J. Embryol. exp. Morph. Vol. 24, 1, pp. 21-32, 1970
Printed in Great Britain
21
Morphogenesis in hydra
II. Peduncle and basal disc formation at the distal end of
regenerating hydra after exposure to low temperatures
By SONDRA C. CORFF 1 AND ALLISON L. BURNETT 2
From the Developmental Biology Center, Department of Biology,
Case Western Reserve University
When Hydra oligactis is excised below the tentacles and incubated for a short
time in concentrations of colchicine that inhibit spindle formation in dividing
cells, a peduncle and basal disc subsequently form at the cut distal end, where
hypostome and tentacles normally form CCorfF & Burnett, 1969). Since recent
reports suggest a similarity in the action of colchicine and low temperature, in
this study the effects of low temperatures on regenerating hydra were investigated. High hydrostatic pressure and low temperature have been shown to act
synergistically with colchicine to inhibit the first mitotic division in sea urchin
eggs (Marsland, 1968). Colchicine and cooling have also been shown to cause
disintegration of the microtubule system in Actinosphaerium (Tilney, 1965).
We have previously discussed peduncle and basal disc formation at the distal
end in terms of colchicine inhibition of cell division and the possible action of
colchicine on the nervous system (Corff & Burnett, 1969). In the present study,
we demonstrate that low temperature can initiate the same responses in regenerating hydra as those initiated by colchicine. Evidence provided by recent
studies (Burnett, 1961; Burnett & Diehl, 1964; Burnett, Diehl & Diehl, 1964)
on the effects of cold on hydra, specifically on the nervous system and growth,
permit greater insight into the mechanisms involved in the production of
peduncle and basal disc at the distal end of regenerating hydra. In general, the
evidence presented here supports our original conclusions regarding the action
of colchicine in initiating this response.
1
Author's address: University of Pennsylvania, School of Medicine, Department of
Anatomy, Philadelphia, Pennsylvania 19104, U.S.A.
2
Author's address: Northwestern University, Department of Biological Sciences, Evanston,
Illinois, 60201, U.S.A.
22
S. C. CORFF AND A. L. BURNETT
MATERIALS AND METHODS
Culturing procedures
Hydra oligactis were cultured according to the method of Loomis & Lenhoff
(1956) modified by the use of distilled water in place of tap water. Mass cultures
were maintained at 18-20 °C. The animals were fed daily on newly hatched
Artemia salina.
Experimental procedures
Hydra were starved for 24 h before each experiment. Depending on the experiment, the hypostome and tentacles were excised from each animal either before
or after exposure to cold (8-12 °C). After the excision, hydra were placed in the
cold or retained at room temperature (18-20 °C).
Assaying procedures
The response of each regenerating hydra to cold treatment was classified
within one of the following categories. This same classification had been used
in recording the response of hydra to colchicine treatment (Corff & Burnett,
1969).
1. Hydra formed a hypostome and tentacles at the distal end. (Regeneration
was frequently delayed. Most of the animals completed normal distal structures;
however, a number of animals regenerated only one or two tentacles or abnormally placed tentacles around the hypostome.)
2. Hydra formed a peduncle and basal disc at the distal end.
3. The wound healed over, but no regeneration of distal structures occurred.
4. Disintegration occurred within 24-48 h after excision.
RESULTS
Hydra exposed to cold (12 °C) prior to excision of
hypostome and tentacles
Hydra exposed to cold (12 °C) for varying times (6-96 h) before excision of
hypostome and tentacles were retained at room temperature (18-20 °C) or
returned to the cold for 5-8 days following the excision.
Hydra retained at room temperature regenerated as shown in Table 1. None
of the animals formed a peduncle and basal disc at the distal end of the body
column unless they had been exposed to cold for a minimum of 48 h before
excision. These animals responded similarly to colchicine-treated hydra. The
animals either regenerated normally, formed fewer tentacles, or a single medial
tentacle, did not regenerate or disintegrated (Table 1). In a group of five hydra
exposed to cold for 72 h prior to excision, one developed a basal disc at its distal
end. This animal also produced a bud with a basal disc at its distal end. This bud
was positioned between two other buds, both of which had formed tentacles.
