The novel signal peptides, pedibin and Hym-346, lower positional value thereby enhancing foot formation in hydra.

Signaling molecules affecting patterning processes are usually proteins and rarely peptides. Two novel peptides, pedibin and Hym-346, that are closely related to one another have been isolated from Hydra vulgaris and Hydra magnipapillata. Several experiments indicate that both cause a reduction in the positional value gradient, the principle patterning process governing the maintenance of form in the adult hydra. The peptides cause an increase in the rate of foot regeneration following bisection of the body column. Treatment of animals with either peptide for an extended period of time resulted in an apical extension of the range of expression of CnNk-2 along the body column. Such an extension is correlated with a decrease in positional value. Transplantation of tissue treated with Hym-346 results in an increase in the fraction forming feet, and aggregates derived from Hym-346 tissue form more feet and fewer heads. The latter two experiments provide a direct measure of the lowering of positional value in the treated tissue. These results suggest that peptides play signaling roles in patterning processes in cnidaria and, plausibly, in more complex metazoans as well.

Isolation and characterization of two new morphogenetically active peptides from Hydra vulgaris.

Foot-specific differentiation processes in hydra are controlled by activating and inhibiting potentials. In an attempt to understand the molecular mechanisms underlying these processes, two substances were isolated from Hydra vulgaris that stimulate foot-specific differentiation measured as acceleration of foot regeneration. These substances were shown to be peptides of 13 and 21 amino acids, respectively, with sequences that bear no significant homology to known peptides or proteins. Polyclonal antibodies were raised against both peptides. The data obtained by biological and radioimmunoassays show that the shorter peptide, pedin, is an excellent candidate for a major component of the 'foot-activating potential'.

Presence and expression of G2 cyclins in the coelenterate hydra.

In hydra all cell-cycle control occurs in the G2/M transition. Cyclins acting at this restriction point in the cell cycle belong to the cyclin A and B families. In agreement with this we isolated cDNAs coding for a cyclin A and a cyclin B from the multiheaded mutant of Chlorohydra viridissima and a cyclin B from Hydra vulgaris. The two B-type cyclins from hydra show 85.6% identity at the amino acid level, and 84.8% at the nucleotide level. The relatedness is less extensive than that found for mammals, e.g. human and mouse, and is evidence that the two hydra species diverged early in evolution. From each hydra species only one B-type cyclin was found, showing equal relatedness to the B1 and B2 subtypes of cyclins, hinting at a role as common ancestor before the split into B1 and B2 cyclins occurred. All three hydra cyclins contain regulation signals typical for G2/M cyclins, such as a ubiquitin destruction box at the amino terminus, needed for rapid degradation of the protein, and translation and polyadenylation elements in the 3' untranslated region to regulate RNA storage and RNA degradation. In hydra cell-cycle times vary depending on feeding regime and growth conditions. Cyclin B RNA expression was found to precede the daily mitotic rhythm induced by feeding. During head regeneration cyclin B expression showed the expected drop early during regeneration and an increase later. At the cellular level strongest expression of cyclin B RNA and protein was detected in interstitial cells which possess with one day the shortest cell-cycle time in hydra. Epithelial cells with a three-day cell-cycle rhythm showed variable, and differentiated cells no cyclin B expression. Regions of hydra containing high numbers of proliferating cells, such as developing buds exhibited elevated levels of cyclin B expression.

Neuronal control of development in hydra.

Hydra is an excellent model system for developmental biology, because pattern formation processes can be easily studied in regeneration, transplantation, and reaggregation experiments. At the cellular level hydra has the advantage that it contains only a few basic cell types and that differentiation pathways are short. Two types of signals, produced and released by nerve cells, control the spatial and temporal patterns of differentiation. Positive signals induce specific local differentiation events, and negative signals inhibit the spread of such inductions to larger areas. Head-specific growth and differentiation are controlled by head activator and head inhibitor, food-specific processes are regulated by foot activator and foot inhibitor. The activators are peptides, the inhibitors are low-molecular-weight substances. The sequence of the head activator is known, and it is conserved throughout the animal kingdom. At the cellular level head activator exerts three types of effects in hydra. It stimulates cells to divide, and it is responsible for the determination and the final differentiation of nerve cells and head-specific epithelial cells. For nerve-cell differentiation the cAMP pathway is used as second messenger system. Components of this pathway were identified in hydra. In mammals head activator is produced by nerve and neuro-endocrine cells, and it acts as mitogen on cells of neural origin. It is present early in neural development and in abnormal neural development, such as brain and neuroendocrine tumours.

Analysis of a foot regeneration deficient strain of Hydra oligactis.

A foot regeneration deficient strain of Hydra oligactis with altered size regulation was investigated. Analysis of the concentration of four morphogenetically active substances in Hydra oligactis showed that the foot regeneration deficiency was mainly due to a drastically reduced foot activator concentration. Foot activator was distributed as a very steep gradient in Hydra oligactis leading to a more severe impairment of foot regeneration the closer to the head cutting was done. The concentration of foot inhibitor was comparable, that of head activator slightly increased and that of head inhibitor reduced compared to Hydra vulgaris. Studies at the cellular level implied that the enlarged gastric region was due to an elevated level of inhibition hinting at a shift in the ratio between bound and free head inhibitor in this animal.

Characterisation of two types of head activator receptor on hydra cells.

