/. Embryol. exp. Morph. 78, 141-168, (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Genetic analysis of developmental mechanisms in
hydra. VIII. Head-activation and head-inhibition
potentials of a slow-budding strain (L4)
By JUN TAKANO 1 AND TSUTOMU SUGIYAMA 1
From the Department of Biology, Nagoya University, and National Institute of
Genetics, Mishima, Japan
SUMMARY
Morphogenetic potentials involved in head formation were examined in a hydra strain (L4)
which has a very low budding rate and a large polyp size, and the results were compared to
those of the normal strain (105).
Hydra tissue has two types of antagonistic morphogenetic potentials which are thought to
play important roles in head structure formation: the head-activation potential and the headinhibition potential. Lateral tissue transplantation (Webster & Wolpert, 1966) was used to
compare the levels of the two potentials in L4 and the normal strain. It was found that the
intact polyp of L4 has a nearly normal or slightly lower head-activation potential but a significantly higher head-inhibition potential than the normal strain. During the course of
regeneration after head amputation, the levels of the two potentials in L4 changed in significantly different patterns from those in the normal strain. These abnormalities of the two
potentials may be responsible for the developmental and morphological abnormalities in L4.
The significance of these observations is discussed in light of the current hydra pattern formation models (Wolpert, Hornchurch & Clarke, 1974; Meinhardt & Gierer, 1974).
INTRODUCTION
Hydra can multiply by asexual budding. A well-fed mature polyp produces
buds at a nearly constant rate. In general, the budding rates vary little among
individual polyps within the same strain, but vary considerably among different
strains. Availability of these strains may be an asset in elucidating the mechanisms involved in bud formation.
Strain L4 has the lowest budding rate and the largest polyp size among the 10
strains of Hydra magnipapillata examined by Sugiyama & Fujisawsa (1979). Its
budding rate is about one-half and its polyp size is about twice that of the
standard wild-type strain (105).
We initially thought that the low budding rate of L4 was caused by the low
mitotic activities of the cells in this strain. However, an examination of the cell
cycle lengths of L4 and 105 showed that the two strains have nearly the same
1
Authors' address: National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411,
Japan.
142
J. TAKANO AND T. SUGIYAMA
average cell cycle lengths of both the large interstitial cells (about 1-3 days) and
the epithelial cells (3 to 4 days) (Takano, Fujisawa & Sugiyama, 1980). This
indicates that L4 produces a sufficient number of cells to form buds at about the
same rate as 105, but apparently not all the cells are used for this purpose.
Hydra tissue shows two types of antagonistic morphogenetic potentials. They
are the potential to stimulate or activate the formation of head structures and the
potential to inhibit head structure formation (hereafter referred to as headactivation and head-inhibition potential, respectively). According to current
views, these potentials play crucial roles in hydra pattern formation (Wolpert et
al. 1974; Meinhardt & Gierer, 1974; MacWilliams, 1983a,b). In normal tissue,
the two potentials are in balance and the head-activation potential is unexpressed
due to the counteracting force of the head-inhibition potential. Under some situations, however, the balance between the two potentials may be lost and the activation potential may become more than a threshold value higher than the inhibition
potential. Formation of head structures such as in budding, head regeneration
and head induction by grafted tissue is thought to occur under such situations.
Previously we examined the head-activation and the head-inhibition potentials of two other developmental mutants, a multiheaded strain (mh-1) and a
regeneration-deficient strain (reg-16), and found that they both have highly
abnormal levels of the two potentials (Sugiyama, 1982; Achermann & Sugiyama,
1983a). The former strain has a higher activation and a lower inhibition potential
whereas the latter has a lower activation and a higher inhibition potential than
the standard wild-type strain (105). Rubin & Bode (1982) also reported altered
potentials in a mutant strain of//, attenuata, termed the aberrant. This strain has
a significantly higher head-inhibition potential than the wild type, but a nearly
normal head-activation potential. These findings support the view that the two
potentials play important roles in hydra morphogenesis, and suggest that an
imbalance of these potentials leads to developmental abnormalities.
We thought that the abnormality of the potentials may be also responsible for
the developmental abnormalities of L4. In the present study, we examined the
head-activation and the head-inhibition potentials of L4 by the lateral grafting
procedure described by Webster & Wolpert (1966). It will be shown that L4 has
a nearly normal or slightly lower head-activation but a significantly higher headinhibition potential than the normal strain (105). In addition, the pattern of
changes of the two potentials is highly abnormal during head regeneration after
amputation of the original head in this strain. The significance of these findings
will be discussed in light of the current models of hydra pattern formation.
MATERIALS AND METHODS
Strains
Two strains belonging to H. magnipapillata were used in the present study.
Strain 105 is the standard wild-type strain. L4 has a significantly lower budding
Morphogenetic potentials in hydra
143
rate and a significantly larger polyp size than 105 (Sugiyama & Fujisawa, 1979).
Both strains were derived from single polyps originally collected from outdoor
ponds, and maintained and propagated as clonal lines by asexual budding under
laboratory conditions for more than eight years.
Culture
It was previously described that lateral tissue grafting experiments carried out
with different batches (but presumably the same strain) of hydra sometimes
produced significantly different results apparently due to long-term fluctuations
in the state of hydra (MacWilliams, Kafatos & Bosset, 1970; Hicklin, Horbruch
& Wolpert, 1975). In order to minimize such a complication, animals used in the
present study were all cultured under rigorously controlled and standardized
conditions (except for animals used in determining the budding rate (see
below)). Stocks of fully matured polyps were cultured at a constant density of 20
polyps per 400 ml of the culture solution (see below) in a 500 ml beaker. From
these stock cultures, newly dropped young polyps were collected daily and they
were cultured at a density of 10 polyps per 200 ml of the culture solution in a
200 ml beaker. These animals were daily examined and the animals showing their
first bud protrusion (termed 'standard polyps') were collected and used in the
various experiments.
