/. Embryol. exp. Morph. Vol. 29, 1, pp. 39-52, 1973
39
Printed in Great Britain
Distribution of the head-activating
substance in hydra and its localization in
membranous particles in nerve cells
By H. SCHALLER 1 AND A. GIERER
From the Max-Planck-Institut fur Virusforschung
Molekularbiologische Abteilung, Tubingen
SUMMARY
The low-molecular-weight substance activating head and bud formation in hydra is
shown to occur in the animal as a gradient decreasing from the hypostomal to the basal
region. The concentration of head-activating substance increases during head regeneration
and during bud initiation.
Most of the low-molecular-weight head-activating substance is present in the animal in a
structure-bound form. More than 90% was sedimentable; 70% was recovered in a highly
purified fraction consisting of membranous particles of ~ 1200 A diameter. This implies
that in the animal only a minor portion of the total activating activity is freely diffusible, i.e.
present in the low-molecular-weight form.
The head-activating substance is mainly produced by and/or stored in nerve cells or a
subgroup of the nerve cells. Nerve cells were enriched tenfold in a fraction containing most
of the head-activating substance in a more than 10 times higher specific activity than in the
animal. In addition, it is shown that only the nerve cells are positively correlated with the
distribution of head-activating activity both with regard to localization within the animal as
to time sequence of appearance during head regeneration and bud formation.
INTRODUCTION
In the previous paper (Schaller, 1973), it was shown that from crude extracts
of hydra a low-molecular-weight substance, probably an oligopeptide, can be
isolated which activates head and bud formation in hydra. Preliminary evidence
suggested that the head-activating substance is present in the animal in a
structure-bound form. The aim of this study was (1) to measure the distribution
of the head-activating activity in different regions of the animal and during
different states of morphogenesis, and (2) to show to which cell type and to
which cell structure the activating substance is bound.
1
Author's address: Max-Planck-Institut fur Virusforschung,
Abteilung, 74 Tubingen, Spemannstr. 35, Germany.
Molekularbiologische
40
H. SCHALLER AND A. GIERER
MATERIALS AND METHODS
Biological assay. Mass cultured Hydra attenuata was used for all experiments. As described in the previous paper (Schaller, 1973), the activation (A)
was assayed by incubating regenerating gastric pieces with or without the
extracts to be tested and measuring after 2 days the percentage increase in
tentacle number of the treated sample (T) over that of the control (C):
A = 100 x ( r - C ) / C ( % ) . Serial dilutions were assayed for each extract. The
amount of substance necessary to achieve a 5-8 % increase in an assay sample
containing 10 ml medium and 25-30 regenerating pieces was arbitrarily defined
as one biological unit (BU). The specific activity is expressed as BU/o.D.280.
The significance of an increase or a difference in tentacle number was ascertained
by comparing the mean tentacle numbers obtained in different dishes of one
sample with those of the control or of other samples by means of the t test of
significance o r - a t higher levels of activation - by comparing the tentacle
numbers directly by means of the ^-test.
Maceration of hydra tissue. As described by David (1972), pieces of hydra or
whole animals were dissociated into cells by incubation and shaking in 7 %
glycerol, 7 % acetic acid. The total number of cells was determined in a
Neubauer cell counter, depth 0-1 mm; the distribution of cell types was counted
with phase-contrast optics according to the criteria described by David (1972).
Separation of cell types. The macerated cells were collected by centrifugation
at lOOOg for 5 min and layered on discontinuous glycerol (50, 30, 25, 20 and
15 % glycerol in 7 % acetic acid) or sucrose gradients (60, 40, 20, 10 and 5 %
sucrose in 1 % acetic acid). These gradients were centrifuged at 500 g for
3-4 min and the fractions collected from the top. After determining the cell
distribution for each fraction the cells were washed in 15 % sucrose, and
resedimented at 3000 g for 10 min. The sediment was dissolved in distilled H2O,
sonicated for 5 min with an MSE sonicator and assayed for head activation.
Nitrogen-mustard treatment. Hydra from the mass culture were incubated in
0-01 % nitrogen mustard for 10 min (Diehl & Burnett, 1964), washed several
times in fresh hydra medium, and subsequently treated as normal animals.
