Development 99, 211-220 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
211
Homarine (/V-methylpicolinic acid) and trigonelline (N-methylnicotinic
acid) appear to be involved in pattern control in a marine hydroid
S. BERKING
Zoologisches Institut der Universitat Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg, FRC
Summary
A morphogenetically active compound has been isolated from tissue extract of Hydractinia echinata and
identified to be iV-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, A'-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 fiM-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 /JM 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.
Key words: homarine, TV-methylpicolinic acid,
trigonelline, N-methylnicotinic acid, pattern control,
hydroid.
controlled by morphogenetically active, signalling
substances, including activators and inhibitors.
In an attempt to isolate morphogenetically active
Hydractinia echinata (Athecata) is a colonial marine
inhibitors
from coelenterate tissue, I have used inhihydroid. Planula larvae develop from eggs and then
bition
of
metamorphosis
of Hydractinia as an assay.
transform during metamorphosis into primary polyps
Anthopleura
(Anthozoa)
was used as a source of
bearing stolons at the basal end (see Figs 1, 6).
Hydractinia,
because it is difficult
material
instead
of
During growth stolons elongate and develop latHydractinia tissue
to
collect
sufficient
quantities
of
eral branches (stolon tips) and secondary polyps
for
these
sorts
of
experiments.
Two Anthopleura
(hydranths). New polyps, as well as new stolon tips,
a
weaker metamorfractions
with
strong
and
one
with
arise at specific minimal distances from preexisting
phosis-inhibiting
activity
were
detected.
One of the
ones, indicating the inhibitory influence of an existing
strong
metamorphosis-inhibiting
fractions
contains
structure on the formation of a new one (Plickert,
iV-methylpicolinic acid (homarine) as the active prinHeringer & Hiller, 1986; Muller & Plickert, 1982).
ciple. The compound is also present in Hydractinia
Indeed, removing a polyp or only a polyp's head
(Berking, 1986).
allows the formation of a new polyp closer to a
Preliminary investigations indicate that homarine
preexisting one (Plickert et al. 1986). Based on
has properties expected of a morphogen in Hydracanalogous experiments with Hydra, models of pattern
tinia: it affects pattern formation and it is present in
formation (McWilliams & Kafatos, 1968; Wolpert,
sufficient amounts within the animal. In Hydra, by
1969; Gierer & Meinhardt, 1972; Schaller, Schmidt &
comparison, homarine appears to be absent if the
Grimmelikhuijzen, 1979; Berking, 1979; Meinhardt,
animals are fed with a homarine-free diet, and it has
1982; Muller, 1982; Kemmner, 1984) have been
only a weak influence on pattern formation in this
proposed in which such patterns of differentiation are
species. However, a chemically related compound,
Introduction
212
5. Berking
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
iV-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).
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 1-6x145 cm) 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 (/V-methylnicotinic 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
0-5x5cm), 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 1-6x150 cm and 2-6x100cm, respectively).
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 Anemia 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 lml were
triggered to undergo metamorphosis by application of CsCl
(final concentration 96min) for 3 h (Muller & 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 10mM-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, Munchen, FRG; column size 55cm, 5-5ml)
and eluted with 0-lM-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
Chemicals used as references
Nicotinic acid and nicotinamide (obtained from Serva,
Heidelberg, FRG); N-methylnicotinic acid (trigonelline),
7V-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 ^-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 fan) and larvae (length 800 ^m, maximal
diameter 100//m) 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
Homarine and trigonelline
25
-
•
•
'oo
c
A
| 20
A
ra
f
•
E
o
X
A
•
•
15
213
concentration than stolons. Small polyps may contain
a higher overall concentration than large 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.
8d
40d
Larva
^
Triggered
Id
2d
3d
Primary polyp
Fig. 1. 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.
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±40ng
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
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.
Since polyp tissue contains about 4^g protein
30 ill"1 (data not shown) and 10 ng homarine /ig"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.
