/ . Embryo/, exp. Morph. Vol. 21, 3, pp. 417-43, June 1969
417
Printed in Great Britain
Morphogenesis in hydra
I. Peduncle and basal disc formation at the distal end of
regenerating hydra after exposure to colchicine
By SONDRA C. CORFF 1 & ALLISON L. BURNETT 1
Developmental Biology Center, Western Reserve
University, Cleveland
The regenerative ability of hydra has been frequently demonstrated since
Trembley (1744) first witnessed the regeneration of a hydranth from the proximal
half of an excised animal. Under normal conditions, the hypostome and
tentacles always regenerate from the distal cut end, and proximal structures, the
peduncle and basal disc, from the proximal cut end. This pattern of regeneration
reflects the rigid polarity of hydra. Normal polarity and regeneration can be
altered, however, by several techniques. Various graft combinations induce
distal structures from regions destined to produce peduncle (Wetzel, 1898;
King, 1903; Browne, 1909; Koelitz, 1911; Mutz, 1930; Burnett, 1961). An interspecific graft between the peduncle region of Hydra oligactis and the distal
end of Hydra pirardi can result in the production of tentacles at the proximal
end of the peduncle, which normally would form a base, and the inversion
of polarity (Burnett, 1961). An extract prepared from an homogenate
of hydra can induce multiple head and tentacle formation and inversion of
polarity in gastric annuli (Lesh & Burnett, 1964; Lentz, 1965a; Lesh &
Burnett, 1966). It appears therefore that regions proximal to the hypostome and
tentacles are not irreversibly determined, but susceptible to redirection under
appropriate stimuli. Although distal structures have been produced from proximal regions, no report exists of peduncle and basal disc formation at the distal
end of an excised hydra.
Colchicine has been frequently used in studies of cell proliferation, regeneration, and metamorphosis (Eigsti & Dustin, 1955; Flickinger, 1959; Flickinger &
Coward, 1962; Ilan & Quastel, 1965). Sturtevant, Sturtevant & Turner (1951)
described the effect of low concentrations of colchicine (3-3 x 10~7 % to 3-3 x
10~3%) in inhibiting and reducing tentacle formation in regenerating hydra.
Ham & Eakin (1958) confirmed these general results and suggested further that
colchicine had an 'all or none' effect on inhibition of regeneration. Both studies
1
Authors' address: Developmental Biology Center, Department of Biology, Case Western
Reserve University, Cleveland, Ohio, U.S.A.
27-2
418
S. C. CORFF & A. L. BURNETT
suggested that colchicine acted as a mitotic inhibitor. When hydra were exposed
to colchicine at various time periods after the excision, the time period of maximum inhibition of regeneration did not correspond to the time period for maximum activity of various nerve depressants but to the time of increased mitotic
activity, suggesting that colchicine did not act as a nerve-damaging agent (Ham
& Eakin, 1958). Colcemid, an JV-acetyl derivative of colchicine, induced multiple
distal structures on regenerating hydra, but not on non-regenerating animals
(Webster, 1967). Colcemid treatment of hydra did not inhibit fragments of
hypostome taken from these animals from inducing a secondary axis when
transplanted to a host animal.
In normal hydra, a correlation exists between the gross morphology of the
animal and the distribution of the various cell types along the body column.
Distinct changes in gross morphology, cell type distribution, and metabolic
activity are observed when the gastric region is compared with the peduncle
(Child & Hyman, 1919; Burnett, 1959; Burnett, 1966).
The effects of colchicine on the morphology of hydra at the organismal and
cellular levels have been investigated. In this paper we will present evidence
demonstrating that a peduncle and basal disc can be induced to form at the
distal end of regenerating hydra after colchicine treatment. Chemical gradients
have been suggested as controlling factors of hydra morphology (Burnett, 1966).
In this study, histological observations have suggested that migration, phagocytosis, and detachment of cells and villi from the mesoglea may be involved
in the process of peduncle formation.
MATERIALS AND METHODS
Hydra oligactis were grown in low-density cultures (1-5 hydra/10 ml) in
culture water prepared by adding 5 ml of 4% CaCla to a liter of distilled water
containing 5 ml of a Versene solution (1 % Versene, 2% NaHCO3) (Loomis &
Lenhoff, 1956). Cultures were fed daily on newly hatched larvae of Artemia
salina. The temperature was maintained between 18-20 °C for both experimental and non-experimental hydra. In order to obtain a more uniform adult
population, large hydra possessing two or more buds were selected for all
experiments except one, in which only hydra possessing stage I buds were used
in order to mark the original polarity of the animal. Stage I buds have no
tentacles and usually occur at the bud-peduncle junction when older buds are
not present. All animals were starved 24-36 h prior to experimentation.
Colchicine treatment
A 0-05 % stock solution of colchicine (Fisher Scientific Company) was
prepared in normal culture water. All concentrations of colchicine used in the
experiments were prepared by dilution of stock solution with normal culture
water.
Hydra selected for regeneration experiments were allowed to extend fully in a
Morphogenesis in hydra
419
large Petri dish containing normal culture water. The hypostome and tentacles
were excised just below the tentacle bases with a sharp scalpel. The animals were
then transferred in groups of 5, 10, 15, or 20, depending on the experiment, to
various dilutions of colchicine for varying periods of incubation. At the end of
the incubation period the hydra were removed from the colchicine, rinsed three
times in 25 ml aliquots of normal culture water, and transferred to a clean
Petri dish containing 15 ml of normal culture water. The culture water was
changed daily, and the animals observed for at least 5 days. Hydra excised
below the tentacles and at the bud-peduncle junction or at the bud-peduncle
junction only were treated similarly. Control animals were treated in the same
manner as experimental animals, but without exposure to colchicine. No
peduncle or basal disc formation was ever observed at the distal end of these
animals after excision of the hypostome and tentacles. In another series of
controls, hydra were exposed to 0005 % colchicine for varying periods of time.
Hydra exposed for a long time to this concentration never developed a peduncle
and basal disc at the distal end of the excised animal. In addition, histological
studies indicated that at this concentration mitotic spindles were present in
dividing cells.