Isolation of particles containing head-activating activity. The following buffers
were used-buffer A: 0-4M sucrose, 10" 2 M Tris-HCl, pH 7-4, 10-3M-MgCl2,
4x 10~3M-CaCl2; buffer B: 0-2M-NaCl instead of 0-4M sucrose, the other
constituents as in buffer A. Since sucrose inhibits head regeneration (rate and
tentacle number) in concentrations above 10~ 3 M, dilution in buffer B and
recentrifugation was always used as a last step to facilitate the biological assay.
To release the low-molecular-weight activating substance, particulate fractions
were freeze-thawed and shocked osmotically by dilution in hydra medium
before testing.
Homogenization: Concentrated hydra (approximately 500 hydra/ml) were
mixed with an equal volume of 2 x buffer A or B. This mixture was
Characterization
of activating substance
41
homogenized gently at 5 °C in a glass or teflon homogenizer until complete
disintegration of tissue and cells was achieved (as checked under the microscope).
Centrifugation: The homogenate was centrifuged at 5 °C in a Spinco (Model
L) ultracentrifuge using a No. 30 rotor for 10 min at 10000 rev/min and the
supernatant for 60 min at 20000 rev/min (35000 g SEDIMENT). The respective
pellets were resuspended in buffer A for further purification or dissolved in
buffer B to be stored frozen for the bioassay.
Equilibrium sedimentation in discontinuous sucrose density gradient:
Sucrose solutions contained 10-2M-Tris-HCl, pH 7-4, 10-3M-MgCl2, 4x 10~ 3 MCaCl2. On to 10 ml of 50 % (w/w) sucrose were layered 10 ml of 40 %, 5 ml of
30 % sucrose+ 5 ml of the 35000g SEDIMENT in buffer A (0-4 M sucrose). The
gradient was centrifuged at 5 °C in a SW 25/1 rotor at 20000 rev/min for 2 h.
Fractions were collected from the bottom of the tube, the sucrose density
determined with a refractometer, each fraction diluted with buffer B, and
concentrated by recentrifugation at 35000 g for 60 min (FRACTION A).
Equilibrium sedimentation in continuous sucrose gradient: A continuous
20-30 % sucrose gradient was layered on a 50 % sucrose cushion (0-2 ml).
1-5 ml of FRACTION A was added and centrifuged in a SW 25/3 rotor at 5 °C and
20000 rev/min for 6-9 h. Fractions were collected from the bottom of the tube,
diluted with buffer B and concentrated by centrifugation at 35000 g for 60 min
(FRACTION B).
Sedimentation velocity centrifugation: 1 ml of FRACTION B was layered on to
a preformed linear gradient of 5-20 % sucrose and centrifuged in a SW25/3
rotor at 5 °C and 12000 rev/min for 70 min. All fractions were washed in
buffer B, recentrifuged and stored frozen (FRACTION C).
RESULTS
l(tf). Distribution of head-activating activity in hydra
To determine the distribution of the substance activating head and bud
formation in hydra, animals without buds were divided into four segments and
the concentration of activating activity measured for each. As Table 1 shows,
the activating substance is present in hydra as a gradient from the hypostomal
to the basal region. The differences between each region are significant (t test,
P < 0-05). The head region, including hypostome and tentacles, contains almost
50 % of the total activity. In it the activating substance is more concentrated
than in any other region. The concentration of activating activity in the tentacles
(2-5 BU/O.D.280) is lower than in the total head region, indicating that the
activating substance may be even more concentrated in the hypostome. The
remaining 50 % of the activity are found in the body column with an
approximately twofold higher specific activity in the upper than in the lower
half. This is in agreement with the finding presented in the previous paper,
0-3 ± 0 1
0-5 ± 0 1
0-9 ±0-2
1-5 + 0-5
004
004
002
(O.D.280)
004
(O.D.280)
l-2±0-3
0-7 ±0-2
2-1 ±0-4
3-7+1-1
(BU/O.D.280)
Specific
activity
15
5
30
50
(%)
Distribution
of activating
activity
11000
4500
10500
12000
of cells
350
550
450
1150
15
20
20
45
(%)
of
nerve cells
No. of
nerve cells
no.