The standard polyps were not fed on the day of the experiment. All the other
polyps were fed heavily once daily in the morning, and a few hours later transferred to new beakers containing fresh culture solution. Newly hatched brine
shrimp nauplii (Nissei brand, Nippon Jisei Sangyo, Tokyo) were used as food.
Shrimps stained with Evans blue (Wilby & Webster, 1970) were used as food to
obtain vitally stained polyps. Modified 'M'-solution described by Muscatine &
Lenhoff (1965) containing 1 mM-NaCl, 1 mM-CaCk, 0-1 mM-KCl, 0-1 mM-MgSO4
and 1 mM-tris-(hydroxymethyl)-aminoethane (adjusted to pH7-6 with HC1) was
used as the culture solution. Culture and all the experiments were carried out in
a constant temperature room maintained at 18 ± 1 °C.
Head regeneration
Head and foot were removed from the standard polyps by amputation at oneforth of the length from hypostome to bud protrusion and at two-thirds of the
length from basal disk to bud protrusion (position 1 and 4, respectively, at far left
in Fig. 1). The remaining body column was left in the culture solution for seven
days without feeding, and the number of tentacles regenerated was counted daily
and used as the index of the head regeneration.
Polyp length
A single standard polyp was placed in a 50 mm plastic Petri dish containing
about 10 ml of the modified M-solution, and the dish was left without disturbance
144
J. TAKANO AND T. SUGIYAMA
under a dissecting microscope. The polyp, which was initially in a tightly contracted state by the handling, generally stretched itself slowly within a few minutes.
When it was fully extended, the lengths from hypostome to basal disk and from
hypostome to bud protrusion were determined using an ocular scale in the
dissecting microscope. The same measurement was repeated twice more, each
time after transferring the polyp to a new container with fresh culture solution,
and the average of the three measurements were obtained.
Cell number per polyp
The total number of cells per standard polyp was determined using David's
(1973) maceration and cell identification procedure. Tentacles of a single animal
were removed by amputation at their bases. The remaining tissue was macerated
into single cells in a small test tube containing 0-5 ml of maceration solution
(glycerine: glacial acetic acid: water, 1:1:13). The density of cells in the resulting
cell suspension was determined under a phase contrast microscope using a
hemocytometer. The same counting procedure was repeated twice more, and the
total number of cells in the animal was calculated from the average of the three
data.
Determination of budding rate
The budding rate was determined using individually cultured polyps as
described previously (Sugiyama & Fujisawa, 1978, 1979). From an actively
growing population, small polyps just detached from their parents were selected
and each was placed in a 50 mm plastic Petri dish containing about 10 ml of the
culture solution. The animals were fed heavily once daily, and a few hours after
each feeding they were transferred to new dishes with fresh culture solution. The
buds produced were discarded after they detached and only the original polyps
were maintained. The number of buds produced by each was plotted against the
day of culture, and budding rate (buds/day/polyp) was calculated from the slope
of the line.
Lateral grafting of tissue
Head-activation and head-inhibition potentials were assayed by the lateral
grafting of tissue following the procedure described by Webster & Wolpert
(1966) as modified by Sugiyama (1982). The procedure is shown schematically
in Fig. 1.
The body column of a well-stretched standard polyp from the hypostome to
the bud protrusion was divided into four equal lengths, the column from the bud
protrusion to the basal disk was divided into the ratio of 1:2 and the four
positions thus obtained were numbered from 1 to 4 as shown in far left in Fig. 1.
In addition, the position immediately below the tentacle ring was numbered 0.
Lateral tissue grafting was carried out using these five positions as the sources of
Morphogenetic potentials in hydra
145
Fig. 1. Schematic representation of the lateral grafting of tissue between animals. A
ring of tissue is cut from position 1 of polyp vitally stained with Evans blue (Wilby
& Webster, 1970), cut into two pieces, and one piece is grafted to position 1 of one
animal while the other is grafted to position 4 of another animal. In the standard wildtype strain (105), the tissue grafted to the same position as the donor is normally
absorbed by the host (upper), whereas the tissue grafted to a more proximal position
frequently induces head formation (lower).
the donor tissue and the four positions from 1 to 4 as the recipient sites. (Position
0 was not used as the recipient site due to technical difficulties of the experiment
involving this recipient site).
A ring of tissue was excised from one of the five positions of a donor polyp,
cut into two or three pieces containing approximately 5000 cells each, and one
of the pieces was grafted onto one of the four positions of a host polyp. In order
to follow the fate of the grafted tissue (see below), it was generally obtained from
146
J. TAKANO AND T. SUGIYAMA
polyps vitally stained with Evans blue (see above), and grafted onto unstained
hosts. In some instances, however, unstained donor tissue was grafted onto
stained hosts. Control experiments showed that the staining had little, if any,
effect on the outcome of the grafting experiments.
The grafted animals were kept individually in small plastic dishes. They were
not fed on the day of the grafting but starting the next day they were fed heavily
once daily for the following seven days. During this period, several types of
structures were produced as a result of the grafts. These structures were classified
into the following six types by Webster & Wolpert (1966): (1) a complete head
with a hypostome and some tentacles, (2) one or two tentacles without a hypostome, which were stable for more than four days, (3) the same as (2), but
tentacles disappeared within three days after they were formed, (4) a small
protrusion without any other structures, (5) complete absorbance into host
tissue, and (6) a small protrusion having an air bubble or sticking to the surface
of the plastic dish at its tip. In the present study, we classified (1) and (2) as head
induction, (3), (4) and (5) as no induction, and (6) as foot induction.
The changes in the potentials of the regenerating animals after head amputation were examined by a modification of the procedure described above, as
shown schematically in Fig. 2. To examine the head-activation potential of the
regenerating animals, heads were removed from the standard polyps by amputation at position 1. At various times thereafter, the donor tissues were
obtained from the distal regenerating tips of the beheaded animals, and they
were grafted into position 1, 2 or 3 of intact 105 polyps (Fig. 2A). To examine
the head-inhibition potential of the regenerating animals, heads were similarly
removed from the standard polyps. At various times thereafter, a donor tissue
obtained from position 1 of intact 105 polyps was grafted to the beheaded
polyps at a position one third of the length from the regenerating tip to the
bud protrusion (corresponding to position 2 of the original polyp) (Fig. 2B).