Morphogenesis in hydra. II
23
Two other buds from this group of five, exhibited abnormally placed basal discs.
One detached bud had two basal discs positioned at its gastric-peduncle junction
and one bud still attached to a non-regenerating animal exhibited a basal disc
at its gastric-peduncle junction.
Table 1. Hydra placed in cold (12 °C)for varying times prior to removal of
hypostome and tentacles, and then transferred to room temperature
Regeneration at distal end
•\
Duration
No. of
hydra
treated
Peduncle
and basal
disc (%)
Medial
tentacle
4
6
8
12
16
24
24
24
24
36
48
48
48
60
72
72
72
26
5
31
35
28
31
22
20
5
38
40
22
5
34
36
25
5
—
—
—
—
—
—
—
—
—
—
—
—
—
3
3
12
20
—
—
3
—
—
—
—
—
—
3
—
5
—
—
—
4
—
84
44
2
1
96
23
4
at
12°C(h)
(%)
Abnormal Normal
distal
distal
regenera- regeneration (%) tion (%)
—
—
—
—
—
—
—
—
20
2
—
—
20
9
—
—
20
—
—
88
100
84
86
93
81
95
100
40
87
65
95
60
61
47
56
40
73
35
No regen- Disinteeration
gration
(%)
—
—
—
—
3
5
—
20
—
—
—
—
12
—
28
20
9
61
(%)
12
—
13
16
7
16
—
—
20
8
35
—
20
15
50
—
—
9
—
None of the hydra returned to the cold directly after excision of hypostome
and tentacles regenerated while in the cold. These animals were therefore
returned to room temperature after 5 or 8 days in the cold. The results of this
transfer to room temperature are shown in Table 2. The frequency of peduncle
and basal disc formation at the distal end was higher than in animals exposed to
cold only prior to excision. After 24 h at room temperature, two out of five
animals previously exposed to cold for 6 h before excision and then returned to
the cold for 5 days, regenerated tentacles (one medial tentacle and two small
tentacles). After 3 days at room temperature, however, both of these animals
exhibited basal discs at the distal end of the animal and the tentacles had disappeared. Twelve hydra exposed to the cold for 6 h prior to excision were
returned to the cold for 5 days. No regeneration occurred in the cold and these
at 12 °C
after
excision
(days)
8
5
5
5
8
8
8
8
8
8
8
8
8
8
8
8
at 12 °C
before
excision
4
6
Peduncle
and basal
disc (%)
40
50
33
—
—
13
80
—
—
40
4
8
40
3
—
No. of
hydra
treated
22
5
6
6
23
20
23
5
26
24
5
27
24
5
35
48
* + R T = plus six hours at room temperature
8
12
16
24
24
36
48
48
60
72
72
84
+ RT6*
+ RT6
(h)
JL/UldllLMl
J_-/ U L u U U l l
T"^l 1 YCk \\C\X\
—
—
—
—
—
—
—
—
—
—
—
—
—
3
2
Medial
tentacle
(%)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Abnormal
distal
regeneration
(%)
A
100
—
33
17
83
85
87
20
77
75
—
66
76
40
43
64
(%)
Normal
distal
regeneration
Regeneration at distal end
60
17
50
13
10
—
—
—
4
60
19
8
20
17
15
(%)
No regeneration
Table 2. Hydra placed at 12 °C before and after excision of hypostome and tentacles
and then transferred to room temperature
—
—
—
4
5
—
—
23
21
—
11
8
—
34
19
(%)
Disintegration
H
zw
c
•
to
Z
O
>
5
>
o
O
on
Morphogenesis in hydra. II
25
animals were transferred back to room temperature. Five animals subsequently
initiated peduncle and basal disc formation at the distal end of the body
column.
