epithelial cell (Fig. 1) with the cilium projecting into the surrounding environment (Westfall and Kinnamon, 1978). Further, as shown in Figure 2, the epidermal sensory cells exhibit a specific spatial distribution within the head. In the tentacles they are uniformly distributed with one epidermal sensory cell per epithelial cell, whereas in the hypostome there are fewer and all are clustered at the apex surrounding the mouth (Kinnamon and Westfall, 1981; Dunne et al., 1985). In contrast, the other type of sensory cell is found in the endoderm of the body column, and differs from the epidermal sensory cell in two respects (Davis, 1972). First, the cell body lies between neighboring epithelial cells instead of being enveloped by a single epithelial cell. Second, the apical end of the nerve cell body and cilium extend towards but do not reach the surface of the endoderm (Fig. 1). Recently the use of antibodies has revealed additional complexity to the nervous system. Grimmelikhuijzen found that antisera to six different neuropeptides (FMRFamide, CCK, bombesin, Substance P, neurotensin, and oxytocin/vasopressin) each bound to a different subset of nerve cells (reviewed in Grimmelikhuijzen, 1984). In each case the subset was located in a particular part of the head, and in some cases also in or near the foot. For example, an antiserum to FRMFamide bound to nerve cells of the tentacles, hypostome, and the lower end of the peduncle (Fig. 3). Similarly, monoclonal antibodies have been generated which bind to different subsets of the ganglion cells (Bode et al., 1985; Yaross et al., 1986) as well as to subsets of the epidermal sensory cells (Koizumi and Bode, 1988). Thus, the two types of neu- 1055 HYDRA NERVE NET FIG. 2. Distribution of ganglion cells (circles) and epidermal sensory cells (triangles) in the tentacles and hypostome. One tentacle shown in full, while others have been truncated. rons defined by morphology are composed of subsets that differ in antigenic expression. BODY X COLUMN DYNAMICS OF THE NERVE NET In adult hydra the epithelial cells of the body column are continuously undergoing cell division (Campbell, 1967a; David and Campbell, 1972). The size of the animal, however, remains unchanged, as tissue is continuously lost from the body column by sloughing at the extremities, or is shunted into developing buds (Campbell, 19676; Otto and Campbell, 1977). As illustrated by the arrows in Figure 3, a result of this steady state of production and loss is that individual epithelial cells of both layers are continually changing their axial location (Campbell, 19676; Wanek and Campbell, 1982). Since the nerve net is intertwined among the epithelial cells, logically one would expect it to undergo a similar behavior. Individual nerve cells would be continually changing their location within the animal as they are displaced, for example, up the body column onto a tentacle, along the tentacle, and eventually lost at the end. Recently, direct evidence for this was obtained using a monoclonal antibody, JD1, which is specific for the epidermal sensory cells (Dunne et al, 1985). As epithelial cells are displaced onto the tentacle from the body column in normal animals, new epidermal sensory cells are added into the net, usually one with each epithelial cell (Dunne PEDUNCLE — BASAL DISK FIG. 3. Overall structure of a hydra and the distribution of the subset of neurons exhibiting FRMFamide-like immunoreactivity. The regions of the animal are identified by name. Arrows running along the body column indicate the direction of tissue displacement. The location of the FRMFamide neurons and their processes are indicated by the network of dots and connecting lines. et al., 1985). This results in the observed steady-state distribution of JD1 + neurons. If, however, no nerve cell precursors were present, no new sensory cells could be added. With time the distribution of epidermal sensory cells would change as the nerve net is displaced with the epithelial cells from tentacle base to tentacle tip. As illustrated in Figure 4, one would expect a gap to appear in the pattern of JD1 + sensory cells at the tentacle base shortly after removal of the precursors. In time the gap should increase in size, and eventually the tentacle would be devoid of cells stained with the antibody. Yaross et al. (1986) carried out this experiment and exactly this pattern was 1056 H. R. BODE ET AL. v-/ Treat with HU or NM to remove nerve cell precursors 3 days after treatment 6 days after treatment 10 days after treatment FIG. 4. Experiment illustrating the displacement of epidermal sensory neurons (triangles) along the tentacles with time in