Migration of multipotent interstitial stem cells in Hydra

Stem cells in Hydra represent one of the phylogenetically most ancient stem cell systems and, therefore, provide information for reconstructing the early history of stem cell control mechanisms. Hydra's interstitial stem cells are multipotent and differentiate into both somatic cell types and germ line cells. Although it is well accepted that cells of the interstitial cell lineage are migratory, the in vivo migratory potential of multipotent interstitial stem cells has never been explored. Combining in vivo tracing of genetically labeled interstitial stem cells and tissue transplantation, we show that in contrast to precursor cells, multipotent interstitial stem cells are stationary. Only when exposed to tissue depleted of the interstitial cell lineage, interstitial stem cells start to migrate and to repopulate emptied stem cell niches. We conclude that multipotent interstitial stem cells in Hydra are static and that microenvironmental cues including signals derived from the interstitial cell lineage or from niche cells can trigger a shift in collective stem cell behavior to start migration.     

The fine structure of differentiating interstitial cells in Hydra

Summary Interstitial cells of hydra are small undifferentiated cells containing an abundance of free ribosomes and few other cytoplasmic organelles. They are capable of differentiating into epitheliomuscular, digestive, glandular, nerve cells, and cnidoblasts. Developing epitheliomuscular and digestive cells acquire bundles of filaments, 50 Å in diameter, which later are incorporated into the muscular processes. Early gland cells develop an elaborate rough-surfaced endoplasmic reticulum and one or more Golgi apparatus. Secretory granules originate in the Golgi region eventually filling the apex of the cell. Neurons are recognized first by the presence of an elaborate Golgi apparatus, absence of a well-developed endoplasmic reticulum, and later the appearance of cytoplasmic processes. The most striking feature of nematocyst formation by cnidoblasts is the presence of a complex distribution system between protein synthesizing rough-surfaced endoplasmic reticulum and the nematocyst. This system consists of connections between cisternae of the endoplasmic reticulum with smooth Golgi vesicles which in turn are connected to minute tubules, 200 Å in diameter. The tubules extend from the Golgi region around the nematocyst finally entering the limiting membrane of the nematocyst. It is suggested that the interstitial cells of hydra represent a model system for the investigation of many aspects of cell differentiation.This work was supported by grants from the National Cancer Institute (TlCA-5055) and from the National Institute of Arthritis and Metabolic Diseases (AM-03688), National Institutes of Health, Department of Health, Education and Welfare.The author is indebted to Dr. Russell J. Barrnett for his guidance and interest throughout this investigation.     

Distribution of interstitial stem cells in Hydra

The distribution of interstitial stem cells along the Hydra body column was determined using a simplified cloning assay. The assay measures stem cells as clone-forming units (CFU) in aggregates of nitrogen mustard inactivated Hydra tissue. The concentration of stem cells in the gastric region was uniform at about 0.02 CFU/epithelial cell. In both the hypostome and basal disk the concentration was 20-fold lower. A decrease in the ratio of stem cells to committed nerve and nematocyte precursors was correlated with the decrease in stem cell concentration in both hypostome and basal disk. The ratio of stem cells to committed precursors is a sensitive indicator of the rate of self-renewal in the stem cell population. From the ratio it can be estimated that <10% of stem cells self-renew in the hypostome and basal disk compared to 60% in the gastric region. Thus, the results provide an explanation for the observed depletion of stem cells in these regions. The results also suggest that differentiation and self-renewal compete for the same stem cell population.     

Gland cells arise by differentiation from interstitial cells in Hydra attenuata

The origin of the gland cells in asexually reproducing adult hydra is unclear. There is evidence suggesting that the gland cells are a self-renewing population as well as contrary evidence suggesting that they must arise from another cell type. We have reexamined the question and found the latter to be the case. Analysis of ectoderm/endoderm chimeras in which the ectoderm was labeled with [3H]thymidine indicates a precursor for gland cells in the ectoderm which migrates into the endoderm. Analysis of grafts between labeled lower halves and unlabeled upper halves of animals indicates the migratory precursor is either a large or a small interstitial cell. Measurement of the cell cycle times of the gland cells and the epithelial cells provided further support. The cell cycle time of the gland cells appears to be longer than that of the epithelial cells of the endoderm throughout the animal. This means that in the steady-state growth condition of hydra tissue, the gland cells cannot maintain their population size simply by cell division. These results and other data suggest the following dynamics for the gland cell population. Gland cells arise by differentiation from large interstitial cells, undergo a limited number of cell divisions, and then become postmitotic.     

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).     

A head signal influences apical migration of interstitial cells in Hydra vulgaris

Although interstitial cells of hydra can migrate either apically or basally along the body column, there is a distinct bias toward apical cell accumulation. This apical bias could be produced by a local vectorial property of the tissue or it may be controlled by a more global property, such as a signal from the apical head region. The migration behavior of BrdU-labeled interstitial cells was examined in several types of grafts to distinguish between these two general types of migration control. Grafting BrdU-labeled midgastric region tissue into a host in either the normal or the reverse orientation had no effect on the apical bias, indicating that a local vectorial cue was probably not guiding cells apically. In grafts with heads or with feet at both ends of the body column, there was no directional bias in migration if the labeled tissue was equidistant from both ends. In the two-headed grafts, if the labeled tissue was closer to one end, there was a bias in the direction of the closer head. The results suggest that a graded signal emanating from the head creates the apical bias and may attract cells via chemotaxis. The apical bias is enhanced in decapitated animals regenerating a head, indicating that the attracting signal is present and is possibly stronger in regenerating heads. The signal for cell migration may be involved in a patterning process underlying head regeneration.     

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.     

