Wilhelm Roux's Archives 188, 133-139 (1980) Roux's Archives of Developmental Biology �9 by Springer-Verlag 1980

Properties of the Foot Inhibitor From Hydra

Tobias Schmidt* and H. Chica Schaller**

European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany

Summary. A substance was isolated from crude ex- tracts of hydra that inhibi ts foot regenerat ion. This substance, the foot inhibi tor , has a molecular weight of < 500 daltons. It is a hydrophi l ic molecule, slightly basic in character and it has no peptide bonds. The pruified substance acts specifically and at concentra- t ions lower than 10-7 M. At this low concent ra t ion only foot and not head regenerat ion is inhibited. Hyd- ra are sensitive to purified foot inhib i tor between the second and eight hour after in i t ia t ion of foot regener- a t ion by cutting. In no rma l animals the foot inhibi tor is most likely produced by nerve cells. A substance with similar biological and physico-chemical prop- erties is found in other coelenterates.

Key words: Hydra - Morphogenet ic substances - Re- generat ion - Pa t te rn fo rmat ion - Sea anemones.

Introduction

Morphogenes is in hydra seems to be control led by at least four morphogenet ic substances that activate or inhibi t head or foot format ion (Schaller 1973; Berking 1977; Gr immel ikhui jzen and Schaller 1977; Schmidt and Schaller 1976). Previously we have de- scribed the basic propert ies of one of these substances, the foot inhibi tor . This substance was isolated f rom hydra tissue and inhibi ted foot fo rmat ion in regener- ates. In the an imal it was present as a gradient descen- ding f rom the foot to the head, paral lel ing the foot- inhibi t ing potent ia l found in t r ansp lan ta t ion experi- ments by MacWil l iams and Kafa tos (1974). In this paper, further in fo rmat ion on the properties of the

Present address: * Hopkins Marine Station, Department of Bio- logical Sciences, Stanford University, Pacific Grove, CA 93950, USA �9 * Max-Planck-Institut f/ir Medizinische Forschung, Abteilung Biophysik, Jahnstr. 29, 6900 Heidelberg, F.R.G.

foot inhibi tor is presented and compared to the head inhibitor.

Materials and Methods

Culture Conditions

Hydra attenuata were mass cultured in a medium derived from that of Lenhoff and Brown (1970) consisting of 1 mM CaCI2, 0.1 mM KC1, 0.1 mM MgCI2 and 2 mM NaHCO3, pH 7.8. The water temperature was kept at 19_+2 ~ C. The animals were fed daily between 10 and 11 a.m. with nauplii of Artemia salina and washed 6-8 h after feeding.

Animals starved for at least 24 h were used for the biological assays.

Biological Assays

Assay for the Foot Inhibitor. Hydra without buds were cut transver- sely into two halves of equal length. The upper halves were trans- ferred immediately after cutting to dishes containing 10 ml of medi- um with or without foot inhibitor. For each concentration 3 dishes with 25 animals were used. The animals were incubated for 20 h, then washed and transferred to new dishes containing hydra medi- um only. Foot regeneration was monitored at roughly hourly inter- vals from 21 28 h. It was considered to be complete when the animals were abte to stick to the surface of the dish or the water with their newly formed foot. Inhibition was determined when 75% of the control animals had regenerated a foot and was defined a s

l = % ~ x 100(%),

where T is the percentage of animals that have regenerated a foot in the treated sample. A 25% inhibition was significant (Z2-test; P< 0.05). The amount of material necessary to achieve this effect in 10 ml of medium was defined as one biological unit (BU: Schmidt and Schaller 1976).

Assay for the Head Inhibitor. Hydra without buds were decapitated at time 0 and transferred immediately after cutting to dishes con- taining 10 ml of medium with or without head inhibitor. For each concentration 3 dishes each containing 25-30 animals were used.

0340-0794/80/0188/0l 33/$01.40

134 T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra

From 20 h after cutting the number of regenerates with clearly visible outgrowths o f at least two tentacles was registered at roughly hourly intervals. As in the foot-inhibitor assay inhibition was deter- mined when 75% of the control animals had tentacle bmnps and was defined as:

I = ~ x 100(%),

where T is the percentage of animals with tentacle bumps in the treated sample. A significant inhibition of 25% ()~2-test; P < 0.05) was obtained with 0.5 BU of the head inhibitor as defined in Schaller et al. (1979). The head inhibitor was purified according to Schaller et al. (1979).

