/. Embryol. exp. Morph. Vol. 29, 1, pp. 27-38, 1973
27
Printed in Great Britain
Isolation and characterization
of a low-molecular-weight substance activating
head and bud formation in hydra
By H. CHICA SCHALLER 1
From the Max-Planck-Institut fur Virusforschung
MoJekularbiologische Abteilung, Tubingen
SUMMARY
From crude extracts of hydra, a substance activating head formation was isolated and
enriched at least lOOOOO-fold. The molecular weight was determined to be approximately
900. Sensitivity against proteases suggests that it is a peptide. The substance acts at a
concentration equivalent to the extract of 1 hydra per ml or at a concentration of less than
10 10M. In its highly purified form the substance activates head and bud formation.
INTRODUCTION
Morphogenetic processes can be studied at different organizational levels: at
the cellular level as determination and/or differentiation of single cells and at
the tissue level as pattern formation. These processes may be related in so far as
the signals or substances that determine the differentiation of single cells also
determine changes of form.
Hydra seems to be an ideal system to study both aspects of the problem. It
has a relatively simple morphology and consists of only a few cell types.
Throughout its (eternal) life hydra contains multipotent cells, the interstitial
cells, which probably differentiate in a one- or two-step process into nerve cells,
the three or four types of nematocytes, the sex cells, and perhaps also the gland
or mucous cells (for a review see Kanaev, 1952, and Lentz, 1966). The high
regenerative capacity of the animal as well as the process of asexual reproduction by budding provide easy means to study morphogenesis at the tissue
level of organization.
Pieces of hydra tissue are known to regenerate head and foot according to
their original polarity (for a review see Kanaev, 1952, and Webster, 1971).
Many models have been presented to explain polarity. One possibility is that it
is due to an unequal distribution of one or several substances along the body
axis of the animal. Several attempts have been made to isolate such substances.
1
Author's address: Max-Planck-Institut fur Virusforschung,
Abteilung, 74 Tubingen, Spemannstr. 35, Germany.
Molekularbiologische
28
H. C. SCHALLER
Thus, Lesh & Burnett (1964, 1966), Lentz (1965), Miiller (1969) and Miiller &
Spindler (1971) presented evidence that a substance is present in crude extracts
of hydra that may determine head formation.
The aim of the present study was to isolate and characterize this substance
and to show that it is relevant for the biological effect observed. This includes
the evidence that the purified substance acts at very low concentration, i.e. is a
specific morphogen influencing head and bud formation in hydra.
MATERIALS AND METHODS
(1) Culture. Hydra attenuata, obtained from P. Tardent, Zurich, in 1966,
was used for all experiments. The animals were mass cultured at a density of
approximately 2-5 hydra/ml in a medium consisting of 10~3 M-CaCl2,
1-25 x 10~5 M EDTA in tap water, at a temperature of 18 ±2 °C. They were
fed daily with Artemia nauplii and washed about 4 h later.
(2) Assay system. 24 h starved hydra without buds were selected from the
mass culture and washed thoroughly. The hypostome with tentacles was cut
off, as was the basal disc, since it was often contaminated with fungus. 25-40
gastric pieces were put into a Petri dish (diameter 6 cm) containing 10 ml of
hydra medium with and without the extracts to be tested. 4-8 h later the pieces
were transferred to dishes with fresh hydra medium. 2 days after cutting the
number of tentacles was counted. Serial dilutions were assayed for each extract.
Routinely three dishes (approximately 100 pieces) were assayed in the control
and in each dilution step (threefold dilutions). At lower levels of activation
(5-10 % increase in tentacle number) the average tentacle numbers obtained in
different dishes containing a given concentration of extract were compared
with those of the control or of other concentrations by means of the t test of
significance. Depending on the standard deviation of the average tentacle
number of each dish from the mean of the whole sample, 3-6 dishes in each
sample sufficed to ensure that the differences observed were significant
(probability of error (P) < 0-05). At higher levels of activation (10-15 % increase
in tentacle number) approximately 50-100 pieces in each sample were sufficient
to ascertain that the effect observed was significant (P < 0-05). In this case the
tentacle numbers were directly compared by means of the #2-test of significance.