Distribution
Total
Serial dilutions were assayed for each extract to determine ithe concentration leading to a 5 % increase in tentacle
number (1 BU). The distribution of nerve cells is taken from Bode et al. (1972)i.
Head region
Upper gastric
region
Lower gastric
region
Basal region
Region
Mass/region
Concentration
of extract
for 1 BU
Table 1. Distribution of the activating activity and nerve cells in freshly dropped buds
tn
tn
o
O
>
>
tn
r
>
SCH
i
Characterization of activating substance
43
Extract from
3-5 pieces/ml
8
12
16
20
24
Time of regeneration (hours)
20
Extract from
2 pieces/ml
8
12
16
20
24
Time of regeneration (hours)
Fig. 1. Activity of crude extracts of pieces regenerating a head at different hours
after removal of the original head.
(a) The extracts from equal-sized gastric pieces (approximately 0-1 o.D.280/piece)
were assayed at two concentrations: x — x, extract corresponding to
0-35 o.D.280/ml or 3-5 regenerating pieces/ml; A
A, extract corresponding to
0-2 O.D. 280/ml or 2 pieces/ml (two independent experiments).
(b) O
O, Extract from lower gastric regions, the concentration corresponded
to 01 o.D.280/ml or 2 pieces/ml (two independent experiments). • - - • ,
Extract from basal regions, the concentration corresponded to 004O.D.280/ml or
2 pieces/ml.
where the upper gastric region of animals starved for 3 days contained
approximately 2-5 times more of the activating activity than the lower gastric
region. The specific activities of these animals were for all regions approximately
2 times higher than for freshly dropped buds which is in agreement with a
higher percentage of nerve cells (as shown by Bode et al. 1972), and as will be
discussed later.
The continuous decrease in concentration of activating activity from
hypostomal to basal region is only found in freshly dropped buds or in animals
without buds starved at least for 3 days. If well-fed animals without buds are
used which would produce buds during the next 1 or 2 days, the specific activity
44
H. SCHALLER AND A. GIERER
Table 2. Activity of crude extracts derived from
basal regions regenerating a head
Source of tissue
for crude extract
Regenerate at 0 h
Regenerate at 24 h
Regenerating surface at 24 h
Tissue below regenerating surface
at24h
Concentration of
extract
(o.D.280/ml)
Activation
(%)
Specific
activity
0-15
012
004
004
6±2
0±2
10 ±3
15 + 3
>2-5
>2-5
004
3+2
<2-5
(BU/O.D. 280)
0-7
of the lower gastric region rises significantly from 1-1 ±0-4 in animals without
bud Anlage to 2-5 ±0-8 BU/o.D.280 in animals with bud Anlage. The activity
of this future budding region is higher than that of the body region above it
(t test, P < 005).
1 (b). Changes in concentration of head-activating activity during regeneration
To observe small differences in content of activating activity the concentration
of extract should be in the quasi-linear part of the activation curve (see fig. 1,
Schaller, 1973), where small changes in concentration cause relatively large
changes in tentacle number. Fig. 1 (a) (this paper) shows how the appropriate
concentration was determined for the first 24 h of head regeneration for relatively
large pieces from the body column. In the course of head regeneration two types
of changes in the concentration of the activating activity were observed.
(1) There is a slight, but measurable drop in activity 4-8 h after the onset of
head regeneration {t test, P < 0-05). To determine the extent of this reduction
in activating activity, serial dilutions were assayed of pieces 6 h after removal
of the original head. It was found that the extract from four regenerating
gastric pieces or 0-11 o.D.280/ml were needed at 6 h to achieve the same effect
as from three pieces or 0-08 o.D.280/ml at 0 h. Activating substance is released
from the regenerating surface into the surrounding medium. This was measured
either by incubating other regenerates in the medium in which regeneration had
occurred or by determining the minimal volume by which a regenerating piece
influenced itself. Incubating a regenerating piece in less than 0-2 ml medium
lead to an activation corresponding to a 5-8 % increase in tentacle number.