the absence of new nerve cell differentiation. observed. Further, they observed that the rate of displacement of epithelial cells and nerve cells along the tentacle was the same, providing evidence that the two cell types move in concert. Since the sensory cells are an integral part of the nerve net, it is most likely that all neurons of the net are displaced at the same rate as their surrounding epithelial cells towards one of the extremities and eventually sloughed. The important qualitative question in the face of the continuous displacement of neurons is how are the regionally specific subsets of neurons defined by morphology or antigenic expression maintained? The explanations of both the quantitative and qualitative aspects lie in the details of nerve cell differentiation, which will be described in the next two sections. Nerve cell differentiation pathway GENERATION OF NEURONS AND THE MAINTENANCE OF THE NERVE NET The continuous expansion of tissue and resulting displacement of individual neurons raise important problems as to how the nerve net is quantitatively maintained in adult hydra. (1) The loss of neurons by sloughing at the extremities is continuous, and yet, the size of the nerve cell population remains unchanged. This suggests that new neurons must be constantly produced to maintain their population. (2) In the body column the density of neurons (number of neurons/epithelial cell) remains constant even though new epithelial cells are constantly being added by cell division. Therefore, new neurons must be inserted into the net throughout the column at a similar rate to prevent their dilution. (3) The neuron density is higher in the head and foot than in the body column. Simple displacement of the nerve net from the body column into the head and foot cannot account for this. The increase in densities must arise by adding neurons into the net at higher rates in the extremities than in the body column. Nerve cells in hydra, as in other animals, do not undergo cell division. Pulse-labelling experiments using [3H]thymidine indicate that new nerve cells arise by differentiation throughout the animal (David and Gierer, 1974; Yaross and Bode, 1978). Hence, new nerve cells are constantly being produced. Calculations suggest the rate of production in sufficient to balance the rate of loss, and thereby maintain the steady state (David and Gierer, 1974). The constant tissue growth in hydra as well as the continuous production of neurons indicates that the nerve cell precursor has the properties of a stem cell. Morphological and ultrastructural studies indicate that neurons arise by differentiation from the interstitial cells (reviewed by Davis, 1974). It is assumed that neurons are derived from the multipotent stem cells among the population of large interstitial cells (David and Gierer, 1974; David and Murphy, 1977; Yaross and Bode, 1978). A large interstitial cell committed to nerve cell differentiation undergoes one or more cell divisions to form smaller interstitial cells. These are morphologically very similar to neurons, dif- HYDRA NERVE NET fering only in the absence of neural processes (Heimfeld and Bode, 1984a, b). They also resemble intermediates in the nematocyte pathway (David, 1973). The nerve differentiation intermediates are distinguished from the nematocyte precursors in that the latter always occur in syncytial clusters of 8, 16 or 32 cells (Slautterback and Fawcett, 1959; David and Gierer, 1974), while the former occur as single cells or in pairs (Bode and Chow, in preparation). As the little interstitial cells of the nerve cell pathway are also capable of one or more cell divisions (Bode and Chow, in preparation), the number of nerve cells arising from a single committed large interstitial cell is at least 4, and possibly more. The quantitative distribution of neuron differentiation is position-dependent The most important quantitative feature of the maintenance of the nerve net that must be explained is the regional differences in the density of the nerve net. The initial answer is simple. There is a striking correlation between the axial distribution of nerve cells and the spatial pattern of nerve cell differentiation. The rate of nerve cell differentiation is high in the head, low in the body column, and intermediate in and near the basal disk (Yaross and Bode, 1978). This corresponds directly with the relative densities of nerve cells in these regions (Bode et al., 1973). Hence, the regional pattern of densities is the result of different levels of neuron differentiation in the several regions. This observation leads to the more intriguing question as to what is the nature