ABSTRACT
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-methylnic-otinic acid (trigonelline), has also been identified in Hydractinia tissue at concentrations about one-third that of homarine.
Incubation of larvae in 10 to 20 μM-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 μM 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.
Introduction
Hydractinia echinata (Athecata) is a colonial marine hydroid. Planula larvae develop from eggs and then transform during metamorphosis into primary polyps bearing stolons at the basal end (see Figs 1, 6). During growth stolons elongate and develop lateral branches (stolon tips) and secondary polyps (hydranths). New polyps, as well as new stolon tips, arise at specific minimal distances from preexisting ones, indicating the inhibitory influence of an existing structure on the formation of a new one (Plickert, Heringer & Hiller, 1986; Müller & Plickert, 1982). Indeed, removing a polyp or only a polyp’s head allows the formation of a new polyp closer to a preexisting one (Plickert et al. 1986). Based on analogous experiments with Hydra, models of pattern formation (McWilliams & Kafatos, 1968; Wolpert, 1969; Gierer & Meinhardt, 1972; Schaller, Schmidt & Grimmelikhuijzen, 1979; Berking, 1979; Meinhardt, 1982; Müller, 1982; Kemmner, 1984) have been proposed in which such patterns of differentiation are controlled by morphogenetically active, signalling substances, including activators and inhibitors.
Homarine content (per animal) of eggs, larvae and primary polyps. Each symbol represents the analysis of a batch of about 70 tissue pieces. Different symbols refer to different batches of eggs. The animals were not fed. 8d, 8-day larva; etc.
In an attempt to isolate morphogenetically active inhibitors from coelenterate tissue, I have used inhibition of metamorphosis of Hydractinia as an assay. Anthopleura (Anthozoa) was used as a source of material instead of Hydractinia, because it is difficult to collect sufficient quantities of Hydractinia tissue for these sorts of experiments. Two Anthopleura fractions with strong and one with a weaker metamorphosis-inhibiting activity were detected. One of the strong metamorphosis-inhibiting fractions contains N-methylpicolinic acid (homarine) as the active principle. The compound is also present in Hydractinia (Berking, 1986).
Preliminary investigations indicate that homarine has properties expected of a morphogen in Hydractinia: it affects pattern formation and it is present in sufficient amounts within the animal. In Hydra, by comparison, homarine appears to be absent if the animals are fed with a homarine-free diet, and it has only a weak influence on pattern formation in this species. However, a chemically related compound, termed inhibitor I, has been isolated from Hydra tissue and shown to be involved in the control of pattern formation (Berking, 1979). Inhibitor I affects pattern formation in Hydractinia as well as in Hydra (Berking, 1984).
The aim of the present study was to gain a better understanding of the role of homarine in Hydractinia pattern-forming processes (1) by analysing the homarine content of Hydractinia during its life cycle and (2) by analysing the effects of homarine on pattern formation. In the course of this analysis N-methylnicotinic acid (trigonelline), a compound closely related to homarine, was also detected at significant concentrations in Hydractinia tissue. Like homarine, trigonelline inhibits metamorphosis of Hydractinia (Berking, 1986).
Materials and methods
Hydractinia echinata colonies, procured in reproductive condition from the Biologische Station Helgoland, FRG, were used as parental generation to obtain larvae and primary polyps. Colonies were grown from primary polyps by feeding them with either Artemia nauplii or pieces of Tubifex (Spindler & Muller, 1972). All experiments were done at 18 °C.
Metamorphosis assay
Larvae in groups of 100 per 3 ml and 25 per 1ml were triggered to undergo metamorphosis by application of CsCl (final concentration 96mM) for 3 h (Müller & Buchal, 1973). One day after treatment the larvae have transformed into primary polyps. Application of homogenates of Hydractinia tissue to larvae immediately after triggering metamorphosis delays or even prevents development into polyps. High concentrations cause the animals to round up into spheres. They remain spheres as long as the treatment lasts, but resume development into polyps when treatment stops. The effects of low concentrations were quantified by comparing the percentage of animals bearing tentacles 24 h after application of the substances to be tested.
