Shaping of colony elements in Laomedea flexuosa Hinks (Hydrozoa, Thecaphora) includes a temporal and spatial control of skeleton hardening.

The colonies of thecate hydroids are covered with a chitinous tubelike outer skeleton, the perisarc. The perisarc shows a species-specific pattern of annuli, curvatures, and smooth parts. This pattern is exclusively formed at the growing tips at which the soft perisarc material is expelled by the underlying epithelium. Just behind the apex of the tip, this material hardens. We treated growing cultures of Laomedea flexuosa with substances we suspected would interfere with the hardening of the perisarc (L-cysteine, phenylthiourea) and those we expected would stimulate it (dopamine, N-acetyldopamine). We found that the former caused a widening of and the latter a reduction in the diameter of the perisarc tube. At the same time, the length of the structure elements changed so that the volume remained almost constant. We propose that normal development involves a spatial and temporal regulation of the hardening process. When the hardening occurs close to the apex, the diameter of the tube decreases. When it takes place farther from the apex, the innate tendency of the tip tissue to expand causes a widening of the skeleton tube. An oscillation of the position at which hardening takes place causes the formation of annuli.

Pattern regulation properties of a Hydra strain which produces additional heads along the body axis.

The multiheaded one (mh-1) strain, isolated from inbred crossings of wild type Hydra magnipapillata, develops additional heads along the body axis. This strain reproduces asexually by budding like the wild type (wt) does. We found that young polyps have a wt-like shape and display wt-like properties. When they grow in size and before they produce extra heads along the body axis, the tissue between the head and the budding zone changes its property: in this region, where later on the extra heads preferentially form, foot regeneration is significantly delayed while head regeneration remains unaffected. Further, following various transplantations additional heads form under conditions under which the wild type did not. The observed changes in pattern control and regulation indicate a two-step process of pattern formation. Morphogenetic signalling is suggested to cause the positional value to increase slowly in the form of patches and preferentially in the region between the head and the budding zone. This increase causes an altered morphogenetic signalling, which is eventually responsible for additional head formation.

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.

The protein phosphatase inhibitor cantharidin induces head and foot formation in buds of Cassiopea andromeda (Rhizostomae, Scyphozoa).

The polyps of Cassiopea andromeda produce spindle shaped, freely swimming buds which do not develop a head (a mouth opening surrounded by tentacles) and a foot (a sticky plate at the opposite end) until settlement to a suited substrate. The buds, therewith, look very similar to the planula larvae produced in sexual reproduction. With respect to both, buds and planulae, several peptides and the phorbolester TPA have been found to induce the transformation into a polyp. Here it is shown that cantharidin, a serine/threonine protein phosphatase inhibitor, induces head and foot formation in buds very efficiently in a 30 min treatment, the shortest yet known efficient treatment. Some resultant polyps show malformations which indicate that a bud is ordinary polyp tissue in which preparatory steps of head and foot formation mutually block each other from proceeding. Various compounds related to the transfer of methyl groups have been shown to affect head and foot formation in larvae of the hydrozoon Hydractinia echinata. These compounds including methionine, homocysteine, trigonelline, nicotinic acid and cycloleucine are shown to also interfere with the initiation of the processes which finally lead to head and foot formation in buds of Cassiopea andromeda.

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.

Control of formation of the two types of polyps in Thecocodium quadratum (Hydrozoa, Cnidaria).

Thecocodium quadratum (Werner, Jber. Biol. Anst. Helgoland, 1965) is a colonial hydroid which produces 2 different types of polyps: gastrogonozooids and dactylozooids. The mouthless dactylozooids bear tentacles and catch the prey, which is then taken over and swallowed by the gastrogonozooids which have no tentacles. It is obvious that for a colony to survive both polyps must exist simultaneously arranged in a certain spatial pattern. Our experiments indicate that the formation of polyps in a growing culture is governed by at least 3 principles: (1) short range inhibition between polyps irrespective of their differentiation; (2) long range specific inhibition between gastrogonozooids; and (3) long range supporting influence (lateral help, Meinhardt, H., Models of Biological Pattern Formation, 1982) between gastrogonozooids and dactylozooids.

Effects of the anticonvulsant drug valproic acid and related substances on developmental processes in hydroids.