This indicates that quite a considerable amount of the substance present in the
regenerate is released during the first hours of head regeneration (approximately
10-30 %). Pieces of tissue regenerating a basal region did not show such a drop
in head activating activity.
(2) 16-24 h after cutting, the concentration of activating activity increases.
This increase in activity at 24 h was found to be independent of the size or the
origin of the regenerating tissue, i.e. does not seem to be dependent on the time
Characterization
of activating
45
substance
Table 3. Procedure for the purification of particles
containing activating activity
.1. Homogenization in buffer A ( 0 4 M sucrose, 10~ 2 M Tris-HCI, pH 7-4,
10 3 M-MgCl2, 4x 10^3 M-CaCl2), centrifugation at 10000£ at 5 °C for 10 min;
resuspension of sediment in buffer A containing 10~2 M EDTA, homogenization,
centrifugation at 10000g for 10 min; centrifugation of the two supernatants
at 35000 g for 60 min to give 35000 g SEDIMENT.
2. Centrifugation of the 35 000 g SEDIMENT in a discontinuous 30-50% (w/w)
sucrose gradient at 50000 g for 2 h (5 °C) to give FRACTION A (reddish band at
interphase to 30 % sucrose).
3. Equilibrium sedimentation of FRACTION A in a continuous 20-30 % (w/w)
sucrose-density gradient at 50000g (5 °C) for ca. 8 h (overnight) to give FRACTION
B (corresponding to 20-5-22 % sucrose).
4. Sedimentation of FRACTION B (after suspension in 02 M-NaCl and resedimentation) in a 5-20% (w/w) sucrose-density gradient (SW 25/3, 13000g
.120000 rev/min, 75 min, 5 °C) to give FRACTION C (migration of activity from
7-7-9-7 cm radial distance corresponding to 7-8-5 % sucrose). Fig. 2 shows an
electron micrograph of FRACTION C.
Table 4. Purification of particles containing the
head-activating activity
Mass
(o.D.280)
Crude extract
(10000 hydra)
35000 g sediment
Fraction A
Fraction B
Fraction C
Low-molecular-weight
activator after G-10
Specific
activity
Yield Purification
(x -fold)
BU
(BU/O.D.280) (%)
700
60
7
<1
2500
2000
2000
1800
1500
1-4
30
33
250
<001
1500
1750
1500
100
80
75
70
70
1000
>150000
70
>100000
1
2
20
180
From fraction C the low-molecular-weight head-activating substance was released by
osmotic shock (dist. H2O) and subsequent sonication. After centrifugation at 35000 g for
60 min the supernatant was chromatographed on a Sephadex G-10 column (as described by
Schaller, 1973).
required for head regeneration. Whereas the gastric pieces used for Fig. l(a)
regenerated a head in 2 days, the pieces used in Fig. l(b) were sections (as
diagrammed) from the lower part of the body column, which have a slower rate
of head regeneration, i.e. they needed 3 and 4-5 days, respectively, to regenerate
a head. The relative increase in specific activity is especially high for a piece with
an originally low level of activating activity, e.g. a piece from the basal region
showed a more than threefold increase in concentration of activity at 24 h as
compared to 0 h (Table 2). This increase in concentration of activity is mainly
46
H. SCHALLER AND A. GIERER
2001) A
Fig. 2. Electron micrograph of particles containing the head-activating substance.
A drop of FRACTION C was placed on a grid and negatively stained in I % aqueous
uranyl acetate.
due to a higher specific activity of the regenerating surface. In tissues
regenerating basal structures no increase in concentration of head-activating
activity was observed at 24 h, indicating that the changes measured in the
course of head regeneration are specific for head regeneration.
2. Localization of the low-molecular-weight head-activating substance in
membranous particles
In the previous paper (Schaller, 1973) it was shown that the head-activating
substance stimulates head formation in a concentration which is far below that
present in the regenerating tissue itself. It acts in a concentration corresponding
to approximately 1/1000 of that in the whole animal. This is only explicable by
Characterization of activating substance
47
assuming that large amounts of the low-molecular-weight substance are not
freely diffusible in the animal, but are present in a stored form, i.e. probably
structure-bound. Furthermore, Lentz (1965) recovered head-activating activity
from a particulate fraction.