Extraction of tissue and chromatography
Pieces of Hydractinia tissue were collected in sea water and spun down at low speed to obtain a pellet. This pellet was extracted in 10 mM-acetic acid by pressing it repeatedly through the narrow orifice of a syringe (diameter: 0·65 mm). Stolons were scraped from the dishes and disintegrated by the same procedure. After centrifugation the supernatants were concentrated by evaporation, applied to a Bio-Gel P-2 column (200–400 mesh, from BioRad Laboratories, München, FRG; column size 55cm, 5·5ml) and eluted with 0·1M-acetic acid. Homarine elutes from such columns in an isolated peak with a u.v. spectrum identical to that of commercially purchased homarine (Berking, 1986). To determine the homarine content of an extract the area covered by the homarine peak was compared to a homarine standard. Separate batches of tissue pieces were used to determine the homarine and the protein content of the tissue. The latter was carried out according to the method of Lowry, Rosenbrough, Farr & Randall (1951).
Determination of trigonelline content
Several hundred pieces of Hydractinia were macerated and extracted as described. The extract was applied to Bio-Gel P-2 (column size 1·6×l45cm) and eluted with 0·lM-acetic acid. The amount of trigonelline was estimated by measuring the optical density of the appropriate fractions at 265 nm and comparing the value obtained with measurements of commercially available trigonelline (A-methyl-nicotinic acid). For a more detailed analysis the fractions in question were applied to the ion exchange resin Dowex 1-X8 OH− form (Serva, Heidelberg, FRG; column size 0·5×5cm), and to Sephadex G 10 (Pharmacia, Uppsala, Sweden; in 40% methanol/60% 0·02M-ammonium hydroxide or 80% methanol/20% 0·lM-acetic acid, column size 1·6×150 cm and 2·6×100cm, respectively).
Chemicals used as references
Nicotinic acid and nicotinamide (obtained from Serva, Heidelberg, FRG); N-methylnicotinic acid (trigonelline), A-methylnicotinamide iodide and picolinic acid (obtained from Sigma, Taufkirchen, FRG), A-methylpicolinic acid (homarine), isonicotinic acid and isonicotinamide (obtained from Ega-Chemie, Steinheim, FRG).
Analysis of colony growth
Colonies with one to four polyps and up to 20 stolons were . drawn on successive days by means of a drawing tube mounted on a stereomicroscope (M5 from Leitz, Wetzlar, FRG). The area covered by stolons was determined by weighing the paper covered by the drawing of the stolons.
Statistical analysis
The number of stolons refers to the number of stolon tips and anastomoses. The experimental error was calculated either by means of the binomial distribution, the χ2-analysis or the Fisher-Yates test.
Results
Analysis of the temporal and spatial distribution of homarine in Hydractinia
(1) Embryogenesis, metamorphosis and early colony development
Eggs, larvae and primary polyps contain about the same amount of homarine (Fig. 1). Both eggs (diameter 200 μm) and larvae (length 800 gm, maximal diameter 100 μm) have a volume of about 4·2 nl and the overall homarine concentration is estimated to be about 25 mM.
Large amounts of homarine are not released during metamorphosis. Homarine was not detected in medium in which larvae had been incubated for 24 h, or in which larvae had been triggered to undergo metamorphosis or in which metamorphosing larvae were incubated for 24 h after onset of metamorphosis. The assay methods were sensitive enough to detect 5 % or more of the homarine found in larvae.
(2) Distribution of homarine in larvae
About 1000 larvae (of one batch) were sectioned transversely and the homarine content of anterior and posterior parts analysed. The larval posterior, which forms head and upper gastric region of the polyp after metamorphosis, contains a slightly lower amount of homarine (5·25 ±0·78 ng homarine and 270 ±40 ng protein) than the anterior (6·18 ±0-23 ng homarine and 270 ± 30ng protein). The difference, however, is not significant. Cutting the larvae did not lead to a substantial loss of homarine because the total amount of homarine and protein was found to be equal in both sectioned and intact larvae. The overall homarine content in the batch of larvae used in this experiment was somewhat lower than usual (see Fig 1).
(3) Distribution of homarine in colonies
Colonies grown from primary polyps have a highly variable appearance depending on the age, the culturing conditions and the genotype. In some colonies the stolons branch close to one another, in others at much larger distances. Reflecting this variability, different colonies were used for the analysis of homarine content. The results in Fig. 2 show that, though the size of the polyps can vary by a factor of 10, the homarine concentration is roughly constant. Polyps contain homarine in the same or in a higher concentration than stolons. Small polyps may contain a higher overall concentration than large polyps.