of this position-dependent pattern of differentiation. There are two straightforward explanations. First, this might reflect a pattern of position-dependent commitment of the stem cells. They would "read" a positional cue and differentiate accordingly. Or, the pattern could be due to the selective migration of large interstitial cells already committed to nerve differentiation, and/or the migration of the differentiation intermediates. Although the position-dependent commitment of inter- 1057 stitial cells was initially assumed to be the answer (e.g., Bode and David, 1978), recent evidence indicates the second explanation is more probable. Both large and small interstitial cells are capable of migration as single cells, and possibly in pairs (Tardent and Morgenthaler, 1966; Campbell, 1967c; Herlands and Bode, 1974; Heimfeld and Bode, 1984a). The number that migrate are substantial (Heimfeld and Bode, 1984a). On average 250 large interstitial cells and 350 small interstitial cells emigrate from any region of the body column (a region is a sixth of the column length) within a day. For the large interstitial cells this amounts to 15% of their total in any region. Also, for both large and small interstitial cells migration is biased since 2-3 times as many cells move in an apical direction as in a basal direction. The numbers migrating are sufficient to account for the observed levels of nerve cell differentiation. Some of these migrating cells must be nerve cell precursors since nerve cells derived from the migrating cells appear within 8 hr of emigration (Heimfeld and Bode, 1984a; Fujisawa, personal communication). Several pieces of evidence indicate that the small interstitial cells are the precursors. One is the distribution of emigrated cells. The rate of migration of small interstitial cells is greater than that of the large ones resulting in a faster and larger accumulation of the smaller ones in the head region within 24 hr. Their rate of accumulation is correlated with the rapid rise in nerve cells in the head (Heimfeld and Bode, 1984a). Second, when the cell composition of the host is altered, the rates of large interstitial cells immigrating into the host is unaffected. In contrast, different cell compositions result in corresponding 2-3-fold differences in both the rate of immigration of small interstitial cells and subsequent nerve cell differentiation derived from migrating cells (Heimfeld and Bode, 19846). Finally, there is a correlation based on the labelling indices of the migrating cells and subsequently derived nerve cells. Pulse-labelled migrating large and small interstitial cells had labelling 1058 H. R. BODE ET AL. indices of 53-56% and 61-64%, respectively, while the derived nerve cells had an index of 63% (Heimfeld and Bode, 19846). Since single small interstitial cells capable of cell division are known to be intermediates in the nerve cell pathway (Bode and Chow, in preparation), a fraction of the migrating small interstitial cells are most likely such intermediates. Calculations based on the numbers of migrating small interstitial cells and subsequently formed nerve cells suggest that the fraction is greater than 50% of this migrating population. This does not exclude the possibility that some of the migrating large interstitial cells may also contribute to nerve cell formation. Indeed, the migrating large interstitial cell population contains half as many stem cells as do the non-migrating large interstitial cells indicating that the majority of these migrating cells are already committed to a particular differentiation pathway (Heimfeld and Bode, 19846). Thus, the simplest view as to how the position-dependent pattern of nerve cell differentiation occurs is the following. Stem cells among the large interstitial cells become committed to nerve cell differentiation anywhere along the body column. A committed cell divides one or more times to form small interstitial cells which are nerve cell differentiation intermediates. These cells migrate and eventually settle somewhere along the body column or accumulate in the extremities, where they finish differentiating into nerve cells. Accumulation in the extremities accounts for the higher densities found there as compared to the body column, and the preferred apical direction of migration accounts for the highest density being in the head. PLASTICITY OF THE DIFFERENTIATED STATE OF NERVE CELLS The foregoing described the production and distribution of nerve cells in terms of numbers but did not address the question as to how a migrating small interstitial cell forms a particular type of nerve cell. In principle, there are two