Crude Extract

Hydra or hydra tissue was sonicated in H20 under ice cooling for 3 x 20 s with a Branson B12 untrosonicator at 50 W. The con- centration of this 'c rude extract ' was determined by measur ing the absorption at 280 n m or by protein determination according to Diamant et al. (1967).

Enzymatic Digestion

Fifty BU each of a Sephadex DE AF A-25 purified extract (see Results) containing 1 mg/ml protein were incubated for 1 h at 37 ~ C with trypsin (1 mg/ml), pronase (1 mg/ml), and proteinase K (0.2 mg/ml) as well as with DNase and RNase (20 gg/ml). After incubation the enzymes (Boehringer, Mannheim) were removed by ultrafiltration with Diaflo U M 2 filters.

Acid Hydrolysis

Two hundred and fifty BU of a Sephadex G-10 pruified extract (see Results) were freeze dried, dissolved in 6 N HC1 and kept at 110 ~ C for 24 h. Prior to analysis the HC1 was evaporated and the material dissolved in H20. The pH was adjusted to 7.8 with NaOH.

Molecular Weight Determination

Five hundred BU of a Sephadex G-10 purified extract were dis- solved in 90% methanol and filtered through the Diaflo filters U M 10, U M 2 and U M 05 (Amicon, Holland) with a cut off level of 10,000, 1,000, and 500 daltons respectively. The filtrates were tested for activity after evaporation of the methanol.

Cell Preparation

Tissue was macerated in a mixture of 7% acetic acid and 7% glycerol and the cells were classified according to David (1973)

Cell Separation

Of a cell suspension obtained by dissociation of hydra tissue in macerat ion medium (7% acetic acid; 7% glycerol) 1 ml portions were layered on a discontinuous gradient consisting of 1 ml each of 60%~ 50%, 40%, 30%, 20%, and 15% glycerol in 7% acetic acid. After 5 min centrifugation at 3,000 g, 0.65 ml fractions were carefully taken from the top of the gradients, the respective frac-

tions pooled and a small sample taken for cell counts (Bode et al. 1973). The pooled fractions were then centrifuged at 3,000 g for t0 rain. The sediments were suspended in a solution of 200 m M NaCI and 35 m M Tris-HC1, pH 7.6, and centrifuged again to re- move the dissociation medium. These sediments were dissolved in HaO and sonicated (Grimmelikhuijzeu 1979).

Nitrogen-Mustard Treatment

Hydra, starved for 24 h, were incubated in 0.015% nitrogen-mus- tard for 10 min. (Diehl and Burrnett 1964), washed several times and from the next day on treated as normal animals. Eight days after treatment, the cell distribution was determined and the ani- mals sonicated in H 2 0 to obtain crude extract.

Sea Anemones

Anthopleura elegantissima were obtained from Pacific Bio-Marine Laboratories, Inc., California. Tealia felina were collected in The Netherlands and Anemonea sulcata in Greece. Crude extracts were prepared and assayed either directly or after purification for con- tent of foot inhibitor.

Results

Purification and Yield

Hydra were homogenized in methanol, the suspension was centrifuged at 3,000 g for 5 min and the pellet reextracted three times with methanol. The pooled supernatants were extracted three times with petrol ether. The methanol-water phase was concentrated by evaporation and applied to a Sephadex G-10 col- umn and then eluted with distilled water (Fig. 1 a). The active fractions were pooled, brought to pH 7.6 with NaOH and applied to a Sephadex DEAE A-25 column equilibrated with 35 mM Tris-HC1, pH 7.6 (Fig. lb). The flowthrough that contained all foot- inhibitory activity was applied to a Dowex AG 50-W- X8 column equilibrated with water. A linear HC1 gradient (0-6 N HC1) was applied. The foot inhibitor eluted at 3-4 N HC1 (Fig. 1 c). The active fractions were pooled, the HC1 evaporated, and the residue dissolved in methanol and chromatographed on a Se- phadex LH-20 column using methanol as the eluent (Fig. 1 d).