Protein and homarine content of polyps and stolons. The graph shows the homarine content of full-grown polyps and stolons normalized to the protein content of the probe (ordinate). Each symbol represents a measurement. Different symbols refer to different batches of polyps or stolons removed from different colonies. The symbols at the right margin refer to measurements of stolon tissue; all other symbols refer to measurements of polyp tissues. The homarine content of polyps is plotted against their protein content (abscissa) as a measure of its size. Batches of 16 to 54 polyps were analysed. One colony was fed for 4 weeks (•) and 7 weeks (○), respectively, with Tubifex instead of Artemia prior to preparation of polyps.
Protein and homarine content of polyps and stolons. The graph shows the homarine content of full-grown polyps and stolons normalized to the protein content of the probe (ordinate). Each symbol represents a measurement. Different symbols refer to different batches of polyps or stolons removed from different colonies. The symbols at the right margin refer to measurements of stolon tissue; all other symbols refer to measurements of polyp tissues. The homarine content of polyps is plotted against their protein content (abscissa) as a measure of its size. Batches of 16 to 54 polyps were analysed. One colony was fed for 4 weeks (•) and 7 weeks (○), respectively, with Tubifex instead of Artemia prior to preparation of polyps.
One colony was fed for more than one month with Tubifex which contains little or no homarine (Berking, 1986). Polyps of this colony (circles on Fig. 2) contain levels of homarine within the normal range for polyps fed with Artemia. Since the colony increased at least threefold in size during the experiment this indicates that homarine can be synthesized by Hydractinia.
Fig. 3 shows that the homarine concentration of head tissue (including tentacles and hypostome) is about twofold higher than the mean concentration of the whole polyp including the head.
The homarine content of head tissue. Abscissa: the relative size of isolated apical pieces is given in relation to the size of the respective polyps, as calculated from protein content determinations. Ordinate: ratio of the homarine content (ng homarineμg−1 protein) of the apical piece to that of whole polyp. Batches of full-grown polyps from different colonies were analysed. Symbols indicate different batches. Symbols as in Fig. 2.
The homarine content of head tissue. Abscissa: the relative size of isolated apical pieces is given in relation to the size of the respective polyps, as calculated from protein content determinations. Ordinate: ratio of the homarine content (ng homarineμg−1 protein) of the apical piece to that of whole polyp. Batches of full-grown polyps from different colonies were analysed. Symbols indicate different batches. Symbols as in Fig. 2.
Since polyp tissue contains about 4μg protein 30 nl−1 (data not shown) and 10 ng homarine μg−1 protein (Fig. 2) the overall homarine concentration of polyp tissue is about 6mM, which is four times lower than in eggs or in larvae.
Hydractinia contains N-methylnicotinic acid
Homarine is not the only metamorphosis-inhibiting compound present in Hydractinia tissue. Fig. 4 shows the elution profile of a crude extract of larvae recovered from a Bio-Gel P-2 column. When the fractions were assayed for their metamorphosisinhibiting activity, two distinct peaks with inhibiting activity were found. The first peak contains three inhibitory activities which are not completely separated from one another: the A-peak substance (Berking, 1986), homarine and a third compound. This third compound has now been identified as N-methylnicotinic acid (trigonelline) based on (1) its u.v. spectrum, which is almost identical with that of trigonelline (Fig. 5), (2) its ability to inhibit metamorphosis which is the same as commercially purchased trigonelline, and (3) its chromatographic behaviour on Dowex 1 and Sephadex G10, which is the same as commercially purchased trigonelline.
Elution profile of crude extract of Hydractinia larvae applied to Bio-Gel P-2. The hatched areas indicate fractions with metamorphosis-inhibiting activity. Fractions of the left area were tenfold more active than fractions of the right area. The elution position of the A-peak activity (Berking, 1986) is indicated by A. Elution positions of standard substances applied to the same column are also shown: dextran blue (DB), homarine (H), trigonelline (T), isonicotinic acid (IN), nicotinic acid (N), picolinic acid (P), N-methyl nicotinamide (MNA), sodium chloride (Cl−), isonicotinamide (INA), and nicotinamide (NA).
Elution profile of crude extract of Hydractinia larvae applied to Bio-Gel P-2. The hatched areas indicate fractions with metamorphosis-inhibiting activity. Fractions of the left area were tenfold more active than fractions of the right area. The elution position of the A-peak activity (Berking, 1986) is indicated by A. Elution positions of standard substances applied to the same column are also shown: dextran blue (DB), homarine (H), trigonelline (T), isonicotinic acid (IN), nicotinic acid (N), picolinic acid (P), N-methyl nicotinamide (MNA), sodium chloride (Cl−), isonicotinamide (INA), and nicotinamide (NA).