(3) Crude extracts. A suspension containing up to 500 hydra per ml was
sonicated for 3-5 min with a MSE ultrasonicator at 1-5-2 A. The absorption
at 280 nm was measured to estimate the concentration of the crude extract.
The crude extract from one hydra without buds corresponds to an absorbance
at 280 nm of 0-10-0-15, the extract from a hydra with buds to 0-2-0-3.
(4) Column chromatography. The following gels were used: Bio-Gel P-2
(from BioRad Laboratories), Sephadex G-10, G-25, G-50 and DEAE A 25
(from Pharmacia, Uppsala).
Isolation of head-activating substance
29
(5) Diaflo ultrafiltration. An Amicon ultrafiltration cell was used with the
filters UM 05, UM 2, and UM 10.
(6) Sedimentation equilibrium centrifugation. 2500 biological units (BU, as
defined in Results) of the purified material in 2 ml of 0-5 M-NaCl were centrifuged
at 60000 rev/min in a SW 65 rotor for 8 days at 5 °C. Aliquots of 0-4 ml were
collected and tested for biological activity. The concentration of biologically
active substance in each fraction was determined (BU/ml = c) (for explanation
of BU see page 30). The molecular weight M was determined by plotting In c
against r2, where r is the distance from the rotor axis. M is calculated from the
slope according to the equation
, cx M.
j\-r\
where c\ and c2 refer to the concentration of activating substance in different
fractions and rx and r2 to their respective distances from the centre of rotation;
the partial specific volume v was assumed to be 0-7.
(7) Enzymic digestions. The following enzymes were used: trypsin, 2 x cryst.
(Worthington Biochem. Corp.), pronase B grade (Calbiochem), DNase,
RNase, lipase, hyaluronidase (all Worthington Biochem. Corp.). Since most of
the enzymes affected the regenerating pieces in the assay, either a further gel
filtration was included or the concentration of substance and enzyme chosen in
such a way that the dilution of the enzyme was great enough to minimize its
direct effect in the assay.
RESULTS
1. Assay system and its relevance for the substance activating head formation
1. Assay
Lesh & Burnett (1964, 1966) and Lentz (1965) used the reversal of polarity
or the production of heteropolarity as a test for head activation. In this paper
the number of tentacles regenerated per animal is used for a quantitative
analysis. Gastric pieces of non-budding animals were incubated with varying
amounts of hydra crude extract and the number of tentacles counted 2 days
after cutting. Since the number of tentacles regenerated per animal varied in the
untreated controls depending on state of growth, temperature, and feeding
condition, the activation (A) was defined as the percentage increase in tentacle
number of the treated sample (T) over that of the control (C):
A =
The addition of increasing amounts of crude extract to the test pieces caused
the effects demonstrated in Fig. 1.
At a concentration of crude extract corresponding to half a hydra per ml (or
007o.D.280/ml) the average tentacle number is increased by 5-8%. In a
30
H. C. SCHALLER
16
14
1:
12
~ 10
I
s
'% 6
4
0-4
0-6
0-7
0-5
01
0-2
0-3
Concentration of crude extract (o.D.280/ml)
Fig. 1. Activation of head formation by increasing concentrations of crude extract.
Hydra were sonicated in distilled water, the concentration of the extract was
estimated by measuring the absorbancy at 280 nm. The crude extract corresponding
to 1 hydra (without buds) in 1 ml resulted in an absorbancy at 280 nm of 012-015.