The grafted polyps were maintained in the same way as described above, except
that the beheaded animals were not fed during the entire period of observation.
The structures induced by the grafts were classified as head, foot or no
induction.
RESULTS
Polyp size and budding rate
Sugiyama & Fujisawa (1979) previously showed that L4 had a significantly
larger polyp size and a significantly lower budding rate than 105. This was confirmed in the present experiment. Table 1 shows that the standard polyp (young
polyp showing its first bud protrusion) of L4 is nearly twice as long as and
contains about 2-5 times more cells per polyp than 105. The table also shows that
the budding rate of L4 is less than half that of 105.
Morphogenetic potentials in hydra
105,L4
147
.
105
\
1-
VI
105
Fig. 2. Schematic representation of the procedures used to follow the changes of the
potentials during regeneration. (A) Head-activation potential. Donor tissues were
obtained from the distal regenerating tips of regenerating animals at various times
after head amputation, and their head-inducing capacities were examined by grafting
them to intact 105 polyps. (B) Head-inhibition potential. The levels of the headinhibition potential in the decapitated polyps were examined by grafting the donor
tissue from position 1 of intact 105 polyps to the decapitated polyps at various times
after head amputation.
No. of
polyps
examined
20
20
Strain
105
L4
A
Length
(mm)
± S.D.
5-4 ±0-6
11-1 ±2-5
s.
Length from hypostome
to basal disk
20
20
No. of
polyps
examined
A
3-5 ±0-4
8-2 ±1-9
± S.D.
Length
(mm)
Length from hypostome
to bud protrusion
10
10
No. of
polyps
examined
96 ± 4
236 ± 9
(X104)
Cell no.
per polyp
Total cell no. per
polyp
Table 1. Polyp size and budding rate of 105 and L4
A
s.
Buds per day
per polyp
± S.D.
1-1 ±0-1
0-5 ±0-1
Budding rate
No. of
determinations
6
6
r
oo
d
C/3
KANO AND 1
GIYAMA
Morphogenetic potentials in hydra
149
Head-regenerative capacity
Figs 3 and 4 show the results of experiments comparing the head regenerative
capacity of 105 and L4.105 polyps originally had an average of 6-4 tentacles per
polyp (Fig. 3A). After head and foot amputation, tentacles were resotred in
significant numbers by the second and in nearly the original numbers by the third
day of regeneration. Strain L4 had originally an average of 7-9 tentacles per
polyp (Fig. 3B). After head and foot amputation, this strain showed a generally
similar but slightly retarded time course of regeneration than 105. Tentacles
appeared in significant numbers by the 3rd day (instead of the 2nd day) and in
nearly the original numbers by the 5th (instead of the 3rd) day of regeneration.
A more significant difference between 105 and L4 was found when the tentacle
regenration of the two strains were compared in individual animals (Fig. 4).
After head and foot amputation, all the 105 polyps examined, without exception,
regenerated tentacles in about the same numbers as, or slightly higher numbers
than, the original animals (Fig. 4A). In contrast, about 10% of the L4 polyps
examined did not produce any tentacles at all. The rest produced tentacles which
ranged from about the original to twice the original numbers (Fig. 4B).
These results indicate that L4 has a slightly impaired regenerative capacity.
0h
4
6
0
Days after amputation
2
4
6
Fig. 3. Tentacle regeneration after head and foot amputation by 105 (A) and L4 (B).
See Materials and Methods for the experimental procedure. Sample size was 60 in
both A and B. Vertical bars represent standard deviations.
150
J. TAKANO AND T. SUGIYAMA
A
30 -
20 -
10 -
B
ill
[1
30 -
20 -
10 -
1.
. lllll.l,..
5
10
Tentacle numbers per polyp
15
Fig. 4. Variation in the tentacle numbers of individual animals of 105 (A) and L4
(B). The tentacle number distributions are shown for animals before head and foot
amputation (white columns) and after seven days of regeneration (dark columns).
Head-activation and head-inhibition potentials of intact polyps
The head-activation and the head-inhibition potentials of 105 and L4 were
examined by the lateral tissue grafting procedure described in Materials and
Methods (also see Fig. 1). The results obtained are summarized in Table 2.
By examining the individual results shown in the table, it is possible to compare the levels of the morphogenetic potentials in 105 and L4. For example, the
1
2
3
4
Position 0
—
(44)
(20)
(18)
(17)
0
0
0
0
(45)
(23)
(20)
(20)
25 %(32)
2
0
0
0
39 %(33)
Position 1
59 %(34)
11 (38)
0 (30)
0 (19)
0 (21)
83 %(29)
35 (43)
2 (42)
0 (18)
0 (17)
2
3
74 %(34)
(38)
(38)
(24)
(19)
21
8
0
0
90 %(30)
57 (42)
33 (36)
0 (23)
0 (17)
Strain 105
(Numbers in parenthesis show the numbers of grafts made).
Strain L4
Strain 105
1
2
3
4
Position 0
^~"--\^
Donor
•
Recipient
^"""^--^^^
100 %(17)
54 (37)
20 (35)
3 (33)
0 (21)
100 %(17)
91 (43)
47 (36)
29 (34)
0 (24)
4
0 %(17)
0 (41)
0 (23)
0 (23)
0 (24)
Position 1
0l %>(16)
0 (33)
0 (28)
0 (23)
0 (22)
2
3
1 2 % (26)
6 (35)
0 (22)
0 (22)
0 (20)
Strain L4
Table 2 . Percentages of head-structure induction by lateral grafting of tissue
62 %(26)
(37)
(35)
(37)
(23)
24
9
5
0
4
a.
a
©
Oq
©
a-
152
J. TAKANO AND T. SUGIYAMA
donor tissues from position 0 of 105 and L4 induced heads at 39 % and 25 %,
respectively, when grafted to position 1 of 105. Head induction is thought to
occur when the head-activation potential of the donor tissue is more than a
threshold value higher than the head-inhibition potential of the recipient tissue
(Wolpert et al. 1974; Sugiyama, 1982; MacWilliams, 1983a,6). Since the same
recipient site was used, the results demonstrate that the head-activation potential
is slightly higher in 105 than in L4 at position 0.