Histology
Histological observations were made on regenerating hydra excised below
the tentacle bases and fixed after 1, 2, 3, or 4 h of incubation in colchicine
(0-005-0025 %). One group of animals was incubated in colchicine for 4 h and
then placed in normal culture water for 1 h before fixation. Histological observations were also made on non-excised hydra treated similarly to the experimental animals excised below the tentacles. In order to ensure exposure of all
the gastrodermis to the colchicine, the hydra were placed in colchicine and the
same concentration injected into the gastric cavity, through the mouth, by
means of a braking pipette. The animals were relaxed in 3 % urethane, prior to
fixation in Bouin's. They were then dehydrated in absolute alcohol, embedded
in paraffin, and sectioned transversely at 6JLL. Sections were stained with 0-001 %
toluidine blue at pH 8, dehydrated and mounted in Permount. Control hydra,
not exposed to colchicine, were treated and prepared for histological examination in the same manner as colchicine-treated animals.
EXPERIMENTS AND RESULTS
The effect of colchicine on distal regeneration of hydra exposed
immediately after the removal of hypostome and tentacles
In this series of experiments, hydra were excised just proximal to the tentacle
bases and transferred in groups of 5, 10, or 20 to various concentrations of
colchicine for varying periods of incubation. At the end of the incubation period,
the animals were rinsed three times and transferred to normal culture water in a
clean Petri dish.
420
S. C. CORFF & A. L. BURNETT
The results obtained from these experiments are summarized in Table 1. All
non-colchicine-treated hydra regenerated a hypostome and tentacles at the
distal end. Incubation for 1-4h in 0005 % colchicine did not affect normal
distal regeneration of hypostome and tentacles. Incubation for 24-31 h at this
concentration delayed or completely inhibited regeneration, but no peduncle or
basal disc was formed at the distal end of the regenerating animal.
Incubation in 001 % to 0025 % colchicine produced varied effects depending
on the concentration and length of incubation. The response of each hydra was
classified within one of the following categories.
(1) Hydra formed a hypostome and tentacles at the distal end, although
regeneration was delayed. Most of the animals completed normal distal structures; however, a number of animals were observed to have only one or two
tentacles or abnormally placed tentacles around the hypostome.
(2) Hydra formed a peduncle and basal disc at the distal end (Fig. 1 A).
(3) The wound healed over, but no regeneration of distal structures occurred.
(4) Disintegration occurred within 24 h after the initial excision.
A basal disc and peduncle were produced at the distal end in 50 % of the
animals incubated for 6 h or more in a concentration of 001 % colchicine. At
higher concentrations (0-015-0025 %) a shorter incubation was sufficient either
to induce basal disc and peduncle formation at the distal end of the animal or to
inhibit any distal regeneration. Histological observations of hydra incubated
for 7 h in 0-01 % colchicine indicated that cells in division still contained mitotic
spindles. Although the results varied depending on the experiment, in one case
(0-015 % for 6 h) 80 % of the treated animals formed peduncles at the distal end.
Usually, at concentrations of 0-015-0025% colchicine, treatment for 3-6h
induced peduncle and basal disc formation in 40-60 % of the animals.
When hydra were incubated in colchicine (0-015-0-025 %) for only 1 h
immediately after the excision, no peduncles were formed at the distal end of the
animal, although a delay or inhibition of regeneration was observed at concentrations of 0-02 % and 0-025 %.
The percentage of hydra regenerating a hypostome and tentacles at the distal
end decreased with increasing concentration and length of exposure to colchicine (Tables 1, 2). Although no detailed counts were made, with increasing
concentration and length of exposure there was a reduction in the number of
tentacles regenerated and tentacles were abnormally positioned around the
hypostome. With increasing periods of colchicine incubation, at concentrations
of 0-015-0-025%, the percentage of hydra disintegrating increased, and a
higher percentage of the surviving animals formed a peduncle and basal disc
at the distal end, or did not regenerate (Table 2).
Morphogenesis in hydra
Fig. 1. Composite photograph of one histological section (l.s.) of a colchicinetreated hydra (A) and a line drawing of the same animal (B). Note characteristic
peduncle (C) and basal disc (B) at the distal end of the column where hypostome
and tentacles normally regenerate. Normally positioned peduncle (C) and basal disc
(B') at the proximal end of the column, x 64. A and A' = cap of mucous and
sloughed cells, D = gastric region, E — bud.
All
422
S. C. CORFF & A. L. BURNETT
Table 1. The effect of colchicine on distal regeneration of hydra incubated
immediately after the removal ofhypos tome and tentacles
Length
Cone.
of
of
colch.
incubation
(%)
(h)
*
0*
0005
0-025
0020
0015
0-025
0020
0015
0025
0020
0015
0025
0020
0015
0020
0015
0010
0025
0020
0020
0015
0010
0020
0-015
0010
0010
Peduncle Hypostome
1
2
3
4
24
31
1
2
3
4
5
6
7
No. of
hydra
treated
11
10
11
11
7
2
2
9
10
10
10
10
10
10
11
9
16
10
9
10
5
5
5
5
No
and
and
basal disc
tentacles
(%)
(%)
(%)
(%)
—
—
—
—
—
—
—
—
—
—
—
—
20
—
—
89
25
—
—
60
80
40
—
—
100
100
100
100
100
—
100
67
70
100
10
10
60
—
—
—
—
—
100
—
33
30
—
—
—
—
—
—
—
—
—
—
—
70
90
20
20
—
—
40
9
11
—
—
—
—
—
—
—
—
—
—
—
—
—
—
30
91
—
30
—
—
6
—
100
—
69
100
—
50
8
3
10
5
24
5
5
2
33
30
60
—
—
50
31
2
—
regenera- Disintegration
tion
20
60
—
—
—
20
40
—
—
50
50
40
—
—
100
100
67
50
—
100
100
—
—
* Control.
The effect of a second excision of the distal end on regeneration of hydra incubated
in colchicine immediately after the removal of hypostome and tentacles
The following experiments were performed in order to examine whether or
not colchicine affects the entire animal and its ability to regenerate. Hydra
were excised below the tentacles and the proximal pieces incubated in various
concentrations of colchicine for 1-4 h. Immediately after removal from colchi-
Morphogenesis in hydra
423
cine the hydra were rinsed 3 times and transferred to a Petri dish containing
normal culture water. The animals were allowed to extend and approximately
1 mm of the distal end of the regenerating animal was removed. After the second
excision, regenerative changes of both pieces were studied.
