Monoclonal Antibodies againstAntigens of Hydra vulgaris and Hydra oligactis

The goal of the study was to obtain a panel of monoclonal antibodies (MAb) against antigens of freshwater polyps of the genus Hydra. Hybrid mice F₁(Balb/c × SJL/J) were immunized with cell membrane fraction of H. vulgaris and three months later their splenocytes were fused with cultured mouse myeloma cells 653A. Testing of culture fluids in ELISA with immobilized H. vulgaris cells, 82 hybridomas producing MAb were revealed. Study of MAb specificity in ELISA with H. vulgaris and H. oligactis cells indicated that 22% of them recognized only H. vulgaris antigens. About 50% of MAb recognized equally antigens of the both species. The rest of MAb reacted with H. vulgaris and H. oligactis antigens to different degree. Eight hybridomas producing MAb of all three above groups were adapted for growth as ascitic tumors. The distribution of antigens binding these MAb was studied in indirect immunofluorescence on fixed polyps, living or fixed cells, and on paraffin- embedded sections. Among the best studied MAb, of the greatest interest were the following reagents. One of them (1A10) revealed an antigen on surface membranes of ectodermal epithelial cells of H. vulgaris. The second one (1G10) was specific of the antigen located in mesoglea and basal cytoplasmic areas of ectodermal and entodermal epithelial cells of the both hydra species. The MAb 4G3 interacted with cytoplasmic antigen of ectodermal epithelia-muscular cells of the both hydra species. MAb 4H1 revealed nematocytes in H. vulgaris and H. oligactis. The data obtained indicate that in two species of hydra the epitopes binding the same MAb might be located in cells of different types.     

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.     

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.     

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.     

Maintenance and Regeneration of the Nerve Net in Hydra

SYNOPSIS. Due to the tissue dynamics, the entire nervous systemof a hydra is in a steady state of production and loss of neuronsNeurons arise by differentiation from the interstitial cellsin the ectoderm Nerve cell intermediates migrate along the bodycolumn, settle and complete differentiation The type of neuronformed is a function of axial or regional location in whichthe differentiation intermediate settles Another result of thetissue dynamics is that all neurons are constantly changingtheir location As a consequence, many neurons switch from onetype of nerve cell to another in accord with positional changes     

Life history traits and previous exposure predict resistance to UV irradiation in the freshwater cnidarian Hydra oligactis

Abiotic stress is an important source of mortality for cnidarians and is likely to be a major factor shaping their life histories. In freshwater hydra, the ability to withstand exogenous sources of stress varies between species and populations, but little is known about the factors responsible for this variation. Here, we investigated resistance to UV irradiation in Hydra oligactis, a common temperate freshwater cnidarian. We collected polyps from 12 populations and propagated these asexually under standard conditions in the laboratory to obtain 69 laboratory clonal lines with a total of 324 polyps of different age. We measured the size of polyps and recorded their budding rate. In addition, a subset of animals was exposed to hormetic treatment, where experimental animals received a short, sublethal irradiation 2 d before testing their resistance to a higher dose. We investigated how life history traits (age, size, and budding rate), hormetic treatment, and the interaction between life history traits and hormetic treatment relate to the ability of hydra polyps to tolerate high doses of UV irradiation. In multivariate models controlling for the effect of other variables, stress tolerance was positively related to age (lower tolerance in freshly detached buds compared to adult hydra) and size (higher tolerance in polyps with a large body column). Budding rate was negatively associated with stress tolerance. Hormetic treatment increased resistance to UV irradiation, but we found no evidence for an interaction between hormetic response and any of the life history traits, suggesting that the ability to upregulate physiological defense mechanisms after exposure to mild stress does not depend on the life history background of the individuals.     

Suppression of Head Regeneration by Accelerated Wound Healing in Hydra

Published evidence suggests that tissue injury is important for head regeneration in hydra [MacWilliams, 1982, 1983a,b; Kobatake and Sugiyama, 1989]. To investigate this problem in more detail, two experimental manipulations, decapitation and mirror-image grafting, were carried out. In the latter, two decapitated polyps were axially grafted to each other to make the wound openings of the two polyps juxtaposed on each other. In normal regenerates, the wound opening closed and healed in 4 to 5 hr, while in mirror-image grafts it healed in about 1 hr. The percentage of head regeneration was lower in mirror-image grafts than that after decapitation. The effect of mirror-image grafting on morphogenetic potential levels was examined using a lateral transplantation technique. Head inhibition levels dropped in both types of regenerates to a similar extent. Head activation levels rose more in normal regenerates than in mirror-image grafts. These results show clearly that the drop in head inhibition level is due to removal of the head and is not affected by grafting. They also show that the increase in head activation levels and in the percentage of head regeneration is affected substantially by the grafting. These observations are consistent with the view that decapitation produced a greater injury effect than mirror-image grafting, and this injury effect raised the head activation level whereas it did not alter the head inhibition level. The fact that the wound remained open for a longer time in normal regenerates than in the grafts suggests that the injury effect depends not on tissue injury itself but on the length of time the wound is open.     