The same potential level comparison can be carried out in a systematic manner
using all the data shown in Table 2. This is done by adopting 105 as an arbitrary
standard of the potentials, and by using the positions in 105 as a way to represent
the potential levels at various positions of both 105 and L4. The actual procedure
is explained below using an example shown in Fig. 5. The data used in the
following part (Figs 5-8) are all taken from Table 2.
Fig. 5 shows the percentages of head induction which were observed when the
five donor tissues from 105 were grafted to a common recipient site at position
4 of 105. Head induction occurred at the highest percentage with the donor tissue
from position 0 (100%), at progressively lower percentages with the donor
tissues from position 1, 2 and 3, and at the lowest percentage with the donor
tissue from position 4 (0 %). This indicates that the head-activation potential is
the highest at position 0 and the lowest at position 4, forming a 'gradient' between these two positions in 105. This agrees well with the results of similar
analysis previously done (Sugiyama, 1982; Rubin & Bode, 1982; MacWilliams,
1983£). In the figure, a line is drawn by connecting the percentages of the head
induction obtained with the five donor tissues. This line is used as a standard line
to determine the 105 positions which have the same head-activation potential as
the five L4 positions. For example, the donor tissue obtained from position 1 of
L4 induced heads at 54 % when grafted to the same recipient site. Application
of this value to the standard line (indicated by the dotted lines) shows that
position 1-8 tissue of 105 would also produce the same percentage of head induction when grafted to the same site. This indicates that position 1 of L4 and
position 1-8 of 105 have the same level of the head-activation potential. In the
present procedure, we use position 1-8 of 105 to represent this level of the
potential at position 1 of L4.
The same procedure is repeated using the results of grafting experiments
involving other common recipient sites. Standard lines similar to the one shown
in Fig. 5 are produced from the results of grafting the five donor tissues from 105
to position 3, 2 or 1 of 105. When grafted to these recipient sites, the position 1
tissue of L4 induced heads at 21 %, 11 % and 0 %, respectively. Application of
these values to the respective standard lines indicates that position 1 of L4 has
the same level of the head-activation potential as position 2-4,1-7 and 2-0 of 105,
respectively.
These differences in the results obtained are presumably produced in part by
the statistical fluctuation of the results and in part by the differences of the
Morphogenetic potentials in hydra
153
recipient sites used to examine the potentials. Averaging these results (1-7,1-8,
2-0 and 2-4) shows that position 2-0 of 105 best represents the potential level at
position 1 of L4.
The same procedure can be also used to determine the 105 positions which
represent the levels of the head-activation potential at the other L4 positions. It
is possible in this way to represent the potential levels at various positions of L4
(or any other strains) all by the positions in 105. Fig. 6 is produced in this manner
to compare the head-activation potential levels in 105 and L4. Since 105 is used
as the standard, its potential levels are represented by a straight (dotted) line in
this figure. As compared to this line, the potential levels in L4 are slightly lower
0 -
0
1
2
Donor position
3
Fig. 5. A standard line to determine the 105 positions which represent the headactivation potential levels at the five positions in L4. A standard (solid) line is
produced from the percentages of head induction which were observed when the five
donor tissues obtained from 105 were grafted to position 4 of 105 (Table 2). The
donor tissue from position 1 of L4 induced heads at 54 % when grafted to the same
site (Table 2). Application of this value to the standard line shows that this donor and
the donor from position 1.8 of 105 have the same head-inducing capacity (indicated
by dotted lines). The latter therefore can repreent the potential level of the former.
154
J. TAKANO AND T. SUGIYAMA
a
3
0
1
2
3
Axial position
4
Fig. 6. Comparison of the head-activation potential levels at the five positions of 105
and L4. The abscissa represents the axial positions of 105 (dotted line) and L4 (solid
line). The ordinate represents the head-activation potential levels which are expressed using 105 as the standard of the potential. For example, the potential in this
strain is the highest in position 0 and the lowest in position 4, and these levels are
shown on the ordinate by 0 and 4, respectively. Since 105 is used as the standard, the
potential levels at the five positions of this strain is shown by the straight (dotted) line
(see Discussion). The potential levels at the five positions of L4 can be represented
by the 105 positions which have the same head-inducing capacities (as determined
in Fig. 5). The figure shows these levels which are determined using position 1 (open
circles), position 2 (closed circles), position 3 (open triangles) or position 4 (closed
triangles) of 105 as the common recipient site to compare the head-inducing
capacities of the two strains.
than in 105 from position 0 to position 3. The potential level at position 4 of L4
cannot be shown exactly. This is because the donor tissue obtained from this
position induced no heads at any recipient sites (Table 2). This indicates that the
potential at this position in L4 is approximately the same as the level at position
4 of 105. The level, however, may be even low.