Table 2. The effect of colchicine on distal regeneration of hydra
incubated immediately after the removal of hypostome and tentacles
Duration and
concentration
of colch.
Peduncle
Hypostome
No
and
and
No. of hydra
surviving
basal disc
tentacles
regeneration
(%)
(%)
(%)
29/29
28/30
27/30
14/35
6/10
10/10
0/10
11/18
2/12
2/ 2
129/186
—
7
22
29
100
60
—
64
50
—
37
79
29
30
—
—
—
—
—
50
50
26
21
64
48
71
—
40
—
36
—
50
37
29/50
38/64
48/58
9/14
28
21
21
43
24
21
50
7
48
58
29
50
43/43
11/11
—
—
83
100
17
001-0025 %
lh
2
3
4
5
6
7
8
24
31
1-31 h
0025 %
0020 %
0015%
0-010 %
0005 %*
1-31 h
0%*
—
* Controls.
The results are summarized in Table 3. None of the forty-four proximal
pieces treated produced peduncles at the distal end. Hypostome and tentacles
regenerated at the distal end in 40 % of the proximal pieces while 43 % did not
regenerate any distal structure. The distal ends removed from hydra treated
with colchicine for 1 or 2 h after the first excision were able to regenerate
tentacles. The distal ends removed from hydra incubated in colchicine for 3 or
4 h after the first excision produced a small peduncle and basal disc in 2060 % of the cases. No hypostome or tentacles were observed on the small distal
pieces forming peduncle and basal disc. None of the distal or proximal pieces
obtained from the control hydra (0005 % colchicine or no colchicine) produced
peduncles or basal discs.
incub.
(h)
1
colch.
(%)
0025
0020
0015
0025
0020
0015
0025
0020
0015
0015
0025
0020
0015
0005*
0*
of
of
1
2
3
4
3-5
4
3
2
Length
Cone.
5
5
5
5
5
5
10
10
10
4
10
10
10
5
5
5
5
5
No. of
hydra
treated
r
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Prox.
piece
(%)
Distal
piece
(%)
—
—
—
—
—
—
40
60
20
—
—
—
40
—
—
—
—
—
Peduncle and
basal disc
* Control.
80
40
100
60
40
100
20
30
10
25
—
10
10
100
80
100
20
60
(%)
Prox.
piece
60
60
80
60
60
80
—
—
20
75
—
—
—
60
80
100
80
80
(%)
Distal
piece
Hypostome and
tentacles
20
60
—
40
60
—
60
70
80
75
—
40
60
—
20
—
80
40
(%)
Prox.
piece
A
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
20
(%)
Distal
piece
No regeneration
\
c
—
—
—
—
—.
100
50
30
(%)
—
—
—
—
—
—
20
—
10
—
Prox.
piece
A
40
40
20
40
40
20
60
40
60
25
100
100
60
40
20
—
20
—
(%)
Distal
piece
Disintegration
Table 3. The effect of colchicine on distal regeneration of hydra excised before and after incubation
\
w
H
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&
d
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Tl
T!
o
O
O
0025
0020
0015
4h
0025
0020
0015
3h
0025
0020
0015
Control
4h
0025
0020
0015
3h
0-025
0020
0015
2h
cone. (%)
of colch.
and
Duration
5
5
5
5
5
5
20
20
20
27
20
20
20
20
20
20
No. of
hydra
treated
—
15
30
7
10
—
30
15
35
40
(%)
—
—
—
—
—
—
—
—
5
—
(%)
Distal
peduncle
and basal
disc only
—
—
20
75
—
—
15
—
10
15
Distal
hypostome
and
tentacles
only
(%)
20
20
—
—
—
—
20
20
—
20
—
—
—
—
—
—
—
—
—
—
—
—
—
Hydra excised at the bud-peduncle junction only
—
5
30
—
—
10
10
—
25
5
(%)
and
Proximal
peduncle
proximal
peduncle and and basal
disc only
basal disc
Distal
—
20
—
—
—
—
—
11
—
—
—
—
—
—
Normal
regeneration
(%)
—
60
60
—
40
60
—
—
20
7
15
5
40
20
15
35
(%)
regeneration
No
80
—
20
80
40
40
100
80
—
—
75
85
5
65
10
5
(%)
Disintegra
tion
Table 4. The effect oj colchicine on regeneration at the proximal end oj hydra excised below the
tentacles and at the bud-peduncle junction
£
426
S. C. CORFF & A. L. BURNETT
The effect of colchicine on regeneration at the proximal cut end of hydra
The hypostome, tentacles, and peduncle were excised from hydra possessing
a stage I bud only. The large gastric pieces were incubated in colchicine (00150-025 %) for 1-4 h. The stage I bud marked the proximal end of the gastric
column and, consequently, the original polarity of the gastric piece.
The results of this series of experiments are summarized in Table 4. Peduncle
formation occurred at the distal end in 5-30 % of the pieces, depending on the
concentration and length of incubation. Hypostome and tentacles, however,
were never formed at the proximal end of any of the regenerating pieces. As in
the previous experiments, some animals regenerated a hypostome and tentacles
at the distal end.
In one experiment hydra were excised at the bud-peduncle junction only.
Only peduncle and basal disc formation occurred at the proximal end. Hypostome and tentacles were never formed at the proximal end of the gastric
column, but a peduncle and basal disc could form at the distal end of the piece,
although hydra were not excised at the distal end.
The effect of colchicine on regeneration of mid-gastric annuli
A series of experiments were performed in order to determine whether
colchicine would exert a different effect on regeneration of small pieces of gastric
region, where the gradient between distal and proximal ends is small. Hydra
were allowed to extend in a Petri dish and two annuli, 1-2 mm in length, were
excised from the mid-gastric region. These annuli were then transferred to
various concentrations of colchicine for varying periods of time.