The purification and respective yields for the foot inhibitor during the preparation are given in Table 1. Purification is expressed in terms of absorption at 280 rim. Although is is not yet clear whether the foot inhibitor absorbs at that wavelength, the O.D.~8o gives an estimate of the degree of purity. The five purification steps gave 14,000 fold enrichment with a yield of 80% compared to hydra crude extract.

This purification procedure also separated the foot inhibitor from the other three morphogens of hydra (Schaller et al. 1979).

T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra 135

02 0.1

. 20 /*0 60 80 100 120 I(,0

10

!!I c//4

I0 20

u Z

c

b 08 -~ . . - " (IJ

/I ...................

~ _ ~ . . . . F ~ , , , " - - - - -_~o2 Z 0 10 20 30 t,O

05 C 0.4 - -- 0.3 . - 6 Z 0.2 L~

"~ S I0 15 20 25 30 35 1 0 ~ ._

e ~ d .~ o 03

0.2 f

30 ~o so 60 7o ao 90

Fraction

Fig. 1.a-d. Purification of the foot inhibitor (F/). a Chromatogra- phy of a methanol extract of hydra on Sephadex G-10. Eluent, H~O; Column size, 340mi; length, 50cm. Fraction size, 9 ml. b Chromatography of fractions 10-20 from a on Sepahdex DEAE A-25. Eluent, 35 mM Tris-HC1, pH 7.6 (in the left part of the graph) and with an additional NaC1 gradient (from 0 1 M NaC1, dotted line) in the right part of the graph. Column size, 28 ml, 16 cm. Fraction size, 3.5 ml. e Chromatography of the flowthrough from b on Dowex AG 50-W-X8. Eluent, H20 followed by a linear HCl-gradient (from 0-6 N HC1). Elution of the foot inhibitor between 3 and 4 N HCI (fractions 22 25). Column size, 35 ml, 19 cm. Fraction size, 4.6 ml. fl Chromatography of fractions 22-25 from e on Sephadex LH 20. Eluent, methanol. Column size, 180 ml, 150 cm. Fraction size, 2 ml

Table 2. Molecular weight determination of the foot inhibitor

Filter Retentivity Activity in filtrate (% of total)

UM 10 > M W 10,000 >95 UM 2 > MW 1,000 >75 UM 05 > MW 500 < 35

when chromatographed in the presence of 500 mM NaC1, with a KD of 1.%2.2. As the exclusion limit of G-10 is 700 daltons, this position close to salt would correspond to a molecular weight lower than 700. Ultrafiltration (see Materials and Methods) gave a similar result. The foot inhibitor passed through filters that retain molecules larger than 10,000 and 1,000 daltons (Table 2); 35% passed through a filter with a cut off level of about 500 daltons. This cut off level also depends on the charge of the molecule. From this we conclude that the foot inhibitor has a molecular weight of around or lower than 500 dal- tons.

Ionic Charge. Hydra crude extract purified on Sepha- dex G-10 was chromatographed on ion exchangers under various conditions. The foot inhibitor did not bind to anion exchangers, at least not up to a pH of 10. It did bind to cation exchangers, however only at very low pH (Dowex AG 50-W-X8, H + form, pH<4). This means that the foot inhibitor has a weakly positive overall charge.

Stability. Foot inhibitor was subjected to enzymatic and chemical hydrolysis as described in Material and Methods. It was found to be stable against trypsin, pronase, proteinase K, DNAase and RNAase. Also the foot inhibitor was not destroyed by acid hydroly- sis (6N HC1 at 110 ~ C for 48 h). It is therefore very unlikely that it contains peptide bonds.