The activation is given as % increase in tentacle number of the treated sample over
that of the control. This activation curve has been obtained repeatedly in many
independent experiments and over an extended period of time (5 years).
normal assay (100-200 pieces or 3-6 dishes in each sample) this 5-8 % increase
in tentacle number is significant as ascertained by the t test of significance and a
probability of error of less than 5 % (P < 005). A maximal activation or a
plateau is reached at about the tenfold concentration. At this maximal activation
the treated pieces regenerate on the average about 15 % more tentacles than the
controls, e.g. seven instead of six tentacles. The increase in tentacle number with
concentration is not linear. At concentrations of crude extract higher than those
shown in Fig. 1 tentacle number and rate of regeneration start to decrease until
they drop below the control at concentrations above 3 o.D.280/ml or 20 hydra/
ml. This indicates that the crude extract contains inhibitory and/or toxic
components as well.
To compare the activities of different extracts with one another, a 5-8 %
increase was chosen as a reference point. At this level of activation the curve is
steep, i.e. small changes in concentration of the active component lead to
relatively large changes in the average tentacle number. The amount of
substance necessary to achieve a 5-8 % increase in an assay sample containing
25-30 regenerating pieces in 10 ml medium was arbitrarily defined as one
biological unit (BU). The specific activity of a given extract was defined as
BU/O.D.280.
2. Biological relevance of the assay
The experiments have shown that crude extracts of hydra contain a substance
that causes an increase in the number of tentacles formed by regenerating
animals. To prove that this increase in tentacle number can be used as an assay
31
Isolation of head-activating substance
Table 1. Rate of head regeneration, number of tentacles regenerated, and activity
of crude extracts of two body regions
Speed of regeneration:
regenerates with
developing tentacles
A
No. of
,
»
test pieces At 25 h
At 33 h
Average no.
of tentacles
regenerated
per head
at 48 h
Specific
activity
(BU/O.D.280)
Upper segment
139
93
133
6-14 ±0-27
5-5 ±1-5
Lower segment
138
1
85
5-60 ± 0 1 8
2-1 ±0-4
Budless animals were selected from the mass culture, starved for 3 days, and animals
without buds used for the experiments. Four dishes containing 30-40 pieces were assayed
for each region to determine speed of regeneration and average tentacle number. Serial
dilutions of the respective crude extracts were assayed to determine the specific activities.
To achieve a 6 % increase in tentacle number (1 BU) 020 ± 0 0 6 O.D.280 of crude extract
were needed from the upper segment, 0-5 ± 0 1 from the lower segment.
Table 2. Capacity for head regeneration and activity of
crude extracts of differently fed animals
Animals
Fed daily
Fed every
second day
Fed
once a week
Time required for
head regeneration
Average no.
of tentacles
regenerated per head
Specific activity
3 days
6-11 ± 0 0 5
2-1 ±0-4
3-5 days
5-62 + 0-14
1-2 + 0-3
4 days
4-82 ± 0 1 1
0-5 ± 0 1 5
BU/O.D.280
Three dishes (approximately 100 pieces) were assayed for each sample to determine speed
of regeneration and average tentacle number. Serial dilutions of the crude extracts from
whole animals were assayed to determine the specific activities. To achieve a 5 % increase in
tentacle number 0-5 ± 0 1 O.D.280 of crude extract were needed from the daily fed animals,
0-9±0-2 from those fed every second day, and 2 0 ± 0 - 5 from the starved animals.
for a substance influencing head formation, the following two lines of argumentation are presented:
(1) Tentacle number and rate of head formation are positively correlated.
Pieces of hydra tissue with a low capacity for head formation, i.e. which need
a long time to regenerate the head, also produce fewer tentacles than pieces
which regenerate the head faster.
(2) Specific activity and capacity for head formation are positively correlated.
A piece of tissue with a low capacity for head regeneration also contains low
32
H. C. SCHALLER
Table 3. Procedure for the purification of the low-molecular-weight form of
the head-activating activity
1. 30000 hydra are collected and suspended in buffer (01 M NaCl, 001 M Tris-HCl,
pH 7-4) to give approximately 100 ml. This mixture is homogenized by
sonication (5 min) and termed CRUDE EXTRACT.