U.v. spectrum of pure trigonelline (0·1 mM) and of the trigonelline peak from the Bio-Gel P-2 elution profile in Fig. 4. Both spectra in 0·1 M-acetic acid.
U.v. spectrum of pure trigonelline (0·1 mM) and of the trigonelline peak from the Bio-Gel P-2 elution profile in Fig. 4. Both spectra in 0·1 M-acetic acid.
Larva, primary polyp and half-metamorphosed animal. The figure shows (A) a planula larva, (B) a primary polyp (20 h old) bearing yet four short tentacles and (C) an animal with stolons at its base while the distal end has remained a larval posterior (latter photograph by Plickert). Bar, 100μm.
Larva, primary polyp and half-metamorphosed animal. The figure shows (A) a planula larva, (B) a primary polyp (20 h old) bearing yet four short tentacles and (C) an animal with stolons at its base while the distal end has remained a larval posterior (latter photograph by Plickert). Bar, 100μm.
Quantitative measurements indicate that larvae as well as polyps contain trigonelline at a concentration about one-third that of homarine. When a crude extract of polyp tissue was applied to the same column used to analyse the larval extract, two additional peaks of inhibitory activity were found: one elutes close to the salt peak and one elutes much later (not shown).
The second peak of metamorphosis-inhibiting activity in Fig. 4 probably contains picolinic acid and nicotinic acid. However, since concentrations of 0T HIM of these compounds have a very weak inhibitory influence on metamorphosis, most of the activity of these fractions is probably due to other compounds. Nicotinamide inhibits metamorphosis as much as homarine and trigonelline on a per weight basis (Berking, 1986). However, at the position at which nicotinamide elutes from the P-2 column no inhibitory activity could be detected (Fig. 4). The overall concentration of nicotinamide in larvae is probably much lower than that of homarine and trigonelline.
Homarine and trigonelline interfere with growth and pattern formation
(1) Embryogenesis
Batches of about 300 eggs in 15 ml medium were exposed to various concentrations of homarine and trigonelline. The culture medium was renewed once a day. Homarine (10 and 60μM) and trigonelline (10 and 60μM), applied immediately before the first cleavage, did not slow cleavage at least up to the 8-cell stage (4h). 60 μM-trigonelline led to disintegration of embryos over a period of 4 days. Concentrations of 10 μM-trigonelline and 10 and 60 μM-homarine retarded development. By day 4 control embryos have developed into normal larvae while most of the treated embryos were elongated but slightly less so than normal larvae. When these larvae were triggered to undergo metamorphosis (in the absence of homarine or trigonelline), they showed a reduced capability of transforming into polyps.
Metamorphosing animals pretreated with 10μM-homarine formed stolons and tentacles like control animals but their development was retarded, metamorphosing animals pretreated with 60 μM-homarine or 10μM-trigonelline transformed into spheres and maintained this state for about 2 days. Then most of them completed metamorphosis while some 20% remained larvae. The hydranths formed by treated larvae were in general much smaller than those of untreated control animals, while the basal plate and stolons were longer. In the group treated with 60 μM-homarine about one-quarter of the larvae formed stolons only; the posterior part of such larvae failed to develop a hydranth. Such animals are rarely obtained in normal metamorphosis although a few batches of larvae frequently give rise to such animals (Fig. 6).
Moreover, in some batches of embryos treated with homarine no animals of such appearance were obtained.
Homarine (1μM) and trigonelline (1μM), respectively, did not interfere with embryogenesis.
(2) Metamorphosis
Homarine and trigonelline prevent the onset of metamorphosis when applied simultaneously with CsCl, the triggering agent (Berking, 1986). High concentrations of both compounds also block reversibly the process of metamorphosis itself while low concentrations simply delay development (Fig. 7). A pulse treatment, however, leads to a completely different result: fewer tentacles and more stolons are formed by the animals treated with homarine and trigonelline from the third to the sixth hour after triggering metamorphosis. The number of tentacles decreased with increasing concentration but the effect was not pronouced. A concentration of 1mM-homarine or trigonelline caused a reduction of the mean number from 6·7 to 5·9 and from 6·9 to 6-3, respectively (χ2-analysis: P<0·01). The effect on stolon development was more pronounced (Fig. 8A). Concentrations as low as 10 μM-homarine or trigonelline caused significant increase in stolon number (Fisher-Yates test: P<0·05). Fig. 8B shows the shift in the frequency distribution of primary polyps with respect to the number of stolons grown at their base while Fig. 9 shows that more of the available tissue formed stolons (χ2-analysis: P < 0·01) following homarine treatment. Although pulse treatment with 5 to 10 mM-homarine or trigonelline blocked development for more than one day, by the third day all larvae had resumed metamorphosis.