To show that most of the low-molecular-weight head-activating substance is
structure-bound, hydra were homogenized gently (glass or teflon homogenizer,
iso-osmolarity, 4 °C), centrifuged at 35000g for 1 h (0-2M-NaCl, 5 °C), and
sediment and supernatant assayed separately for activity. Under these conditions
only 5—10 % of the activating substance were found in the supernatant, i.e. in a
low-molecular-weight form, whereas more than 90-95 % were structure-bound.
Since some breakage is unavoidable even under mild homogenization conditions, in the animal the actual percentage of structure-bound head-activating
substance is probably higher.
To further purify and characterize this particulate fraction, the procedure
outlined in Table 3 was developed. By equilibrium density and velocity centrifugation the activity was enriched about 1000-fold in a fraction containing 70 %
of the original activity (Table 4). As shown in the electron micrograph in Fig. 2,
this fraction consists mainly of particles of approximately 1200 A diameter.
This particle size is consistent with a sedimentation coefficient of approximately
800 S calculated from the sedimentation velocity in the sucrose gradient and a
density of 1-09 as determined by the density equilibrium centrifugation. The
density of 1-09 is characteristic for membranous structures with a high lipid
content, especially for constituents of the smooth endoplasmic reticulum, e.g.
products of the Golgi complex. From these membranous particles the lowmolecular-weight substance is released quantitatively (Table 4) by osmotic
shock, ultrasonication, or repeated freeze-thawing.
3. Localization of the head-activating substance in nerve cells
Lentz (1965) and Lesh & Burnett (1966) discussed nerve cells as possible
sources for substances responsible for the polarity of hydra. Muller & Spindler
(1971) postulated that the head-inducing substance may be a product of the
nematocytes.
To exclude nematocytes as sources for the head-activating activity, mustardtreated animals (Diehl & Burnett, 1964) - which, as Table 5 shows, contain less
than 2 % of the normal complement of nematoblasts and nematocytes, very
few interstitial cells, but quite normal numbers of nerve cells - were assayed for
their content of head-activating activity. It was found that they still contained
70 % of the activity of normal animals: the extract from 1-2 mustard-treated
animals was as active as the extract from 0-8 normal animals. Therefore
nematocytes and also interstitial cells are excluded as major sources for the headactivating activity.
To prove that nerve cells produce and/or store the head-activating substance
the attempt was made to isolate or enrich them. A suspension of macerated cells
21000
Animals 9 days after treatment
/o
No.
/o
No.
24
7000
73
15000
20
14
6000 4000
—
200
28
8000
1
200
7
2000
10
2000
5
1500
14
3000
500
1
200
}
}
65
15
25
50
60
75
5
50
25
<5
<5
5
30
35
50
45
35
20
Large cellsf
(%)
( B U / O . D . 280)
0-8
100
3-3
0-2
0-3
0-3
100
6
6
27
31
30
Specific
activity
(%)
(O.D.280)
f ih
100
60
20
5
5
10
of activity
(7o)
TV
X-/lolllUU LIUI1
The cells accumulate at the interphase of the different glycerol concentrations. Fraction 1 contains the cells collected
from the uppermost interphase, i.e. between 10 and 20 % glycerol (see Materials and Methods). Fraction 5 was rich in
tissue clumps.
* Including interstitial cells, nematoblasts, and nematocytes.
t Including epithelial, mucous, and gland cells.
Unseparated cells
Fraction 1
Fraction 2
Fraction 3
Fraction 4
Fraction 5
Origin of cell mixture
Small cells*
(%)
A
Distribution of cell types in each fraction
Nerve cells
(%)
c
Distribution
of total
cell mass
trm
0-83 + 0-26
1-25 ±0-25
Epithelial Big Little Nematocytes
Specific
and
inter- interand
activity
digestive stitial stitial nematoblasts Nerve Gland Mucous (BU/O.D. 280)
Table 6. Distribution of activating activity and cell types after maceration and separation
in a glycerol gradient
29000
Normal animals
Total
cell
no.