The same principle used to compare the head-activation potentials of 105 and
L4 described above can be also used to compare the head-inhibition potentials
of the two strains. Fig. 7 shows a standard line which is used to determine the 105
Morphogenetic potentials in hydra
155
oRecipient site
Fig. 7. A standard line to determine the 105 positions which represent the headinhibition potential levels at the four positions in L4. The solid line shows the percentages of head induction which were observed when the position 1 tissue from 105 was
grafted to the four recipient sites on 105 (Table 2). The same donor tissue induced
heads at 24 % when grafted to position 4 of L4 (Table 2). The dotted lines inducate
that the level of the inhibition potential at this site can be represented by position 1-7
of 105.
positions which have the same head-inhibition potentials as the four L4
positions. This line is produced from the percentages of head induction which
were observed when the common donor tissue from position 1 of 105 was grafted
to the four recipient sites on 105. Head induction occurred at the lowest percentage at position 1 (2 %) and at progressively higher percentages at more proximal
positions, indicating that the head-inhibition potential also forms a gradient from
head to foot. This agrees well with the results of similar previous studies
(Sugiyama, 1982; Rubin & Bode, 1982; MacWilliams, 1983a). The same donor
tissue induced heads at 24 % when grafted to position 4 of L4. Application of this
156
J. TAKANO AND T. SUGIYAMA
value to the standard line (shown by the dotted lines) indicates that head induction would also occur at the same percentage if the same donor tissue is grafted
to position 1-7 of 105. This shows that position 1-7 of 105 and position 4 of L4
have the same level of the head-inhibition potential. Therefore, the potential
level at position 4 of L4 can be represented by position 1-7 of 105.
Standard lines similar to the one shown in Fig. 7 are also produced from the
results of grafting the donor tissues from position 0, 2 or 3 of 105 to the four
recipient sites of 105, and the four standard lines are similarly used to determine
the 105 positions which can represent the head-inhibition potential of the four
Fig. 8. Comparison of the head-inhibition potential levels at the four positions of 105
and L4. The abscissa represents the axial positions of 105 (dotted line) and L4 (solid
line). The ordinate represents the head-inhibition potential levels which are expressed using 105 as the standard of the potential. Since 105 is used as the standard,
the potential levels at the four positons of this strain is shown by the straight (dotted)
line (see Discussion). The potential levels at the four positions of L4 can be represented by the 105 positions having the same head-inhibition potentials (as determined
in Fig. 7). The figure shows these levels which were determined using position 1
(open circles), position 2 (closed circles), position 3 (open triangles) or position 4
(closed triangles) of 105 as the common donor tissue to compare the potentials of the
two strains. The arrows attached to the symbols show that the potential levels are
significantly higher than the level indicated, but the exact levels cannot be shown.
Morphogenetic potentials in hydra
157
positions (position 1 to 4) of L4. The results obtained are presented in Fig. 8.
The figure shows that the levels of the head-inhibition potential at position 1
and 2 of L4 are very high and above the highest level of the standard (position
1 of 105). This means that none of the donor tissues used was able to induce heads
when grafted to these positions (see Table 2). The levels at position 3 and 4 of
L4 are also significantly higher than the levels at the corresponding positions in
105.
Grafts from L4 to L4
The donor tissues obtained from L4 can be grafted to the recipient sites on L4.
The approximate percentages of head induction in such grafts can be predicted
from the relative potential levels shown in Figs 6 and 8 and the standard lines
used to produce them. Table 3 shows the predicted and the experimentally
observed values of the results of such grafts. No significant differences are
present between the two values. This indicates that the functioning of morphogenetic potentials does not show any significant strain specificity between 105 and
L4, and that the relative potential levels of L4 are accurately represented in Figs
6 and 8. The absence of strain specificity in the lateral grafting of tissue was also
previously noted between 105 and mh-1 (Sugiyama, 1982) and between 105 and
reg-16 (Achermann & Sugiyama, 1983a).
Comparison of the potentials on the scale models
The results presented in Figs 6 and 8 show that L4 has a slightly lower headactivation and a significantly higher head-inhibition potential than 105. This
conclusion is based on the comparison of the potentials at the corresponding
relative positions along the body axis of the two strains.
Table 3. Percentages of head-structure induction by grafting the L4 donor tissues
to the L4 recipient sites
Donor
Recipient
Predicted
Observed
Position 0
position 1
2
3
4
0
0
0-29
40-82
Position 1
2
3
4
0
0
0-44
0(24)
3(35)
17 (36)
43 (35)
0(22)
0(28)
16 (32)
Position 2
3
4
CD CD
% Head induction
0(24)
3(33)
(Numbers in parenthesis show the numbers of grafts made).
EMB78
158
J. TAKANO AND T. SUGIYAMA
\
\
4
...
2 mm
L4
Fig. 9. Comparison of the potential levels on the scale models of 105 and L4. The
regions which have the same levels of the head-activation potential (A) and the headinhibition potential (B) are indicated. The potential levels in and near the bud
protrusion are not considered (see Discussion).
However, a slightly different conclusion is obtained if the polyp size difference
of the two strains is taken into consideration. As shown in Table 1, the average
standard polyp of L4 is nearly twice as large as 105. Fig. 9 shows the scale models
of 105 and L4, and the potentials of the two strains are compared on these
models. Fig. 9A shows that, when compared on the basis of the equal physical
distance from the hypostome, the difference of the head-activation potential in
L4 and 105 is very small, particularly in the regions near the hypostome. Position
2-1 in L4 and position 4 in 105 are located approximately the same distance away
Morphogenetic potentials in hydra
159
Table 4. Percentages of head-structure induction by the donor tissues obtained
from the distal regenerating tips of the decapitated animals
Donor
A
^
r
Strain
105
L4
Hours between
decapitation and
grafting
0
6
12
18
24
0
6
12
18
24
36
48
60
% Head induction on the recipient (105)
A
N.
i
Position 1
2
3
0%(23)
38 (24)
85 (27)
100 (22)
100 (14)
0 %(28)
3 (34)
6 (48)
9 (33)
16 (49)
31 (49)
51 (47)
82 (33)
30% (30)
63 (24)
100 (22)
100 (22)
100 (14)
0,%(31)
17 (35)
24 (35)
31 (35)
45 (48)
47 (49)
56 (48)
88 (33)
52% (29)
76 (25)
100 (22)
100 (22)
100 (14)
20% (29)
56 (36)
65 (46)
64 (36)
56 (48)
54 (46)
67 (45)
89 (35)
(Numbers in parenthesis show the numbers of grafts made).
from the hypostome. In these regions, L4 appears to have a slightly higher headactivation potential than 105.