The percentage of pieces disintegrating increased with increasing concentration and duration of incubation (Table 5). Peduncle formation occurred in
approximately 30 % of the surviving pieces after incubation in colchicine for
1-4 h (Table 6). A higher percentage of annuli formed peduncles and basal
discs in 0-025 % colchicine (44 %) than in 0-015 % colchicine (23 %). It was
not possible, however, to determine whether peduncle formation occurred at
the distal or proximal ends of the annuli. In control annuli, not exposed to
colchicine, only 4 % of the eighty-six pieces treated produced peduncles and
basal discs. In the control annuli, exposed to 0005 % colchicine, 3 % of the
pieces produced peduncles and basal discs. One piece produced a single tentacle
at both ends of the annulus. Peduncle formation was significantly higher in
colchicine-treated annuli (Tables 5, 6). Usually, only one peduncle and basal
disc was formed on each annulus, although 3 out of 60 annuli incubated for 2 h
formed a peduncle and basal disc at both ends.
o%*
2
3
4
0-005 %*
1h
0-025 %
0020
0015
4h
0-025 %
0020
0015
3h
2h
0025 %
0020
0015
0-025 %
0020
0015
lh
Duration
and cone.
of colch.
(%)
hydra
22
21
22
21
86
25
20
20
20
60
20
20
20
60
20
20
20
60
9
—
—
—
2
4
—
—
5
2
—
—
—
—
10
25
20
—
5
5
15
8
basal disc
no.
20
21
20
61
and
Total
Peduncle
9
5
18
5
9
—
—
—
—
—
—
—
—
—
_
—
—
—
—
—
10
3
(%)
Tentacles
only
* Control.
5
19
5
5
9
20
—
—
—
—
—
—
—
—
_
—
—
—
10
—
10
7
(%)
tentacles
and
Hypostome
5
—
23
28
14
—
—
—
—
—
—
—
5
2
_
—
5
2
—
—
5
2
(%)
Distal and
proximal
structure
18
—
9
—
7
24
—
—
—
—
—
—
5
2
_
—
—
—
—
10
—
3
(%)
Buds
formed
No
54
76
45
62
59
52
—
—
10
3
—
5
10
5
_
25
45
23
10
25
25
20
(%)
regeneration
Table 5. The effect of colchicine on regeneration of mid-gastric annuli
—
—
—
—
—
—
100
100
85
95
100
95
85
91
90
50
30
57
75
60
35
57
(%)
Disintegration
|
S!'
-&>
hyd
0%*
0025
0020
0015
0005 %*
1-4 h
2
3
4
0015-0025 %
1h
9
—
44
32
23
3
4
7/80
19/81
34/80
86/86
25/25
* Controls
—
6
19
42
—
33
8
20
28
—
6
15
4
—
—
(%)
(%)
(%)
8
—
—
—
tentacles
and
basal disc
Hypostome
and
Tentacles
only
Peduncle
26/61
26/60
5/60
3/60
Duration and
concentration No. of pieces'
of colchicine
surviving
14
—
—
9
4
—
20
—
(%)
Distal and
proximal
structure
28
58
53
59
52
7
24
H
w
fcd
5
o
o
(%)
46
54
60
67
O
/-•fc
C/5
No structures
10
3
8
—
20
—
(%)
Buds
formed
Table 6. The effect of colchicine on regeneration of mid-gastric annuli
OO
Morphogenesis in hydra
429
Time period of maximum effectiveness of colchicine incubation
after excision of the hypos tome and tentacles
In order to correlate the time and duration of exposure after the excision
with the percentage of hydra exhibiting altered regeneration, the following
experiments were performed. Animals were excised just below the tentacles,
Table 7. Time of maximum effectiveness of colchicine incubation
after excision of hypostome and tentacles
Time of
incubation
after
Duration and
concentration excision
(h)
of colchicine
1 h
0-025 %
0020
0015
0-025 %
0020
0015
0025 %
0020
0015
0-025 %
0020
0015
0-025 %
0020
0015
0-025 %
Peduncle Hypostome
No. of
hydra
treated
and
basal disc tentacles
(%)
(%)
67
40
40
87
80
80
67
73
60
67
53
0020
15
0015
Controls
15
15
—
—
—
—
—
—
7
—
—
—
—
7
—
—
—
—
—
—
—
15
15
15
15
15
15
15
15
18
15
15
15
15
15
15
27
—
—
13
—
—
—
—
—
—
—
—
—
—
—
0-1
1-2
2-3
3-4
4-5
5-6
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
and
60
73
60
87
87
60
73
80
No
regeneration
(%)
Disintegration
(%)
33
60
60
13
13
20
26
27
40
33
47
33
27
40
13
13
40
27
20
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
53
40
53
40
67
67
67
40
44
—
33
40
47
67
40
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2h
0-025 %
0020
0015
0-025 %
0020
0015
0-025 %
0020
0015
0-025 %
0020
0015
0-025 %
0020
0015
0-2
1-3
2-4
3-5
4-6
20
60
47
47
33
33
33
60
56
100
67
60
53
33
60
430
S. C. CORFF & A. L. BURNETT
and pulsed with colchicine at concentrations of 0015 %, 0020 %, and 0025 %
for 1 or 2 h at different times after the excision. The results are shown in Table 7.
A pulse of 1 h at 0-5 h after the excision of hypostome and tentacles did not
induce peduncle or basal disc formation, with the exception of two out of 270
animals treated. The most effective time period for complete inhibition of
regeneration occurred 0-1 h after excision. Regeneration was delayed and the
number of tentacles reduced to one or two in some cases. Animals occasionally
regenerated only a single median tentacle.
A pulse of 2 h induced peduncle formation at the distal end in six out of 225
animals treated. These peduncles were induced by colchicine treatment at 0-1
and 1-3 h after the excision. Incubation in colchicine 4 h or more after the excision did not induce peduncle formation at the distal end after either a 1 or 2 h
pulse.
The effect of colchicine on non-excised hydra
Non-excised hydra were placed in concentrations of colchicine (00150025 %) for 2-5 h of incubation. They were then rinsed 3 times and transferred
to normal culture water. At this time, all the animals appeared normal. At 24 h
after colchicine treatment the tentacles on all the animals were affected to some
extent. Most of the animals retained short tentacles or tentacle bases. In a few
instances, however, the distal end was rounded over, and no hypostome could
be detected. These animals had completely lost their tentacles. By 72 h after the
colchicine treatment, over 95 % of the hydra had regenerated tentacles. In some
cases the tentacles were fused or reduced in number. A single medially placed
tentacle originating from the center of the hypostome was observed on several
hydra.
