Diacylglycerol (DAG) mediates transmembrane transduction for a wide variety of extracellular signals. Though pattern formation in multicellular organisms is, as a rule, based on intercellular signalling, reports on the participation of DAG in pattern-forming processes are lacking. Here evidence is presented for the involvement of DAG in pattern control in Hydra. Upon daily exposure to 1,2-dioctanoyl-sn-glycerol, wild-type polyps form ectopic heads along the gastric column in a periodic pattern and transform into phenocopies of a multiheaded mutant. The appearance of ectopic head structures is preceded by a (wave-like) increase in the positional value. Long before ectopic tentacles appear in the intact animal and, beginning with the first pretreatment, excised segments progressively fail to regenerate feet, form heads also at their lower end and eventually over the entire segment. DAG is the first physically defined substance found to induce, in hydra, an increase in the positional value and to evoke ectopic head formation.

The freshwater polyp Hydra was named so by Linné after the ancient nine-headed water snake which replaced each severed head by two. Yet, in contrast to the archetype of the myth, present day hydras faithfully restore the original pattern and regenerate only one head.

In the last decades, studies on Hydra have contributed much to widely discussed theories of biological pattern formation. Two major results are pertinent to the present study. (1) In Hydra, positional information is provided constantly to direct the perpetual cell renewal in these potentially immortal animals. (2) The strictly maintained polarity resides in graded scalar properties which ensure the dominance of the upper end in head formation (review: Bode & Bode, 1984). In the models of Wolpert (Wolfert et al. 1974) and of Gierer and Meinhardt (Meinhardt, 1982; MacWilliams, 1983a,b,c), positional information is provided by diffusible morphogens, by a single morphogen ‘S’, or by the ratio of an ‘activator’ and an ‘inhibitor’. This information is used to adjust a more stable graded tissue property, the ‘positional value P’ or ‘source density’, which acts as a positional memory and stabilizes the polarity.

In attempts to substantiate such theoretical concepts, the functions of activating or inhibiting morphogens have been assigned to substances that accelerate or inhibit the reappearance of tentacles or the outgrowth of latent buds by virtue of their mitogenic or antimito-genic action (Schaller et al. 1979; Schaller & Bodenmüller, 1981). The development of ectopic heads, i.e. of heads outside the normal position, was not among the effects that were reported for these putative morphogens.

In the hydrozoan Hydractinia, we extracted a factor that causes segments of polyps to form heads not only at their upper end but also at their lower end. This factor could be replaced by tumour-promoting phorbol esters known to activate protein kinases of the C-type (Müller, 1985). The same kinases are stereospecifically activated also by permeant 1,2-diacyl-glycerols composed of fatty acyl moieties of medium length (reviews: Kikkawa & Nishizuka, 1986; Nishizuka, 1986; Berridge, 1987; Castagna, 1987). Therefore, the initial aim of the present study was to examine whether externally applied di acylglycerols (DAG) might cause double-head formation in Hydractinia, but the study was then extended to Hydra, a genus known to maintain its polarity rigorously.

Animals

H. magnipapillata, wild-type strain 105 and the mutant strain mh-1 were kindly supplied by Tsutomu Sugiyama and grown in a modified M-solution (Sugiyama & Fujisawa, 1977) at 20°C. In order to start experiments with uniform material, budding specimens were selected from mass cultures, kept, as a rule singly, in 2 ml of medium per animal and first adjusted to a budding rate of 0·5–0·6 new buds per day by daily feeding the animals at 8 a.m. with brine shrimps. Polyps were vitally stained by feeding them with brine shrimps previously grown in a suspension of Evans blue (Wilby & Webster, 1970).

Hydractinia echinata is a marine colonial hydroid routinely raised in our laboratory.