However, the conclusion for the head-inhibition potentials of the two strains
remains unchanged whether the potential comparison is made on the basis of the
relative positions (Fig. 8) or equal physical distances from the hypostome (Fig.
9B). L4 has a significantly higher inhibition potential than 105.
Potential changes during regeneration
After head removal, the head-activation and the head-inhibition potentials in
the hydra tissue show drastic and dynamic changes during the course of regeneration (Webster & Wolpert, 1966; MacWilliams, 1983a,6).
The changes of the potentials after head amputation were examined in 105
and L4 according to the procedure described in Materials and Methods (also
see Fig. 2). The results obtained are shown in Tables 4 and 5. In order to
systematically compare the patterns of the changes of the potentials in the two
strains, the same principle used above to compare the potentials of the intact
polyps (Fig. 6 and 8) was used again. The results shown in Tables 4 and 5 were
applied to the standard lines used to produce Figs 6 and 8, and the levels of the
potentials in the regenerating animals were all expressed by the positions in the
intact 105 polyps having the same potentials. The results obtained are presented
in Fig. 10.
160
J. TAKANO AND T. SUGIYAMA
Table 5. Percentages of head-structure induction on the decapitated hosts*
% Head induction on the decapitated host
Hours between decapitation
and grafting
intact polyp
0
6
12
18
24
36
48
60
72
84
96
r
105
]L4
30 %(30)
96 (26)
74 (27)
78 (23)
78 (22)
54 (26)
42 (26)
16 (26)
-
0%(33)
6 (34)
20 (35)
40 (52)
58 (36)
60 (57)
84 (61)
73 (60)
44 (32)
31 (48)
22 (32)
17
(48)
* The donor tissue was obtained from position 1 of intact 105 polyp.
(Numbers in parenthesis show the numbers of grafts made).
Fig. 10A shows the changes of the two potential in 105. It shows that the headactivation potential was at the level of about 1-1 at time zero (immediately after
head amputation). The level rose to about 0-3 in 6 h and to 0 or higher in 12h.
Thereafter, the level apparently became significantly higher (Table 4), but the
exact levels cannot be shown since they were above the highest level of the
standard used (position 0 of 105).
The head inhibition potential of 105 was originally at the level of about 1-9
(Fig. 10A). It dropped drastically to the level of about 4 immediately after head
amputation, and then returned gradually to nearly the original, or higher than
the original, level in 36 to 48 h. These patterns of the changes of the two potentials in the regenerating 105 are similar to the results of similar studies previously
reported by other workers (Webster & Wolpert, 1966; Wolpert et al. 1974;
MacWilliams, 1983a,fc).
Fig. 10B shows the potential changes in L4 after head amputation. The
patterns of the change of the two potentals in L4 are significantly different from
those in 105 shown in Fig. 10A. The head-activation potential was initially at
the level of about 2-4, and during the first 6 h of regeneration it rose significantly to the level of about 1-2. Thereafter, the level became higher at a very slow
rate.
The head-inhibition potential of L4 was initially at some level higher than 1-0
(the exact level is unknown, see Fig. 8). It dropped to the level of about 1-1
immediately after head amputation, and then continued to drop very slowly for
the next 36 h. Thereafter, it returned very gradually to nearly the original level
in 72 to 96 h.
Morphogenetic potentials in hydra
,2
ff
°
w
24
48
72
161
96
Hours after amputation
Fig. 10. Changes of the potential levels during regeneration of 105 (A) and L4 (B).
The abscissa represents the time after head amputation. Time 0 represents immediately after head amputation. The ordinate represents the potential levels which
are expressed using 105 as the standard of the potentials. The head-activation potential levels were determined by grafting the donor tissues from the distal regenerating
tips of the decapitated animals to position 1 (closed circles), position 2 (closed
triangles) or position 3 (closed squares) of the intact 105 polyps. The head-inhibition
potential levels were determined by grafting the position 1 tissue of 105 to the
decapitated animals at the site which corresponds to position 2 of the intact animals
before amputation (open circles). The arrows attached to the symbols show that the
potential levels are significantly higher than indicated but that the exact levels cannot
be shown. The levels of the head-activation potentials in the intact polyps are not
shown. However these levels are exactly the same as those at time 0. This is because
the operations involved in obtaining the donor tissue from the intact polyp are
exactly the same as the operations involved in first decapitating and then obtaining
the donor tissue from the distal regenerating tip of the decapitated animals immediately after head amputation.
DISCUSSION
Comparison of the potential levels
In a previous paper, Sugiyama (1982) used the 'topographic mapping' method
to compare the head-activation and the head-inhibition potentials of the standard wild-type strain (105) and a multiheaded mutant strain (mh-1). In the
162
J. TAKANO AND T. SUGIYAMA
present study, a different method was used to compare the potentials of 105 and
L4. This change was made because the previous method, although effective in
comparing the potential levels at various positions of different strains, cannot be
applied directly to follow the potential changes in the regenerating animals. In
contrast, the new method can be used to analyse the potentials of both the intact
and the regenerating animals. However, the basic principles involved in the two
methods are similar and exactly the same conclusion is reached when the results
of the previous study is analysed by the previous or by the new method.
In their model of the positional information theory, Wolpert etal. (1974) used
two straight lines to represent the gradients of the head-activation and the headinhibition potentials along the hydra body axis. (The terms positional value and
positional signal were used by these workers as parameters corresponding to the
two potentials). In the present study, we also used two straight lines to represent
the gradients of the two potentials in 105 (Figs 6 and 8). This, however, does not
mean that the two potentials actually form linear gradients in the hydra body
column. These lines are used only to indicate that the potential levels are high
near the head and become progressively lower toward the foot. At present, the
exact shapes of the gradients of the potentials are unknown since the lateral
tissue grafting procedure can compare the relative levels, but cannot determine
the absolute levels, of the potentials.