Some animals formed a peduncle and basal disc at the distal end. In one series
of experiments, six out of 135 hydra treated with colchicine formed a peduncle
and basal disc at the distal end of the animal. In another series of experiments,
four out of 180 animals formed a peduncle and basal disc at the distal end.
In experiments described previously, where adult hydra were excised before
incubation in colchicine, it was noted that some of the buds, still attached to the
adult hydra, occasionally formed a peduncle and basal disc at the distal end.
We did not determine in these experiments whether the bud possessed tentacles
before the initiation of a peduncle and basal disc.
Histological observations of normal Hydra oligactis
Histological characteristics of the six morphologically distinct regions of
hydra have been described (Semal-van Gansen, 1954; Burnett, 1959). We will
therefore discuss only observations pertinent to this study, with emphasis
on characteristics of the bud-peduncle region.
In the hypostome region the gastrodermis is oriented in folds or 'villi'
(McConnell, 1931) which extend into the gastric cavity. The digestive cells in
the villi are long and thin and appear to maintain a small area of contact with
Morphogenesis in hydra
431
the mesoglea. Between the villi, more rounded digestive cells are observed,
maintaining what seems to be a larger area of contact with the mesoglea. The
tentacles emerge from between the villi. A similar positioning of tentacles has
been observed in Tubu/aria (Campbell & Campbell, 1968).
Examination of more proximal sections reveals an increase in the number of
villi until the bud-peduncle junction is reached. In the gastric region villi do not
extend as far into the gastric cavity, but it is still possible to observe rounder or
shorter cells between them. Cell divisions are more frequently found in these
areas between the villi than in cells located in the villus.
In the bud-peduncle region a sharp decrease in the number of villi occurs,
along with a decrease in cell density. In the peduncle, villi completely disappear.
Distinct changes are observed in cell morphology and distribution of cell types
between sections of the gastric region and sections of the peduncle region. The
epitheliomuscular cells of the epidermis are similar in the gastric and peduncle
regions. At the basal disc, however, these cells are characterized by the presence
of numerous mucous granules and the secretion of an acid mucopolysaccharide.
The number of cnidoblasts and interstitial cells sharply decreases at the budpeduncle junction. The number of nerve cells, however, increases in the peduncle
and basal disc. Characteristic food inclusions and lipid droplets in digestive
cells of the gastric region are lost in digestive cells of the peduncle. In the
peduncle these cells are also highly vacuolated. Gland and mucous cells, numerous in the gastric region of H. oligactis, almost completely disappear in the
peduncle. When the bud-peduncle region is examined carefully it becomes
evident that large clumps of villi have detached from the mesoglea and lie freely
in the gastric cavity.
Histological observations: the effect of colchicine on hydra excised
below the tentacles prior to incubation
In order to determine the effects of colchicine on the cellular level of organization, hydra were excised below the tentacles and incubated in various concentrations of colchicine for 1-4 h. The animals were then fixed and sectioned as
described previously. One group of animals was incubated in colchicine for
4 h, rinsed, and transferred to culture water for 1 h before fixation.
The histology of hydra incubated in colchicine (0005-0025 %) for 1 h prior
to fixation appears identical to that of control hydra (Fig. 2 A). Sections of
hydra incubated for 2 or more hours in 0005 % colchicine closely resembled
control sections, but as the incubation period increased, the number of cnidoblasts in the gastrodermis increased.
Incubation in 0-015-0025% colchicine for 2 or more hours initiated
striking changes in histology of the animals (Fig. 2B). Serial sections revealed
that a large number of digestive cells in the distal region of the animal had
detached from the mesoglea and rounded up within the gastric cavity. The
nuclei of these cells appeared normal. Many digestive cells contained several
28
1EEM 21
432
S. C. CORFF & A. L. BURNETT
Morphogenesis in hydra
433
developing nematocytes, cnidoblasts, mucous and gland cells (Fig. 2C). These
phagocytosed cells still retained their nuclei. The nucleus was normal in some
cells but in others it was either in colchicine-metaphase or pyknotic. In these
latter cells, no nuclear membrane was observed around the condensed mass of
chromatin. Many interstitial cells and developing cnidoblasts were present in
the gastrodermis of the animals, although normally few of these cells are present
in this layer.
In some areas of the distal sections the mesoglea appeared to be absent, and
as a result the epidermis and gastrodermis seemed to be in direct contact. In one
instance several epitheliomuscular cells of the epidermis, still containing
mucous droplets at their borders, were observed in the gastrodermis. Although
a large number of digestive cells had rounded up within the gastric cavity of the
distal sections, at least one layer of cells remained attached to the mesoglea in
all regions.
Histological observations of peduncle and basal disc formed at the
distal end of excised hydra after colchicine treatment
There was no observable difference between a peduncle and basal disc formed
at the proximal end of normal hydra and at the distal end of colchicine-treated
animals. The characteristic morphology of a normal peduncle and basal disc
was present in both (Figs. 3 A, B). Digestive cells of the gastrodermis appeared
vacuolated. The epidermal cells of the basal disc contained numerous mucous
granules. Toluidine-blue staining revealed a high concentration of metachromatic material indicative of the epidermal mucous cell of the basal disc.
The effect of colchicine on mitosis in Hydra oligactis
Hydra were exposed to various concentrations for varying periods of time,
fixed, and sectioned. Histological examination of hydra exposed to 0005 %,
0-075 %, or 0-01 % colchicine for approximately 7 h revealed that cells in
division still contained mitotic spindles. Before fixation these animals showed no
sign of depression.
After incubation in 0-015% colchicine for 7 h dividing cells in both the
gastrodermis and epidermis were in colchicine-metaphase. No spindles were
observed in these cells. Before fixation the tentacles and peduncles of these
animals appeared normal, but a few sloughed cells were observed in the Petri
dish.
Fig. 2. (A) Section through upper gastric region of non-colchicine-treated hydra
2 h after hypostome and tentacles were excised from the distal end. x 160. (B)
Section through upper gastric region of hydra incubated in 0020 % colchicine
for 2 h following excision of hypostome and tentacles. Note digestive cells (D),
detached from the mesoglea, are rounded-up within the gastric cavity. Some of
these cells contain secretory cells (S), cnidoblasts with immature nematocysts (IN),
or cnidoblasts with mature nematocysts (MN). x 160. (C,D) Digestive cells, detached
from the mesoglea, in the gastric cavity of colchicine-treated hydra. The cells contain
secretory cells (S), immature nematocysts (IN), and mature nematocysts (MN).
x400.
