Thomas W. Holstein

Pattern of epithelial cell cycling in hydra

We have investigated the spatial pattern of epithelial cell cycling in a mutant strain of Hydra magnipapillata (sf-1). This strain has temperature sensitive interstitial stem cells and thus polyps containing only epithelial cells can be obtained by growth at the restrictive temperature. Epithelial animals were pulse labeled with the thymidine analog 5-bromo-2-deoxyuridine (Brdu) and stained with anti-Brdu antibody to visualize S phase cells. Our results indicate that Brdu-labeled cells are broadly and fairly evenly distributed along the body column. Feeding stimulates a rapid decrease and then an increase in labeled cells in gastric tissue; labeled cells in the head are not affected. Starvation leads to a twofold decrease in labeled cells in the gastric region; the density of labeled cells in head tissue remains similar to that in well-fed animals. During bud formation the number of labeled epithelial cells increases significantly in the evaginating bud. During head regeneration the number of labeled cells declines sharply during the first 12 hr and then increases to a density typical of head tissue by 24-36 hr of regeneration. The results indicate the release of signals by feeding and regeneration which inhibit mitosis. By contrast head tissue and developing buds express signals stimulating mitosis. Thus changes in epithelial cell cycling in hydra are closely correlated with morphogenetic events as well as with feeding stimuli.

Cell sorting during the regeneration of Hydra from reaggregated cells

The role of cell sorting in the reorganization of Hydra cell reaggregates was studied. We quantitatively labeled ectodermal and endodermal cells by incubating whole animals in fluorescent beads or by injecting the beads into the gastric cavity. Beads were stably incorporated into the cells by phagocytosis. Our data show that dramatic cell sorting processes drive the formation of ectoderm and endoderm within the first 12 hr of reaggregation. After the ectoderm is established, no further rearrangement could be observed. We also tested the ability of cells to sort out with respect to their original position in Hydra by dissociating labeled apical and basal pieces of Hydra and measuring the clumping of labeled cells during reorganization. There was no increase in the clumping of cells during reorganization indicating that cell sorting is not involved in the formation of early activation centers. There was also no preferential incorporation of apically derived (presumptive head) tissue into tentacles that subsequently formed, indicating that after dissociation into single cells there is no predisposition of erstwhile presumptive head tissue to form heads.

Nerve cell differentiation in hydra requires two signals

Endogenous signals controlling nerve cell commitment in hydra were investigated using an assay for committed nerve precursors. Extracts of hydra tissue were prepared and tested for their ability to induce nerve cell commitment. The active component in such extracts was identified as a neuropeptide, the head activator [H. C. Schaller and H. Bodenmuller (1981) Proc. Natl. Acad. Sci. USA 78, 7000–7004], based on its chromatographic properties and reaction with anti-head activator antibody. In addition, synthetic head activator (10−13–10−11 M) was shown to cause nerve cell commitment. Additional experiments demonstrated that committed nerve precursors require a second signal to differentiate nerve cells. Committed precursors induced by treatment of hydra with head activator do not differentiate in whole hydra; but do differentiate when pieces of treated tissue are explanted or when whole animals are simply injured with transverse cuts. The injury stimulus is long-lived. It cannot be replaced with head activator (10−12–10−10 M) but is contained in a methanol extract of hydra tissue.

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.

Bilateral symmetry in the cnidocil-nematocyst complex of the freshwater medusaCraspedacusta sowerbii Lankester (Hydrozoa, Limnomedusae)

Abstract We have investigated the fine structure of the cnidocil apparatus and the nematocyst in the hydrozoan Craspedacusta sowerbii using both light and electron microscopy. The structures are intimately connected and form a highly polarized, bilaterally symmetrical structure, the cnidocil-nematocyst complex. The cnidocil apparatus consists of a modified cilium, about 12 stereocilia, and an inner ring of short microvilli surrounding the nematocyst and cnidocil. The stereocilia enclose the cnidocil circularly in its distal region but in a crescent-like pattern in the proximal region; thus the nematocyst is not encompassed by the stereocilia, but only by the inner ring of short microvilli. We present a new reconstruction of the cnidocil apparatus and discuss the physiological significance of the various elements of the cnidocil-nematocyst complex.

The primitive metazoan Hydra expresses antistasin, a serine protease inhibitor of vertebrate blood coagulation: cDNA cloning, cellular localisation and developmental regulation

We have isolated and characterized cDNAs from Hydra which encode antistasin, a potent inhibitor of factor Xa in the vertebrate blood clotting cascade. Hydra antistasin is expressed in gland cells and represents a major class of transcripts from Hydras head. Sequence analysis revealed that Hydra antistasin contains 6 internal repeats of a 25–26 amino acid sequence with a highly conserved pattern of 6 cysteine and 2 glycine residues identical to that in leech antistasin. Conservation of antistasin in a lower metazoan provides a potential link between the vertebrate and invertebrate coagulation systems.

The properties of nerve cell precursors in hydra

Two signals, the head activator and an injury stimulus, control differentiation of nerve cells from uncommitted stem cells in hydra [Th. Holstein, H. C. Schaller, and C. N. David, (1986) Dev. Biol. 115, 9–17]. The time of action of these signals in the precursor cell cycle was determined. Methanol extracts of hydra containing 10−13 M head activator cause nerve cell commitment in S phase of the precursor cell cycle. Committed precursors complete the cell cycle, divide, and arrest in G1. Injury relieves the G1 block and precursors differentiate nerve cells. Under these conditions the time from commitment to nerve differentiation is 12 hr, the time from the end of S phase to nerve differentiation is 9 hr, and the time from the G1 block to nerve differentiation is 4 hr. Committed precursors blocked in G1 are unstable, decaying with a half-life of 12 hr if not stimulated to differentiate by an injury stimulus.

A quantitative method for separation of living hydra cells

SummaryWe describe a rapid method for the isolation of large numbers of livingHydra cells of defined cell type in an isotonic cell medium (Gierer et al. 1972). Intact animals are enzymatically dissociated into a single cell suspension and the various cell types separated in less than one hour by counterflow centrifugation elutriation. Cell loss is minimal. RNA isolated from various fractions can be probed with cell type specific cDNA-clones.

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.

Hydra braueri Bedot 1912 (Cnidaria: Hydrozoa): investigations into the taxonomic status of an enigmatic species

Hydra braueri Bedot, 1912, described 100 years ago by August Brauer, has influenced the development of hydra taxonomy but it has been observed rarely and its characters are puzzling. Hydra braueri has some characters normally associated with H. circumcincta Schulze, 1914 and others associated with H. oligactis Pallas, 1766. We show that the original description of H. braueri was derived from a mixture of these two species. This conclusion is based on an analysis of the original descriptions, on the succeeding literature on the animal, on examination of some of the early specimens still preserved in museums, and from study of other species of European hydra. Thus, H. braueri is a junior synonym of H. circumcincta, but some of the characters associated with the name H. braueri represent H. oligactis.