Developmental processes suitable for the detection of effects of drugs used in human therapy were investigated in the freshwater polyp Hydra and in the marine hydroid Hydractinia. The organisms were treated with the anticonvulsant drug valproic acid (VPA), a suspected human teratogen causing exencephaly in mice, and 12 related substances. In Hydractinia the acquisition of the normal larval shape in the course of embryogenesis and the developmental step from the larval to the adult stage (metamorphosis) were studied. When applied during embryogenesis, the test substances were found to affect cellular adhesiveness. The greater the concentration, the greater the number of groups of cells that failed to integrate within the spindle-shaped larval body. The most potent substance was octanoic acid, while diethyl-acetic acid had almost no effect. In experiments on metamorphosis, 2-propyl-pentanol was the most potent in reducing the frequency of induction of metamorphosis, whilst 4-pentanoic acid was the least effective. Long treatments (24 hr) with low concentrations of VPA in Cs(+)-enriched seawater reduced, and short treatments (3 hr) with high concentrations increased, the frequency of induction of metamorphosis. VPA itself failed to induce metamorphosis. A possible explanation for this may be that VPA and the other potent compounds affect the turnover of potassium ions in Hydractinia larvae. In Hydra, head regeneration and its inhibition by the drugs was studied. Head regeneration was inhibited by low concentrations (less than 0.1 mm) of VPA, 4-en-valproic acid and octanoic acid, while valproic acid amide (valpromide) and diethyl-acetic acid had almost no effect. The order of potency of these test substances varies greatly with different mammalian in vivo and in vitro test systems. Therefore, a correlation between potency in hydroids and in mammalian test systems is not possible. However, some similarities are explored.

Homarine (N-methylpicolinic acid) and trigonelline (N-methylnicotinic acid) appear to be involved in pattern control in a marine hydroid.

A morphogenetically active compound has been isolated from tissue extract of Hydractinia echinata and identified to be N-methylpicolinic acid (homarine). When applied to whole animals, homarine prevents metamorphosis from larval to adult stage and alters the pattern of adult structures. The concentration of homarine in oocytes is about 25 mM. During embryogenesis, metamorphosis and early colony development the overall homarine content does not change. Adult colonies contain a fourfold lower homarine concentration than larvae. The polyp's head contains twofold more homarine than the gastric region and the stolons. A second, similarly active compound, N-methylnicotinic acid (trigonelline), has also been identified in Hydractinia tissue at concentrations about one-third that of homarine. Incubation of larvae in 10 to 20 microM-homarine or trigonelline prevents head as well as stolon formation. If the compounds are applied in a pulse during metamorphosis, a large part of the available tissue forms stolons. Since microM concentrations of homarine and trigonelline are morphogenetically active, whereas mM concentrations are present in the tissue it appears that both substances are stored within the tissue.

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.

Commitment of stem cells to nerve cells and migration of nerve cells precursors in preparatory bud development in Hydra.

Budding in Hydra starts as an evagination of the double-layered tissue in the parent animal's gastric region. Five hours later the density of nerve cells in the bud's tissue doubles, representing the first detectable difference from the cellular composition of the surrounding tissue. These new nerve cells derive from multipotent stem cells which are in S-phase one day before evagination starts. Some of the bud's new nerve cells derive from stem cells which have migrated into the future bud's tissue after their commitment, apparently attracted by the bud anlage. The bud anlage recruits precursors of nerve cells even during starvation, during which nerve cell production ceases in other parts of the body. Furthermore, the bud anlage controls the duration of the development from commitment to final differentiation of the resulting nerve cells. Experiments with an inhibitor purified from hydra tissue indicate a tight correlation between stages of preparatory bud development and stages of recruitment of nerve cells for the bud. Whether or not precursors of nerve cells are involved in the control of bud formation in normal hydra, as compared to epithelial hydra which still bud though consisting of epithelial cells only, will be discussed.

Control of nerve cell formation from multipotent stem cells in Hydra.

Feeding of starved animals provides a very short signal which determines stem cells to differentiate into nerve cells after the next mitosis. Only those stem cells become determined which are just in the middle of their S-phase at the time of feeding. Stem cells of any other stage of the cycle do not become determined. Nerve cell determination is suppressed by very low concentrations of an endogenous inhibitor. The inhibitor exerts its effect only during the first half of the S-phase, not before and not after this period. Based on these finding it is proposed that stem cells are susceptible to 2 different signals during the first half of their S-phase; one signal allows the development into nerve cells, the other prevents this development. Within this period the decision whether to become a nerve cell or not is reversible. It becomes fixed at the end of this period.

Quantitative analysis of cell types during growth and morphogenesis in Hydra.

Tissue maceration was used to determine the absolute number and the distribution of cell types in Hydra. It was shown that the total number of cells per animal as well as the distribution of cells vary depending on temperature, feeding conditions, and state of growth. During head and foot regeneration and during budding the first detectable change in the cell distribution is an increase in the number of nerve cells at the site of morphogenesis. These results and the finding that nerve cells are most concentrated in the head region, diminishing in density down the body column, are discussed in relation to tissue polarity.