alternatives. In one, the differentiation fate (e.g., sensory cell, or specific neuropeptide expression) of a migrating small interstitial cell may have been determined before it began to migrate. Once determined, it would migrate selectively to the region where that particular type of neuron occurs and would differentiate there. In the other alternative, a migrating small interstitial cell may be committed to nerve cell differentiation in general, but not to a particular type of neuron. Its final location would determine the type, and would, therefore, be a position-dependent decision. Experiments based on a consideration of how a specific subset of neurons arises in the context of the dynamics of the nerve net indicate the latter alternative is probably correct. Plasticity of neuropeptide expression Two facts lead directly to the idea that neurons are not irreversibly committed to a particular differentiated state, which is the heart of the issue. First, each nerve cell is continually changing its position in the animal in concert with the pattern of displacement of the two epithelial layers. Second, the spatial distribution of a subset of neurons is constant. Thus, at any time one can demonstrate that the subset of neurons defined, for example, by their immunoreactivity with an antiserum against FRMFamide, occur throughout the head, but are not found in the upper body column (Grimmelikhuijzen et al., 1982; Koizumi and Bode, 1986). To maintain this constant pattern, nerve cells exhibiting FLI (FRMFamide-like immunoreactivity) must be continuously added at the base of the tentacles and hypostome to replace the ones that have been recently displaced further out on the tentacle, or up the hypostome. Otherwise the distribution of FLI + neurons would change in time as was observed (Fig. 4) for the JD1+ epidermal sensory cells of the tentacles after removal of the nerve cell precursors (Yaross et al., 1986). The new FLI+ neurons that are added to the net could arise by differentiation from interstitial cells, or by conversion of FLI" neurons. The evidence suggests the latter possibility is correct. All nerve cell precursors (large and small interstitial cells) can be removed from an animal by treating it with nitrogen mustard or hydroxyurea 1059 HYDRA NERVE NET Treat with HU or NM to remove nerve cell precursors Decapitate nerve cell precursors Head regeneration FIG. 5. Experiment illustrating the conversion of FLI~ neurons to FLI+ neurons during head regeneration. Circles and triangles represent FLI+ ganglion and FLI+ epidermal sensory cells respectively. (Diehl and Burnett, 1964;Bode etal., 1976). When such animals are examined for FLI binding for up to 3 wk after removal of the precursors, the spatial distribution of FLI+ neurons is the same as in normal animals (Koizumi and Bode, 1986). Were maintenance of the pattern dependent on new differentiation, changes in the distribution with time would have been expected as in Figure 4. The constancy of the pattern suggests that FLI~ neurons are converted into FLI+ neurons as they are displaced from the upper body column onto a tentacle or the hypostome. Another experiment, illustrated in Figure 5, provided more direct evidence that changing the position of a neuron could change its neuropeptide expression. Removal of the head results in the regeneration of a new head by the tissue at the apical tip of the decapitated animal. The process is morphallactic in that it involves the remodeling of the existing tissue and is independent of cell division (Hicklin and Wolpert, 1973; Cummings and Bode, 1984). Thus, epithelial cells and neurons formerly in the body column become part of a tentacle or the hypostome, and have in effect changed their axial location. Animals devoid of all nerve cell precursors were bisected in the upper body column to remove the head and all the FLI+ neurons of the apical end of the animal. Following regeneration of a head they once again exhibited FLI+ neurons (Koizumi and Bode, 1986). Such neurons could only have arisen from either FLI~ neurons or epithelial cells since these were the only two cell types in the ectoderm. As epithelial animals, which consist only of epithelial cells, do not form nerve cells (Marcum and Campbell, 1978) the reappearance of the FLI + neurons must be due to the conversion of FLI" neurons of the body column into FLI + neurons as they are incorporated into the head after tissue reorganization. Hence, a change in the location of a neuron can alter its neuropeptide expression. The same has also been demonstrated for the subset of neurons expressing vasopressin-like immunoreactivity (Koizumi and Bode, in preparation). These