Identification of caspases and apoptosis in the simple metazoan Hydra.

Apoptosis is a normal process by which cells die and are eliminated from tissue by phagocytosis [1]. It is involved in regulating cell numbers in adult tissues and in eliminating 'excess' cells during embryogenesis and development. Apoptosis is mediated by activation of caspases, which then cleave a variety of cellular substrates and thereby cause the characteristic morphology of apoptotic cells (rounded cells, condensed chromatin, susceptibility to phagocytosis) [2]. Although apoptosis has been well documented in nematodes, insects and mammals, it is not yet clear how early in evolution apoptosis or its component enzymes arose. In the simple metazoan Hydra vulgaris, cell death regulates cell numbers [3] [4] [5]. In starved animals, for example, epithelial cell proliferation continues at a nearly normal rate although the tissue does not increase in size; the excess cells produced are eliminated by phagocytosis. Cell death can also be induced in wild-type hydra by treatment with colchicine [6] or in a mutant strain (sf-1) by temperature shock [7]. Here, we show that cell death in hydra is morphologically indistinguishable from apoptosis in higher animals, that hydra polyps express two genes with strong homology to members of the caspase 3 family, and that caspase-3-specific enzyme activity accompanies apoptosis in hydra. The occurrence of apoptosis and caspases in a member of the ancient metazoan phylum Cnidaria supports the idea that the invention of apoptosis was an essential feature of the evolution of multicellular animals.     

Spermatogenesis in Hydra oligactis. II. How temperature controls the reciprocity of sexual and asexual reproduction

Hydra oligactis undergo two mutually exclusive modes of reproduction: at warm temperatures (18-22 degrees C) animals reproduce asexually by budding, while at cold temperatures (10-12 degrees C) gamete differentiation occurs. Using a monoclonal antibody which is specific for cells of the sperm lineage, it was discovered that under conditions where sperm differentiation does not occur (18-22 degrees C), cells continually enter the sperm pathway but progression down the pathway is prematurely halted, effectively blocking the production of sperm. To elucidate the mechanism by which completion of sperm differentiation is controlled, the cell cycle times of interstitial cells entering the sperm pathway at both the restrictive (18 degrees C) and permissive (10 degrees C) temperatures were examined. It was envisaged that at the restrictive temperature the cell cycle times of committed cells would lengthen as they proceeded down the pathway, leading to dilution and eventual loss of cells at later stages of sperm differentiation. This did not occur. Although cells of the sperm lineage were found overall to divide more slowly at 18 degrees C than at 10 degrees C, at both temperatures the cell cycle times shortened as cells proceeded further down the pathway, making a dilution mechanism untenable. The effect of high temperature on the survival of cells was then tested by subjecting animals to a heat shock. Within 12 hr of the increase in temperature, the total number of sperm lineage interstitial cells dropped 10-fold while the total numbers of epithelial and somatic interstitial cells remained virtually unchanged. A distinct consequence of this cell loss was the disappearance of cells furthest down the sperm pathway. It is proposed that as cells move down the sperm pathway, they become increasingly sensitive to high temperature which adversely affects their survival; the higher the temperature, the earlier in the pathway cells die. The lethal effect is abolished by lowering the temperature, allowing sperm differentiation to continue to completion. The possible adaptive advantages of temperature controlling gametogenesis are discussed.     

Genetic analysis of developmental mechanisms in hydra: XIV. Identification of the cell lineages responsible for the altered developmental gradients in a mutant strain,reg-16

The developmental gradients of six chimeric strains of hydra produced from a normal strain (105) and a regeneration-deficient strain (reg-16) were analyzed. The reg-16 mutant has been shown to have a lower gradient of head-activation potential and a higher gradient of head-inhibition potential than the normal 105 strain. The chimeric animals consisted of different combinations of the three self-renewing cell lineages found in hydra (the ectodermal and endodermal epithelial cell lineages and the interstitial cell lineage) from each of the parental origins. To identify the cell lineages responsible for the abnormal gradients in reg-16, the head-activation and head-inhibition potentials of these cell lineage chimeras were assayed by lateral transplantation of tissue. The results obtained have provided evidence which indicates that the defect responsible for the low head-activation potential in reg-16 resides in its ectodermal and endodermal epithelial cell lineages, whereas the defect responsible for its high head-inhibition potential resides in its endodermal epithelial and interstitial cell lineages. The cellular localization of these defects is not identical but very similar to the cellular localization of the regenerative defects in reg-16. This finding is consistent with and supports the view that the abnormalities of the developmental gradients are correlated to the reduced head regenerative capacity in reg-16.     

Hydra, a versatile model to study the homeostatic and developmental functions of cell death

In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered.     

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.     

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.     

A SEM analysis of DMSO treated hydra polyps

Summary— Scanning electron microscopy revealed that exposure of hydra polyps to DMSO at concentrations used for permeabilizing tissue results in striking changes in epithelial cell morphology. Epithelial cells from treated polyps rounded up in shape and formed numerous large blebs at the cell surface. Along the borders of epithelial cells numerous small projections became detectable. The DMSO-induced changes at the cell surface corresponded to drastic changes in the intracellular organization. No evidence could be found for DMSO induced opening of cell junctions and/or opening of the interstitial space. The results demonstrate that DMSO affects the morphology and intracellular organization of hydra epithelial cells. Thus, caution is necessary in interpreting cell behavior in DMSO treated tissue.     

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.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.     

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     

A quantitative method for maceration of hydra tissue

Summary A method is described for the maceration (dissociation) of hydra tissue into single cells. The cells have characteristic morphology such that all basic types — epithelial, gland, mucous, interstitial, nematoblast, and nerve — can be distinguished. Criteria are given for identifying each cell type by phase contrast microscopy. It is shown that maceration quantitatively recovers cells from hydra tissue.     