2. After addition of acetic acid (to 025 %) the solution is centrifuged at 35000g
for 60 min to give SUPERNATANT.
3. The SUPERNATANT is adjusted to pH 7 by adding N H 4 0 H , concentrated by
evaporation, and chromatographed on a 4 x 60 cm Bio-Gel P-2 column. The
activity is found in fractions 30-37 (FRACTION P-2a, Fig. 2a).
4. FRACTION P-2a (90 ml) is concentrated by evaporation and rechromatography
on one or two smaller 1 x 60 cm P-2 columns. Fractions 12-15 give FRACTION
P-2b.
5. FRACTION P-2b (8-15 ml) is evaporated and chromatographed on a 1-5x17 cm
Sephadex G-10 column. Fractions 17-22 give FRACTION G-lOa (Fig. 26). In
steps 3-5 the buffer consists of 0 1 M-NaCl, 0 0 1 M Tris-HCl, pH 7-4.
6. FRACTION G-lOa (12 ml) is evaporated and desalted on a small 1 x 1 0 cm
Sephadex G-10 column, which is eluted with distilled water. Fractions 13-17
give FRACTION G-lOb (Fig. 2c).
7. FRACTION G-lOb (5 ml) is applied to a 0-5x3 cm column filled with 1 ml of
DEAE A 25 (Sephadex) and equilibrated with Tris-HCl to pH 7-4. After
washing with the same buffer (001 M Tris-HCl, pH 7-4) the column is eluted
either stepwise or with a linear gradient of NaCl to give FRACTION DEAE.
8. The DEAE FRACTION is desalted on a small column of P-2 or G-10.
concentrations of the head activating substance. Both these correlations are
demonstrated (a) in Table 1 for pieces of tissue taken from different positions
of the body axis and (b) in Table 2 for pieces of tissue taken from differently
fed animals.
(a) As diagrammed in Table 1, two approximately equal-sized pieces were
excised from the upper and lower gastric region of hydra without buds. The
size was estimated visually and by measuring the optical density of the respective
crude extracts. The pieces from the upper half regenerate the head faster
(#2-test, P < 0-001), and they produce on the average about half a tentacle
more per regenerated head than the pieces from the lower segment (t test,
P < 005). When crude extracts of such pieces are compared, the upper segment
contains more than twice as much (2-6 + 0-9 times more) of the activating
substance than the lower segment.
(b) Three sets of animals were produced by differential feeding over 2
months: animals were fed daily, every second day, or once a week. Gastric
regions were excised from animals without buds and assayed for speed of head
regeneration and number of tentacles formed per regenerate. As Table 2 shows,
the more starved the animals were, the longer they needed to regenerate a head.
Also the number of tentacles regenerated per animal decreased the less well fed
33
Isolation of head-activating substance
NaCl
Blue dcxtran
200 ~
£ 50
c
JN
40
u
8 30
100 G
X>
< 10
« * i
10
20
30
40
50
60
Fraction number
(c)
(b)
B.D. NaCl
B.D. NaCl
:
- 1000
•0 £ OS
c
A*
- ! \1
j 0-4 -
0-2 -
5
10 15 20
Fraction number
25
U
0-2 -
500 >
«
r; -°
H 2000
1
_ 1000 >>"
1
5
10 15 20
Fraction number
Fig. 2. Purification by chromatography of the low-molecular-weight activating
substance. #
• , Absorbance at 255 nm; hatched areas, biological activity;
B.D., blue dextran.
(a) Chromatography of the SUPERNATANT on Bio-Gel P-2. Buffer 0-1 M NaCl,
001 M Tris-HCl, pH 7-4. Fraction volume = 11 -5 ml. Vt = 660ml; RF = 0-65.
(6) Chromatography of FRACTION P-2b on Sephadex G-10. Buffer 0-1 M NaCl,
001 Tris-HCl, pH 7-4. Fraction volume = 1-9 ml. Vt = 25 ml; RF = 0-3;
Kr-^ 31.