Substances to be tested

1,2-sn-dioctanoyl-glycerol (1,2-sn-diC8), 1,2-rac-diC8, 1,3-rac-diC8, and l-oleoyl-2-acetylglycerol (OAG) were purchased from Sigma. 5 mg were dissolved in 145 μl methanol or ethanol. 10 ml of this alcoholic solution was mixed into 10 μl of culture medium by means of a sonifier to obtain an emulsion having a nominal concentration of 0·1 mm. The same amount of alcohol was given to the controls. Because the DAG emulsion is unstable and DAG may accumulate at the water surface, the polyps were covered with a layer of emulsion only. 0·3 cm deep in glass bowls with a flat bottom. The bowls were gently shaken and protected from light. Incubation was begun 3–4 h after the feeding. The incubation time was 0·5–1 h on day one and 2–3 h on the following days. After incubation the polyps were washed and transferred into new glass dishes.

New batches of DAG were, as a rule, first tested using the metamorphosis assay in Hydractinia (Leitz & Müller, 1987).

(A) Ectopic head formation in regenerating gastric segments

(1) Ectopic heads are evoked by known activators of protein kinase C

The traditional procedure to assay morphogenetic effects of substances in Hydra is to select young budless animals, to remove the head and the foot, and to expose the excised gastric segments to the solutions to be tested for 4h. A single exposure of excised segments to DAG emulsions retards regeneration but does not result in dramatic alterations of the polar pattern. Segments of Hydra formed double heads (Fig. 1) in response to 1,2-sn-diC8 (nominal dose 0·1 OIM). However, although the effect could be observed, of 1176 segments only 2·1% formed complete ectopic heads (control 0 out of 798) and additional 1·8% formed merely ectopic tentacles. Moreover, a number of the ectopic heads might have originated from latent buds that failed to form a foot as they emerged and, hence, were unable to detach.

Fig. 1.

Monitoring of the regenerative capacities of excised gastric segments after repeated pretreatment of intact animals at 0 to 8 successive days. (A) Schematic illustration of the experimental design. Whole, budding polyps were treated with 0·1 mm-DAG for 2–3 h per day. The treatment was iterated at successive days as indicated by the numbers. Subsequently, the upper gastric region was excised. The arrows point to the most frequent outcome. Actual data are given in Table 2 and Fig. 2. (B) After 4 pretreatrnents, the regenerates formed a head also at their lower end instead of a foot. 33 out of 48 displayed this phenotype.(C) After 8 pretreatments, ectopic tentacles and mouth cones were formed all around. 40 out of 48 displayed this phenotype, the remaining 8 were biheaded as shown above in B.

Fig. 1.

Monitoring of the regenerative capacities of excised gastric segments after repeated pretreatment of intact animals at 0 to 8 successive days. (A) Schematic illustration of the experimental design. Whole, budding polyps were treated with 0·1 mm-DAG for 2–3 h per day. The treatment was iterated at successive days as indicated by the numbers. Subsequently, the upper gastric region was excised. The arrows point to the most frequent outcome. Actual data are given in Table 2 and Fig. 2. (B) After 4 pretreatrnents, the regenerates formed a head also at their lower end instead of a foot. 33 out of 48 displayed this phenotype.(C) After 8 pretreatments, ectopic tentacles and mouth cones were formed all around. 40 out of 48 displayed this phenotype, the remaining 8 were biheaded as shown above in B.

Because of the (initially) low yield and intrinsic difficulty in identifying the origin of additional heads, it appeared advisable to check the specificity of the DAG effect in Hydractinia. In this species, the polyps do not produce buds, as the capacity to bud is restricted to the stolons, and clones can be found with a reduced stability of the regeneration polarity.

Nutritive polyps of Hydractinia, which superficially resemble small hydras, were isolated from such a clone. Excised gastric columns constituting two thirds of the body were exposed to one of the diacylglycerols for 2 h.

The established order of effectiveness (Table 1) corresponds to the known capacity of these lipids to activate protein kinase C in living cells (Nishizuka, 1986).

Table 1.