Homarine and trigonelline interfere with metamorphosis. Groups of 100 to 150 larvae were exposed to homarine and trigonelline, respectively, beginning immediately after onset of metamorphosis. The compounds were not removed. The percentage of animals with stolons and tentacles was scored when about 70 % of the control animals (*) have formed at least one stolon and one tentacle, respectively (about one day after triggering metamorphosis).
Homarine and trigonelline interfere with metamorphosis. Groups of 100 to 150 larvae were exposed to homarine and trigonelline, respectively, beginning immediately after onset of metamorphosis. The compounds were not removed. The percentage of animals with stolons and tentacles was scored when about 70 % of the control animals (*) have formed at least one stolon and one tentacle, respectively (about one day after triggering metamorphosis).
Pulse treatments with homarine or trigonelline increase the number of stolons. (A) 3h after the end of the triggering treatment animals were exposed for a period of 3 h to homarine or trigonelline, respectively. At the 26th h (homarine) and the 28th h (trigonelline) the number of stolons was scored. The experiments were performed with larvae from different batches. (B) Cumulative frequency distribution of primary polyps with respect to the number of stolons formed at their base. Trigonelline was applied in a pulse as described in A. The standard deviation is in the range of the size of the symbols; 255 to 400 animals were analysed in each group.
Pulse treatments with homarine or trigonelline increase the number of stolons. (A) 3h after the end of the triggering treatment animals were exposed for a period of 3 h to homarine or trigonelline, respectively. At the 26th h (homarine) and the 28th h (trigonelline) the number of stolons was scored. The experiments were performed with larvae from different batches. (B) Cumulative frequency distribution of primary polyps with respect to the number of stolons formed at their base. Trigonelline was applied in a pulse as described in A. The standard deviation is in the range of the size of the symbols; 255 to 400 animals were analysed in each group.
Pulse treatment with homarine increases stolon mass of primary polyps. A homarine pulse (1 HIM) was applied to metamorphosing larvae as described in Fig. 8A. Abscissa: area covered by the stolons and the bases of primary polyps at 28 h after triggering. Ordinate: percentage of total polyps. Controls: 57 animals; treated group 56 animals, χ2-analysis: P<0·01.
Pulse treatment with homarine increases stolon mass of primary polyps. A homarine pulse (1 HIM) was applied to metamorphosing larvae as described in Fig. 8A. Abscissa: area covered by the stolons and the bases of primary polyps at 28 h after triggering. Ordinate: percentage of total polyps. Controls: 57 animals; treated group 56 animals, χ2-analysis: P<0·01.
In summary: when applied during metamorphosis homarine and trigonelline affect processes that determine stolon and tentacle number. Pulse-treated animals use up a larger part of their tissue to form stolons.
(3) Polyp formation from stolons
When a founder polyp is removed from small colonies consisting of one polyp and a few stolons, most colonies develop a new polyp within 2 days while some even form two or three. By comparison, colonies from which the founder polyp is not removed do not form a further polyp. In almost all cases the new polyps do not develop at the wound surface, indicating that they have not been formed by regeneration from some residual polyp tissue. When homarine or trigonelline is applied to colonies from which the founder polyp has been removed, polyp formation is inhibited at concentrations as low as 0·1 μM (Fig. 10). However, about 10μM is necessary to mimic the inhibitory influence of a polyp.
Homarine and trigonelline inhibit polyp formation. Larvae were triggered to undergo metamorphosis. 48 h later the founder polyp of each of the resultant small colonies was removed and (A) homarine or (B) trigonelline was applied. The ordinate gives the percentage of colonies that formed at least one polyp 48 h after sectioning. A significant difference (χ2-analysis: P<0·05) is indicated by a closed symbol. Each symbol represents 60 to 80 animals. Different symbols refer to experiments performed on different days with larvae of the same batch.
