The germ plasm component Vasa allows tracing of the interstitial stem cells in the cnidarian Hydractinia echinata.

Germ cells in hydrozoans arise lifelong from multipotent interstitial stem cells. To discover if a true germline-soma segregation exists in these species, we studied gametogenesis in Hydractinia echinata using in situ hybridization and immunohistochemistry for the germ cell marker Vasa. We could show that Hevas is a zygotic transcript, present in embryos from the gastrula stage onward. In the planula larva, Hevas is expressed in the interstitial stem cells located in the endoderm. During metamorphosis, Hevas-expressing cells appear in the ectoderm in the lower half of the polyp. While the Hevas transcript is not detectable in developing gametes, the protein accumulates during oogenesis. Vasa containing granules are detectable at the polar-body-forming pole after fertilization. These results suggest that, in Hydractinia, maternal Vasa protein, but not the mRNA, is a maternal constituent of a germ plasm and might be involved in the specification and maintenance of interstitial stem cells.

Automated in situ detection (AISD) of biomolecules.

In order to facilitate in situ detection of biomolecules in large sample series the processing of whole-mount specimens has been automated. A freely programmable liquid handling system is described by which embryos or similar biological materials are processed. Possible applications include in situ hybridization (ISH), immunocytochemistry (ICC) or reporter gene assays. Process times required for the preparation of whole-mount in situ hybridizations in Drosophila, Xenopus, Gallus and in hydroids were - in part - significantly reduced as compared with manual processing. Application of automated in situ detection (AISD) in random screening is demonstrated in hydroids. Potential further applications are discussed.

X-ray microanalysis of ion contents in vacuoles and cytoplasm of the growing tips of a hydroid polyp as related to osmotic changes and growth pulsations.

Growth pulsations (GP) in hydroid polyps are associated with changes in vacuolar patterns which can be imitated by altering external osmolarity. With the use of X-ray spectroscopy we measured the elemental contents in the vacuoles and cytoplasm of the growing tips of a hydroid polyp, Podocoryne carnea, under various tonicity conditions. Under hypertonic condition which arrested the samples at the retraction phase of normal GP, the elemental content within the vacuolar compartment appeared to be similar to that of the external medium, confirming our previous conclusion about the dehermetization of the vacuolar compartment under these conditions. Under hypotonical condition which arrested samples at the extension GP phase (vacuoles isolated) element ratio data displayed an obvious bimodality. At least one of the data groups could be characterized by a significant increase in the concentrations of sodium and potassium, as related to Cl, Ca and Mg, and in comparison to the same ratios in hypotonical samples and those in the external medium. We suggest that under hypotonical conditions the isolated vacuolar compartment is formed by influx of sodium and potassium ions. These cations are accompanied by anions other than chloride. Potassium appears to be transferred into the vacuoles from the cytoplasm while the sodium derives from the external environment.

The proportion altering factor (PAF) and the in vitro transdifferentiation of isolated striated muscle of jellyfish into nerve cells.

The effect of proportion altering factor (PAF) on the transdifferentiation of isolated striated muscle into RFamide-positive nerve cells was investigated. The factor reduces incorporation of 3H-thymidine into replicating DNA; the effect is concentration-dependent and reversible. Transdifferentiation to nerve cells increases by up to 60% if PAF is applied shortly before or at the time of initiation of DNA synthesis. When treatment was terminated 4 h before the start of S-phase or when PAF was applied at the peak of S-phase no increase in nerve cell formation was observed.

Proportion-altering factor (PAF) stimulates nerve cell formation in Hydractinia echinata.

The low-molecular weight proportion altering factor (PAF) from colonial hydroids has general animalizing effects on morphogenesis in hydroid development. On the cellular level, the factor stimulates the formation of nerve cells in several developmental stages of Hydractinia echinata. Treatment during metamorphosis yielded a fivefold increase in the number of nerve cells with FMRF-amide-like immunoreactivity. In non-metamorphosing planula larvae, PAF triggered the differentiation of nerve cells that normally develop only upon initiation of metamorphosis and that occur in the polyp stage. After post-metamorphic treatment of young primary polyps, nerve cell numbers increased to 2.5 times the normal value.

Transdifferentiation from striated muscle of medusae in vitro.

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.

Proliferation kinetics and cell lineages can be studied in whole mounts and macerates by means of BrdU/anti-BrdU technique.

S-phase cells in intact animals of the coelenterate species Eirene viridula, Hydractinia echinata, Hydra attenuata, and Hydra magnipapillata incorporate the thymidine analogue bromodeoxyuridine (BrdU) into newly synthesized DNA. BrdU-labelled nuclei divide and cells appear to undergo normal differentiation. Whole-mount preparations and macerated tissues were screened for S-phase cells by means of immunocytochemical detection of BrdU (Gratzner, 1982). It is demonstrated that spatial patterns of DNA replication can be evaluated easily. Cell lineages and pathways of cell migration could be traced.

Cell proliferation and early differentiation during embryonic development and metamorphosis of Hydractinia echinata.

The early embryonic development of Hydractinia lasts about 2.5 days until the developing planula larva acquires competence for metamorphosis. Most embryonic cells stop cycling on reaching the larval stage. In older larvae of Hydractinia, cells that are still proliferating occur exclusively in the endoderm in a typical distribution along the longitudinal axis. During metamorphosis, proliferation activity begins again. The number of S-phase cells has increased by the 9th hour after induction of metamorphosis. Proliferative activity starts in the middle gastric region and in basal parts of primary polyps. Tentacles and stolon tips are always free of replicating cells.

Regeneration in Hydrozoa: distal versus proximal transformation in Hydractinia.

Polyps of mature colonies of Hydractinia echinata obey the "rule of distal transformation" by regenerating heads but not stolons. However, this rule is not valid for young polyps as these regenerate stolons from proximal cut ends. Also, small cell aggregates and even small fragments excised from full-grown polyps are capable of stolon formation. Aggregates produced from dissociated cells undergo either distal or proximal transformation depending on their size, speed of head regeneration in the donor used for dissociation and the positional derivation of the cells. The latent capability of stolon formation is released under conditions that cause loss of morphogens and depletion of their sources. However, internal regulative processes can also lead to gradual proximal transformation: regenerating segments of polyps sometimes form heads at both ends and the distal pattern is duplicated. Subsequently, all sets of proximal structures, including stolons, are intercalated. In contrast to distal transformation, proximal transformation is a process the velocity of which declines with the age and size of the cell community.