Effectiveness of various diacylglycerols in causing ectopic head formation in Hydractinia polyps

Effectiveness of various diacylglycerols in causing ectopic head formation in Hydractinia polyps
Effectiveness of various diacylglycerols in causing ectopic head formation in Hydractinia polyps
Table 2.

Frequency of ectopic head structures formed during regeneration as a function of iterated pretreatment

Frequency of ectopic head structures formed during regeneration as a function of iterated pretreatment
Frequency of ectopic head structures formed during regeneration as a function of iterated pretreatment

(2) The yield is increased from 2 to 100% by periodic pretreatment of the polyps before cutting

To increase the yield in hydra and to establish clear criteria for identifying the origin of the ectopic heads, I chose intact budding specimens H. magnipapillata, wild-type strain 105. In this strain, buds are generated in a narrow ring-like zone in the lower part of the body column. The animals were pretreated daily with 1,2-sn-diC8 and, at regular intervals, samples of 48 animals each were removed, the upper gastric regions excised (Fig. 1) and their regenerative behaviour monitored.

The incidence of ectopic head structures increased dramatically as a function of the number of previous applications (Table 2, Figs 1, 2) and reached the 100% level after 6–8 days when the first supernumerary tentacles began to appear also in the intact polyps (section B). At the lower end of the excised columns, tentacles sprouted in close vicinity to the foot, which progressively decreased in size, and eventually the foot was totally replaced by complete heads (Figs 1B, 2). After 6–8 pretreatments, most of the excised probes were completely transformed into head structures and studded with disorderly arranged tentacles and mouth cones over the entire surface (Fig. 1C). Thus, the gastric segments of the periodically treated polyps displayed an increased capacity to form head structures when released from the inhibitory dominance of the original head.

Fig. 2.

Iterated pretreatment with DAG causes a decrease in the capacity of the gastric region to regenerate a foot when the region is excised (left part of the diagram). Simultaneously, the capacity to form ectopic head structures proves to be increased (right diagram). The data shown were scored 5 days after excision of the probes. The data on the frequency of ectopic head structures are taken from Table 1, last lane.

Fig. 2.

Iterated pretreatment with DAG causes a decrease in the capacity of the gastric region to regenerate a foot when the region is excised (left part of the diagram). Simultaneously, the capacity to form ectopic head structures proves to be increased (right diagram). The data shown were scored 5 days after excision of the probes. The data on the frequency of ectopic head structures are taken from Table 1, last lane.

(B) Transformation of intact hydras into phenocopies of a multiheaded mutant

The periodic exposure of intact animals to DAG resulted in a progressive transformation of the polyps into multiheaded forms with a growing number of ectopic heads. The animals elongated slowly but continuously by a factor of 1·5 per week. After the first week of treatment, the animals had reached a length of 11·25 ±1·5 mm whereas the untreated controls preserved a steady state length of 7·5 ± 1·6 mm. A diminished export of cells contributed to this elongation as existing buds detached prematurely and new buds were initiated with increasing time lags (data not shown). After 6–10 applications, a second, disordered set of long tentacles grew out from the gastric region (Fig. 3). Later, 1–4 mouth cones emerged amidst the clusters of tentacles and eventually gave rise to secondary axes. This sequence of events is clearly different from budding. In the process of bud formation first a lateral protuberance evaginates and 5–7 short tentacles are subsequently formed near the distal pole of the protruding cone.

Fig. 3.

Development of phenocopies of multiheaded mutants from intact, uninjured wild-type hydras caused by daily treatment of the polyps with DAG. (A) After 8 days of treatment the first whorl of ectopic tentacles appears; a second cluster is announced by a first single tentacle. Near the foot is a small bud. (B) A nine-headed hydra. The specimen has been treated with DAG for two weeks; thereafter, the treatment stopped. Now, a week later, the monster is about to separate into one-headed individuals. The first normalized individual will soon detach. (C) Summary and interpretation in terms of positional value P. The value P takes the shape of a standing wave. Tentacles appear when the interrupted line is reached, a mouth cone emerges when the dotted line, i.e. the highest level, is reached.