A subset of cells in the nerve net of Hydra oligactis defined by a monoclonal antibody: its arrangement and development

A monoclonal antibody, termed JD1, was generated that bound to a subset of the nerve cells in the hypostome and tentacles of Hydra oligactis. Using a whole-mount technique the spatial pattern of the subset of nerve cells and their processes could be clearly visualized using indirect immunofluorescence. The subset largely corresponds to the epidermal sensory cells. Using the same technique the development of the pattern during head regeneration and budding was examined. The appearance of the nerve cells coincides with the formation of both the tentacles and hypostome. When head regeneration does not occur, JD1+ cells do not appear suggesting the differentiation of JD1+ cells is an integral event in head formation dependent on antecedent patterning processes.     

Seasonal variation of genotypes and reproductive plasticity in a facultative clonal freshwater invertebrate animal (Hydra oligactis) living in a temperate lake

Facultative sexual organisms combine sexual and asexual reproduction within a single life cycle, often switching between reproductive modes depending on environmental conditions. These organisms frequently inhabit variable seasonal environments, where favorable periods alternate with unfavorable periods, generating temporally varying selection pressures that strongly influence life history decisions and hence population dynamics. Due to the rapidly accelerating changes in our global environment today, understanding the population dynamics and genetic changes in facultative sexual populations inhabiting seasonal environments is critical to assess and prepare for additional challenges that will affect such ecosystems. In this study, we aimed at obtaining insights into the seasonal population dynamics of the facultative sexual freshwater cnidarian Hydra oligactis through a combination of restriction site‐associated sequencing (RAD‐Seq) genotyping and the collection of phenotypic data on the reproductive strategy of field‐collected hydra strains in a standard laboratory environment. We reliably detected 42 MlGs from the 121 collected hydra strains. Most of MLGs (N = 35, 83.3%) were detected in only one season. Five MLGs (11.9%) were detected in two seasons, one (2.4%) in three seasons and one (2.4%) in all four seasons. We found no significant genetic change during the 2 years in the study population. Clone lines were detected between seasons and even years, suggesting that clonal lineages can persist for a long time in a natural population. We also found that distinct genotypes differ in sexual reproduction frequency, but these differences did not affect whether genotypes reappeared across samplings. Our study provides key insights into the biology of natural hydra populations, while also contributing to understanding the population biology of facultative sexual species inhabiting freshwater ecosystems.     

A Comparative Analysis of Gene Expression Profiles during Skin Regeneration in Mus and Acomys

The African spiny mouse (Acomys spp.) can heal full thickness excisional skin wounds in a scar-free manner with regeneration of all dermal components including hair and associated structures. Comparing Acomys scar-free healing from Mus scarring identifies gene expression differences that discriminate these processes. We have performed an extensive comparison of gene expression profiles in response to 8mm full-thickness excisional wounds at days 3, 5, 7 and 14 post-wounding between Acomys and Mus to characterize differences in wound healing, and identify mechanisms involved in scar-free healing. We also identify similarities with scar-free healing observed in fetal wounds. While wounding in Mus elicits a strong inflammatory response, wounding in Acomys produces a moderated immune response and little to no increase in expression for most cytokines and chemokines assayed. We also identified differences in the ECM profiles of the Acomys wounds, which appear to have a collagen profile more similar to fetal wounds, with larger increases in expression of collagen types III and V. In contrast, Mus wounds have very high levels of collagen XII. This data suggests that an overall lack of induction of cytokines and chemokines, coupled with an ECM profile more similar to fetal wounds, may underlie scar-free wound healing in Acomys skin. These data identify candidate genes for further testing in order to elucidate the causal mechanisms of scar-free healing.     

Protein kinase modulators interfere with bud formation in Hydra vulgaris

The fresh water polyp Hydra can reproduce asexually by forming buds. These buds separate from the parent animal due to the development of foot tissue in a belt-like region and the formation of a constriction basal to that region. A single pulse treatment with activators of protein kinase C, including 1,2-dioctanoyl-rac-glycerol and 12-o-tetradecanoylphorbol-13-acetate, and inhibitors of various protein kinases, including staurosporine, H-7 and genistein, interfered with foot and constriction formation. The buds did not separate. Therewith, branched animals were formed, some of which bore a lateral foot patch. Simultaneous treatments with an activator and inhibitor led to a higher amount of branched animals than treatments with one of these agents alone. Based on the different specificities of the activators and inhibitors used we propose that activation of a protein kinase C and/or inhibition of a probably non-C-type protein kinase interfere with the decrease of positional value at the bud's base, a process necessary to initiate the pattern forming system leading to foot formation. Correspondence to: F. Perez     

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.