Table 3. Hydra placed in cold (8 °C)for varying times prior to removal of
hypostome and tentacles, and then transferred to room temperature
Regeneration at distal end
A
Duration
at
8 °C (h)
4
8
12
16
17
24
36
40
48
60
72
84
No. of
hydra
treated
Peduncle
and basal
disc (%)
28
30
33
37
20
29
55
31
35
46
46
33
—
—
—
—
—
—
16
17
17
—
21
Medial
tentacle
(%)
4
—
—
—
—
—
—
—
—
7
11
3
Abnormal Normal
distal
distal No regen- Disintegration
regenera- regenera- eration
tion (%) tion (%)
(%)
(%)
—
—
—
—
—
—
—
—
—
—
—
82
100
100
100
85
100
85
6
83
41
32
6
7
—
—
—
15
—
4
10
—
28
50
46
7
—
—
—
—
—
11
68
—
7
7
24
Hydra exposed to cold (8 °C) treatment prior to excision of
hypostome and tentacles
Hydra were exposed to cold (8 °C) for varying periods of time prior to
excision of hypostome and tentacles. One group of animals was retained at
room temperature after the excision. The results are shown in Table 3. Hydra
exposed for 40 or more h prior to excision formed a high percentage of peduncles
and basal discs at the distal end of the body column. However, in the group of
46 hydra exposed for 72 h, no distal basal discs were formed, but a high percentage of animals formed a single medial tentacle. A larger percentage of animals
disintegrated after treatment at 8 °C.
All hydra returned to 8 °C after the excision of hypostome and tentacles
disintegrated within 48 h.
Hydra excised below tentacles prior to exposure to cold (12 °C)
Hydra excised below the tentacles were exposed to cold (12 °C) for varying
periods of time and returned to room temperature. The results of this treatment
are shown in Table 4. None of the animals formed a peduncle and basal disc
at the distal end of the body column. One hydra in the group of 10, exposed for
7 h, regenerated one tentacle after 48 h at room temperature, but no hypostome.
This animal subsequently regenerated normally.
26
S. C. CORFF AND A. L. BURNETT
Hydra excised below the tentacles prior to exposure to cold (8 °C)
Hydra excised below the tentacles were exposed to cold (8 °C) for varying
periods of time and returned to room temperature. Three formed a peduncle
and basal disc at the distal end of the body column when exposed to the cold
for 48-60 h after the excision (Table 4). More animals formed medially placed
tentacles after exposure to 8 °C than after exposure to 12 °C.
Table 4. Hydra excised and placed in cold (12 °C or 8 °C)for varying times
before transfer to room temperature
Regeneration at distal end
r
Duration
Abnormal Normal
distal
distal
No regen- Disinteregenera- regenera- eration
gration
tion (%) tion (%)
(%)
(%)
Peduncle
and basal
disc (%)
Medial
tentacle
12°C(h)
No. of
hydra
treated
4
7
8
12
16
24
36
48
60
72
84
21
10
21
25
21
23
22
22
26
23
23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4
—
—
—
—
—
—
—
—
15
—
4
100
100
95
84
100
96
100
100
70
83
79
—
5
—
—
4
—
—
15
13
17
—
—
16
—
—
—
—
—
—
—
21
21
21
24
22
23
20
11
—
—
—
—
—
—
5
18
—
5
—
4
5
—
5
—
—
—
—
—
—
—
—
18
100
95
100
96
86
96
75
46
—
—
—
—
9
4
15
18
—
—
—
—
—
at
(%)
Duration
at8°C
4
8
12
16
24
36
48
60
—
—
Hydra excised below tentacles and placed at room temperature (18-20 °C)
for varying times before transfer to cold (9-10 °C)
Hydra excised and retained at room temperature for varying periods of time
subsequently regenerated tentacles as shown in Table 5. Regeneration was
considerably delayed (ca. 48 h) when compared with controls at room temperature, regardless of the length of time regeneration had proceeded at room
temperature. It is apparent, however, that hydra retained at room temperature
for 20-24 h are less affected by cold, since 73 and 95 % of the animals, respectively, regenerated at least one tentacle by 72 h after the excision. Hydra retained
1
2
3
4
5
6
Control
4h
4
8
12
18
20
24
10 min
(h)
Duration at
room temp.