Fig. 3.

Development of phenocopies of multiheaded mutants from intact, uninjured wild-type hydras caused by daily treatment of the polyps with DAG. (A) After 8 days of treatment the first whorl of ectopic tentacles appears; a second cluster is announced by a first single tentacle. Near the foot is a small bud. (B) A nine-headed hydra. The specimen has been treated with DAG for two weeks; thereafter, the treatment stopped. Now, a week later, the monster is about to separate into one-headed individuals. The first normalized individual will soon detach. (C) Summary and interpretation in terms of positional value P. The value P takes the shape of a standing wave. Tentacles appear when the interrupted line is reached, a mouth cone emerges when the dotted line, i.e. the highest level, is reached.

The response to continued reiteration of the treatment was the intercalation of more tentacle whorls in the gastric region. Their regular spacing indicates that the capacity to form head structures took the form of a standing wave with an increasing number of wave crests. The time course in which these structures arose reflected the order of their normal spatial position: first there appeared the (lower positioned) tentacles and only later the (higher positioned) mouth cones.

After about two weeks of chronic treatment, all of the remaining 48 uncut animals had formed at least 3 supernumerary heads, and some possessed 9 heads just as did the archetype of the myth (Fig. 3B). A week later about 18 heads were formed in several specimens. Since heads can give rise to secondary axes which again may branch, the number of possible heads is virtually unlimited. Occasional interruption of the treatment facilitates the outgrowth of branches.

Prolonged daily application of DAG produces morphologically perfect copies of a nonbudding, multiheaded mutant of H. viridis (Novak & Lenhoff, 1980).

In this mutant, heads appear along the body column but the outgrowth of secondary axes is retarded.

The phenotype of another multiheaded mutant, the strain mh-1 of H. magnipapillata, could not be reliably reproduced. Only occasionally were the multiheaded forms produced by the DAG treatment indistinguishable from the mutants. The mutant mh-1 is, as a rule, more compact and, although additional heads are often announced by the sprouting of tentacles, other ectopic heads may arise from latent buds (Sugiyama, 1982).

(C) Normalization after termination of treatment

Pattern regulation, in hydra, is a perpetual process. Therefore, aberrant forms and phenocopies are stable only as long as the treatment is continued. After its termination, budding is resumed in an overshooting response (data not shown) and basal discs (feet) are formed near the convergence of the body axes. The common basal discs segregate and the axes separate from each other. Thus, the monsters disassemble into normal wild-type hydras in the course of several weeks or months.

The process of normalization can most clearly be followed by observing bipolar forms with a head at each end (Fig. 4). Initially, these consist of a mirror-image duplication of the upper or ‘distal’ regions. The missing lower or ‘proximal’ body parts are not inserted until treatment with DAG has finished. Then, first a budding zone is formed exactly halfway between the two heads. Subsequently, this zone splits into two and the next buds emerge in two symmetrically located zones, whilst the former central budding zone acquires the characteristics of a stalk. Finally, in the centre, a basal disk is formed. It splits into two and two complete wild-type animals separate from each other.

Fig. 4.

Process of normalization. Starting forms were biheaded regenerates as shown in Fig. 1B. (A,B,C) Actual development in the course of 2–4 weeks. (D) Summary and interpretation in terms of positional value P. The P values were arbitrarily divided in 10 units. The mouth is defined by the value 10, the budding zone by the value 3–4, the basal disk (foot) by the value 0. The P-value decreases in the middle as if a sink were inserted halfway between the two heads.

Fig. 4.

Process of normalization. Starting forms were biheaded regenerates as shown in Fig. 1B. (A,B,C) Actual development in the course of 2–4 weeks. (D) Summary and interpretation in terms of positional value P. The P values were arbitrarily divided in 10 units. The mouth is defined by the value 10, the budding zone by the value 3–4, the basal disk (foot) by the value 0. The P-value decreases in the middle as if a sink were inserted halfway between the two heads.