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
0-lniM 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
214
S. Berking
Protein (/zg)
Fig. 2. 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 (O), respectively, with Tubifex instead of
Artemia prior to preparation of polyps.
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 60 JXM) and trigonelline (10
and 60/XM), applied immediately before the first
cleavage, did not slow cleavage at least up to the
8-cell stage (4h). 60 /iM-trigonelline led to disintegration of embryos over a period of 4 days.
Concentrations of 10 /iM-trigonelline and 10 and
60 /zM-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 10/iMhomarine formed stolons and tentacles like control
animals but their development was retarded, metamorphosing animals pretreated with 60 jUM-homarine
or 10/xM-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 /iMhomarine 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).
Homarine and trigoneUine 215
Homarine (1/XM) and trigoneUine (1/XM), respectively, did not interfere with embryogenesis.
O
1-8
C
I 1-6
u
c
c
I 1-4
o
tive
.c
-
A
•
o
1-0
*
•
30
40
Relative size (%)
50
Fig. 3. 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 homarine /ig"1 protein) of the
apical piece to that of whole polyp. Batches of full-gTOwn
polyps from different colonies were analysed. Symbols
indicate different batches. Symbols as in Fig. 2.
Moreover, in some batches of embryos treated with
homarine no animals of such appearance were obtained.
(2) Metamorphosis
Homarine and trigoneUine 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
trigoneUine 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 lmMhomarine or trigoneUine caused a reduction of the
mean number from 6-7 to 5-9 and from 6-9 to 6-3,
respectively (^-analysis: P<0-0l). The effect on
stolon development was more pronounced (Fig. 8A).
Concentrations as low as 10/iM-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
0-4
IN
N P
MNA Cl~ INA NA
I III I I I t
It I I
DB
0-2
Z///////V//////////A
40
50
60
70
80
Fraction number
90
100
110
Fig. 4. 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), trigoneUine (T), isonicotinic
acid (IN), nicotinic acid (N), picolinic acid (P), N-methyl nicotinamide (MNA), sodium chloride (C\~), isonicotinamide
(INA), and nicotinamide (NA).
216
S. Berking
0-5
240
0-4
Trigonelline
0-3
0-2
01
230
250
270
290 (nm)
Fig. 5. U.v. spectrum of pure trigonelline (0-1 ITIM) and
of the trigonelline peak from the Bio-Gel P-2 elution
profile in Fig. 4. Both spectra in 0-1 M-acetic acid.
formed stolons (^-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.
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
Fig. 6. 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, 100fan.
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
Homarine and trigonelline
217
Tentacles
Stolons
100
! , • Homarine
> Trigonelline
Trigonelline
Homarine
*
50
c
<
0-1
1
0
Concentration
0-1
Fig. 7. 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).
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/iM (Fig. 10). However, about 10IJM is necessary
to mimic the inhibitory influence of a polyp.
(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 1/XM
caused fewer colonies to form more than one polyp
within this period (Fisher- Yates test: P < 005); while
treatment with 10 I*M caused fewer colonies to form a
branch compared to untreated control colonies and
colonies treated with 1(JM (P<0-05). Elongation of
existing stolons was apparently unaffected.
0-01
0-1
1
10
Concentration (ITIM)
100
50
1
2
3
4
5
6
Number of stolons per animal, n
7
Fig. 8. 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.
218
30 .
S. Berking
Control
20 •
10
.
30
1
Homarine
I
I
11
1
ii i
20
10
•
-
1
11
1II .
004
0-08
0-12
Area covered by stolons (mm2)
Fig. 9. Pulse treatment with homarine increases stolon
mass of primary polyps. A homarine pulse (1 DIM) 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, ^-analysis: P<0-0l.
Discussion
Two compounds with strong morphogenetic activity
have been isolated from Hydractinia tissue and identified to be iV-methylpicolinic acid (homarine) and
Af-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.
0-01
1
Trigonelline (/ZM)
Fig. 10. 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
(j^-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.
