A possible role for the cnidarian homologue of serum response factor in decision making by undifferentiated cells.

We have isolated the serum response factor (SRF) homologue from two hydrozoans, the freshwater polyp Hydra vulgaris and the marine colonial Hydractinia echinata; we have termed the Hydra gene HvSRF and the Hydractinia gene HeSRF. The MADS-box of both genes is identical in sequence and more similar to SRFs of other organisms than to non-SRF MADS-box-containing proteins from other organisms. Within the N terminus of the predicted protein, a motif of 14 amino acids is nearly identical between Hydra and Hydractinia. This motif is absent from other known SRF sequences. In the adult Hydra polyp, SRF is predominantly expressed in cells of the interstitial cell (I-cell) lineage. Expression of SRF ceases when I-cells differentiate into nerve cells, nematocytes, or gland cells. In the course of sexual reproduction in Hydractinia, SRF is expressed in female germ cells. During embryogenesis, SRF transcripts are observed in all blastomeres. Later on, SRF expression is turned off in cells forming the ectodermal layer but further on is expressed in cells of the central cell mass, from which the endodermal epithelial cells and the I-cell lineage originate. Expression eventually becomes restricted to the I-cell lineage. We conclude that hydrozoan SRF is expressed in all these cells, which still have the property for differentiation. In adult Hydra, the abundance of SRF transcripts varies during the day. The pacemaker of this diurnal rhythm is the feeding regime. HvSRF expression decreases by 4 h after feeding and returns to the initial level 12 h after feeding. When feeding is stopped, the cycle of SRF expression persists through the first day when the animals are not fed. It has been shown that feeding partly synchronizes the cell cycle of the epithelial cells but not that of the I-cells. We suggest that the epithelial cells affect SRF expression in I-cells and thereby influence the decision of I-cells to enter a differentiation pathway.

Deceiving appearances: signaling by "dead" and "fractured" receptor protein-tyrosine kinases.

The mechanisms by which most receptor protein-tyrosine kinases (RTKs) transmit signals are now well established. Binding of ligand results in the dimerization of receptor monomers followed by transphosphorylation of tyrosine residues within the cytoplasmic domains of the receptors. This tidy picture has, however, some strange characters lurking around the edges. Cases have now been identified in which RTKs lack kinase activity, but, despite being "dead" appear to have roles in signal transduction. Even stranger are the cases in which genes encoding RTKs produce protein products consisting of only a portion of the kinase domain. At least one such "fractured" RTK appears to be involved in signal transduction. Here we describe how these strange molecules might function and discuss the questions associated with their evolution. BioEssays 23:69-76, 2001.

Induction of segmentation in polyps of Aurelia aurita (Scyphozoa, Cnidaria) into medusae and formation of mirror-image medusa anlagen.

Polyps of Aurelia aurita can transform into several medusae (jellyfish) in a process of sequential subdivision. During this transformation, two processes take place which are well known to play a key role in the formation of various higher metazoa: segmentation and metamorphosis. In order to compare these processes in bilaterians and cnidarians we studied the control and the kinetics of these processes in Aurelia aurita. Segmentation and metamorphosis visibly start at the polyp's head and proceed down the body column but do not reach the basal disc. The small piece of polyp which remains will develop into a new polyp. The commitment to the medusa stage moves down the body column and precedes the visible onset of segmentation by about one day. Segmentation and metamorphosis can start at the cut surface of transversely cut body columns, leading to a mirror-image pattern of sequentially developing medusae.

A gene whose major transcript encodes only the substrate-binding domain of a protein-tyrosine kinase.

We have identified a novel protein-tyrosine kinase gene family in the simple multicellular animal Hydra vulgaris that consists of at least three members. Two of the genes encode receptor protein-tyrosine kinases. The third member of the family is unusual in that in non-sexual animals, the only transcripts that it produces encode polypeptides lacking all or nearly all of the ATP-binding lobe. Characterization of multiple cDNA clones and hybridization mapping of genomic DNA indicate that the gene, which we have termed Hinterteil (Hint), undergoes alternative cis-splicing, alternative trans-splicing, and alternative polyadenylation. In-situ hybridization analysis shows that expression of the gene is upregulated during spermatogenesis. Sexual males also produce an additional Hint transcript that is larger than the transcript seen in non-sexual animals, but still not large enough to encode a receptor.

