Stefan Berking

Quantitative Analysis of Cell Types during Growth and Morphogenesis in Hydra

SummaryTissue 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.

Bud formation inHydra: Inhibition by an endogenous morphogen

SummaryFrom crude extracts ofHydra tissue a substance has been purified which prevents or retards the asexual reproduction by budding. The molecular weight is in the range of 300 to 1000 daltons. Inhibition of bud formation can be observed with concentrations equivalent to the extract from one hydra per 4 ml, that is, to a more than 10,000-fold dilution of the initial crude extract of a hydra. The purified inhibitor is active at a concentration of less than 10−8 M.Most of the inhibitor present inHydra is bound to cells. Within the cells the substance is mainly bound to particulate structures which sediment at 10,000 g. Its concentration is highest in the hypostomal region and decreases in the direction of the tentacles and peduncle. A second, lower, peak has been found in the basal disc. Treatment of the animals with a toxic agent (nitrogen mustard) which depletes the animal of interstitial cells, nematocytes and nematoblasts excludes the possibility that the inhibitor is present to any great extent in these cells. In conjunction with cell separation experiments by centrifugation of fixed cells in suspension, these results indicate that nerve cells are the most likely sites of storage of the inhibiting substance, although epithelial cells are not excluded as sources for the inhibitor.

Analysis of head and foot formation inHydra by means of an endogenous inhibitor

SummaryIn tissue regenerating the head, the ability to initiate head formation in a host increases with the time allowed for regeneration before grafting, while the foot-initiating ability decreases concomitantly. The reverse was found for tissue about to regenerate a foot. The early divergent changes thus indicated are counteracted in both head and foot regeneration by treatment with an inhibitor (Berking, 1977) in low concentrations.The inhibitor also interferes with processes which determine wether or not hypostome and tentacles are formed, and how many tentacles (if any) appear. The circumferential spacing of the tentacles was regular whether their number was normal or below normal.Secondary axes caused by implanted tissue either detach after having formed a head and a foot (i.e. behave like buds) or do not detach, having only formed a head. This alternative depends on the origin and amount of the implanted tissue and on the position of the implant within the host.The following model based on these findings is proposed: Head and foot formation start with pre-patterns which cause a continuously increasing change of the tissues ability to initiate a head or a foot. Along the body axis this ability is determined by a graded distribution of “sources”. As development progresses, the high source density which accumulates in the head region causes the formation of a hypostome and tentacles; the angular spacing of tentacles is also dependent on source density. At a certain low source density foot-formation is initiated. The inhibitor counteracts the increase of source density in head-forming tissue as well as the decrease of source density in foot-forming tissue. It thus appears to be part of the mechanism which controls morphogenesis in hydra.

Ammonia tetraethylammonium barium and amiloride induce metamorphosis in the marine hydroid hydractinia

SummaryIn Hydractinia metamorphosis from the swimming larval stage to the sessile polyp stage has been found to be inducible by several agents, including Li+, K+, Cs+, Rb+, diacylglycerol (DG), tetradecanoyl-phorbol-acetate (TPA) and some other tumour-promoting phorbol esters. Induction is antagonized by ouabain and compounds which are able to increase the internal level of S-adenosylmethionine (SAM). Based on the finding that Hydractinia larvae contain such compounds in a stored form, including N-methylpicolinic acid, N-methylnicotinic acid and N-trimethylglycine, as well as on the results of experiments with antagonists of SAM production and transmethylation, it has been argued that regulation of the internal SAM level plays a key role in the control of metamorphosis. However, it remains to be clarified whether the inducing agents act by decreasing the SAM level or by via different pathways. In the present study, substances chemically related to the substances known to induce or inhibit metamorphosis were tested for their metamorphosis-inducing abilities. Some were found to be effective, including NH4+, methylamine, tetraethylammonium ions (TEA+), ethanolamine, Ba2+, Sr2+ and the diuretic, amiloride. It is of particular interest that in many organisms TPA and DG increase cytoplasmic pH while amiloride prevents a rise in pHi. Several of the substances known to trigger metamorphosis may increase the internal NH4+ concentration by hindering the export of the constantly produced NH4+ through K+ channels or through the Na+-H+ antiport. Treatment with Cs+ for 1 h increases the internal level of NH4+. Produced and applied ammonia, as well as applied methylamine and ethanolamine, may act by accepting methyl groups, thus reducing the SAM level.