Physico-Chemical Properties

Molecular Weight Determination. The size of the foot inhibitor was determined by gelfiltration and by ultra- filtration. On Sephadex G-10 the foot inhibitor eluted,

Biological Properties

Specificity of the Purified Foot Inhibitor. Transplanta- tion experiments have shown that the inhibition

Table 1. Enrichment and yield of the foot inhibitor from a preparation of about 30,000 hydra

Fraction Total activity Total O.D.2s0 Specific activity Overall yield Overall purification (BU) (BU/O.D.2so) (%) (fold)

Crude extract 4,200 5,040 0.8 100 0 Methanol extract 4,100 890 4.6 98 5.8 Fraction G-10 3,900 760 5.3 95 6.6 Fraction DEAE-A25 3,700 80 47.3 90 59.1 Fraction AG50-W-X8 3,500 1 3,182 85 3,978 Fraction LH-20 3,300 0.3 11,000 79 13,750

136

60i r

20

FOOT HEAD REGENERATION REGENERATION

0.5 1 5 10 50 100 Concentration of foot inhibitor (BU)

o= 8 0

:5 60

- /+0

20

HEAD FOOT REGENERATION REGENERATION

"/~ b

Concentration of heed inhibifor(BU)

Fig. 2a and b. Specificity of action of foot inhibitor and head inhibitor, a Effect of purified foot inhibitor on foot and head regeneration, b Effect of purified head inhibitor on head and foot regeneration

T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra

Table 3. The distribution of foot and head inhibitor in hydra. The content is given as % of total inhibitor present in the animal.

Basal disc Rest of body (%) (%)

Foot inhibitor 65 35 Head inhibitor 20 80

A

50

--= 30

20

10

1'0 1'2 1'/+ ll6 ll8 Time offer cuffing (h)

Fig. 3. The foot inhibitory effect as a function of incubation time. 3.5 BU of purified foot inhibitor were used. Start and end of incu- bation during foot regeneration

emanating f rom the foot is different from that of the head region. To prove that the foot inhibitor is a substance specific for foot inhibition we investi- gated its influence on foot as well as on head regenera- tion (Fig. 2a). Purified head inhibitor (Schaller et al. 1979) was also tested for its influence on the regenera- tion of both structures (Fig. 2b). These experiments show that the foot inhibitor as well as the head inhibi- tor act specifically. At low concentrations only the regeneration of the respective structure is inhibited. These results are somewhat in contradiction to those of Berking (1979) who described a factor that inhibits head and foot formation equally well. In addition, Berking's inhibiting factor has two maxima, one in the head and another smaller one in the foot (Berking 1977). In the following we show that head and foot indeed contain both inhibitors but that the ratio is different in head and foot. The head region predomi- nantly contains head inhibitor, the foot region foot inhibitor. The experimental approach was as follows. Basal discs of hydra were cut off and the two resultant pieces (basal disc and rest of body) were assayed for their content of foot and head inhibitor. This was done after acid hydrolysis of the crude extracts and chromatography on Dowex A G 50-W-X8. The crude extracts were subjected to acid hydrolysis to destroy the peptidic head and foot activator (Schaller 1973; Grimmelikhuijzen 1979) and to chromatography on Dowex A G 50 to separate head from foot inhibitor. Table 3 shows that the foot inhibitor is most concen- trated in the foot region. The basal disk which repre-

sents approximately one tenth of the total animal contains 65% of the foot inhibitor present in an ani- mal. The head inhibitor, on the other hand, shows a different distribution; 80% of the total activity is present in the body regions above the basal disk.

Action of the Purified Foot Inhibitor on Foot Regenera- tion. Similar dose-response curves were obtained for hydra crude extract and for purified foot inhibitor. However, to achieve the same effect the incubation time could be reduced f rom 20 h for crude extract to 8 h for purified foot inhibitor. To determine at what time the foot inhibitor is active during foot regenera- tion, upper halves of hydra were incubated with a low concentration of foot inhibitor for different per- iods after cutting. The duration as well as the start of incubation was varied. Figure 3 shows that the foot-inhibitor sensitive phase is between the second and eight hour after initiation of foot regeneration by cutting. I f new foot inhibitor is provided every two hours during the sensitive period (2-8 h) the inhi- bitory effect is significantly enhanced (not shown).