(c) Desalting on G-10 with distilled water as eluant. Fraction volume = 095 ml.
Vt = Sm\;RF= 0-27; Kd = 5-5.
the hydra were. Each difference in tentacle number is significant {t test,
P < 0-05). Coincident with this decrease in tentacle number there was a
continuous decrease in the specific activity of the crude extracts.
Both experiments show a direct correlation between speed of regeneration,
number of tentacles formed per regenerate, and specific activity of the
corresponding crude extracts. It is therefore assumed that an assay based on
inducing additional tentacles in regenerates is a relevant one for isolating a, or
the, substance activating head formation in hydra.
EMB 29
34
H. C. SCHALLER
Table 4. Yield and enrichment of activating substance from a
preparation of 30000 hydra
Total
Total
activity
Specific activity
Fraction
O.D.280
BU
BU/O.D.280
Crude extract
Supernatant
Fraction P-2a
Fraction P-2b
Fraction G-10 a
Fraction G-l Ob
Fraction DEAE
Fraction G-lOc
9000
925
240
32
0-2
005
< 001
<001
12000
11000
10000
9500
8000
8000
7000
7000
1-3
12
42
300
40000
160000
>700000
>700000
Overall
Overall
yield purification
(x -fold)
(%)
100
90
85
80
65
65
60
60
10
30
230
30000
120000
> 500000
>500000
II. Isolation of the low-molecular-weight activating substance
The different steps of the isolation procedure are summarized in Table 3.
The yield for each step and the final enrichment for a preparation from
approximately 30000 hydra (60 ml of hydra tissue) are given in Table 4.
After homogenization, acetic acid precipitation and centrifugation about
90-95 % of the original activity of the crude extract were found in a lowmolecular-weight form in the supernatant. By gel filtration on Bio-Gel P-2 and
Sephadex G-10 all those substances were removed which are not in the same
molecular weight range and which do not show the same absorbing properties
as the active component. On Sephadex G-10 the activating substance was
absorbed strongly in dist. H2O as eluant (Kd = 5-5), less strongly but still
markedly in 0-1 M-NaCl, Tris-HCl, pH 7-4 buffer (Ka = 3-1). This step proved
to be very useful for desalting.
By rechromatography on these two types of gels a final enrichment of
100000 times was achieved. As a further step a DEAE ion-exchange column
was used. The activating substance eluted at pH 7-4 in the range of 0-2-0-4 MNaCl in a linear gradient or with a 0-5 M-NaCl step.
Starting with approximately 30000 hydra (1 g of protein) the purified
material had an absorbance at 260-280 nm of less than 001 corresponding to
less than 10 /*g of protein. In section III.2 it will be shown that the active
substance is pronase- and trypsin-sensitive. Assuming that the absorbance of
the substance is similar to that of other proteins or peptides it can be concluded
that the above purification procedure led to an at least 100000-fold
enrichment compared to the crude extract with about 50 % yield of biological
activity. 1 BU is due to less than 10~9 g of material.
Isolation of head-activating substance
35
Table 5. Activity after Diaflo ultrafiltration
Membrane
MW of permeable
molecules
% of
i activity
in retentate
% of activity
in filtrate
UM10
UM 2
UM05
10000
1000
500
0
1-5
100
100
95
0
Table 6. Distribution of activity in centrifuge tube after
equilibrium sedimentation
Fraction
Radial distance:
(cm)
Concentration
(BU/fraction)
1
2
3
5
8-6
8-25
7-88
7-1
2000
400
80
8
III. Characterization of the activating substance
1. Molecular weight
The chromatographic properties of the activating substance suggested that
the molecular weight is low. The active component eluted from Bio-Gel P-2
had an RF of 0-65, which corresponds to a molecular weight of about 350
daltons. On Sephadex G-10 the activating substance eluted after salt, indicating
that it contains strongly absorbing groups. Since such absorption effects play a
role in gel nitration on P-2 as well, the RF of 0-65 can only be used to roughly
estimate the molecular weight as being greater than 350, but of the same order
of magnitude.