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.     

In the multiheaded strain (mh-1) of Hydra magnipapillata the ectodermal epithelial cells are responsible for the formation of additional heads and the endodermal epithelial cells for the reduced ability to regenerate a foot

Hydra consist of three self-renewing cell lineages: the ectodermal epithelial, endodermal epithelial and interstitial cell lineages. The role of these cell lineages in head formation and foot regeneration in Hydra magnipapillata was studied by comparing the multiheaded strain mh-1 with the wild-type. Adult polyps of this strain show a reduced ability to regenerate a foot in the apical body half several days before additional heads are formed there. Cell lineage chimeras were produced, and it was found that in mh-1, the ectodermal epithelial cell lineage is responsible for the formation of additional heads, whereas the endodermal epithelial cell lineage and, to a lesser extent, the derivatives of the interstitial cell lineage, are responsible for the reduced ability of foot regeneration.     

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.     

Influence of intercellular contacts in epithelial sheets on the capacity of the cell surface for adhesion and phagocytosis of particles]

The influence of intercellular contacts on the ability of the upper cell surface to adsorb and to phagocytose particles was studied in different types of cultured cells of mouse origin. In cultures of the MPTR strain, cells formed firm contacts which remained unbroken during the epithelial sheet migration into the wound. The contact inhibition of phagocytosis was found in these cultures. The phenomenon involved a low phagocytic activity of the sheet cells which made intercellular contacts in all directions, and of high phagocytic activity of marginal cells which had activity moving free edges. Other epithelial cultures, such as explants of normal kidney and hepatoma 60, behaved similarly. Cultured embryo fibroblasts and hepatoma 22a cells did not form firm intercellular contacts and migrated into the wound one by one. In these cultures most cells had high phagocytic activity. It is suggested that the formation of intercellular contacts alters the upper cell surface ability to adhesion and phagocytosis of particles.     

Growth regulation in Hydra: Relationship between epithelial cell cycle length and growth rate

The relationship between epithelial cell production and growth rate was investigated in Hydra attenuata under different feeding regimes. The increase of epithelial cell number was compared to the duration of the epithelial cell cycle using standard methods of cell cycle analysis. The results indicate that cell cycle changes accompanying changes in feeding regime are not sufficient to explain the altered growth rate. Under heavy feeding regimes, epithelial cell production equals tissue growth rate. At low feeding level or under starvation conditions the epithelial cell cycle lengthens and growth rate of epithelial cell population is slowed. However, the cell cycle changes are insufficient to account for the reduction in tissue growth and thus there is an effective overproduction of epithelial cells amounting to 10% per day. Evidence suggests that these excess cells are phagocytized by neighboring cells in the tissue. Thus phagocytosis is directly or indirectly involved in regulating the growth of hydra tissue.     

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.     

Ultrastructural changes in the gastrodermal phagocytic cells of the planarian Dugesia gonocephala s.l. during food digestion (Plathelminthes)

Summary In the present report the functional morphology of the planarian gastrodermal phagocytic cells is examined in feeding animals. A functional interpretation of some of the morphological findings is given. The events in the fine-structure modifications of the phagocytic cells in the course of phagocytosis and intracellular digestion of food particles were followed through five post-feeding stages in the planarian Dugesia gonocephala. Light and electron microscopical observations demonstrate that there is preliminary intraluminal digestion of food particles; their phagocytosis takes place quickly.Beef hepatocytes that served as food are found engulfed at first in food vacuoles near the apical border of the phagocytic cells, and are clearly recognizable. The vacuoles increase in number to occupy most of the cytoplasm of these cells. Progressive breakdown and disappearance of phagocytosed hepatocytes occurs. In time the vacuoles move deeper into the cells, their contents lose their identity, and condense to homogeneous or heterogeneous residual bodies. These are returned to the distal surface of the cells, and then voided into the intestinal lumen. At the same time, synthesis and accumulation of numerous lipid droplets occurs, probably as a final product resulting from metabolism of the digested material. When feeding is over, the phagocytic cells are filled with lipid droplets, acquiring their typical appearance.It is suggested that disintegration of phagocytic cells during starvation is balanced by proliferation and differentiation of neoblasts into new phagocytic cells during the feeding-starvation cycle.     

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.     

In vitro effect of temperature on phagocytic and cytotoxic activities of splenic phagocytes of the wall lizard, Hemidactylus flaviviridis.

The in vitro effect of temperature on phagocytosis, nitric oxide production and interleukin-1 (IL-1) secretion by splenic phagocytes isolated from the wall lizard (Hemidactylus flaviviridis) demonstrated that changes in temperature altered non-specific defenses. The LPS-induced percentage phagocytosis and phagocytic index were recorded maximum at 25 degrees C. The phagocytic activity declined considerably when the phagocytes were incubated at low (7 and 15 degrees C) or high (37 degrees C) temperatures. The presence of bacterial lipopolysaccharide (LPS) in the incubation medium could considerably enhance the phagocytic activity of splenic phagocytes. A similar temperature-related effect was also observed on LPS-induced cytotoxic activity of phagocytes. LPS could stimulate the nitrite release indicating nitric oxide production only at 25 degrees C. Likewise, the proliferative responses of immature rat's thymocytes to LPS-induced phagocyte-conditioned medium suggest that IL-1 secretion was enhanced when phagocytes were cultured at 25 degrees C. This suggests that 25 degrees C is the optimal temperature for phagocyte functions in H. flaviviridis. The decrease or increase in temperature other than at 25 degrees C dramatically suppressed the phagocyte activities.