Differentiation of the interstitial cell line in hydrozoan planulae

Repopulation of epithelial (colchicine-treated) planular tissue by interstitial cells, nematoblasts/nematocytes, and ganglionic cells was examined via grafting. Seventy-two-hour epithelial planular head pieces were grafted to 72-hour control labelled planular tail pieces, left in contact for 24 h, separated, and the head pieces were analyzed for interstitial cells and their derivatives. The reciprocal experiment of grafting 72-hour epithelial planular tails to 72-hour control labelled planular heads was also done and the tail pieces were examined. Repopulated planular head pieces contained interstitial cells, ganglionic cells and a reforming neural plexus but few nematoblasts/nematocytes. Reconstituted planular tail pieces contained interstitial cells and nematoblasts/nematocytes but no ganglionic cells. Results possibly suggest that the migrating interstitial cell population of 72-hour planulae is rich in committed precursors.     

The interstitial cells control the sexual phenotype of heterosexual chimeras of hydra

The three stem cell populations in hydra, the epithelial cells of the ectoderm and endoderm, which make up the body of the hydra, and the interstitial cells, which give rise to nerve cells, nematocytes, and gametes, were tested for their effects on determining the sexual phenotype of individuals. This was done by creating epithelial hydra, which are devoid of interstitial cells and their derivatives, of one sexual type and repopulating them with interstitial cells from individuals of the other sexual type. The resulting heterosexual chimeras were found in all cases to display the same sexual phenotype as that of the interstitial cell donor, indicating this cell type is responsible for the sex of the animal. The epithelial tissue had no influence in determining which gamete type was produced.     

Characterization of interstitial stem cells in hydra by cloning.

A procedure has been developed for cloning interstitial stem cells from hydra. Clones are prepared by introducing small numbers of viable cells into aggregates of nitrogen mustard-inactivated host tissue. Clones derived from added stem cells are identified after 1–2 weeks of growth by staining with toluidine blue. The incidence of clones increases with increasing input of viable cells according to one-hit Poisson statistics, indicating that clones arise from single cells. After correction for cell losses in the procedure, about 1.2% of the input cells are found to form clones. This compares with estimates from in vivo experiments of about 4% stem cells in whole hydra [David, C. N., and Gierer, A. (1974). Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci.16, 359–375.]Differentiation of nematocytes and nerve cells in clones was analyzed by labeling precursors with [³H]thymidine and scoring labeled nerves and nematocytes 2 days later. Nine clones examined in this way contained both differentiated nerve cells and nematocytes, demonstrating that the interstitial stem cell is multipotent. This result suggests that the observed localization of nerve and nematocyte differentiation in whole hydra probably occurs at the level of stemcell determination. The observation that differentiated cells occur very early in clone development suggests that a stem cell's decision to proliferate or differentiate is regulated by shortrange feedback signals which are already saturated in young clones.     

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.     

Bud formation inHydra: Inhibition by an endogenous morphogen

Summary From crude extracts ofHydra tissue a substance has been purified which prevents or retards the asexual reproduction by budding. The molecular weight is in the range of 300 to 1000 daltons. Inhibition of bud formation can be observed with concentrations equivalent to the extract from one hydra per 4 ml, that is, to a more than 10,000-fold dilution of the initial crude extract of a hydra. The purified inhibitor is active at a concentration of less than 10^–8 M.Most of the inhibitor present inHydra is bound to cells. Within the cells the substance is mainly bound to particulate structures which sediment at 10,000 g. Its concentration is highest in the hypostomal region and decreases in the direction of the tentacles and peduncle. A second, lower, peak has been found in the basal disc. Treatment of the animals with a toxic agent (nitrogen mustard) which depletes the animal of interstitial cells, nematocytes and nematoblasts excludes the possibility that the inhibitor is present to any great extent in these cells. In conjunction with cell separation experiments by centrifugation of fixed cells in suspension, these results indicate that nerve cells are the most likely sites of storage of the inhibiting substance, although epithelial cells are not excluded as sources for the inhibitor.     

Pulses of ammonia and methylamine induce down-regulation of nematocyte and nerve cell populations in Hydrozoa (Hydra; Hydractinia)

Summary Hydrozoa replace used-up nematocytes (cnidocytes) by proliferation and differentiation from interstitial stem cells (i cells). Repeated pulsed exposure ofHydra to elevated levels of unprotonated ammonia leads to successive loss of the various types of nematocytes: first of the stenoteles, then of the isorhizas and finally of the desmonemes. The loss is due to deficits in supply; the number of nematoblasts and differentiating intermediates is reduced. In the hydroidHydractinia the main process leading to numerical reduction was observed in vivo: mature nematocytes as well as precursors emigrate from their place of origin into the gastrovascular channels where they are removed by phagocytosis. This is a regular means by which these animals down-regulate an induced surplus of nematocytes. With lower effectiveness, pulses of methylamine, trimethylamine and glutamine also induce elimination of the nematocyte lineages. In the long term the population of nerve cells, which are permanently but slowly renewed from interstitial neuroblasts, decreases, too. After 2 months of daily repeated treatment the density of the Arg-Phe-amide-positive nerve cells was reduced to 50% of its normal level. Thus, ammonia induces down-regulation of all interstitial cell lineages. The temporal sequence of the ammonia-induced loss reflects the diverse rates with which the various i cell descendants normally are renewed.     