results raised a second question: Is the reverse possible? Can FLI + neurons turn off FLI expression if they enter a FLI~ region? Normally neurons are displaced from the FLI" body column basally into the FLI+ lower peduncle, and later into the basal disk, which is again FLI" suggesting this reversal might occur. This possibility was examined by transplanting lower peduncle regions into the middle of the body column. In all grafts in which the transplanted tissue took on the character of the body column, FLI disappeared. In those grafts in which the transplanted tissue maintained its lower peduncle character, FLI+ neurons were observed (Koizumi and Bode, 1986). Apparently expression of the neuropeptide can be turned on or off depending on the axial location of the neuron. Conversion of ganglion cells into sensory cells The reappearance of FLI + neurons in the regenerated hypostome also suggests another type of switch in neuron phenotype was taking place. Both FLI + ganglion and FLI + epidermal sensory cells occur in the hypostome (Koizumi and Bode, 1986). As there are no epidermal sensory cells in 1060 H. R. BODE ET AL. Nv 1 Li t Nv 2 I m — Bi Nv 3 \ Li Nv4 FIG. 6. Nerve cell differentiation pathway with the minimum in complexity. Im: multipotent stem cell; Bi: large interstitial cell committed to nerve cell differentiation; Li: small interstitial cell = differentiation intermediate; Nvl-Nv4: specific types of neurons. the body column (Westfall, personal communication), and neurons such as endodermal sensory cells do not move across the mesoglea (Smid and Tardent, 1984), the FLI + epidermal sensory cells probably arose from ganglion cells in the body column ectoderm. The use of two monoclonal antibodies provided more direct evidence for this deduction. The monoclonal antibody, TS33, binds only to epidermal sensory cells of the hypostome, and to no other neurons in the animal. To demonstrate the conversion of ganglion cells to sensory cells, the same experimental design described for conversion of FLI~ neurons to FLI + neurons (Fig. 5) was used. Animals devoid of interstitial cells were decapitated, allowed to regenerate, and found to exhibit TS33 + epidermal sensory cells in the regenerated hypostome (Koizumi and Bode, 1988). Because of the absence of sensory cells in the body column ectoderm, TS33 + epidermal sensory cells must have arisen from TS33— ganglion cells. Evidence for intermediates undergoing this transition was obtained by combining the use of TS33 with a second antibody, TS26, which binds to ganglion cells in the body column and the head. If ganglion cells were being converted to sensory cells during regeneration, one might expect to see cells in transition that bind both antibodies. The experiment was repeated and regenerates were double-labelled with the two antibodies using indirect immunofluorescence and two fluorochromes. On average, 2 double-labelled epidermal sensory cells were found per regenerate (Koizumi and Bode, 1988). As regeneration proceeded this number remained constant, while the number of TS33+ epidermal sensory cells per animal increased. This is consistent with the idea that the doublelabelled cells are in transition from a ganglion to an epidermal sensory cell. After obtaining this result, careful examination of normal animals revealed that a small number of double-labelled sensory cells were present suggesting that this conversion also occurs normally. It is plausible that the conversion from ganglion cells to sensory cells is a normal maturation process, and not due to a change in position. The following experiment renders this unlikely. Regeneration of the head at the apical tip of the lower half of an animal following bisection in the middle of the body column resulted in the formation of epidermal sensory neurons in the new head (Koizumi and Bode, 1986). As all the neurons that formed the head were originally destined to be displaced in a basal direction, none of them would ever have formed an epidermal sensory cell. Therefore, the appearance of new epidermal sensory cells could not have been due to maturation, but was due to a change in position. Hence, a consequence of neurons continuously changing their location as the epithelia are displaced is that they can alter their differentiated state from one type of neuron to another. A direct implication of this is that the type of differentiation undertaken by a migrating small interstitial cell depends on the region in which it settles. If it stops in the head, it will "read" its location, and form, for example, a neuron that expresses FLI + . CONCLUSIONS There are now sufficient data to draw a first