28-2
434
S. C. CORFF & A. L. BURNETT
At concentrations of 002 % and 0025 % colchicine all dividing cells observed
were in c-metaphase. At these concentrations sloughing of cells into the Petri
dish was more pronounced. With increasing concentration and duration of
Fig. 3. Higher magnification of peduncle and basal disc at the proximal (A) and
distal (B) ends of the hydra shown in Fig. 1. Note characteristic mucous cells of the
basal disc (A) and vacuolated digestive cells of the peduncle (JB) in both figures.
xl60.
Morphogenesis in hydra
435
incubation a greater degree of sloughing occurred. After 7 h of colchicine treatment at these concentrations, tentacles were clubbed or in the process of disintegrating. The normally smooth appearance of the epidermal surface of the
animal disappeared. The epidermis looked rough and individual cells or groups
of cells could be seen protruding from the surface.
Mitotic spindles reappeared in colchicine-treated hydra transferred to normal
culture water after a delay period influenced by concentration and duration of
exposure. Spindles were clearly observed in hydra incubated in 0-02 % colchicine
for 3 h and rinsed in culture water for 3 h. No spindles were observed in hydra
treated similarly in 0-025 % colchicine. Spindles were seen in animals incubated
in 0-025 % colchicine for 2 h and rinsed for 2 or 4 h. No spindles were observed
in animals treated similarly, but incubated for 4 h before rinsing.
DISCUSSION
The higher the concentration of colchicine the shorter the exposure
time needed to affect normal regeneration. When hydra are excised and placed
in normal culture water for 0-5 h before transfer to colchicine, the longer
regeneration proceeds in normal culture water the lower the percentage of
hydra forming peduncle and basal disc at the distal end or exhibiting no
regeneration of any structure at the distal end. This suggests that colchicine
interferes with the regeneration process occurring immediately after the initial
excision. Higher concentrations of colchicine (0015-0025 %) are more effective
in preventing normal regeneration than lower concentrations (0-005-0-01 %).
This correlates with studies in which the lower concentrations did not disrupt
the mitotic spindle in dividing cells. At concentrations of 0-015 % and above,
however, no mitotic spindles were observed and colchicine-metaphase figures
were present. In hydra removed from colchicine, mitotic spindles reappeared
after a delay period related to concentration and length of exposure to
colchicine. Many hydra incubated in colchicine for a short time (1-4 h) disintegrate within 24 h after removal from colchicine, suggesting that colchicine
interrupts not only the regeneration process but some mechanisms involved in
general maintenance of the entire animal. Colchicine does not permanently
block the capacity of the remainder of the body column to regenerate hypostome
and tentacles. When hydra are excised before incubation in colchicine and again
after incubation, fewer animals disintegrate than when excised only prior to
incubation. Hydra did not form peduncles at the distal end after the second
excision, although the distal piece removed at this time, in some cases, formed a
peduncle and basal disc. These results also suggest a localization of the effects
of colchicine. Peduncle and basal disc regenerated at the proximal end of the
body column whether or not the hypostome and tentacles were excised prior to
colchicine treatment. Mid-gastric annuli, 1-2 mm long, normally form hypostome and tentacles before any other structure. When mid-gastric annuli are
436
S. C. CORFF & A. L. BURNETT
exposed to colchicine, a higher percentage form peduncle and basal disc, or do
not regenerate any structure.
Non-excised hydra placed in colchicine partially or totally lose their tentacles
within 24 h after the incubation period. Concentrations of colchicine effective
in altering normal regeneration in excised hydra are less effective in blocking
normal regeneration and maintenance of non-excised animals.
Histological studies of excised hydra incubated in colchicine (0-015-0-025 %)
for 2 h or more reveal extreme changes in the cell organization, particularly in
the distal area. Digestive cells detach from the mesoglea and round up within
the gastric cavity. This detachment of digestive cells is similar to a response
reported by Miszurski (1949), who observed that the first effect of colchicine on
hanging-drop cultures of fibroblasts was to initiate withdrawal of cell processes
from the substratum. In colchicine-treated hydra many of the detached digestive
cells contain cnidoblasts, nematocysts, mucous, or gland cells. The gastrodermis
contains a large number of epidermal cells (cnidoblasts, interstitial cells, and
nematocysts). It appears therefore that although colchicine prevents pseudopod
formation and phagocytosis in leukocytes (Malawista & Bensch, 1967), it does
not inhibit migration and phagocytosis in hydra.
Hydra do not respond to colchicine in an 'all or none' fashion as suggested
by Ham &Eakin(1958). After colchicine treatment the distal end of regenerating
hydra can assume organismal and cellular characteristics of regions found
proximally along the body column of normal hydra. Although some hydra form
peduncle and basal disc at the distal end, others regenerate normal hypostome
and tentacles or abnormal structures characterized by a reduction in tentacle
number, abnormal positioning of tentacles around the hypostome, or the
emergence of a single medially placed tentacle. Hydra often completely disintegrate or do not form any structure at the distal end. The frequency of these
responses is related to (1) the concentration of colchicine, (2) the length of
exposure to colchicine, and (3) the duration of regeneration in normal culture
water before transfer to colchicine. Since colchicine appears to inhibit or
repress those factors responsible for normal hypostome and tentacle formation
(Lentz & Barnett, 1963; Burnett, Diehl & Diehl, 1964; Lesh & Burnett, 1964,
1966; Lentz, 1965a, b), the varied morphological forms produced at the distal
end after colchicine treatment suggest that different degrees of response to
colchicine occur in individual animals. At a high level of colchicine repression
(related to high concentration and long exposure), distal tissue produces proximal tissue (peduncle and basal disc). If we assume that the other morphological
forms observed after colchicine treatment reflect lesser degrees of colchicine
effectiveness, and as a result a smaller tendency for production of proximal
tissue, the observed forms can be arranged according to a morphogenetic
hierarchy that indicates not only the different degrees of effectiveness of colchicine action on individual hydra (repression of normal distal structures), but also
the actual disto-proximal distribution of tissue and form in normal animals
Morphogenesis in hydra
437
(Fig. 4). The qualitative changes at the distal end resulting from colchicine
treatment may possibly reflect the proposed quantitative changes (Burnett,
1966) in inducer and inhibitor levels postulated to control polarity, morphogenesis, and cell differentiation along the body column of normal animals. We
can speculate on how colchicine acts to induce the four levels of response, and
in particular, peduncle and basal disc formation. Colchicine could directly or
indirectly affect the ability of cells to function or respond normally to processes
directing regeneration and maintenance of form. Synthesis or activation of
substances needed for these processes could be impaired.