Homarine and trigonelline inhibit polyp formation. Larvae were triggered to undergo metamorphosis. 48 h later the founder polyp of each of the resultant small colonies was removed and (A) homarine or (B) trigonelline was applied. The ordinate gives the percentage of colonies that formed at least one polyp 48 h after sectioning. A significant difference (χ2-analysis: P<0·05) is indicated by a closed symbol. Each symbol represents 60 to 80 animals. Different symbols refer to experiments performed on different days with larvae of the same batch.
(4) Colony growth
The effect of homarine and trigonelline on colony growth is much more difficult to analyse than their effect on metamorphosis because growth is highly variable from colony to colony and regression of stolons and polyps occurs spontaneously. Fig. 11 shows the result of two experiments in which the growth of colonies in the presence of homarine was analysed over a period of 72 h. Treatment with 1μM caused fewer colonies to form more than one polyp within this period (Fisher-Yates test: P< 0·05); while treatment with 10 μM caused fewer colonies to form a branch compared to untreated control colonies and colonies treated with 1 UM (P<0·05). Elongation of existing stolons was apparently unaffected.
Homarine interferes with colony growth. Colonies grown for two weeks were drawn, treated with homarine and drawn a second time 72 h later to determine the number of newly formed polyps (ordinate left graph), the number of stolons (ordinate middle graph) and the growth (mm) of old and newly formed stolons per colony, including elongation and occasional regression of stolons (ordinate right graph). The results of two experiments (•, ▴) are shown performed with 19 to 28 colonies at each homarine concentration including the untreated control colonies. Last feeding was the day before treatment. Homarine was renewed daily.
Homarine interferes with colony growth. Colonies grown for two weeks were drawn, treated with homarine and drawn a second time 72 h later to determine the number of newly formed polyps (ordinate left graph), the number of stolons (ordinate middle graph) and the growth (mm) of old and newly formed stolons per colony, including elongation and occasional regression of stolons (ordinate right graph). The results of two experiments (•, ▴) are shown performed with 19 to 28 colonies at each homarine concentration including the untreated control colonies. Last feeding was the day before treatment. Homarine was renewed daily.
Discussion
Two compounds with strong morphogenetic activity have been isolated from Hydractinia tissue and identified to be A-methylpicolinic acid (homarine) and 7V-methylnicotinic acid (trigonelline). The compounds have also been found to be present in other coelenterates (Ackermann, 1953; Welsh & Prock, 1958; Gupta, Miller & Williams, 1977). Both compounds have properties expected of morphogens in Hydractinia-. they affect pattern formation and are present in sufficient amounts within the animal. When applied to whole animals homarine and trigonelline prevent metamorphosis from larval to adult stage. If applied in a pulse during metamorphosis a large part of the available tissue forms stolons. Applied to colonies the formation of polyps and stolon tips is prevented.
Homarine is present during the entire life cycle of Hydractinia. During embryogenesis and metamorphosis the overall concentration remains constant. In full-grown polyps the homarine concentration is also roughly constant although the size of such polyps can vary by a factor of 10. The head of polyps contains a somewhat higher concentration than other parts of the colony.
Both homarine and trigonelline affect stolon and polyp development in Hydractinia if applied externally at concentrations equivalent to 1/1000 of the animal’s overall internal concentration. Thus, most of the homarine and trigonelline in Hydractinia tissue must be stored in such a way that it cannot reach the targets that affect development. It is not yet known in cnidarians which cells concentrate these compounds or which cells are primarily affected. In the poly-chaetous annelid Myxicola, however, homarine was found to be concentrated (63mM) in nerve cells (Gilbert, 1975).
In view of the high levels of homarine and trigonelline in Hydractinia tissue and the low concentration required to affect morphogenesis, it is tempting to ascribe a regulatory role to these molecules during development. For instance, if the organism could control the release of these molecules from natural stores, the compounds can be involved in maintaining the larval state until an appropriate signal allows metamorphosis or in regulating the body proportion during metamorphosis or in controlling the distances between polyps in a colony. Whether or not the animal actually makes use of the compounds to control these events remains to be seen.
ACKNOWLEDGEMENTS
I wish to thank E. Fischer and G. Günther for excellent technical assistance, the members of our hydrozoan research group for fruitful discussions and C. N. David for reading the manuscript. Support was provided by the Deutsche Forschungsgemeinschaft.