Ultrafiltration with the Diaflo membrane niters UM 10, UM 2 and UM 05
indicated (Table 5) that the activating substance is larger than 500 and probably
smaller than or in the range of 1000 daltons. To support this finding a given
amount of the purified substance (2500 BU) was sedimented to equilibrium by
centrifuging at 60000 rev/min for 8 days. Five equal fractions were collected,
and assayed at various concentrations for activity (Table 6). From the slope in
concentration of the various fractions versus r2 and assuming a partial specific
volume of 0-7, the molecular weight was calculated to be approximately 900.
2. Other properties
The following enzymes had no effect on the activity of the purified substance:
DNase (10/<g/ml), lipase (100/tg/ml), and hyaluronidase (100/tg/ml). The
activity was destroyed by trypsin (100/tg/ml) and by pronase (150/tg/ml)—
the pronase effect is shown in Table 7. The substance is heat-stable. It was
stored frozen for several years without loss of activity.
3-2
36
H. C. SCHALLER
Table 7. Digestion of the purified substance by pronase
Average no.
Average no.
Average no.
of tentacles
of buds
of tentacles per animal
per regenerate per regenerate (bud and regenerated head)
Sample
1 BU substance
1 BU substance
digested with
150/tg/ml pronase
150/ig/ml pronase
Control
6-2
5-8
0-9
0-7
9-4
8-3
5-9
5-7
0-7
0-7
8-2
8-2
One BU of the substance was incubated with and without pronase (150 /*g/ml) for 6 h ;
0-86 /tg pronase were used for this purpose. As control this amount of pronase was tested as
well. The number of tentacles and buds was determined 2 days after cutting. Three dishes
(100-120 pieces) were assayed in each sample.
Table 8. Effect of the purified substance on
head regeneration and budding
Head regeneration
Concentration
of substance
0 (control)
1 BU
3BU
10 BU
30 BU
Bud formation
% of regenerates
with developing
tentacles
at24h
Average no.
of tentacles
per regenerate
at48h
Average
no. of
buds/reg.
at24h
Average
no. of
buds/reg.
at48h
21
44
57
59
65
5-21 ±0-14
5-52 + 0-11
5-80 ±0-18
5-73 ±0-21
5-97 ±0-17
0-58
0-65
0-87
0-90
0-74
1-2
11
1-5
1-4
1-3
Average
of tentacles
per regenerate
no. of
tent./bud (bud •{- head)
at48h
at48h
30
3-2
30
3-5
3-6
8-8
91
10-3
10-6
109
Three dishes (approximately ]100 pieces) were assayed in control and at each concentration.
IV. Mode of action of the purified substance
As Table 8 shows, the purified substance produces the following effects. (1) It
increases the rate of head formation - this is shown in Table 8 as the percentage
of regenerating animals with developing tentacles at 24 h (#2-test, P < 005 for
all concentrations). (2) It leads to an increase in the number of tentacles
regenerated per head (t test, P < 005 for all concentrations) - this increase in
tentacle number is more pronounced if pieces of the column are used which do
not produce buds at the same time. (3) The activating substance stimulates bud
formation. In the test system used the gastric pieces regenerate not only a head,
but form buds as well. Whereas increasing amounts of crude extract lead to a
steady decrease in the number of buds formed, the purified substance stimulates
budding (Tables 7, 8). The increase in number of buds and the increase in the
average number of tentacles per bud are included in the calculation of the
Isolation of head-activating substance
37
average number of tentacles formed by a regenerate (last column of Tables 7
and 8).