Lithium ions interfere with pattern control in Hydra vulgaris

SummaryLiCl in concentrations exceeding 0.5 mM affects morphogenesis in Hydra vulgaris (formerly named H. attenuata) by interfering with the foot-forming system(s). Pulse treatment of Hydra bearing small buds or of animals that develop a bud within 14 h after the end of treatment prevented foot formation at the buds base in a concentration-dependent manner. With increasing concentrations of Li+ or length of treatment in increasing percentage of the buds remained permanently connected to the parent by a bridge of tissue thus forming a stable secondary axis. Instead of the normal ring-shaped foot a patch of basal disc tissue developed or the bud failed to differentiate foot tissue at all. Long-term culture of animals in 1 mM LiCl inhibited budding from the second day of treatment onwards and detachment of existing buds was delayed. After 4 days of treatment 15%–30% of budless or bud-bearing animals developed up to three patch-like basal discs at various positions along the body axis; these usually grew out one above the other on the same side of the animal but never at the same transverse level. Besides these patch feet broad belts of foot tissue were observed in the lower gastric region. After 1 week of treatment half of the animals developed a constriction located usually in the lower two-thirds of the body axis. The tissue adjacent to this constriction and particularly above it differentiated into mucus-secreting foot tissue. Subsequent separation into two morphologically intact polyps occurred occasionally. When treatment was stopped, budding restarted within the next 3 days at several positions along the body axis whether or not secondary feet or a constriction existed. Buds grew out in different budding zones, which persisted for several days. This burst of budding led to up to 7 buds per animal within 3 days. After about 1 week the animals regulated to normality or became epithelial, i.e. they lost their stem cells during and after treatment.

3 Hydrozoa Metamorphosis and Pattern Formation

Publisher Summary This chapter discusses metamorphosis from the larval to the polyp state and pattern formation in larvae, polyps, and colonies. Metamorphosis and pattern formation in larvae and colonies have been studied by experiments with Hydractinia , and pattern formation in polyps, by experiments with Hydra . In Hydractinia , the larval state is stable until external cues trigger the onset of metamorphosis. Hydractinia , a rather typical hydrozoan with a simple structure, is the best studied. There exist female and male animals, which release sperm and oocytes, respectively, into the surrounding water. Following fertilization, cleavage takes place, leading to different cell types and an elongated body shape. Hydrozoa provides an access to some elementary processes, including regulation of cell proliferation and differentiation as well as interaction of and communication among cells. Hydractinia has several technical advantages. It is possible to get thousands of larvae several times a week throughout the year. Metamorphosis can be triggered deliberately. Substances applied to the surrounding seawater are easily taken up by the larvae and influence the development. Various approaches are used to study pattern formation in hydrozoa, including regeneration and transplantation techniques, methods of molecular biology, and biochemical methods. Models play the role of a link between the different fields and the different species.

Metamorphosis ofHydractinia echinata Insights into pattern formation in Hydroids

SummaryHydractinia echinata is a marine, colony-forming coelenterate. Fertilized eggs develop into freely swimming planula larvae, which undergo metamorphosis to a sessile (primary) polyp. Metamorphosis can be triggered by means of certain marine bacteria and by Cs+. Half a day after this treatment a larva will have developed into a polyp. The induction of metamorphosis can be prevented by addition of inhibitor I, a substance partially purified from tissue ofHydra. The larvae ofH. echinata also appear to contain this substance. Inhibitor I appliedafter the onset of metamorphosis blocks its continuation as long as it remains in the culture medium. Cs+ applied within the same period of time also blocks the continuation of metamorphosis. However, these two agents have opposite effects on the body pattern of the resultant polyps. The experiments indicate that application of Cs+ triggers the generation of the pre-pattern. Inhibitor I appears to be a factor of this prepattern. A model is proposed which describes the basic features of head and foot/stolon formation not only forHydractinia but also for other related hydroids.

Is homarine a morphogen in the marine hydroid Hydractinia

SummaryHomogenate of coelenterate tissue interferes with metamorphosis in Hydractinia and pattern formation in both Hydractinia, and Hydra. From the extracts two fractions comprising low-molecular-weight compounds with strong metamorphosis-inhibiting activity were separated. One of these contains, as the active compound, homarine (N-methyl picolinic acid). Homarine concentrations down to 10−6 mol/l stop or retard metamorphosis. High concentrations block the continuation of metamorphosis as long as they are maintained in the culture medium and treatment with homarine during metamorphosis influences the proportioning of the future polyps body pattern. Most of the homarine found in Hydra tissue derives from Artemia given as food. It is not identical with inhibitor I, an activity partially purified from Hydra tissue, which prevents head and foot formation in Hydra.

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.

Heat shock as inducer of metamorphosis in marine invertebrates

SummaryIn most sessile marine invertebrates, metamorphosis is dependent on environmental cues. Here we report that heat stress is capable of inducing metamorphosis in the hydroid Hydractinia echinata. The onset of heat-induced metamorphosis is correlated with the appearance of heat-shock proteins. Larvae treated with the metamorphosis-inducing agents Cs+ or NH4+ also synthesize heat-shock proteins. In heat-shocked larvae, the internal NH4+-concentration increases. This fits the hypothesis that methylation plays a central role in control of metamorphosis. In the tunicate Ciona intestinalis, a heat shock is able to induce metamorphosis too.