20
21
23
20
21
20
21
20
20
21
21
21
22
20
No. of
hydra
treated
r-
—
—
—
—
—
—
100
95
100
95
95
95
100
85
0
—
—
—
—
5
—
5
—
5
5
5
—
5
1
48
20
90
76
83
i
00
47
80
—
—
—
•—
—
—
5
—
—
—
—
—
—
—
5
10
24
17
3
2
48 h after cut.
No. of tentacles
regenerated (%)
t—
—
—
—
—
—
—
90
80
90
71
61
52
27
5
0
—
—
—
—
—
—
10
10
5
19
24
19
23
15
1
—
—
—
—
—
—
10
5
10
10
24
45
60
2
—
—
—
—
5
5
5
20
3
100
100
100
100
100
100
72 h after cut
No. of tentacles
regenerated (^'o)
r-
—
—
—
—
—
—
62
—
40
—
—
5
—
—
0
—
—
—
—
—
—
33
30
15
14
—
10
9
5
1
—
—
—
——
—
5
40
30
24
29
24
27
10
2
96 h after cut.
No. of tentacles
regenerated (%)
—
—
—
—
—
—
30
15
62
71
61
64
85
3
t
—
—
—
—
—
—
19
—
—
—
—
—
—
—
0
—
—
—
—
—
—
10
5
—
—
—
5
—
—
1
i
—
—
—
—
—.
—
19
40
25
—
5
—
—
—
2
N
3
—
—
—
—
—
—
52
55
75
100
95
95
100
100
144 h after cuit.
No. of tentacles
regenerated (%)
Table 5. Hydra excised and placed at room temperature (18-20 °C)for varying times before transfer to cold (9-10 °C)
2^
Co"
lar-
1
2
3
4
5
6
Control
4h
4
8
12
18
20
24
10 min
(h)
Duration at
room temp.
20
21
23
20
21
20
20
20
20
21
21
23
21
21
No. of
hydra
treated
—
—
—
—
—
—
100
100
100
100
95
91
47
19
0
r-
10
24
17
—
48
20
—
—
—
—
—
—
38
42
—
—
—
—
5
9
15
10
—
—
—
—
—
—
2
1
J
48 h after cut.
No. of tentacles
regenerated (%)
90
76
83
100
52
80
—
—
—
—
—
—
—
29
3
—
—
—
—
—
—
75
5
10
5
10
4
5
5
0
r-
—
—
—
—
—
—
5
5
—
—
—
—
5
10
1
—
—
—
—
—
—
5
30
40
71
61
48
52
28
2
72 h after cut.
No. of tentacles
regenerated (%)
100
100
100
100
100
100
15
60
50
24
29
48
38
57
3
—
—
—
—
—
—
20
—
—
—
—
—
—
5
0
f
—
—
—
—
—
—
10
—
—
—
—
—
—
—
1
A
—
—
—
—
—
—
10
10
—
19
33
17
29
24
2
96 h after cut.
No. of tentacles
regenerated (%)
—
—
—
—
—
—
60
90
100
81
67
83
71
71
3
—
—
—
—
—
—
—
—
—
—
—
—
—
c
—
—
—
—
—
—
—
—
—
.—
—
—
—
1
0
f
A
—
—
—
—
—
—
—
—
10
5
—
19
10
2
144 h after cut.
No. of tentacles
regenerated (%)
3
—
—
—
—
—
—
95
100
100
90
95
100
81
90
Table 6. Hydra excised and placed at room temperature (18-20 °C) for varying times before transfer to cold (13-14 °C)
H
zw
d
&
CO
O
>
>
o
!«
n
oo
NJ
Morphogenesis in hydra. II
29
at room temperature for 4-18 h after the excision, showed progressively less
regeneration with decreasing periods of regeneration at room temperature.
