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.     

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.     

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.     

Both the epithelial cells and the nerve cells are involved in the head inhibition properties in Hydra attenuata

The aberrant, a morphological mutant of Hydra attenuata, has been shown to have altered inhibition properties(D. I. Rubin and H. R. Bode, 1982, Develop. Biol. 89, 316–331). Based on transplantation experiments, the inhibition gradient in the aberrant is steeper and higher near the head than that in the normal. Also, the amount of inhibition transmitted by the aberrant head is higher. Both epithelial cells and nerve cells have been implicated in the patterning process of hydra. The cell types involved in the altered inhibition properties of the aberrant were determined by making chimeras consisting of epithelial cells from one strain and nerve cells from the other. Experiments with these chimeras demonstrated that the epithelial cells were responsible for the altered inhibition gradient of the aberrant. In contrast, both the epithelial cells and the nerve cells were involved in the higher amount of inhibition transmitted by the aberrant head. Thus, both cell types are involved in this aspect of patterning.     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Regulation of interstitial cell differentiation inHydra attenuata

Summary The axial position of interstitial-cell (i-cell) differentiation into nematocytes inHydra was studied. Nests of developing nematoblasts of three types of nematocytes were distributed in a non-uniform manner along the body column. Stenotele nematoblasts were distributed in a gradient with a maximum in the peduncle. Desmoneme and atrichous isorhiza nematoblasts were found predominantly in the upper half of the body region. These results suggest that the type of nematocyte differentiation an i-cell undergoes is influenced by the axial position of the i-cell. Because the assayed stage of nematocyte differentiation occurred 6–7 days after beginning of differentiation, the axial position of the anticedent i-cell at the time of commitment was determined by correcting for tissue displacement.     

Homeostatic recovery of interstitial cell populations in Hydra.

The growth of interstitial cell populations in Hydra magnipapillata was examined following transplantation of small numbers of interstitial cells into "epithelial animals" which lacked all cell types in the interstitial cell lineage. The distribution pattern of transplanted interstitial cells during the growth phase was examined by staining whole animals with toluidine blue and cell numbers were determined by maceration. The following results were obtained: (1) Transplanted interstitial cells formed a contiguous patch which spread distoproximally but not circumferentially. (2) The displacement of interstitial cells from parents to buds was a random process; buds incorporated interstitial cells only when they were formed in the vicinity of the patch. (3) Interstitial cells increased exponentially in number with a doubling time of 1.8 days for at least 10 days after transplantation, which is faster than the normal doubling time of 2.8 days. (4) The self-renewal probability at low interstitial cell levels was estimated to be 0.72, which was higher than the normal value of 0.64. This increase was attained by lowering the fraction of nematocyte differentiation. These results indicate that the homeostatic recovery of interstitial cell populations is attained by increasing the self-renewal probability rather than by preferential retention of interstitial cells in parent animals at the expense of buds (Heimfeld, 1985).     

Ultrastructural analysis of nematocyte removal in Hydra

A consecutive series of ultrathin sections through the distal one-third of a Hydra tentacle has revealed at least four categories of nematocytes: (1) normal, mounted nematocytes, in specific arrangements within the battery cells; (2) degenerating nematocytes, within the battery cells; (3) mature nematocytes, enclosed within endodermal cells; (4) a mature nematocyte, in the enteric cavity. The degenerating nematocytes within the battery cells and the nematocytes in the endoderm and enteric cavity appeared to be aging nematocytes undergoing death and removal. The results provide the first ultrastructural evidence for nematocyte degeneration within battery cells and also suggest phagocytosis of mature nematocytes by endodermal cells.     

Disturbing epithelial homeostasis in the metazoan Hydra leads to drastic changes in associated microbiota

Microbes have profound influence on the biology of host tissue. Imbalances in host–microbe interaction underlie many human diseases. Little, however, is known about how epithelial homeostasis affects associated microbial community structure. In Hydra , the epithelium actively shapes its microbial community indicating distinct selective pressures imposed on the epithelium. Here, using a mutant strain of Hydra magnipapillata we eliminated all derivatives of the interstitial stem cell lineage while leaving both epithelial cell lineages intact. By bacterial 16S rRNA gene analysis we observed that removing gland cells and neurones from the epithelium causes significant changes in hydra's microbial community. Absence of interstitial stem cells and nematocytes had no affect on the microbiota. When compared with controls, animals lacking neurones and gland cells showed reduced abundance of β-Proteobacteria accompanied by a significantly increased abundance of a Bacteroidetes bacterium. This previously unrecognized link between cellular tissue composition and microbiota may be applicable to understanding mechanisms controlling host–microbe interaction in other epithelial systems.     

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.     

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.     