Changes in Foot-Inhibitor Concentration During Re- generation. Foot inhibitor diffuses rapidly into the surrounding medium when hydra are cut into pieces (Schmidt and Schaller 1976). To determine the quan- tity released, the time sequence of release and the subsequent synthesis of foot inhibitor during foot re- generation, the following experiment was done. From foot-regenerating halves of hydra, crude extracts were

T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra 137

< I./3

Z

.~ 0.08

o 0.0~ 0

Foot regeneration

0.04 I 0.02 I '

12 10 HOURS AFTER CUTTING

Fig. 4. Content of foot inhibitor during foot regeneration. Upper halves of hydra were kept in medium for varying periods of times and then tested for foot-inhibitor content

024 8

i

i

20 ' 24 ' ~0

prepared at varying periods of time after cutting. These crude extracts were then tested for content of foot inhibitor. As can be seen from Fig. 4 there is a loss of about 20%-25% of the total foot-inhibitory activity from the regenerating animal during the first two hours after cutting. This decrease was restricted to the regenerating tip and not found in the underly- ing tissue (experiment not shown). At 4 h after cutting the foot-inhibitor concentration starts to increase un- til the value for normal animals with mature feet is reached after roughly 28 h of foot regeneration.

Localization of the Foot Inhibitor in Nerve Cells. In order to find the morphogen producing cell type, cell composition and foot-inhibitor content were com- pared in different regions of hydra and in hydra vary- ing in cell composition. As shown in Fig. 5 from the distribution of cell types and of foot inhibitor in the different body regions of hydra it seems unlikely that nematocytes, interstitial cells, gland or mucous cells are the producers of foot inhibitor, since the numbers of these cells are either negatively or not correlated

c~

oJ

-'E 1 .Nerve celts

05 = . . . . . . . .

13tend and mucous cetts I-

05 ~ j ~ 7 - f - / - ? ~ ~ . . . . Head Upper Lower Foot

gastric region Fig. 5. Distribution of cells and foot inhibitory activity in different regions of hydra. The 'density' of a given cell type is defined as the ratio of the number of alls of that cell type to the number of epithelial cells. The concentration of foot inhibitor is expressed as biological units per O.D.280 (BU/O.D.280)

with the foot-inhibitor concentration. This is supprot- ed by the finding that nitrogen-mustard treated hydra (Diehl and Burnett 1964) which one week after treat- ment have lost selectively all their interstitial cells and most of their nematocytes (Table 4), contained normal amounts of foot inhibitor. This suggested that epithelial cells or nerve cells may be the source of foot inhibitor.

To decide between these two alternatives an at- tempt was made to isolate and enrich nerve cells. A suspension of macerated cells (David 1973) which had lost 50% of its original foot-inhibitory activity was separated by density centrifugation in discontin- uous glycerol gradients. The fractionation resulted in an enrichment of nerve cells in the top fractions of the gradient. Figure 6 shows that in fraction one (the top fraction), nerve cells were enriched 10 fold and in fraction two, 8 fold. Epithelial cells were also enriched in these two fractions, namely 1.5 fold in both cases. These two fractions contained almost no other cell types but they contained more than 90% of the total foot-inhibitor activity in the cell suspen-

Table 4. Distribution of cell types in normal and nitrogen mustard treated animals

Epithelial Big Little Nematocytes Nerve cells cells (%) interstitial interstitial and -blasts (%)

cells (%) cells (%) (%)

Gland and Mucous cells (%)

Normal animals 22.2 11.4 14.4 40.5 6.2 6.3

Nitrogen mustard animals 71.7 - - 7.1 8.9 12.3

138 T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra

~ 80[I/nseperate d 601 ceil mixture

20 ==

0iio i i5 40

20

601 Fraction 2

60 t Fraction 3

601 Fraction 4

2O r-'fT]

30 x

20 -~ g

10

O

6

4

2 FL I-L_ _

Nerve epithelia( i-ceils nema~o- gland and cells ceils cyfes mucous ceils

Fig. 6. Enrichment of nerve cells. The distribution and number of the major cell types is given for the unseparated cell mixture and for the four top fractions of the glycerol gradient

sion. From this we conclude that either a subpopula- tion of the nerve cells (22% of total) or a much smaller subpopulat ion of the epithelial cells (4% of total) is responsible for the foot-inhibitory activity.