Periodic treatment with DAG causes wild-type hydras to produce successively ectopic heads along the elongating gastric column. The used wild-type strain 105 of H. magnipapillata is progressively transformed into a phenocopy of a known multiheaded mutant of H. viridis (Novak & Lenhoff, 1980). In this mutant, as well as in the phenocopies produced here, heads arise in a periodic pattern the spacing being not very regular.

In terms of the concepts underlying the classical transplantation studies, ectopic head formation may be caused (a) by a sustained lowering of the head-inhibition level and (b) by increasing the positional value of the tissue and, hence, by enhancing its capacity to form and to induce head structures (Wolpert et al. 1974; MacWilliams, 1983a,b; Sugiyama, 1982; Takano & Sugiyama, 1983; Nishimiya et al. 1986).

In the animals exposed to DAG, it is an increase in the positional value that precedes the visible appearance of head structures. In the present pilot study, this statement is supported by two arguments. (1) The temporal order in which ectopic head structures arise reflects their position along the body column. Tentacles appear first and only later mouth cones. (2) Long before ectopic tentacles sprout in the intact animals, excised segments not only form supernumerary head structures but also lose the ability to regenerate feet.

Extensive transplantation studies, which will be documented in a separate paper, confirm this statement. They clearly show an early increase in the positional value beginning with the first pretreatment. In short, tissue pieces transplanted into the same positional level of untreated recipients progressively induce head formation instead of being integrated whereas pieces imported from untreated donors into DAG-treated hosts form feet.

DAG is the first, and presently only, physically defined substance to induce, in hydra, an increase in the positional value and, hence, ectopic head formation. DAG cannot be replaced by the often-used surrogate for DAG, the phorbol ester TPA, nor by the ‘head activator’ (Schaller & Bodenmiiller, 1981). TPA may evoke ectopic head formation in a small sublethal range of concentrations but it evokes also ectopic foot formation with almost equal frequency (Bernhard Schwoerer-Böhning, personal communication). Animals treated with the ‘head activator’ did not differ morphologically from untreated controls even when this peptide was applied periodically (Michael Kroiher, personal communication).

Explanation

All results presented here can be consistently interpreted by the following hypothesis which is compatible, but not identical, with a more formal model proposed by Berking (1984).

A phosphatidylinositol-diacylglycerol-protein kinase C based signal processing system is involved in the release or the storage of positional information. The uptake of DAG leads to the release of a stimulating factor, or it mimicks the reception of such a stimulating signal by virtue of its role as second messenger. The stimulated cells respond by adjusting their positional memory to a higher value. This memory is closely coupled with position-specific differentiation and encoded in the number of molecules P. These undergo a certain turnover.

To synthesize the stationary P-molecules, cells need and consume mobile precursors, pro-P. Cells endowed with a higher positional value detract pro-P from their neighbours having a lower value. Thus, pro-P and, hence, eventually also P, decrease in the lower body part. A foot is formed by cells exhausted of pro-P. This notion is deduced from (still unpublished) transplantation and regeneration studies. Polyps onto which a second head has been grafted regenerate a foot much faster than do single-headed animals and these regenerate faster than do decapitated polyps.

The process of depletion is slow, and in some hydroids the velocity is almost zero. Such hydroids, e.g. large polyps of the hydroid Hydractinia used in this study (section A), obey the rule of distal transformation, being unable to restore lower body parts and forming heads sometimes also at their lower end (Müller, 1982; Müller et al. 1986). Surprisingly, on daily exposure to DAG, Hydra also obeys this rule. The results are those bi- and multiheaded forms shown in Figs 1 and 4, which fail to regenerate the lost proximal body regions. In terms of the current hypothesis, DAG increases the supply of pro-P and/or reduces the decomposition of the P-molecules.

After the DAG treatment has finished, degradation of P-molecules prevails over their renewal in regions distant from existing heads because these regions become exhausted of pro-P due to the suction by the cells having a higher positional value, and, therefore, a higher turnover rate. In the biheaded forms with heads at the two opposing ends first the middle part is depleted. The concomitant intercalation of proximal structures is visualized in Fig. 4.