A head activator (HA) analogue is described which even at high concentrations does not lose its biological activity. By cross-linking two HA molecules over a C8 spacer, the conformation was sufficiently altered, such that self-inactivation of HA by dimerisation was prevented. In addition, the introduction of a tyrosine instead of phenylalanine in one of the two HA molecules allowed radioactive labelling with iodine. This HA bipeptide was used to investigate the effect of HA at different concentrations and as ligand for HA receptor characterisation. We found that low concentrations (0.1-10 pM) sufficed to stimulate interstitial cell mitosis, and that higher concentrations (10-1000 pM) were required for the determination of interstitial cells to nerve cells. Binding of the radioactive HA ligand to living hydra and to purified membrane fractions was saturable and specific. Binding was compatible with HA analogues with a stable monomeric conformation, but less well with dimerising HA and HA analogues. Scatchard and kinetic analyses revealed the presence of at least two types of binding site in the membrane fraction, one with a 'lower' affinity (Kd = 10(-9) M) and one with a 100-fold higher affinity (Kd = 10(-11) M). Autoradiography showed that interstitial cells were differentially labelled, suggesting that the number or types of HA receptors may vary depending on cell cycle status. A mutant of hydra with a multiheaded morphology contained 6-20-times more HA receptors per mg protein than other hydra species or mutants.

Action of foot activator on growth and differentiation of cells in hydra.

Foot activator is a small peptide found in hydra and specifically activates foot formation. I present a method for the further purification of foot activator by high-pressure liquid chromatography. The morphogenetically active fractions were assayed for their effect at the cellular level. Foot activator acts as a mitogen by pushing epithelial and interstitial cells, which are arrested in G2, into mitosis. In the presence of foot activator, epithelial stem cells are stimulated to differentiate into foot mucus cells, and interstitial nerve precursor cells differentiate into mature nerve cells. The interaction of foot activator with head activator in the development of hydra is discussed.

Role of the neuropeptide head activator for growth and development in hydra and mammals.

In hydra, HA is produced by nerve cells and released into the intercellular space bound to large-molecular-weight carrier(s). By additional interaction with extracellular matrix components and selfinactivation by dimerisation, a local action is ensured. HA acts as a mitogen on all dividing cell types in hydra forcing them to pass through G2, divide, and either start a new round of cell division or terminally differentiate. In addition, HA is required for head-specific determination and differentiation processes. To become a head-specific nerve cell, for example, an interstitial stem cell requires HA in early S-phase to become determined to the nerve cell pathway, in late G2 to progress through mitosis, and/or in G1 to differentiate to a head-, and not to a foot-, specific nerve cell. HA (with identical amino acid sequence) occurs in other animals including mammals. In mammals, it is produced by nerve or endocrine cells and it probably acts, as in hydra, on nerve-precursor cells. On the neural cell line NH15-CA2 and on the pituitary cell line AtT20, HA acts as mitogen by stimulating cells arrested in G2 to enter mitosis. The presence of HA early in neural development and in abnormal neural development, such as in brain and neuroendocrine tumors, are consistent with a function in growth control for HA in mammals.

Production and characterization of monoclonal antibodies recognizing head activator in precursor form and immunocytochemical localization of head activator precursor and head activator peptide in the neural cell line NH15-CA2 and in hydra.

A synthetic gene for the hydra neuropeptide head activator (HA) was used to produce large amounts of an HA bacterial fusion protein. From this protein an HA-containing fragment was cleaved out, attached in high copy number to carrier proteins, and used as an immunogen to produce monoclonal antibodies able to recognize head activator in precursor form. Using such antibodies and others with different specificities for HA epitopes in combination with different fixation procedures, we detected HA immunoreactivity in three locations in the HA-rich neural cell line NH15-CA2. A precursor-like HA immunoreactivity was present in the cytoplasm of cells and detected, independent of fixation procedure, by monoclonal antibodies characterized as HA-precursor-specific. With antibodies specific for the HA peptide, two immunoreactivities could be distinguished, one within cells and one at the outer cell membrane. HA was detected within differentiated cells with long processes when crosslinkers such as carbodiimide or glutaraldehyde were applied together with agents like methanol. HA peptide bound to target cells was restricted to small round cells with an undifferentiated morphology, especially to those in the process of cell division. In hydra HA precursor immunoreactivity was localized in interstitial cells and in developing nerve cells. HA peptide immunoreactivity was present in nerve cells, but was more concentrated on and in target cells such as interstitial cells and epithelial cells. In tissue sections immunoreactive cells were especially abundant in regions of high HA content such as hypostome, subhypostomal region, and the future head region of developing buds.

Differentiation pathways of ectodermal epithelial cells in hydra.

The differentiation pathways of ectodermal epithelial cells in hydra were investigated. We found that under steady state conditions the ectodermal epithelial cells of the foot, the foot mucous cells, and the ectodermal epithelial cells of the tentacles, the battery cells, differentiate from gastric ectodermal ephithelial stem cells. From stem cell to the terminally differentiated state, a single cell cycle is required. The cells undergo a final round of DNA replication, double their genome to 4 n and become arrested in the G2-phase of the cell cycle. The ectodermal ephithelial cells of the hypostome, which like the tentacle cells are part of the head structure, can also arise from gastric ectodermal epithelial stem cells, but do so only during head regeneration and budding. They differentiate from stem cell to hypostomal cell in a single cell cycle, but in contrast to foot mucous and battery cells they remain capable of cell proliferation. Due to this self-renewal potential, they do not require recruitment from the gastric stem-cell pool in steady-state animals.