Normal regeneration
Single medial tentacle
No regeneration at
distal end
Peduncle and basal disc
formation at distal end
Disintegration
Fig. 4. Diagrammatic representation of the four main categories of response to
colchicine arranged according to a morphogenetic hierarchy.
Colchicine is known to damage or disrupt microtubules (Robbins & Gonatas,
1964; Tilney, 1965; Behnke & Forer, 1967), possibly by binding to a 6S protein
subunit (Borisy & Taylor, 1967; Shelanski & Taylor, 1967; Wilson & Friedkin,
1967). Since microtubules are found in hydra nerve cells (Lentz & Barnett,
1963; Davis, Burnett, & Haynes, 1968), colchicine may suppress normal
regeneration by temporarily interfering with the normal functioning of these
cells (Angevine, 1957). Preliminary studies with the electron microscope indicate
that in colchicine-treated hydra, neurosecretory material normally present in
regenerating animals is lacking (L. E. Davis & S. C. Corff, unpublished results).
Colchicine also prevents mitotic spindle formation in hydra. Burnett (1962) and
Ham & Eakin (1958) have observed an increase in cell division after removal of
438
S. C. CORFF & A. L. BURNETT
hypostome and tentacles. In this study colchicine concentrations that prevent
spindle formation are more effective than lower concentrations in altering normal regeneration and initiating peduncle and basal disc formation at the distal
end, suggesting that inhibition of cell division directly or indirectly can cause
peduncle formation. Interference with both the nervous system and the cell
division process may therefore prevent normal regeneration and elicit the production of peduncle and basal disc formation at the distal end.
A theory of peduncle formation in hydra
Although the initiation of a peduncle at a certain level of the body column is
probably controlled by a chemical gradient (Burnett, 1966), the results of the
present study suggest a method for the actual achievement of characteristic
peduncle form and histology.
We propose that the characteristic peduncle structure results from physical
detachment of cells and 'villi' of the gastrodermis at the bud-peduncle junction.
Since interstitial cells differentiate into nerve (Lentz, 1965 c), the increased
number of nerve cells in the peduncle and basal disc may arise from interstitial
cells and account for their depletion in the peduncle epidermis. In colchicinetreated hydra, histological characteristics of the peduncle epidermis (lack of
interstitial cells and cnidoblasts) are achieved in part by the migration of nonepitheliomuscular cells (cnidoblasts and possibly interstitial cells) from the
epidermis into the gastrodermis, where they may be phagocytosed and removed
from the area.
The rationale for this proposal comes from several observations made by us or
reported in the literature. At the bud-peduncle junction of a normal hydra we
have observed large clumps of villi detached from the mesoglea lying freely in
the gastric cavity. McConnell (1929, 1931) first reported the detachment of villi
from the mesoglea and postulated that a process of 'endogenous fragmentation'
occurred, whereby these cells were carried to different regions where they would
liberate food particles to neighbouring cells. Lentz (1966) reports that cytoplasmic fragments found in the gastric cavity at the base of the animal contain
mitochondria, food vacuoles, masses of amorphous material containing dense
structures, and nuclei. These fragments occasionally appear to be attached to
digestive cells by a cytoplasmic neck. Lentz (1966) also has observed structures
resembling disintegrating nematocysts or a type of lysosome in the digestive
cells of starved hydra. He was not able to observe intact nematocysts inside
cnidoblasts in the gastrodermis. Semal-van Gansen (1954) reported that gland
and mucous cells either slough into the gastric cavity of the peduncle region or
do not resynthesize droplets at this region. In regenerating hydra, incubated in
colchicine, the distal end of the gastric cavity becomes filled with detached
digestive cells containing an assortment of epidermal cells and secretory cells
of the gastrodermis. Colchicine treatment may therefore permit us to observe
an enlarged and extended step in the normal process of peduncle formation,
Morphogenesis in hydra
439
that of cell detachment and phagocytosis. In normal hydra one layer of digestive
cells always remains at the bud-peduncle region, even though, villi appear to be
detaching from this region. Although colchicine initiates detachment of cells
from the mesoglea, at least one layer of digestive cells remains attached in all
regions. Colchicine may therefore cause an initial retraction of cell processes in
some cells while others remain attached to the mesoglea. According to a recent
study (Haynes, Burnett & Davis, 1968), epitheliomuscular cells attach to the
mesoglea by either a smooth uniform junction or by a deep attachment, which
results when basal parts of the cells send deep processes into the mesoglea.
These deep processes are frequently apposed by deep attachments of the
digestive cells from the gastrodermal side.
Studies now in progress indicate that most cell divisions in a normal hydra
occur in the area between the villi. Although there is no definitive evidence, the
cells of this area may act as 'stem' cells in the production of villi. Studies of cell
divisions in hydra indicate that not all spindles in dividing cells are oriented
parallel to the longitudinal axis of the animal, but that many spindles are
oriented tangentially to the longitudinal axis (unpublished results), and probably
contribute to the circumferential growth of the gastric region. A correlation
may exist between the 'stem' cells and the cells exhibiting deep attachments to
the mesoglea. The deep attachments would prevent release of cells at the
peduncle.
Inherent in Burnett's hypothesis of an inducer-inhibitor control of growth
and form is the concept that all regions of hydra possess potential for redirection
into another region. Many studies (Lesh & Burnett, 1964; Lentz, 1965a,6;
Lesh & Burnett, 1966) have demonstrated that the addition of a factor present
in the hypostome region can induce distal structures from proximal regions.