Comparison to the vertebrate limb

The terms ‘positional value’ and ‘distal transformation’ are also used in studies on the proximodistal pattern of the vertebrate (and insect) limb. Common terms suggest common formal mechanisms of pattern generation or pattern control. Attempts to look for formal analogies presuppose that an appropriate point of reference is found for defining which end of the longitudinal axis the highest positional value should be assigned to.

Unfortunately, the literature on hydroids on the one hand and limbs on the other hand is infiltrated with discrepancies. In hydra, the highest positional value has been assigned to the upper end (Wolpert et al. 1974) and this end is usually designated as the ‘distal’ one because, in budding, this end grows away from the parent animal. In contrast, in the vertebrate limb the highest value has been assigned to the proximal end (Wolpert, 1978; Maden, 1984).

On the other hand, as pointed out above, many hydroids and, surprisingly, also hydra upon periodic exposure to DAG, obey the ‘rule of distal transformation’ as do the vertebrate limbs (e.g. Maden, 1980; Stocum, 1981). Historically, this rule has first been formulated for hydroids (Rose, 1957). According to this rule, the head of a polyp is equivalent to the hand of a limb. Therefore, the segments of hydra forming a head at each end are formally equivalent to an excised segment of a limb that would regenerate a hand at both ends. Ectopic tentacles were equivalent to ectopic fingers. The effect of DAG on hydra, as shown in this study, is formally inverse to the effect of retinoic acid on the proximodistal axis of the axolotl limb (e.g. Maden, 1984).

Apart from this mere formal inversion DAG promises to become a clue that will help in identifying the biochemical mechanism of positional information transfer and storage as did retinoic acid in case of the vertebrate limb.

