J. Embryol. exp. Morph., Vol. 16, 2, pp. 245-257, October 1966
Printed in Great Britain
245
A study of the effects of colchicine on the
process of morphogenesis and induction
in chick embryos
By B. A. DIWAN 1
From the Department of Zoology, University ofPoona
The importance of sulfhydryl groups in the process of morphogenesis and
induction has been discussed by several workers (Rapkine & Brachet, 1951;
Seilern-Aspang, 1959; Brachet, 1960; Pohl & Quertier, 1963; Brachet, 1964).
Lakshmi (1962a, b) studied the effects of chloroacetophenone on the development of chick embryos and emphasized the importance of sulfhydryl groups
in morphogenesis and induction. Colchicine is known to affect cell division,
probably by its action on sulfhydryl groups of the spindle proteins (Dustin,
1947; Mazia & Dan, 1952). In the present work the effect of colchicine on the
processes of morphogenesis and induction in chick embryos has been studied.
In order to gain further insight into the mechanism of action of the chemical it is
useful to see if its action is reversible. Therefore, an attempt has been made to
reverse the action of colchicine by cysteine.
MATERIALS AND METHODS
Fresh, fertilized eggs of White Leghorn hens were obtained from a local
poultry farm. These eggs were incubated at 37 °C until the desired stage of
development. The glassware and instruments were sterilized and the solutions
to be used for preparation of Compton solution were auto.claved. Colchicine was
dissolved in distilled water and a stock solution of 0-05 g/1. (1-2 x 10~7 M) was
made. This solution was further diluted with Pannett Compton solution to the
desired strengths.
Parti
Experiment 1. Effect of colchicine on chick embryos cultured in vitro
Embryos were explanted from the incubated eggs and mounted in vitro by the
ring technique of New (1955). After some preliminary experiments a concentration of 5 x 10~5 g/1. (1-2 x 10~10 M) colchicine was found to be suitable. Eightyfive embryos at the definitive streak stage and 15 embryos at the head process
1
Author's address: Department of Zoology, University of Poona, Poona-7, India.
246
B. A. DIWAN
stage were treated with this solution and grown for 20-21 h. Thin albumen was
added around the ring. Seventy-five embryos at the definitive streak stage and
15 embryos at the head process stage were kept as controls.
Experiment 2. Reversal of the action of colchicine
In this set of experiments the effects of colchicine on chick embryos have been
nullified by cysteine hydrochloride applied after treatment with colchicine.
Colchicine (0-0025 g/1.; 1 ml per egg) was injected in ovo (Silver, 1957) in 50 unincubated eggs which were then kept at room temperature for 15 h to allow
proper diffusion of the chemical. They were incubated until the definitive
primitive streak stage. These embryos were then explanted in vitro (New, 1955).
Pannett Compton solution (2 ml per embryo) was added ventrally to 15 embryos
(controls) and thin albumen was then added around the ring. The remaining
35 embryos (experimentals) were treated with cysteine hydrochloride (freshly
prepared in Pannett Compton solution, 2-5 jug/ml) instead of plain Compton
solution.
At the end of the treatment the embryos of exps. 1 and 2 were examined in
living condition to measure their lengths and record the gross abnormalities.
They were then fixed in acetic acid alcohol (1:3) for 1-1| h. Some of the fixed
embryos were processed for whole mounts, stained in dilute Delafield's haematoxylin and photographed. All the embryos were then sectioned at 8/t and
studied histologically.
Part II
Experiment 1. Effect of colchicine on the inducing capacity of Henserts node
Colchicine (1 ml per egg) was injected in ovo (Silver, 1957) into the unincubated
eggs which were kept at room temperature to allow proper diffusion of the
chemical. The eggs were incubated to the definitive primitive streak stage. The
nodes of these embryos were then cut out and washed in Pannett Compton
solution three or four times to ensure removal of colchicine.