The budding zone is located between position 3 and position 4 (Fig. 1). It is
known that the tissue at the tip of the bud protrusion has a very high level of the
head-activation potential (Li & Yao, 1945). This, however, is neglected for
simplicity in the representation of the potentials in Fig. 6 since no grafting
experiment involving the budding zone was carried out in the present study.
In spite of these deficiencies, the present method of representing the potential
levels is highly useful to analyse the potential levels of both the intact and the
regenerating animals of 105 and L4.
Abnormalities of the potentials in the intact polyp of L4
When compared by relative positions, L4 has a slightly lower head-activation
and a significantly higher head-inhibition potential than 105 (Figs 6 and 8).
However, the difference in the head-activation potential virtually disappears
when the potential levels are compared by distance from the head (Fig. 9). This
indicates that L4 has an abnormally high head-inhibition potential but a nearly
normal or only slight low head-activation potential.
The mechanisms responsible for the high inhibition potential in L4 is unknown
at present. One possibility is that this strain contains a very high level of the
morphogenetic substance, head inhibitor (Berking, 1977; Schaller, Schmidt &
Grimmelikhuijzen, 1979). When externally applied, this substance suppresses
budding and head regeneration. It exists in a gradient from head to foot along
the body column. A mutant strain (reg-16) contains a higher than normal level
of the head-inhibitor activity in its tissue (Kemmner & Schaller, 1981) and has
Morphogenetic potentials in hydra
163
a higher than normal head-inhibition potential (Achermann & Sugiyama,
1983a). These and other observations suggest that the head inhibitor plays important roles in determining the head-inhibition potential level. The high inhibition potential in L4 may be produced by the high concentration of the head
inhibitor in its tissue.
Alternatively, it is also possible that the head inhibitor is uninvolved in the
high inhibition potential in L4. A mutant strain, aberrant, has a higher than
normal head-inhibition potential (Rubin & Bode, 1982), but has a lower than
normal head-inhibitor activity in its tissue (Schaller, Schmidt, Flick & Grimmelikhuijzen, 1977). This suggests that the high inhibition potential can be
produced by some mechanisms not directly related to the level of the head
inhibitor. The levels of the morphogenetic substances in L4 have not been
analysed.
Polyp size and budding rate of L4
L4 has a significantly larger polyp size and a significantly lower budding rate
than 105 (Table 1). These characters may be produced by the high headinhibition potential in this strain.
In normal hydra, the inhibition potential level is high in the head and becomes
gradually lower toward the foot as already mentioned. A variety of experiments
suggests that this gradient affects the occurrence and location of bud formation.
Budding zone is located some distance away from the head. Budding is enhanced
by the removal of the head (Tardent, 1972), and suppressed when the head is
artificially moved nearer to the budding zone (Burnett, 1961). As already mentioned, external addition of the head inhibitor suppresses budding (Berking,
1977; Schaller et al. 1979). In a mutant strain (mh-1) which has a lower than
normal head-inhibition potential (and a higher than normal head-activation
potential), abnormal budding frequently occurs along the body column in
regions away from the normal budding zone and very close to the head
(Sugiyama, 1982). These observations are all consistent with the view that budding is suppressed by the high head-inhibition potential, and that the budding
zone is located on the body column where the level of the head-inhibition potential becomes sufficiently low.
In L4, the length from the head to the budding zone, and hence also the total
length from the head to the basal disk, are significantly longer than those in 105
(Table 1). It is conceivable that this is produced because the budding zone is
moved further away from the head due to the high head-inhibition potential in
this strain. The low budding rate (Table 1) may be also produced by the high
head-inhibition potential.
There is, however, one aspect which remains unresolved in this explanation.
If the inhibition potential affects both the budding location and budding rate, a
mutant strain with a very high inhibition potential may have (1) a very large polyp
size but a normal budding rate, (2) a normal polyp size but a very low budding
164
J. TAKANO AND T. SUGIYAMA
rate, or (3) a moderately large polyp size and a moderately low budding rate. L4
appears to have the third combination of the characters. At present we have no
explanation why L4 has this and not the other two combinations. In fact, the
combinations of (1) or (2) have never been found. Sugiyama & Fujisawa (1979)
examined several developmental characteristics of a number of strains of H.
magnipapillata, and found a high negative correlation (r=—0*79, P<0-01)
between the polyp size and the budding rate of these strains. This indicates that
strains which have larger polyp sizes generally have lower budding rates. This
appears to suggest that the strains having large polyp sizes cannot have high
budding rates because the mechanisms determining the polyp size and the budding rate are not independent form, but are mutually closely related to, each
other in some way.
Whether or not the inhibition potential play important roles in determining the
polyp size and the budding rate in normal hydra, and whether or not the high
inhibition potential level in L4 is directly responsible for the large polyp size and
the low budding rate in this strain remain to be verified.
The changes of the potentials during regeneration
L4 shows a slightly impaired head regeneration after amputation of the original heads (Figs 3 and 4). In both 105 and L4, the levels of the head-activation and
the head-inhibition potentials change drastically during the course of regeneration after decapitation, but the patterns of the changes are significantly different
in the two strains (Fig. 10). The head-inhibition potential drops drastically immediately after head amputation in 105, whereas the drop is very gradual in L4.
The head-activation potential rises steadily and significantly for at least 18 h in
105 (Table 4), whereas it rises significantly only during the first 6h of regeneration in L4. After this period, however, the rise continues at a very low rate in this
strain. The head-inhibition potential, after dropping to a very low level, gradually returns to the original level in 36 to 48 h in 105, whereas the return is very
much delayed in L4.
Little is known at present about the exact mechanisms of the potential changes
after head removal. However, two interesting models are available on the roles
of the two potentials in hydra head regeneration. One is the 'positional information' theory proposed by Wolpert and his associates (Wolpert et al. 1974), and
the other is the 'lateral inhibition' theory proposed by Gierer & Meinhardt
(Gierer & Meinhardt, 1972; Meinhardt & Gierer, 1974). Although some important differences exist, the basic concepts involved are very similar in the two
models. It has been shown very recently that the potential levels in the intact and
in the regenerating polyps of a normal strain can be successfully explained both
qualitatively and quantitatively by the Gierer-Meinhardt model (MacWilliams,
1982, 1983a,b). Therefore, it may be worth while to consider the abnormal
potential changes in L4 in view of this model.