approximation of the development and maintenance of the nerve net. In a normal adult the net is maintained in a steady state by the continuous loss of neurons at the extremities balanced by a constant pro- 1061 HYDRA NERVE NET duction of new nerve cells throughout most of the animal. During a process such as head regeneration, the net is in part developed anew. Both situations require the generation and appropriate distribution of specific numbers and types of neurons. The current picture as to how neurons are generated is shown in Figure 6, and represents the minimum in complexity. Neurons arise by differentiation from multipotent stem cells in a two-step commitment process. At all times some multipotent stem cells (Im) among the large interstitial cells undergo a commitment to form nerve cells, but the type of neuron is not specified. For two reasons, this initial commitment is probably due to an internal mechanism. It can take place anywhere interstitial cells occur in the animal suggesting that commitment to nerve cells does not require a particular environment. Also, there is no evidence for external control of this step. One can imagine a number of internal mechanisms ranging from genetically fixed programs to stochastic decisions based on a set of probabilities for differentiation. The committed cell (Bi) divides to produce two small interstitial cells (Li), which are differentiation intermediates that are also not committed to type of neuron. These intermediates migrate, and will eventually settle in the body column or in one of the extremities. The type of neuron formed (Nvl -Nv4) is influenced by the final location of the small interstitial cell. This is the second step in the commitment process. The specific distributions of neurons can be explained in part with the same process. The asymmetric densities of neurons are a result of the migration behavior of the differentiation intermediates. Many small interstitial cells accumulate in the extremities resulting in higher rates of neuron differentiation there than in the body column. Since migration occurs preferentially in an apical direction, the highest differentiation rate and density are found in the head. The position-dependent distribution of subsets of types of neurons is due to the intermediates "reading" their final loca- tion, and differentiating accordingly. The constancy of this pattern in the face of the continuous change of location of each neuron can be attributed to the metastability of the differentiated state of many of the neurons. They change their type of differentiation (Nv2 -• Nvl) as they change position during tissue movements. Whether all neurons are plastic in this regard is not known. Although many can arise by conversion from other neurons, some arise only by differentiation from interstitial cells (Nv3, Nv4). An example is the subset of epidermal sensory cells in the tentacles, defined by the monoclonal antibody, JD1, which was not replaced when the precursors were removed (Yaross et al., 1986). Finally, the metastability of the differentiated state of many of the neurons in the hydra nervous system was observed simply because neurons change position within the animal. This raises the possibility that the differentiated state of neurons in other animals may also be metastable rather than inherently terminal. The differentiated state may only appear to be terminal because neurons in other animals seldom change their location. ACKNOWLEDGMENTS We thank Patricia Bode for a critical reading of the manuscript. Some of the research described was supported by research grants from the National Institutes of Health to H.R.B. (HD 08086 and HD 16440), from the National Science Foundation to M.S.Y. (PCM-83-02581), and from the Japanese Ministry of Education and Fukuoka Prefecture to O.K. Current addresses: Shelly Heimfeld, Dept. of Pathology, Stanford University, Stanford, California 94305. Osamu Koizumi, Physiological Laboratory, Department of Science, Fukuoka Women's University, Higashi-ku, Fukuoka, 813 Japan. Marcia Yaross, Pharmacia Intraocular Intermedics, Pasadena, California 91140. REFERENCES Bode, H. R., S. Berking, C. N. David, A. Gierer, H. Schaller, and E. Trenkncr. 1973. Quantitative analysis of cell types during growth and morpho- 1062 H. R. BODE ET AL. genesis in hydra. Wilhelm Roux Arch. EntwMech. Org. 171:269-285. Bode, H. R. and C. N. David. 1978. Regulation of a multipotent stem cell, the interstitial cell of hydra. Prog. Biophys. Molec. Biol. 33:189-206. Bode, H., J. Dunne, S. Heimfeld, L. Huang, L. Javois, O. Koizumi, J. Westerfield, and M. Yaross. 1986. 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