The concentrations of colchicine used were 0-0015 g/1. (3-7 x 10~8 M), 0-0020 g/1.
(5-0 x 10-8 M) and 0-0025 g/1. (6-2 x 10~8 M). The stock solution was diluted with
Pannett Compton solution to the desired concentrations.
The host embryos at the definitive primitive streak stage were explanted in
vitro according to the method of New (1955). The nodes of the donor embryos
were grafted into these untreated host embryos by the technique of Waddington
(1932). In control experiments the donors were treated in ovo with Pannett
Compton solution instead of colchicine and grafting was performed in the same
way as referred to above. In all, 356 grafts were made of which 181 were experimentals and 175 were controls. All the experimental and control embryos
were incubated for 18-20 h after grafting. They were then fixed in acetic alcohol
(1:3), sectioned serially at 8 /i, and stained in dilute Delafield's haematoxylin for
histological study.
Effect of colchicine on morphogenesis
247
Experiment 2. Effect of colchicine on the competence of the host ectoderm
In this set of experiments eggs treated with colchicine in ovo as above were
explanted in vitro and used as hosts. The concentrations used for treatment were
0-0020 g/1. and 0-0025 g/1. Untreated embryos at the definitive primitive streak
stage were used as donors for grafting. In all, 90 grafts were made of which 45
were experimentals and 45 controls. The embryos were incubated for 18-20 h,
then prepared for examination as above.
Experiment 3. Reversal of the action of colchicine on the inducing capacity of
Hensen's node
Eggs treated with colchicine in ovo as described above were incubated to the
early primitive streak stage. Some of these embryos were grown in vitro in
Pannett Compton solution and used as control donors. In the case of experimental donors, cysteine hydrochloride in Pannett Compton solution (0-0025
g/1.) was added (0-02 ml). All these embryos were incubated to the definitive
primitive streak stage.
The nodes of the control and experimental embryos were then cut out, washed
in Pannett Compton solution three or four times and grafted on to the untreated
host embryos, explanted in vitro at the definitive primitive streak stage, by the
technique of Waddington (1932). In all, 65 grafts were made, out of which 35
were experimentals and 30 were controls. All the embryos were incubated for
18-20 h and then prepared for examination.
RESULTS
Parti
Experiment 1. Effect of colchicine on chick embryos cultured in vitro
Out of 85 embryos treated with 5 x 10~5 g/1. colchicine, 71 survived and 14
died. The abnormalities shown by the surviving embryos were recorded and their
analysis is given in Table 1. Fifteen embryos at the head process stage developed
normally.
In the embryo illustrated in Fig. 1A the most anterior region of the brain has
formed a solid mass of neural tissue. Posteriorly the neural tube is a flat plate
(Fig. 1C). The closure of the neural tube thus seems to have been affected by
colchicine treatment. The neural folds are wavy and wide apart. There are seven
pairs of somites lying ventral to the neural folds. The fore-gut is not formed.
Vitelline veins are not fused. The length of the embryo is 3 mm. Length of the
corresponding control embryo (Fig. 1B) is 5 mm.
Experiment 2
The embryo in Fig. 2 A was treated with colchicine in ovo. Here the brain has
differentiated into prosencephalon, mesencephalon and rhombencephalon and
l6
JEEM l6
248
B. A. DIWAN
optic lobes are formed but the region corresponding to midbrain is open (Fig.
2C). The embryo is also bent towards the right side. The neural tube posterior
to the heart is open and presents a wavy appearance. The somites are diffused
and undifferentiated. Vitelline veins are fused, forming a normal heart.