A histological and ultrastructural study of germinal differentiation of interstitial cells arising from gland cells in Hydra viridis

Gland cells of the gastrodermis of Hydra when isolated from the epidermis are capable of dedifferentiating into interstitial cells. Under proper environmental conditions these interstitial cells are capable of undergoing meiotic divisions and forming normal gametes. This dedifferentiation and redifferentiation sequence has been studied at the level of the light and electron microscope. It is concluded that in Hydra there is no specific germinal cell line determined during embryogeny, and that a somatic cell under proper environmental conditions can be induced to undergo meiosis.     

Germ cells in Hydra oligactis males. I. Isolation of a subpopulation of interstitial cells that is developmentally restricted to sperm production

Single clones of interstitial cells were generated and analyzed to determine if one interstitial cell has the capacity to differentiate both somatic and germ cells. Such clones were produced by using hydroxyurea to selectively eliminate interstitial cells from normal Hydra oligactis males. The number of animals devoid of interstitial cells within the population was determined by staining whole animals with toluidine blue which renders the interstitial cells visible. The number of animals containing single clones of interstitial cells was then estimated using single hit Poisson statistics. In treatments which rendered 60-80% of the population devoid of interstitial cells, the majority of the animals containing interstitial cells lost the ability to produce somatic cells, including nerves and nematocytes, but retained the capacity to produce sperm. This result strongly suggests the presence of a separate germ line in hydra.     

The ultrastructure of the zymogen cells in Hydra viridis

Summary The zymogenic secretory cells of Hydra viridis are scattered between the digestive muscle cells of the gastric region. The mature zymogenic cells are located along the apical surface of the gastrodermal epithelium and contain numerous spherical secretory droplets. They appear to differentiate from stem cells located near the mesoglea. These stem cells resemble epidermal interstitial cells and are filled with free ribosomes. They differ from the interstitial cell in that they usually possess a small amount of granular endoplasmic reticulum. During the process of differentiation they elaborate a highly organized system of granular endoplasmic reticulum. This system becomes dispersed into vesicles as the secretory product is synthesized. There is no indication that the Golgi apparatus participates directly in the formation of the secretory droplets, and there is no indication of a membrane bounding the mature secretory droplet.The fate of the zymogenic cell following the discharge of its secretory product was not determined. It is possible that these cells revert back to a stage resembling the stem cell before resynthesizing a new supply of secretion. In this case the normal secretory process would be very similar to the events described in the dedifferentiation of the zymogenic cells during regeneration.This work was supported by Grant number GB-3262 from the National science Foundation.     

Sex determination in hydra: control by a subpopulation of interstitial cells in Hydra oligactis males

The stability of sexual phenotype was examined in a single clone of Hydra oligactis males maintained at two culture temperatures, 18 and 22 degrees C. At these temperatures animals of this species do not reproduce sexually, but reproduce asexually by budding, and males and females are morphologically indistinguishable. When the temperature is lowered to 10 degrees C gametogenesis is induced and sexual phenotype can be assayed. Males cultured for several years at 18 degrees C expressed a stable sexual phenotype when induced to undergo gametogenesis; males remained male. Those cultured at 22 degrees C for 1 year, however, expressed a low frequency of sex reversal from male to female; males ceased sperm differentiation and began producing eggs. Male sex reversal in cultures maintained at the higher temperature was correlated with the loss of a specific subpopulation of interstitial cells, those that bind the monoclonal antibody, AC2, which labels cells specific to the spermatogenic pathway in H. oligactis males. When interstitial cells restricted to this pathway were reintroduced into sex-reversed males (phenotypic females), the male phenotype was reestablished and animals reverted to sperm production. To further investigate the role of AC2+ cells in the masculinization of females, normal males (containing AC2+ cells) and sex-reversed males (lacking AC2+ cells) were grafted to females. In grafts between normal males and females, egg production ceased and sperm differentiation ensued, whereas those between sex-reversed males and females continued to produce eggs. Thus, the presence of AC2+ interstitial cells is strictly correlated with male sexual phenotypes and it is only in their absence that the female phenotype is expressed.     

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     

Germ cells in Hydra oligactis males. II. Evidence for a subpopulation of interstitial stem cells whose differentiation is limited to sperm production

Animals containing germline-restricted interstitial cells were obtained by treating males from a clone of Hydra oligactis with hydroxyurea (HU) to lower the interstitial population to 1 or 2 cells per animal. A 3-day HU treatment produced animals whose interstitial cells did not form somatic cells, but did produce sperm. The isolation of these cells in HU-treated animals has lead us to propose that the interstitial cell population may contain subpopulations which possess different growth dynamics and developmental potentials. Through asexual propagation, we have cloned several animals containing only sperm precursor interstitial cells and have examined the growth and differentiation behavior of these cells in offspring propagated over a 2-year period. Evidence has been obtained which demonstrates (1) the extensive self-renewal capacity of the sperm precursor interstitial cells, and (2) the restricted differentiation capacity of these interstitial stem cells. Factors which affect cells entering and traversing the spermatogenic pathway are also presented.     

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.     

Hydra and the evolution of stem cells

Hydra are remarkable because they are immortal. Much of immortality can be ascribed to the asexual mode of reproduction by budding, which requires a tissue consisting of stem cells with continuous self‐renewal capacity. Emerging novel technologies and the availability of genomic resources enable for the first time to analyse these cells in vivo. Stem cell differentiation in Hydra is governed through the coordinated actions of conserved signaling pathways. Studies of stem cells in Hydra, therefore, promise critical insights of general relevance into stem cell biology including cellular senescence, lineage programming and reprogramming, the role of extrinsic signals in fate determination and tissue homeostasis, and the evolutionary origin of these cells. With these new facts as a backdrop, this review traces the history of studying stem cells in Hydra and offers a view of what the future may hold.     

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.