Foot Inhibitor and Nuerotransmitters. Since the foot inhibitor seemed to be a product of nerve cells, we assayed known and putative transmitters of similar low molecular weight for their action on foot and head regeneration in hydra. We found that at concen- trations f rom 10 -1~ to 10 .6 M, the following sub- stances showed no effects comparable to the head or foot inhibitor: acetylcholine, aspartic acid, dopa- mine, epinephrine, GABA, glycine, glutamic acid, his- tamine, norepinephrine, octopamine, praline seroto- nine, and taurine. F rom this we conclude that the head and foot inhibitor are not identical with any of these substances.

Other Sources for Foot Inhibitor. Other coelenterales, mammal ian brain and intestine were assayed for their

Table 5. Content of foot inhibitor in some sea anemones

Total Total Specific Hydra O.D.280 activity activity equi-

(BU) (BU/O.D.z80) valents

Hydra attenuata 0.15 0.13 0.8 1 Anemonea sulcata 15,000 2,500 0.17 19,200 Tealiafelina 15,000 1,500 0.1 11,500 Anthopleura I6,000 600 0.1 4,300

elegentissima

content of foot inhibitor. F rom the different sources investigated, only sea anemones contained a sub- stance with biological and physico-chemical proper- ties similar to the foot inhibitor from hydra. The content of this substance varied f rom species to spe- cies (Table 5).

Discussion

In a previous paper (Schmidt and Schaller 1976) evi- dence was presented that hydra contain a substance that inhibits foot regeneration. In this paper we show that this substance, the foot inhibitor, is a small basic hydrophilic molecule of about 500 daltons molecular weight. It does not seem to be a peptide, since its activity is not destroyed by proteases or under condi- tions where peptide bonds are cleaved (6N HC1 at 110 ~ C for 48 h). By means of five purification steps, a 15,000 fold enrichment was obtained (in terms of optical density at 280 nm) and it was separated from the three other hydra morphogens (Schaller et al. 1979). This purification and separation is essential for a biological characterisation of the foot inhibitor, but it is not sufficient for the elucidation of its chemi- cal structure. A further pruification will require addi- tional steps, especially steps which allow a separation of the foot inhibitor form other small molecules such as salts. So far no single such step has worked satisfac- torily. Another problem for the chemical analysis is the amount of material available. One hydra contains less than 10 10 mol of foot inhibitor. Since hydra cannot be produced in large quantities other sources may be required. We found that one possible source are sea anomones which contain foot inhibitor and which are available in large quantities.

One of the problems in the biological characterisa- tion of substances like the foot inhibitor is to prove that they are really involved in the control of morpho- genesis in viva. Three lines of evidence support the notion that the foot inhibitor is the morphogen re- sponsible for the inhibition of foot formation in hyd- ra. The foot inhibitor is distributed within the animal

T. Schmidt and H.C. Schaller: Foot Inhibitor in Hydra 139

as a gradient with a maximal concentration in the foot region (Schmidt and Schaller 1976). This agrees with results from transplantation experiments which had shown that a foot-inhibiting principle is most concentrated in the basal disc (MacWilliams and Ka- fatos 1974). From the molecular weight and the pre- sent degree of purity (in terms of dry weight) it can be calculated that the foot inhibitor is active at a very low concentration, less than 10-7 M. Since the preparation is still impure the active concentration may actually be lower. This indicates that a molecule with a very specific action has been enriched. Finally, the foot inhibitor at this low concentration only inhi- bits foot and not head regeneration. Such a specificity is expected, if head and foot formation are governed by independent regulatory systems.

In non-regenerating normal hydra, the foot inhibi- tor seems to have two functions. It prevents the for- mation of a second foot in the vicinity of an existing one, and it may be required for regulating the size of the existing foot. During foot regeneration, at least in the early phase, absence of foot inhibitor seems to be required to allow induction of foot regeneration. This is supported by two findings. First, the foot inhibitor exerts its inhibitory action on foot regenera- tion most drastically during the first 8 h after cutting. The experiments revealed that the inhibitor sensitive phase is between the second and eighth hour after initiation of foot regeneration by cutting and from transplantation experiments it is known that within this time the tissue near the cut is reprogrammed to form a foot (MacWilliams and Kafatos 1968 ; Web- ster 1971). The presence of foot inhibitor during this critical period thus seems to interfere with the induc- tion of foot regeneration. Second, in accordance with this views, we found a drop in foot-inhibitor concen- tration in animals regnerating feet during the first two hours after cutting. This drop was restricted to the regenerating tip and was not found in the adjacent overlying tissue. The concentration of the foot inhibi- tor later increased in the animals regenerating feet until it reached the value found in mature feet when foot regeneration was completed.