The initial rapid drop of the head-inhibition potential in normal hydra is
Morphogenetic potentials in hydra
165
explained by the Gierer-Meinhardt model by the rapid release of the inhibitor
from the wound surface. A substantial experimental evidence now exists to
support this explanation. Schaller (1976) reported that the head inhibitor was
rapidly released by the regenerating normal hydra into the surrounding culture
solution. A mutant strain, reg-16, has a very reduced head-regenerative capacity. In this strain, the head inhibitor is little released (Kemmner & Schaller, 1981)
and the inhibition potential drops only slightly (Achermann & Sugiyama, 19836)
immediately after head amputation. These observations suggest that the head
inhibitor release is responsible for the potential drop.
The small drop of the inhibition potential soon after head amputation in L4 may
be produced by the limited release of the head inhibitor in this strain. The gradual
drop of the inhibition potential occurring later in L4 may be produced by the
gradual release of the remaining head inhibitor or by is gradual decay in the tissue
after wound closure. As mentioned already, however, the levels of the morphogenetic substances in the intact or in the regenerating L4 have not been examined.
The gradual and significant rise of the activation potential after head amputation in normal hydra is explained by the Gierer-Meinhardt model as follows;
The activator has the property to autocatalytically stimulate its own production
and also to cross-catalytically stimulate the inhibitor production, whereas the
inhibitor has the property to cross-catalytically inhibit the activator production.
In normal tissue, the activator production is held in check by the inhibitor. This
check, however, is removed by the release of the inhibitor by head amputation,
leading to the autocatalytic activator amplification and the rise of the activation
potential. This aspect of the model, however, is purely speculative, and there is
no experimental evidence to support it.
In L4, the head-activation potential rises significantly only during the first 6h
of regeneration (Fig. 10B). This rise may be produced in response to the initial
small drop of the inhibition potential. These potential changes thus appear to be
consistent with the model. If this is true, however, the activation potential in L4
should continue to rise as in 105 because the autocatalytic activator production,
once started, should continue at ever accelerating rates until the level of the
inhibitor becomes sufficiently high to hold down the activator production. This,
however, is not the case in L4. Following the initial significant rise, the rate of
the rise of the activation potential becomes very low long before the recovery of
the inhibition potential in L4. In fact, the inhibition potential continues to drop
gradually until approximately 36 h after decapitation in this strain. This should
be an additional factor for the rise of the activation potential during this period.
These patterns of the potential changes in L4 are apparently not readily explainable by the Gierer-Meinhardt model.
Two alternative mechanisms can be considered for this inconsistency between
the model and the observations. First, L4 may have a genetic defect(s) in the
mechanisms for the autocatalytic amplification of the activator and/or the crosscatalytic inhibition of its production by the inhibitor.
166
J. TAKANO AND T. SUGIYAMA
Alternatively, the model may be wrong in its basic assumptions. The model
assumes autocatalytic and cross-catalytic relationships in the activator and inhibitor production. In normal hydra, the initial drop of the inhibition potential
is followed by the rise of the activation potential, which is then followed by the
recovery of the inhibition potential. These sequences of events are conveniently
explained by these relationships. As yet, however, there is no experimental
evidence which directly shows the cause-result relationships in these events. The
activation and the inhibition potentials may change independently from each
other, and in different patterns in 105 and L4.
After dropping to a very low level, the inhibition potential gradually recovers
in the standard wild-type strain (105) (Fig. 10A). This recovery process appears
to occur in two phases: the initial small recovery 6 to 18 h after head removal and
a more substantial recovery later. The initial recovery is very small but probably
significant (see Table 5), and the same two-phase recovery was also observed in
105 by Achermann & Sugiyama (1983b). (This, however, was not observed in H.
attenuata by Mac Williams (1983a)).
These two phases of recovery can be explained by the Gierer-Meinhardt
model as follows. As already described, the inhibitor production is crosscatalytically stimulated by the activator. Therefore, as the activator level
becomes gradually higher during regeneration, stimulation of the inhibitor
production should also become higher. This should be a relatively rapid process,
and may be primarily responsible for the initial small recovery from 6 to 18 h after
head removal.
Another important factor for the inhibitor production in the Gierer-Meinhardt model is the density of the 'source'. The head has a high capacity (a highsource density) to produce the inhibitor. This capacity should be initially very
low in the distal regenerating tissue, but should become gradually higher as the
formation of the new head structure proceeds. This may be primarily responsible
for the slow but substantial recovery starting about 24h after head amputation.
In L4, the recovery of the inhibition potential are not observed until 36 to 48 h
after decapitation. The initial small recovery may be unobservable simply
because it is masked by the gradual but significant drop of the inhibition potential
taking place from 0 to 36 h after decapitation in this strain. The delay in the
substantial recovery in L4 may indicate the slow increase of the source density
in this strain. It may be recalled that the tentacle regeneration in L4 is also slower
than in 105 (Fig. 3).
In parallel with this study, the potential changes after head removal were
also examined in a mutant strain (reg-16) which has a greatly reduced headregenerative capacity (Achermann & Sugiyama, 19836). As discussed above
for L4, some aspects of the potential changes in reg-16 are also not readily
explainable by the Gierer-Meinhardt model. Whether these difficulties arise
from the nature of these mutant strains or from the fault of the model remains
to be seen.
Morphogenetic potentials in hydra
167
We thank Dr J. Achermann for stimulating discussions and Ms P. Bode for critically reading
the manuscript. This work was supported by grants from Ministry of Education, Japan and
Mitsubishi Foundation.
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{Accepted 18 August 1983)