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 polychaetous annelid Myxicola, however, homarine was
found to be concentrated (63mM) in nerve cells
(Gilbert, 1975).
Homarine and trigonelline
219
Growth
Polyps
0-6
:
\
0-3
•
1
10
0
1 10
Homarine
o
1
10
Fig. 11. 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.
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.
I wish to thank E. Fischer and G. Gunther 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.
References
ACKERMANN, D. (1953). Uber das Vorkommen von
Homarin, Trigonellin und einer neuen Base Anemonin
in der Anthozoa Anemonia sulcata. Z. Chemie 295, 1-9.
BERHNG, S. (1979). Analysis of head and foot formation
in Hydra by means of an endogenous inhibitor. Wilhelm
RouxArch. devl Biol. 186, 189-201.
BERKTNG, S. (1984). Metamorphosis in Hydractinia
echinata. Insights into pattern formation in hydroids.
Wilhelm Roux Arch, devl Biol. 193, 370-378.
BERKTNG, S. (1986). Is homarine a morphogen in the
marine hydroid Hydractinia? Wilhelm Roux Arch, devl
Biol. 195, 33-38.
GIERER, A. & MEINHARDT, H. (1972). A theory of
biological pattern formation. Kybernetik 12, 30-39.
GILBERT, D. S. (1975). Axoplasm chemical composition in
Myxicola and solubility properties of its structural
proteins. Biochemistry 59, 301-319.
GUPTA, K. C , MILLER, R. L. & WILLIAM, J. R. (1977).
Isolation of homarine (N-methyl picolinic acid) and
trigonelline (N-methyl nicotinic acid) from the hydroid
Tubularia larynx. LJoidia 40, 303-305.
LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L. &
RANDALL, R. J. (1951). Protein measurement with the
folin reagent. J. biol. Chem. 193, 265-275.
KEMMNER, W. (1984). Model of head regeneration in
Hydra. Differentiation 26, 83-90.
MEINHARDT, H. (1982). Models of Biological Pattern
Formation. New York, Oxford: Academic Press.
MCWILLIAMS, H. & KAFATOS, F. (1968). Hydra viridis:
Inhibition by the basal disc of basal disc differentiation.
Science 159, 1246-1247.
MOLLER, W. A. (1982). Intercalation and pattern
regulation in hydroids. Differentiation 22, 141-150.
MOLLER, W. A. & BUCHAL, G. (1973). Metamorphose-
Induktion bei Planulalarven. II. Induktion durch
monovalente Kationen: Die Bedeutung des GibbsDonnan-Verhaltnisses und der Na + /K + -ATPase.
Wilhelm Roux Arch. EntwMech. Org. 173, 122-135.
MOLLER, W. A. & PUCKERT, G. (1982). Quantitative
analysis of an inhibitory gradient field in the hydrozoan
stolon. Wilhelm Roux Arch, devl Biol. 191, 56-63.
PLICKERT, G., HERTNGER, A. & HILLER, B. (1987).
Analysis of spacing in a periodic pattern. Devl Biol.
(accepted).
220
5. Berking
SCHALLER, H. C , SCHMIDT, T. & GRIMMEUKHUUZEN,
C. J. P. (1979). Separation and specificity of action of
four morphogens from Hydra. Wilhelm Roux Arch, devl
Biol. 186, 139-149.
SPINDLER, K. D. & MOLLER, W. A. (1972). Induction of
metamorphosis by bacteria and by a lithium-pulse in
the larvae of Hydractinia echinata (Hydrozoa). Wilhelm
Roux Arch. EntwMech. Org. 196, 271-280.
J. H. & PROCK, P. B. (1958). Quaternary
ammonium bases in Coelenterates. Biol. Bull. mar.
Biol. Lab., Woods Hole 115, 551-561.
WOLPERT, L. (1969). Positional information and the
spatial pattern of cellular differentiation. J. theor. Biol.
25, 1-47.
WELSH,
(Accepted 28 October 1986)