For the two activators from hydra it was shown (Schaller and Gierer 1973; Grimmelikhuijzen 1979) that they are produced by nerve cells. Likewise we found that in normal animals the foot inhibitor is most likely produced by and/or stored in nerve cells, less likely by epithelial cells. In both cases the foot inhibitor is present in a subpopulation of the cell type, 22% of the nerve cells, 4% of the epithelial cells, respectively, which according to the distribution of the foot inhibitor within the animal must be local- ised in the foot region. This is in accordance with the notion that nerve cells as well as epithelial cells

of specific morphology are found only in the foot region (David 1973; Epp and Tardent 1978).

The data presented, indicate that foot formation in hydra is controlled by two morphogens, the foot inhibitor and the foot activator (Grimmelikhuijzen and Schaller 1977). How these substances interact to control foot formation in hydra remains to be investigated.

Acknowledgement. The authors wish to thank Dr. C.J.P. Grimme- likhuijzen for stimulating discussions, Beate Zachmann for excel- lent technical assistance, Ines Benner for typing the manuscript, and the Deutsche Forschungsgemeinschaft for financial support.

References

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Berking S (1979) Analysis of head and foot information in hydra by means of an endogenous inhibitor. Wilhehn Roux's Archives 186:189~10

Bode H, Berking S, Gierer A, Schaller H, Trenkner E (1973) Quan- titative analysis of cell types during growth and morphogenesis in hydra. Wilhelm Roux's Archives 171:269-285

David CN (1973) Quantitative method for maceration of hydra tissue. Wilhelm Roux's Archives 171:259-268

Diamant B, Redlich D v, Glick D (i967) Comparison of the phen- olreagent and bromsulfalein-binding methods for determination of protein and its measurement in mast cells. Anal Biochem 2i : 135 146

Diehl F, Burnett AL (1964) The role of interstitial ceils in mainte- nance of hydra. I. Specific destruction of interstitial cells in normal, asexual, non-budding animals. J Exp Zool 155 : 253-260

Epp L, Tardent P (1978) The distribution of nerve ceils in Hydra attenuata Pall. Wilhelm Roux's Archives 185:185-193

Grimmelikhuijzen CJP (1979) Properties of the foot activator from hydra. Cell Differ 8:267-273

Grimmelikhuijzen CJP and Schaller HC (1977) Isoiation of a sub- stance activating foot formation in hydra. Cell Differ 6 : 297-305

Lenhoff HM, Brown RD (t970) Mass culture in hydra: an im- proved method and its application to other aquatic inverte- brates. Lab Anita 4:139 154

MacWilliams HK, Kafatos FC (1968) Inhibition by the basal disc of basal disc differentiation in hydra. Science 159:1246-1247

MacWilliams HK, Kafatos FC (1974) The basal inhibition in hydra may be mediated by a diffusing substance. Am Zool 14 : 633-645

Schaller H (1973) Isolation and characterization of a low-molecu- lar-weight substance activating head and bud formation in hyd- ra. J Embryol Exp Morphol 29:27-38

Schaller H, Gierer A (1973) Distribution of head activating sub- stance in hydra and its localisation in membranous particles in nerve cells. J Embryol Exp Morphol 29:39 52

Schaller HC, Schmidt T, Grimmelikhuijzen CJP (1979) Separation and specificity of action of four morphogens from hydra. Wil- helm Roux's Archives 186:139-149

Schmidt T, Schaller HC (1976) Evidence for a foot-inhibiting sub- stance in hydra. Cell Differ 5:151 159

Webster G (1971) Morphogenesis and pattern formation in hyd- roids. BioI Rev 46:1-46

Received December 18, 1979; accepted in revised form February 4, 1980