Table 1. Analysis of the malformations in the colchicine-treated embryos
1 Fore-brain
Abnormal 74-9%
Normal 25-1%
2 Posterior portion of the brain
3 Neural tube
Abnormal 800%
Normal 200%
Abnormal 64-5%
Normal 35-5%
Abnormal 20-0%
Abnormal 67-0%
Normal 130%
17-0%
46-2%
Absent 36-8%
6 Notochord
Abnormal 15-9%
Normal 84-1%
7 Somites
(a) Present
(b) Absent
Abnormal 47-5%
22-0%
Normal 30-5%
8 Length of axis
Subnormal 88-5%
Normal
4 Optic vesicle
(a) Both absent
(b) Both present
5
Heart
(a) Vitelline veins not fused
(b) Vitelline veins fused
flexure
Present
11-5%
Fig. 2B and D represent an experimental embryo protected from the action
of colchicine by subsequent treatment with cysteine hydrochloride (2-5 /*g/ml).
All the organs of the embryo are formed normally. The length of the embryo is
5-5 mm.
Part II
Experiment 1. Effect of colchicine on the inducing capacity ofHensen's node
Out of 356 grafts made, 26 experimentals and 14 controls could not be taken
for analysis. In some cases, the graft was lodged in between the splanchnic mesoderm and endoderm, while in others it was seen in the cavity of the fore-gut or in
the region ofthe heart or in a few cases in the coelom. In some embryos, although
prominent induction was present the graft was either degenerating or could not
be found. Such cases were also discarded since the possibility could not be ruled
out that the induction might be due to a degenerating graft.
Table 2 shows that the percentage of induction has fallen from 80 % to
15-4 % with the increase of colchicine concentration from 0-0015 to 0-0025 g/1.
Apart from this fall in the induction capacity of the graft, the quality of induction has also been affected by colchicine treatment. In order to study the quality
of inductions produced they are classified into strong inductions and moderate
inductions. For example, Fig. 3B obtained from a graft treated with 0-0015 g/1.
colchicine illustrates a strong induction where the graft has induced a neural tube
Effect of colchicine on morphogenesis
249
of considerable dimension. Fig. 3D illustrates a moderate induction obtained
from a graft treated with 0-0020 g/1. colchicine.
It will be seen from Table 2 that the grafts treated with 0-0015 g/1. colchicine
have caused strong inductions in 54-3 % of the cases. When the concentration
was increased to 0-0020 g/1. the grafts showed strong inductions in only 18-2 %
of the cases. When the concentration was increased to 0-0025 g/1. no cases of
strong inductions and only 15-4 % cases of moderate inductions were obtained.
^^fe^siis^fS
®m& c
I
0-2 mm
I
D
Fig. 1. A, Macroscopic view of chick embryo treated with 5 x 10~4 g/1. colchicine at
the primitive streak stage; x 24. B, Control chick embryo for the experimental embryo in A; x 24. C, T.S. passing through the brain region of embryo in A. D, T.S.
passing through the tail region of embryo treated with 5 x 10~4 g/1. colchicine,
showing flattening of neural tube.
The differentiation of the grafts treated with different concentrations of colchicine was studied. Of the grafts treated with 0-0015 g/1. colchicine, 78 % showed
good differentiation. At a concentration of 0-0020 g/1., 45 % of the grafts showed
differentiation and the remaining grafts presented an undifferentiated neural
mass though the cells were quite healthy. At a concentration of 0-0025 g/1.
16-2
250
B. A. DIWAN
0-2 mm
Fig. 2. A, Macroscopic view of chick embryo treated m ovo with 0-0025 g/1. colchicine, x 24. B, Macroscopic view of chick embryo protected from the action of
colchicine by subsequent treatment of cysteine hydrochloride (00025 g/1.), x24.
C, T.S. passing through the mid-brain of embryo in A, showing open neural tube.
D, T.S. passing through the fore-gut region of embryo in B.
Table 2. Analysis of induction capacity ofHensen's node
after colchicine treatment
Concentration
of colchicine
(g/1.)