Regeneration of one or more tentacles in most of these animals did not occur
until 96 h after the excision of the tentacles. Hydra placed in the cold almost
immediately after the excision of the tentacles were most affected and regeneration did not occur until 144 h after the excision.
Hydra excised and placed at room temperature (18-20 °C)for varying times
before transfer to cold (13-14 °C)
Hydra placed in the cold (13-14 °C) following regeneration at room temperature subsequently regenerated tentacles at a faster rate than hydra placed in the
cold (9-10 °C). Regeneration, however, was still delayed by ca. 24 h as compared with controls (Table 6). Hydra placed in the cold (13-14 °C) immediately
after the excision were delayed 48 h compared with controls.
DISCUSSION
Peduncle and basal disc formation at the distal end of
regenerating hydra
The various morphological forms observed at the distal end of regenerating
hydra following exposure to low temperatures parallel those observed in hydra
following exposure to colchicine (Corff & Burnett, 1969). In colchicine-treated
hydra, the various morphological forms were arranged in a morphogenetic
hierarchy that reflected the effectiveness of colchicine action on individual
hydra. This arrangement appears valid for cold-treated animals as well. According to Burnett (1966), polarity, morphogenesis and cell differentiation along the
body column of hydra are controlled by quantitative changes in inducer and
inhibitor levels along the body column. Inducer is thought to be produced by
nerves in the hypostome. The involvement of neurosecretion in regeneration of
hypostome and tentacles has been reported (Lesh & Burnett, 1964; Lentz, 1965;
Lesh & Burnett, 1966). A gradual reduction of neurosecretion in cold-treated
hydra or cold-induced sexual hydra is suggested by Lesh & Burnett (1966) and
Burnett & Diehl (1964). In the present study, with increased duration of exposure and decreased temperature, hydra at room temperature subsequently
regenerate fewer tentacles, abnormally positioned tentacles, or a single medial
tentacle (reflecting lower levels of inducer than normally present at the distal
end), no tentacles (reflecting levels of inducer normally present at the midgastric region) or a peduncle and basal disc (reflecting peduncle levels of inducer). At room temperature, neurosecretory droplets usually increase within
the nerve cells over a 4 h period in regenerating hydra (Burnett et al. 1964). If
hydra are excised and placed in the cold, this build-up of neurosecretory material
may be inhibited or may proceed very slowly. When the animals are returned
to room temperature, growth and regeneration will resume, but the form
30
S. C. CORFF AND A. L. BURNETT
regenerated at the distal end will depend on the level of inducer and inhibitor at
the cut end.
If the inducer-inhibitor levels at the distal end determine morphological form
at the distal end of cold-treated hydra returned to room temperature, why do
animals retained in the cold not form peduncles and basal discs at the distal
end? Several observations on the effects of low temperature on hydra may help
to explain this result. (1) Low temperature inhibits or reduces the rate of
budding, suggesting a reduction of growth (mitosis) at least in the epitheliomuscular cells of the epidermis and the digestive cells of the gastrodermis
(Burnett, 1961). (2) At room temperature, starving hydra cease budding.
(3) Testes on hydra maintained in the cold are stationary. At room temperature,
however, they are displaced down the body column suggesting the resumption
of growth in the body column. (4) Hydra slowly adapted to low temperatures,
will grow and bud normally at temperatures that induce sexuality or inhibit
growth in non-adapted hydra. These observations suggest that when growth is
slowly reduced, normal morphology and polarity can be maintained. Coldadapted or starving hydra must therefore maintain their own gradient of
inducer and inhibitor levels. When hydra are placed in the cold to regenerate,
growth probably occurs slowly enough to permit the establishment of a normal
gradient. When cold-treated hydra are returned to room temperature, in some
cases, growth resumes before the normal gradient can be established.