Table 5. Distribution of cell types and activity in normal and nitrogen mustard-treated animals
w
w
£
>
tn
n
. c/a
oo
Characterization of activating substance
49
was used as starting material. As described in detail by David (1972), all cell
types, including nerve cells, are easily and quantitatively recognizable in such
preparations. The macerated cell mixture still contained 70-80 % of the headactivating activity present in the living animal, and was therefore suitable for
cell separation experiments. The macerated cell mixture was separated by
density centrifugation in discontinuous glycerol or sucrose gradients. The
distribution of cell types in the different fractions after centrifugation and the
specific activity of each fraction is given in Table 6. Whereas the unseparated
cell mixture consisted to only 5 % of nerve cells, fraction 1 consisted to 50 % of
nerve cells, fraction 2 to 25 %, i.e. the separation led to a tenfold enrichment of
nerve cells in fraction 1, fivefold in fraction 2. Concomitantly, fraction 1
containing 60 % of the total activity showed a more than 10 times higher
specific activity than the unseparated cells or a 30-50 times higher specific
activity than the other fractions containing either enriched concentrations of
large or small cells. It seems therefore very unlikely that cell types other than
nerve cells contain major quantities of the head-activating substance. The only
objection to this conclusion is the possibility that the activity may not be
bound to cells in the macerate, but to some larger structures (e.g. pieces of cell
debris) that band in the same region as the nerve cells and are sedimentable in
5 min at 1000 g. However, since fraction 2 contained practically no debris, but
still increased amounts of activating activity in correlation with the increased
percentage of nerve cells, it is more likely that the activity is bound to nerve
cells (or a subgroup of the nerve cells).
This is supported by the finding that of all the cell types only the nerve cells
show a similar distribution (Bode et al. 1972) to the head-activating activity
both with regard to time sequence of appearance during head regeneration and
bud initiation as to localization within the animal. During bud initiation the
first detectable change in the distribution of cell types is a local increase in
density of nerve cells at the site where the bud tip becomes visible. In head
regeneration the density of nerve cells doubles at the regenerating surface
24-28 h after removal of the head. As shown in Table 1 in the animal the
concentration of nerve cells is highest in the head region, with a maximal
density in the hypostomal region, from where it decreases as a gradient down
the body column and into the tentacles. The density of nerve cells increases
again in the basal region. No other cell type follows such a distribution. Interstitial cells, nematoblasts and gland cells have a low density in the hypostomal
region; their density is higher in the gastric region. Nematocytes occur almost
exclusively in the tentacles. Mucous cells of the hypostome are more or less
confined to the head region, and epithelial cells are relatively evenly distributed
over all body regions of hydra.
EMB 29
50
H. SCHALLER AND A. GIERER
DISCUSSION
The low-molecular-weight substance which as shown in the previous paper
(Schaller, 1973) activates head and bud formation in hydra is present in the
animal as a gradient from the hypostomal to the basal region. The graded
distribution of the activating substance is found only in animals without visible
or developing buds. In animals with a developing bud the specific activity of the
budding region is almost as high as that of the hypostomal region, i.e. the
gradient from top to bottom becomes interrupted in the future budding region
by the establishment of a second area of high activator concentration. Together
with the fact that the purified substance stimulates bud formation this suggests
that the head-activating substance plays a role in bud initiation as well. The
concentration of activating activity changes with time in a piece of tissue
regenerating a head. During the first 4-8 h there is a slight, but significant
decrease in the concentration of activating activity, at 16-24h there is a
considerable increase. Neither of these changes are observed during regeneration
of basal structures and are therefore probably specific for regeneration of head
structures.
