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.     

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).     

Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.     

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.     

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.     

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.     

Oogenesis in hydra. II. Cytophotometric determination of DNA concentration in the nuclei of somatic and sex cells

During sexual reproduction in Hydra, interstitial cells in the female sex zone of the body (i-cells) undergo mitotic division and form a thickening in the epiderm. The proliferation of i-cells is accompanied by the increase of cytoplasm volume and by the appearance in the cytoplasm of a great number of membranous structures (rough endoplasmic reticulum, Golgi apparatus and mitochondria), enzymatic granules, lipid inclusions and glycogen. All cells of the epidermal thickening soon (in approximately twenty four hours) acquire the characteristics of typical phagocytes. However it is the cell situated inside the group of syncytially connected ones and adjacent to mesogloea that begins to grow rapidly and phagocytize surrounding cells. The cells of the epidermal thickening, though they are often given the name of oogonia, were found to have a tetraploid DNA content in their nuclei. The presence of four unseparated centrioles of equal size suggests that all preparatory processes for division were completed. A conclusion was drawn that cells of the epidermal thickening undergo premeiotic DNA synthesis prior to their phagocytizing by the growing oocyte and, thus, are oocytes themselves. The oogonial stage in Hydra coincides with the early period of mitotic reproduction of i-cells. The data obtained are discussed from the viewpoint of the formation of the accessory gonad apparatus.     

Totipotent migratory stem cells in a hydroid

Hydroids, members of the most ancient eumetazoan phylum, the Cnidaria, harbor multipotent, migratory stem cells lodged in interstitial spaces of epithelial cells and are therefore referred to as interstitial cells or i-cells. According to traditional understanding, based on studies in Hydra, these i-cells give rise to several cell types such as stinging cells, nerve cells, and germ cells, but not to ectodermal and endodermal epithelial cells; these are considered to constitute separate cell lineages. We show here that, in Hydractinia, the developmental potential of these migratory stem cells is wider than previously anticipated. We eliminated the i-cells from subcloned wild-type animals and subsequently introduced i-cells from mutant clones and vice versa. The mutant donors and the wild-type recipients differed in their sex, growth pattern, and morphology. With time, the recipient underwent a complete conversion into the phenotype and genotype of the donor. Thus, under these experimental conditions the interstitial stem cells of Hydractinia exhibit totipotency.     

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     

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.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

Growth regulation of the interstitial cell population in hydra. III. Interstitial cell density does not control stem cell proliferation

The interstitial cells of hydra contain a stem cell population which produces several classes of differentiated cell types. A model has been proposed which governs the growth rate of the interstitial cell population. This model, based on the density of interstitial cells in the tissue, makes specific predictions about the relationships among this density, the proportion of stem cells in the interstitial cell population, the growth rate of the interstitial cell population, and the amount of nematocyte differentiation. Hydroxyurea treatments were used to experimentally reduce interstitial cell numbers, and the validity of these expected correlations was tested. The results demonstrate that the predictions of the interstitial cell density model were not upheld. Furthermore, the findings suggest that the interstitial cells are a heterogeneous population, containing some cells which are no longer stem cells but which do retain a limited capacity for proliferation. In the following paper (S. Heimfeld and H.R. Bode, 1986, Dev. Biol. 115, 59-68) we have proposed an alternative mechanism to explain the observed correlations, which incorporates this heterogeneity into amplification divisions of interstitial cells already committed to differentiation.     

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.     

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.     

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.     

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.