Hydra regenerating at room temperature for longer periods of time, regenerate tentacles faster than those placed in the cold immediately after the excision.
Since most animals, however, will eventually regenerate in the cold, the temperatures used in this study do not completely prevent growth (cell division) and
the ability of nerves to produce neurosecretory material.
The response of hydra to low temperature and colchicine treatment
Both colchicine and low temperature appear to affect the same processes:
cell division and neurosecretion. Although certain concentrations of colchicine
(0-015-0-025 %) inhibit spindle formation in dividing cells, long exposure to
colchicine for three or more hours at these concentrations frequently disrupts
hydra at the cellular level (Corff & Burnett, 1969). Preliminary experiments with
low concentrations of colchicine (0-005 %) and low temperature suggest that the
two act synergistically. Hydra placed in 0-005 % colchicine in the cold are less
affected than those exposed to the same concentration at room temperature.
This may be due to the subsequent decrease in growth at cold temperatures.
The use of both colchicine and cold treatment, however, may provide a
method of retaining c-metaphase cells in hydra over a longer period of time
without disorganization at the cellular levels, thereby providing a valuable
technique for an approach to the determination of cell cycles in hydra.
Morphogenesis in hydra. II
31
SUMMARY
1. Hydra can form a peduncle and basal disc at the distal end of the body
column when they are exposed to cold for various periods of time either before
or immediately after the excision of hypostome and tentacles. The response is
not 'all or none' and hydra frequently regenerate a smaller number of tentacles,
abnormally placed tentacles, or a single medial tentacle. Some hydra do not
regenerate.
2. The frequency of peduncle and basal disc formation at the distal end
increases with lower temperatures and longer duration of exposure to cold. It
decreases with longer duration of regeneration at room temperature prior to
cold treatment.
3. Hydra do not form a peduncle and basal disc at the distal end when they
are kept in the cold after excision. Normal regeneration is delayed and the
length of delay depends upon the decrease in temperature and the length of time
regeneration proceeded at room temperature before transfer to the cold.
4. Colchicine and low temperature treatment appear to influence basal disc
formation at the distal end in a similar manner. The role of cell division and the
nervous system in regeneration, in relation to the effects of cold treatment, is
discussed.
RESUME
Morphogenese chez Hydra. II. Formation du pedoncule et du disque basal a
Vextremite distale d'une Hydre en regeneration, apres exposition a basses
temperatures
1. Les hydres peuvent former un pedoncule et un disque basal a i'extremite
distale de la colonne somatique quand elles sont exposees au froid pendant des
periodes variees, soit avant, soit immediatement apres l'excision de rhypostome
et des tentacules. La reponse n'est pas 'tout ou rien' et l'hydre regenerefrequemment un plus petit nombre de tentacules, des tentacules en position anormale,
ou un seul tentacule median. Quelques hydres ne regenerent pas.
2. La frequence de formation du pedoncule et du disque basal a I'extremite
distale, augmente avec des temperatures plus basses et une duree accrue
d'exposition au froid. Elle diminue avec une duree plus longue de regeneration
a la temperature de la piece avant le traitement par le froid.
3. Les hydres ne forment pas de pedoncule et de disque basal a I'extremite
distale quand on les maintient au froid apres excision. La regeneration normale
est retardee et la longueur du retard depend de l'abaissement de temperature et
de la duree au cours de laquelle la regeneration a eu lieu avant le transfert au froid.
4. La colchicine et le traitement par une basse temperature apparaissait
influencer de la meme maniere la formation du disque basal a I'extremite distale.
On discute le role de la division cellulaire et du systeme nerveux dans la regeneration, en relation avec les effets du traitement par le froid.
32
S. C. CORFF AND A. L. BURNETT
ACKNOWLEDGEMENTS
This paper was prepared from a thesis submitted in partial fulfilment for the degree of
Doctor of Philosophy. This work was supported by the U.S. Public Health Service training
grant no. HD20 (Corff), and by the National Science Foundation grant no. GB-7345 (Burnett).
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