The head-activating substance stimulates head formation in a concentration
which is far below that present in the regenerating tissue itself. This is only
explicable by assuming that large amounts of the low-molecular-weight
substance are not freely diffusible in the animal, but are present in a stored form,
i.e. probably structure-bound. It could be shown that under mild homogenization
conditions 90-95 % of the total activity are sedimentable. This was already
indicated by Lentz's finding (1965) that head-activating activity can be recovered
from a particulate fraction. By various centrifugation methods 70 % of the
total activity were enriched 1000-fold in a fraction consisting of membranous
particles of ~ 1200 A diameter. From these particles the low-molecular-weight
substance was released quantitatively by osmotic shock or ultrasonication. The
high percentage of total activating activity bound to these particles together
with the high specific activity of this fraction makes it very probable that the
activating substance is located in these structures in the animal also.
Lentz (1966) presented evidence that nerve cells play a role in regulating the
polarity of hydra. Furthermore he showed that one group of nerve cells contains
neurosecretory granules which resemble the particles isolated in size and
properties. To correlate activating activity with nerve cells the distribution of
activity was compared with the distribution of cell types (as measured by Bode
et al 1972).
Of all the cell types only the nerve cells show a positive correlation with the
activating activity both with regard to localization within the animal as to time
sequence of appearance during different states of growth and morphogenesis.
Activity as well as nerve cells are most concentrated in the hypostomal region,
decreasing in density in the tentacles and down the body column. During head
Characterization
of activating substance
51
regeneration and bud initiation increase in specific activity and increase in
nerve cell density again coincide. The high nerve cell density in the basal disc
as well as the increase in nerve cell density during foot regeneration are not
accompanied by an increase in activity, indicating that the nerve cells of the
basal disc do not contain major amounts of the head-activating substance and/or
that they serve a different function.
Since these findings hinted at nerve cells the attempt was made to isolate
them. They could be enriched tenfold in one fraction, fivefold in another.
Together these two fractions contained only 12 % of all the cells but 80 % of the
total activity.
The three lines of evidence, namely (1) the similarity of the isolated particles
with neurosecretory granula, (2) the isolation or enrichment of nerve cells in
a cell fraction containing most of the activity, and (3) the positive correlation in
time and localization of nerve cells and activity, make it very probable that the
nerve cells or a subgroup of the nerve cells are the main site of production and/or
storage of the head-activating substance.
All experiments presented in this and the previous paper (Schaller, 1973) are
consistent with the assumption that the substance activating head and bud
formation is a true morphogen which influences or regulates hydra
morphogenesis, probably in conjunction with other such substances. For the
mode of action of the head-activating substance the following tentative model
is suggested. The low-molecular-weight head-activating substance is mainly or
exclusively produced by nerve cells and is stored there in particles resembling
neurosecretory granules. From the nerve cells the low-molecular-weight
substance is released steadily or dependent on certain stimuli in minute
concentrations. Since the granules containing head-activating activity are
present in hydra as a gradient decreasing from the hypostomal to the basal
region, this release would suffice to build up and maintain a gradient of freely
diffusible head-activating substance. To explain regeneration and budding the
release rate must be assumed to depend not only on granula concentration, but
also on other effects controlling or affecting the release. The experiments have
shown that during the first hours of head as opposed to foot regeneration
increased amounts of the stored substance are released. This increased release
can only be caused by the removal of the head. Probably the normal balance of
morphogenetically active substances is disturbed or the release may be affected
by some other mechanism.
At the cellular level the concentration of head-activating substance together
with the presence or absence of other such morphogens probably determines
what types of differentiations occur. Since during head regeneration the level
of head-activating activity rises due to increased release, a gastric region is
reprogrammed to form head structures, i.e. differentiate into those cell types
characteristic for head as opposed to gastric regions. The cell distribution data
(Bode et al. 1972) indicate that at the regenerating surface, for example, more
4-2
52
H. SCHALLER AND A. GIERER
interstitial cells become determined to differentiate into nerve cells, whereas the
differentiation into nematoblasts seems to be turned down. The concentration
of head-activating substance probably also influences the other cell types:
epithelial cells differentiate into the battery cells of the tentacles, the production
of gland cells is reduced, that of the mucous cells enhanced etc. Since other
morphogens may be involved in this process a detailed analysis of the effects
of the isolated substance on the differentiation of cell types is necessary.
We thank H. Schwarz and Dr H. Frank for taking electron micrographs.
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BODE,
{Manuscript received 24 April 1972, revised 23 June 1972)