Berking
,
S.
(
1984
).
Metamorphosis of Hydractinia echinata, insights into pattern formation in hydroids
.
Wilhelm Roux’s Arch, devl Biol
.
193
,
370
378
.
Berridge
,
M. J.
(
1987
).
Inositol trisphosphate and diacylglycerol: two interacting second messengers
.
A. Rev. Biochem
.
56
,
159
193
.
Bode
,
P. M.
&
Bode
,
H. R.
(
1984
).
Patterning in hydra
.
In Pattern Formation
(ed.
G. M.
Malacinski
&
S. W.
Bryant
), pp.
213
241
.
New York
:
Macmillan
.
Castagna
,
M.
(
1987
).
Phorbol esters as signal transducers and tumor promoters
.
Biol. Cell
59
,
3
14
.
Kikkawa
,
U.
&
Nishizuka
,
Y.
(
1986
).
The role of protein kinase C in transmembrane signalling
.
A. Rev. Cell Biol
.
2
,
149
178
.
Leitz
,
T.
&
Müller
,
W. A.
(
1987
).
Evidence for the involvement of Pl-signaling and diacylglycerol second messenger in the initiation of metamorphosis in the hydroid Hydractinia echinata
.
Devl Biol
.
120
,
82
89
.
Macwilliams
,
H. K.
(
1983a
).
Hydra transplantation phenomena and the mechanism of Hydra head regeneration. I. Properties of the head inhibition
.
Devl Biol
.
96
,
217
238
.
Macwilliams
,
H. K.
(
1983b
).
Hydra transplantation phenomena and the mechanism of hydra head regeneration. II. Properties of the head activation
.
Devl Biol
.
96
,
239
257
.
Macwilliams
,
H. K.
(
1983c
).
Numerical simulations of Hydra head regeneration using a proportion-regulating version of the Gierer-Meinhardt model
.
J. theoret. Biol
.
99
,
681
703
.
Maden
,
M.
(
1980
).
Intercalary regeneration in the amphibian limb and the rule of distal transformation
.
J. Embryol. exp. Morph
.
56
,
201
209
.
Maden
,
M.
(
1984
).
Retinoids as probes for investigating the molecular basis of pattern formation
.
In Pattern Formation
(ed.
G. M.
Malacinski
&
S. W.
Bryant
), pp.
539
555
.
New York, London
:
Macmillan
.
Meinhardt
,
H.
(
1982
).
Models of Biological Pattern Formation
.
New York, Oxford
:
Academic Press
.
Müller
,
W. A.
(
1982
).
Intercalation and pattern regulation in hydroids
.
Differentiation
22
,
141
150
.
Müller
,
W. A.
(
1985
).
Tumor-promoting phorbol esters induce metamorphosis and multiple head formation in the hydroid Hydractinia
.
Differentiation
29
,
216
222
.
Müller
,
W. A.
,
Plickert
,
G.
&
Berking
,
S.
(
1986
).
Regeneration in hydrozoa: distal versus proximal transformation in Hydractinia
.
Roux’s Arch, devl Biol
.
195
,
513
518
.
Nishimiya
,
C.
,
Wanek
,
N.
&
Sugiyama
,
T.
(
1986
).
Genetic analysis of developmental mechanisms in hydra. XIV. Identification of the cell lineages responsible for the altered developmental gradients in a mutant, reg-16
.
Devl Biol
.
115
,
469
478
.
Nishizuka
,
Y.
(
1986
).
Studies and perspectives of protein kinase C
.
Science
233
,
305
312
.
Novak
,
P.
&
Lenhoff
,
H. M.
(
1980
).
Regulation of bud induction and site of tentacle sprouting in a nonbudding strain of Hydra viridis
.
In Developmental and Cellular Biology of Coelenterates
(ed.
P.
Tardent
&
R.
Tardent
), pp.
237
242
.
Amsterdam, New York, Oxford
:
Elsevier/North HolLond Biomed. Press
.
Rose
,
S. M.
(
1957
).
Polarized inhibitory effects during regeneration in Tabularía
.
J. Morph
.
100
,
187
206
.
Schaller
,
H. C.
&
Bodenmüller
,
H.
(
1981
).
Isolation and amino acid sequence of a morphogenetic peptide from hydra
.
Proc, natn. Acad. Sci. U.S.A
.
78
,
7000
7004
.
Schaller
,
H. C.
,
Schmidt
,
T.
&
Grimmelikhuuzen
,
C. J. P.
(
1979
).
Separation and specificity of action of four morphogens from hydra
.
Wilhelm Roux’s Arch, devl Biol
.
106
,
139
149
.
Stocum
,
D. L.
(
1981
).
Distal transformation in regenerating double anterior axolotl limbs
.
J. Embryol. exp. Morph
.
65
,
3
18
.
Sugiyama
,
T.
(
1982
).
Roles of head-activation and head-inhibition potentials in pattern formation of Hydra: Analysis of a multiheaded mutant strain
.
Am. Zool.
22
,
27
34
.
Sugiyama
,
T.
&
Fujisawa
,
T.
(
1977
).
Genetic analysis of developmental mechanisms in hydra. I. Sexual reproduction of Hydra magnipapillata and isolation of mutants
.
Develop., Growth and Differ
.
19
,
187
200
.
Takano
,
J.
&
Sugiyama
,
T.
(
1983
).
Genetic analysis of developmental mechanisms in hydra. VIII. Head-activation and head-inhibition potentials of a slow-budding strain (L4)
.
J. Embryol. exp. Morph
.
78
,
141
168
.
Wilby
,
O. K.
&
Webster
,
G.
(
1970
).
Experimental studies on axial polarity in hydra
.
J. Embryol. exp. Morph
.
24
,
595
613
.
Wolbert
,
L.
(
1978
).
Pattern formation in biological development
.
Sci. Am
.
10
,
124
137
.
Wolfert
,
L.
,
Hornbruch
,
A.
&
Clarke
,
M. R. B.
(
1974
).
Positional information and positional signalling in Hydra
.
Am. Zool
.
14
,
647
663
.