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.     

Nematocyte development in Hydra attenuata is dependent on both the interstitial cells and the epithelial cells

The aberrant, a morphological mutant of Hydra attenuata, has altered patterns of the development and distribution of nematocytes. The number of nematoblasts and nematocytes is higher in the aberrant than in the normal. Stenotele differentiation is incomplete and the numbers of desmonemes and holotrichous isohrizas mounted on the body column are much higher than normal. Because nematocytes arise by differentiation from the interstitial cells, epithelial cell/interstitial cell chimeras between the aberrant and normal strains were made to determine whether the lesion giving rise to the alterations in the mutant was due to the epithelial cells or a cell type in the nematocyte lineage. Only the chimera in which both cell types were derived from the aberrant exhibited the altered nematocyte development. If the chimera contained a normal cell type, either epithelial cell or interstitial cell, nematocyte development was normal. Thus, both epithelial cells and cells of the nematocyte lineage are involved in the control of nematocyte development. A defect in one of the lineages can be compensated for by the other cell type.     

Interstitial cell migration in Hydra attenuata. I. Quantitative description of cell movements

The interstitial cell system of hydra contains multipotent stem cells which can form at least two classes of differentiated cell types, nerves and nematocytes. The amount of nerve and nematocyte production varies in an axially dependent pattern along the body column. Some interstitial cells can migrate, which makes it conceivable that this observed pattern of differentiation is not the result of regionally specified stem cell commitment, but rather arises by the selective movement of predetermined cells to the correct site prior to expression. To assess this latter possibility quantitative information on the dynamics of interstitial cell migration was obtained. Epithelial hydra were grafted to normal animals in order to measure (1) the number of cells migrating per day, (2) the location of these cells within the host tissue, and (3) the axial directionality of this movement. Tissue properties such as axial position and the density of cells within the interstitial spaces of the host were also tested for their possible influence on migration. Results indicate that there is a considerable traffic of migrating interstitial cells and this movement has many of the characteristics necessary to generate the position-dependent pattern of nerve differentiation.     

Non-budding Hydra: Form Regulation and Bud Induction

Form regulation and bud induction were studied in a non-buddingstrain of Chlorohydra viridissima. Regeneration at a cut surfacein a column piece with an existing hydranth was observed andfound to be dependent on the column length Another aspect ofform regulation, formation and control of supernumerary tentacles,was investigated by grafting. Supernumerary tentacle formationin long polyps can be suppressed by implants of hypostomal orsubhypostomal tissue. Non-budding hydra can be induced to bud by implanting smallpieces of normal tissue into their columns. The cellular basisof this process was investigated by means of grafting, radioautography,and histological methods. No differences in the proportionsor appearances of the cell types were observed between non-buddingand normal animals. However, induced buds have higher proportionsof interstitial cells and their derivatives (nerves and nematoblasts)than do normal buds. Many of these interstitial cells and derivativesoriginate from cells in the grafted implant. Normal tissue fromwhich interstitial cells have been previously removed will notinduce buds in non-budding hydra. The non-budding syndrome is probably related to a deficiencyin interstitial cell differentiation. If nerve cells are involvedin bud initiation and form regulation, these results suggestinterstitial cells of non-budding hydra are unable to transforminto sufficiently active and/or numerous nerve cells to controlthose processes.     

Putative intermediates in the nerve cell differentiation pathway in hydra have properties of multipotent stem cells

We have investigated the properties of nerve cell precursors in hydra by analyzing the differentiation and proliferation capacity of interstitial cells in the peduncle of Hydra oligactis, which is a region of active nerve cell differentiation. Our results indicate that about 50% of the interstitial cells in the peduncle can grow rapidly and also give rise to nematocyte precursors when transplanted into a gastric environment. If these cells were committed nerve cell precursors, one would not expect them to differentiate into nematocytes nor to proliferate apparently without limit. Therefore we conclude that cycling interstitial cells in peduncles are not intermediates in the nerve cell differentiation pathway but are stem cells. The remaining interstitial cells in the peduncle are in G1 and have the properties of committed nerve cell precursors (Holstein and David, 1986). Thus, the interstitial cell population in the peduncle contains both stem cells and noncycling nerve precursors. The presence of stem cells in this region makes it likely that these cells are the immediate targets of signals which give rise to nerve cells.     

Role of the cellular environment in interstitial stem cell proliferation in Hydra

Summary The role of the cellular environment on hydra stem cell proliferation and differentiation was investigated by introduction of interstitial cells into host tissue of defined cellular composition. In epithelial tissue lacking all non-epithelial cells the interstitial cell population did not grow but differentiated into nerve cells and nematocytes. In host tissue with progressively increased numbers of nerve cells growth of the interstitial cell population was positively correlated to the nerve cell density. In agreement with previous observations (Bode et al. 1976), growth of the interstitial cell population was also found to be negatively correlated to the level of interstitial cells present. The strong correlation between the growth of the interstitial cell population and the presence of interstitial cells and nerve cells implies that interstitial cell proliferation is controlled by a feedback signal from interstitial cells and their derivatives. Our results suggest that the cellular environment of interstitial cells provides cues which are instrumental in stem cell decision making. Offprint requests to: T.C.G. Bosch     

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.     

Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system

Cnidarians represent the first animal phylum with an organized nervous system and a complex active behavior. The hydra nervous system is formed of sensory-motoneurons, ganglia neurons and mechanoreceptor cells named nematocytes, which all differentiate from a common stem cell. The neurons are organized as a nerve net and a subset of neurons participate in a more complex structure, the nerve ring that was identified in most cnidarian species at the base of the tentacles. In order to better understand the genetic control of this neuronal network, we analysed the expression of evolutionarily conserved regulatory genes in the hydra nervous system. The Prd-class homeogene prdl-b and the nuclear orphan receptor hyCOUP-TF are expressed at strong levels in proliferating nematoblasts, a lineage where they were found repressed during patterning and morphogenesis, and at low levels in distinct subsets of neurons. Interestingly, Prd-class homeobox and COUP-TF genes are also expressed during neurogenesis in bilaterians, suggesting that mechanoreceptor and neuronal cells derive from a common ancestral cell. Moreover, the Prd-class homeobox gene prdl-a, the Antp-class homeobox gene msh, and the thrombospondin-related gene TSP1, which are expressed in distinct subset of neurons in the adult polyp, are also expressed during early budding and/or head regeneration. These data strengthen the fact that two distinct regulations, one for neurogenesis and another for patterning, already apply to these regulatory genes, a feature also identified in bilaterian related genes.     

Growth regulation of the interstitial cell population in hydra. II. A new mechanism for the homeostatic recovery of reduced interstitial cell populations

The interstitial cells of hydra comprise a stem cell population, producing at least two classes of terminally differentiated cell types, nerve cells and nematocytes. Exposure to hydroxyurea (HU) results in selective depletion of interstitial cells from the tissue. The surviving cells subsequently recovered to normal levels, and the mechanisms involved in this repopulation were examined. Hydra were treated for varying times with HU such that interstitial cell numbers were reduced to 7 or 35% of normal. Subsequent growth of the epithelial and interstitial cell populations in these animals was monitored. The results indicate that the growth rates of these two cell types were only slightly different from untreated controls during the 4 weeks after HU exposure, implying that repopulation should not have occurred. However, recovery of the interstitial cell population was observed. Further analysis revealed that the interstitial cells in HU animals, unlike normal hydra, were not uniformly distributed in the body column, and were especially reduced in the budding region. In normal animals a constant fraction of the interstitial and epithelial cells are lost into buds. However, as a consequence of this nonuniform distribution a smaller fraction of the interstitial cells are displaced into HU buds, thereby retaining a higher proportion in the adult tissue. Calculations indicate that this mechanism of increased retention is of sufficient magnitude to account for 40-60% of the observed recovery after HU treatment.     

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.     

Growth regulation of the interstitial cell population in hydra. IV. Control of nerve cell and nematocyte differentiation by amplification of non-stem interstitial cells

The precursors for several differentiated cell types in hydra, such as nerve cells and nematocytes, arise from the interstitial cell population. Previously, it has been suggested that the interstitial cells represent a homogeneous stem cell population, and that both the rate of growth and the amount of differentiation are regulated strictly at the level of stem cell self-renewal and commitment. However, recent evidence does not support this viewpoint. In this paper we have proposed that the interstitial cell population is complex, containing both clonable stem cells and other cells which have a reduced division capacity. In response to hydroxyurea treatment, there is an amplification in the number of divisions that the non-stem interstitial cells undergo before differentiating. This amplification model is consistent with the correlations found in the preceding report (S. Heimfeld and H.R. Bode, 1986, Dev. Biol. 115, 51-58) and fits well with previously published data. An additional experiment which tests two specific predictions of this new model is presented.     

Cell lineages in Hydra: isolation and characterization of an interstitial stem cell restricted to egg production in Hydra oligactis

In an attempt to isolate unipotent stem cells (progenitors to the nerve cells, nematocytes, gland cells, and gametes) from Hydra oligactis females, animals were treated with a drug (hydroxyurea, HU) that preferentially lowers or eliminates the interstitial stem cells, leaving the epithelial tissue intact. In this epithelial environment, interstitial cells remaining after treatment will proliferate and differentiate, permitting a long-term analysis of their developmental capabilities. Following treatment of females with HU, animals were isolated that contained interstitial cells that gave rise to eggs only. Two clones of animals containing these cells were propagated for several years and the growth and differentiation behavior of the interstitial cells examined in their asexually produced offspring. During this time, the cells displayed an extensive proliferative capacity (classifying them as stem cells) and remained restricted to egg differentiation. It is proposed that both the sperm- and the egg-restricted stem cells arise from a multipotent stem cell, which also gives rise to the somatic cells (see above), and that, in hydra, sex is ultimately determined by interactions between cells of the two germ cell lineages.     

The role of nerve cell density in the regulation of bud production in hydra

Summary The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.