This activation of bud formation is also obtained if non-regenerating animals
are treated with the purified substance. Hydra with one just visible bud were
incubated with and without 1 BU of the substance. After 24 h 50 % of the
animals in the treated samples had developed a second bud (519 out of 1015
totals) in contrast to only 30% in the controls (183 out of 519 totals). This
activation of bud formation is significant (#2-test, P < 0001). It also indicates
that the substance acts on whole animals and not only on cut surfaces.
DISCUSSION
In the course of head regeneration a piece of tissue from the body column
differentiates into those types of cells which form the structures typical for the
head as opposed to the foot or the gastric region. Since tentacles are easily
recognizable head structures, a test system was developed which used the
increase in the number of tentacles regenerated per head as a quantitative assay
for a substance activating head formation. Evidence is presented that the
number of tentacles regenerated by a given piece of tissue is directly correlated
with the rate or time required for head regeneration, indicating that the tentacle
number is a relevant criterion for head formation.
By means of this assay crude extracts were examined for their ability to
influence head regeneration. It was shown that the concentration of extract
corresponding to 1 hydra per ml is sufficient to increase the number of tentacles
regenerated per animal significantly. Since the volume of one hydra is about
1 /d, the crude extract acts in a concentration which corresponds to only
1/1000 of that within the animal, indicating that a large amount of the activating
substance is present in hydra in a stored form. The range in which increasing
amounts of crude extract cause an increase in tentacle number is narrow
(between 0-5 and 5 hydra per ml) and the increase is not linear. Higher
concentrations (more than 20 hydra/ml) lead to a decrease in rate of
regeneration and gradually also to a decrease in tentacle number, indicating that
crude extracts contain inhibitory and/or toxic components as well.
From the crude extract a substance was isolated which because of its lability
towards proteases probably is a peptide. The presence of groups other than
amino acids cannot be excluded. By gel chromatography, Diaflo ultrafiltration
and sedimentation equilibrium centrifugation the molecular weight was
determined to be about 900 daltons. This substance was enriched more than
100000-fold. To achieve a positive effect in the assay less than 10~9 g were
needed in 10 ml. On the basis of a molecular weight of approximately 1000 it
can be deduced that the substance is active at a concentration corresponding to
less than lO"10 M.
Unspecific stimulation of tentacle number is obtained by incubating
38
H. C. SCHALLER
regenerating animals with a number of different agents including degradative
enzymes (10~4 to 10~6 M), chelators and salts (lO"1 to 10~3 M), distilled water,
etc. In previous work extracts have been used and tested at high concentrations
(Lesh & Burnett, 1964; Miiller, 1969; Miiller & Spindler, 1971), where such
unspecific effects may have contributed to the effects observed. The very low
concentration at which the purified substance is active excludes such unspecific
action and is consistent with the notion that the substance isolated is a true
morphogen.
In its purified form the substance not only stimulates the number of tentacles
in regenerating animals and the rate of head regeneration, but also the number
of buds and the rate of bud formation. It appears to have no influence on foot
regeneration. The fact that animals with a low capacity for head formation
contain low levels of the activating substance and its uneven distribution within
the animal make it very probable that the substance isolated is part of the
morphogenetic system influencing or determining head and bud formation in
hydra.
I thank Dr Alfred Gierer, in whose laboratory this work was carried out, for support and
discussion, and my colleagues in the hydra research group in Tubingen for constructive and
helpful criticism.
REFERENCES
KANAEV, J. J. (1952). Hydra.
Essays on the Biology ofFresh Water Polyps (ed. H. M. Lenhoff).
Moscow: Soviet Academy of Sciences.
LENTZ, T. L. (1965). Induction of supernumerary heads by isolated neurosecretory granules.
Science, N. Y. 150, 633-635.
LENTZ, T. L. (1966). The Cell Biology of Hydra. Amsterdam: North-Holland Publishing
Co.
LESH, G. E. & BURNETT, A. L. (1964). Some biological and biochemical properties of the
polarizing factor in Hydra. Nature, Lond. 204, 492-493.
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(Manuscript received 24 April 1972, revised 23 June 1972)