In this study, however, we have produced proximal structures at the distal end
of regenerating hydra. These results strongly support the idea that polarization
and differentiation in hydra are not due to an inherent quality of the cells, or to
such factors as aging of cells or position in the body column, but result from
stimuli provided by an outside environment or microenvironment.
SUMMARY
1. The effects of colchicine on hydra morphology at the organismal and
cellular levels were investigated by incubating hydra, excised below the tentacles,
in various concentrations of colchicine for varying periods of time. After incubation in concentrations of colchicine that prevented mitotic spindle formation,
hydra sybsequently formed a peduncle and basal disc at the distal end, where
hypostome and tentacles normally regenerate. The frequency of this response
was correlated with the concentration of colchicine, the duration of exposure,
and the duration of regeneration in normal culture water before transfer to
colchicine. With increasing concentration of colchicine and duration of expo-
440
S. C. CORFF & A. L. BURNETT
sure, a higher percentage of animals formed a peduncle and basal disc at the
distal end. Treatment 0-3 h after the excision was more effective than incubation
4 h after the excision.
2. Peduncle and basal disc formation at the distal end of colchicine-treated
hydra could be prevented by removal of a small piece of the distal end immediately after incubation. The tissue removed by the second excision, however,
occasionally formed a peduncle and basal disc.
3. Hydra did not respond to colchicine in an 'all or none' fashion. The four
main categories of response (abnormal tentacle regeneration, peduncle and
basal disc formation, no regeneration of distal structures, or disintegration)
have been arranged according to a proposed morphogenetic hierarchy, which
may reflect quantitative changes in the inducer-inhibitor ratio at the distal end.
4. Peduncle and basal disc regenerated at the proximal end of the body
column, whether or not the hypostome and tentacles were removed from the
distal end prior to colchicine treatment.
5. Mid-gastric annuli, 1-2 mm long, normally form hypostome and tentacles,
but after colchicine treatment a higher percentage of these pieces formed
peduncle and basal disc.
6. Non-excised hydra are less affected by colchicine treatment. Tentacle
disintegration occurs to some degree on all animals, but only a few hydra form
peduncle and basal disc at the distal end.
7. Histological observations of colchicine-treated hydra indicated that colchicine concentrations that prevented mitotic spindle formation and induced
peduncle formation at the distal end also induced migration of cnidoblasts,
nematocysts, and interstitial cells from the epidermis into the gastrodermis;
phagocytosis of cnidoblasts, nematocysts, and secretory cells by the digestive
cells; and detachment of gastrodermal cells and villi from the mesoglea. The
significance of these processes in peduncle formation is discussed.
8. Possible sites of colchicine action and their significance in the production
and maintenance of form in hydra are discussed.
RESUME
Morphogenese chez Hydra. /. Formation du pedoncule et du disque
a Vextremite distale d'Hydra en regeneration apres
exposition a la colchicine
1. On a etudie les effets de la colchicine sur la morphologie de les hydres aux
niveaux de l'organisme et de la cellule en incubant des hydres, excisees audessous des tentacules, dans des concentrations variees de colchicine pendant
des durees diverses. Apres incubation dans des concentrations de colchicine qui
empechaient la formation du fuseaumitotique, les hydres ont forme un pedoncule
etun disque basal a l'extremite distale, ou l'hypostome et les tentacules regenerent
normalement. La frequence de cette reaction a ete mise en correlation avec la
Morphogenesis in hydra
441
concentration de colchicine, la duree d'exposition, la duree de regeneration
dans l'eau de culture normale avant le transfert dans la colchicine. Avec une
concentration croissante de colchicine et une duree d'exposition accrue, un
pourcentage plus eleve d'animaux ont forme un pedoncule et un disque basal
a l'extremite distale. Un traitement de 0 a 3 h apres l'excision a ete plus efficace
qu'une incubation de 4 h apres l'excision.
2. On a pu empecher la formation du pedoncule et du disque basal a
l'extremite distale d'une hydre traitee a la colchicine, en enlevant un petit fragment de l'extremite distale immediatement apres l'incubation. Le tissu ote par
la deuxieme excision, neanmoins, a forme occasionellement un pedoncule et
un disque basal.
3. L'hydre ne reagit pas a la colchicine selon la loi du 'tout ou rien'. Les
quatre categories principales de reaction (regeneration anormale des tentacules,
formation du pedoncule et du disque basal, pas de regeneration des structures
distales, ou disintegration) ont ete reparties selon une hierarchie morphogenetique proposee, qui peut reflechir des modifications quantitatives dans le rapport
inducteur-inhibiteur a l'extremite distale.
4. Le pedoncule et le disque basal ont regenere a l'extremite proximale de la
colon ne somatique, que l'hypostome et les tentacules aient ou non ete otes de
l'extremite distale, avant le traitement a la colchicine.
5. Des anneaux medio-gastriques, de 1 a 2 mm de longueur, forment normalement un hypostome et des tentacules; mais apres traitement a la colchicine un
pourcentage plus eleve de ces fragments ont forme un pedoncule et un disque
basal.
6. Les hydres non excisees sont moins affectees par le traitement a la colchicine. La disintegration des tentacules survient, a un certain degre, chez tous les
animaux, mais quelques hydres seulement forment un pedoncule et un disque
basal a l'extremite distale.
7. Les observations histologiques faites sur les hydres traitees a la colchicine
ont montre que les concentrations de colchicine qui empechaient la formation
du fuseau mitotique et induisaient la formation d'un pedoncule a l'extremite
distale, induisaient aussi: (a) la migration des cnidoblastes, des nematocystes et
des cellules interstitielles, allant de l'epiderme dans le gastroderme; (b) la
phagocytose des cnidoblastes, des nematocystes et des cellules secretrices par les
cellules digestives; (c) le detachement des cellules gastrodermiques et des
villosites de la mesoglee. On discute la signification de des processus dans la
formation du pedoncule.
8. Les sites possibles de Faction de la colchicine et leur signification dans la
realisation et le maintien de la forme chez l'hydre sont discutes.
This work was supported by the National Science Foundation grant no. GB-7345.
442
S. C. CORFF & A. L. BURNETT
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BEHNKE, O. & FORER, A. (1967). Evidence for four classes of microtubules in individual cells.
/. Cell Sci. 2, 169-92.
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(Manuscript received 12 September 1968)