Morphological chimeras of larvae and adults in a hydrozoan--insights into the control of pattern formation and morphogenesis.

In the marine hydroid Hydractinia echinata, metamorphosis transforms the spindle-shaped larva into a primary polyp. It bears a hypostome with a ring of tentacles at its apical end, a gastric region in the middle and stolons at the base. In nature, metamorphosis is induced in response to external stimuli provided by bacteria. These stimuli can be replaced by artificial inducers, one of which is heat shock. Among heat shock treated stages are those undergoing complete metamorphosis but also specimens forming chimeras of different developmental stages. In the chimeric larvae, the posterior is transformed into the apical hypostome of the adult polyp while the anterior part of the larva persists as larval tissue. After transverse sectioning, these stage chimeras regenerate the missing body parts with respect to the nature of the tissue at the wound surface. This shows that the decision to make larva or polyp morphology depends not on the majority of the tissue in the original body section, but on stage specificity within the regenerating animal part. Single cells can escape from this general rule, since RFamide nerve cells which usually differentiate in polyp tissue appear in regenerated larval tails of sectioned stage chimeras. The results indicate that the pattern-forming system of the larva and of the adult have features in common. The primary signals controlling patterning along the anterior-posterior axis in larvae and the apical-basal axis in polyps arethus likelyto be the same while the interpretation of these primary signals by the individual cells changes during metamorphosis.

In Hydra magnipapillata the activator of protein kinase C diC8 causes multiple head formation along the body axis only when accompanied by feeding, but heavy feeding alone is sufficient to cause multiple head formation.

In Hydra magnipapillata additional head structures can be induced to form by daily feeding accompanied by a daily treatment with diC8, an activator of protein kinase C. Based on these results, it was proposed that the PKC- pathway plays a central role in head formation in hydra. The results described here show that ectopic structures, as well as the ectopic localization of nerve cells, can be induced by heavy feeding alone. Furthermore, diC8 treatment does not induce ectopic head structures in starved animals. DiC8 reduces the rate of budding, leading to an unusual lengthening of the body column in reasonably fed animals.

Metamorphosis and pattern formation in Hydractinia echinata, a colonial hydroid.

There are several reasons why Hydractinia echinata Hydrozoa, Cnidaria) is excellently suited to study developmental processes. In the laboratory fertilization takes place every morning in the seawater in thousands of eggs. Cleavage starting synchronously leads to a ciliated planula larva within 2 to 3 days. Onset of metamorphosis from the larval to the polyp stage must be triggered externally. There are several agents known to induce or to interfere with induction of metamorphosis thus allowing access to the biochemical basis of this process. The pattern of the resultant polyp can be influenced by certain treatments during the process of metamorphosis allowing access to a process of proportioning. The colony develops by elongation of hollow tubes at the base of the polyps, termed stolons on which in more or less regular intervals new polyps emerge. Two (main) types of polyps are formed allowing to study spacing by lateral inhibition and lateral dependence of each other. In the present paper current data and hypotheses concerning all these topics are discussed.

Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography.

The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.

Identification of a gene encoding a novel protein-tyrosine kinase containing SH2 domains and ankyrin-like repeats.

Using the polymerase chain reaction with primers corresponding to conserved regions in the kinase domain of protein-tyrosine kinases, we amplified segments of several protein-tyrosine kinase genes from Hydra vulgaris, a member of the ancient metazoan phylum Cnidaria. Characterization of cDNA clones for one of these genes, HTK16, revealed that it encodes a non-receptor protein-tyrosine kinase with two SH2 domains but no SH3 domain. In this regard the predicted HTK16 protein resembles two mammalian non-receptor protein-tyrosine kinases, the products of the ZAP-70 and syk genes. However, the HTK16 protein contains five ankyrin-like repeats, a structural motif which has not previously been found in protein-tyrosine kinases. The HTK16 protein also contains a potential tyrosine phosphorylation site in its carboxyl-terminal tail which resembles the phosphorylation site in members of the src family. RNA hybridization analysis indicates that the HTK16 gene is expressed in epithelial cells, cells which also express the Hydra homologue of the src protein. Our finding of the HTK16 gene in Hydra indicates that diversification of genes encoding non-receptor protein-tyrosine kinases was a very early event in metazoan evolution.

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.