Arrangement of accessory cells and nematocytes bearing mastigophores in the tentacles of the cubozoan Chironex fleckeri

Nematocytes containing microbasic mastigophores are intimately associated with accessory cells in the epidermis of Chironex fleckeri. Large microbasic mastigophores may be surrounded by seven to nine such cells. Each accessory cell possesses an apical portion containing secretory droplets and a basal portion which carries a radially oriented fibre linking the cell to the underlying mesogloea. The fibre is capable of projecting and retracting the accessory cell. Junctional complexes occur between accessory cells and the apical regions of neighbouring mastigophores. Each nematocyte bearing a mastigophore contains a triggering apparatus consisting of a cnidocil surrounded by microvilli. This apparatus protrudes from an invagination in the apical region of the nematocyte and is exposed when the mastigophore is in the fire-ready position. A basket of filaments which make contact with microvilli surrounds the apical end of the nematocyst like a collar. The basket is linked via fibrous bundles which envelop the mastigophore to radially oriented fibres basally. These fibres are capable of projecting and retracting the mastigophore and its associated triggering apparatus. Up to nine such fibres were observed to be associated with a single large microbasic mastigophore. Microtubules averaging 25 nm in diameter and linked via cross bridges to electrondense material were detected in the radial fibres of both nematocytes and accessory cells. Retraction of the accessory cells and projection of nematocytes result in mastigophores being brought to the firing line and in the exposure of the cnidocil apparatus.     

Cell cycle length, cell size, and proliferation rate in hydra stem cells

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.     

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     

The Notch signaling pathway in the cnidarian Hydra

Many of the major pathways that govern early development in higher animals have been identified in cnidarians, including the Wnt, TGFbeta and tyrosine kinase signaling pathways. We show here that Notch signaling is also conserved in these early metazoans. We describe the Hydra Notch receptor (HvNotch) and provide evidence for the conservation of the Notch signaling mode via regulated intramembrane proteolysis. We observed that nuclear translocation of the Notch intracellular domain (NID) was inhibited by the synthetic gamma-secretase inhibitor DAPT. Moreover, DAPT treatment of hydra polyps caused distinct differentiation defects in their interstitial stem cell lineage. Nerve cell differentiation proceeded normally but post-mitotic nematocyte differentiation was dramatically reduced. Early female germ cell differentiation was inhibited before exit from mitosis. From these results we conclude that gamma-secretase activity and presumably Notch signaling are required to control differentiation events in the interstitial cell lineage of Hydra.     

Impaired stria vascularis integrity upon loss of E-cadherin in basal cells

In the cochlea, sensory transduction depends on the endocochlear potential (EP) and the unique composition of the endolymph, both of which are maintained by a highly specialized epithelium at the cochlear lateral wall, the stria vascularis. The generation of the EP by the stria vascularis, in turn, relies on the insulation of an intrastrial extracellular compartment by epithelial basal cells. Despite the physiological importance of basal cells, their cellular origin and the molecular pathways that lead to their differentiation are unclear. Here, we show by genetic lineage tracing in the mouse that basal cells exclusively derive from the otic mesenchyme. Conditional deletion of E-cadherin in the otic mesenchyme and its descendants does not abrogate the transition from mesenchymal precursors to epithelial basal cells. Rather, dedifferentiation of intermediate cells, altered morphology of basal and marginal cells and hearing impairment due to decreased EP in E-cadherin mutant mice demonstrate an essential role of E-cadherin in terminal basal cell differentiation and their interaction with other strial cell types to establish and maintain the functional architecture of the stria vascularis.     

Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes,epithelial cells,and basement membrane

Summary Nematocytes (stinging cells) of hydra tentacles are anchored to the basement membrane by peculiar complex junctions in which a flattened tongue of an epithelial cell is interposed between the nematocyte and the basement membrane. In this paper we describe the arrangement of these junctions with emphasis on how they are related to the architecture of the epithelial cell. Each epithelial cell, called a battery cell, harbors 10–20 nematocytes and bears muscle processes that extend along the basement membrane. The epithelial cell component of the complex junction is usually a lateral extension of a muscle process. All nematocytes within a battery cell make junctions with muscle processes of the same (resident) epithelial battery cell despite the presence of numerous muscle processes from adjacent (foreign) cells. Some nematocytes make junctions with several resident processes, spanning the foreign processes to do so. Most junctions reside near the proximal ends of the muscle processes. New findings are reported on the substructure of the junctions. They are composed of aggregates of smaller elements, and the cytoskeleton within the complexes has a pronounced longitudinal organization. These observations are consistent with a suggestion that the complex junctions develop by aggregation of smaller junctional units originating elsewhere on the cells.     

Regulation of interstitial cell differentiation in Hydra attenuata. I. Homeostatic control of interstitial cell population size.

Mechanisms regulating the population size of the multipotent interstitial cell (i-cell) in Hydra attenuata were investigated. Treatment of animals with 3 cycles of a regime of 24 h in 10-2 M hydroxyurea (HU) alternated with 12 h in culture medium selectively killed 95-99% of the i-cells, but had little effect on the epithelial cells. The i-cell population recovered to the normal i-cell:epithelial cell ratio of I:I within 35 days. Continuous labelling experiments with [3H]thymidine indicate that the recovery of the i-cell population is not due to a change in the length of the cell cycle of either the epithelial cells or the interstitial cells. In control animals 60% of the i-cell population undergo division daily while 40% undergo differentiation. Quantification of the cell types of HU-treated animals indicates that a greater fraction of the i-cells were dividing and fewer differentiating into nematocytes during the first 2 weeks of the recovery after HU treatment. Therefore, the mechanism for recovery involves a shift of the 60:40 division:differentiation ratio of i-cells towards a higher fraction in division until the normal population size of the i-cells is regained. This homeostatic mechanism represents one of the influences affecting i-cell differentiation.