00015
00020
0-0025
Control for
00015
Control for
00020
Control for
0-0025
No. of
grafts
made
No. of
No. of
Strong Moderate Absence of Overall
grafts not
grafts
taken for taken for induction induction induction induction
analysis
analysis
(%)
(%)
(%)
(%)
Overall % of
inductions by
corresponding
control grafts
40
64
77
40
5
9
12
4
35
55
65
36
54-3
18-2
—
94-5
25-7
27-3
15-4
—
20
54-5
84-6
5-5
800
45-5
15-4
—
94-5
90-9
94-4
—
60
5
55
90-9
—
91
—
—
75
5
70
94-4
—
5-6
—
—
Effect of colchicine on morphogenesis
251
only 15 % of the grafts showed differentiation and the remaining grafts presented
a mass of loose disaggregated cells (Fig. 3E).
Thus it can be seen from Table 2 that increase in the concentration of colchicine used for treatment has affected the differentiation of the graft. It was
suggested by Waddington (1932) that the differentiation of grafts into neural
tissue is a requisite for normal induction. However, the present results indicate
that there is no correlation between the quality of differentiation of the graft and
the quality of the induction. Fig. 3 B illustrates a case where the graft treated with
0-0015 g/1. colchicine differentiated well and also caused good induction, while
Fig. 3C illustrates a case where the graft treated with 0-0015 g/1. colchicine has
differentiated, but has not caused good induction. Thus, loss in the inducing
capacity observed in the majority of the experimental grafts (Fig. 3E) may not
be taken as due to the non-differentiation of the grafts.
Experiment 2. Effect of colchicine on the competence of the host ectoderm
Out of the 45 experimental grafts only 31 could be taken for analysis, as in the
remaining cases the grafts either degenerated or were not in contact with the host
ectoderm. The results presented in Table 3 seem to indicate that colchicine has
no effect on the ectodermal competence of the host. Fig. 3 G and H illustrate
strong inductions produced by this series.
Table 3. Analysis of inductions produced by normal node in
hosts treated with colchicine
Concentration No of exof colchicine perimental
grafts
(g/1.)
00020
00025
9
22
Strong
inductions
(%)
Moderate
inductions
(%)
No. of
control
grafts
77-88
72-72
22-22
27-28
25
20
Strong Moderate
inductions inductions
(%)
(%)
84
16
85
15
Experiments. The reversal of the action of colchicine on the inducing capacity of
Henserts node
Out of 65 grafts made, 18 experimental grafts and 8 control grafts could not
be taken for analysis for the reasons mentioned above. It will be seen from Table
4 that subsequent treatment with cysteine hydrochloride has a strong effect on
the inducing capacity of the node previously treated with colchicine. The percentage of induction has increased from 11 • 1 % to 70 % after subsequent
treatment with cysteine hydrochloride.
Both the experimental and control grafts were studied from the point of view
of differentiation. It was seen that 65 % of the control grafts (treated with
0-0025 g/1. colchicine) did not show any differentiation at all, while the remaining
grafts presented a loose disaggregated appearance (Fig. 4E). However, the
experimental grafts (Fig. 4B, D, F) showed differentiation in 85 % of the cases.
GR
INT
Fig. 3. A, T.S. of control induction for experiments of node treated with 00015 g/1.
colchicine. B, T.S. through a strong induction produced by node graft treated with
00015 g/1. colchicine. C, T.S. through a case showing absence of induction reaction
to node treated with 00015 g/1. colchicine. D, T.S. showing moderate induction produced by node treated with 00020g/1. colchicine. E, T.S. through case showing
absence of induction reaction to node graft treated with 00025 g/1. colchicine. F, T.S.
showing control induction produced by normal (untreated) graft in the untreated
host. G, T.S. through case showing strong induction produced by untreated node
graft in host treated with 0-0025 g/1. colchicine. H, T.S. showing strong induction
produced by untreated node graft in host treated with 0-0025 g/1. colchicine. HNT,
Host neural tissue; INT, induced neural tissue; GR, node graft.
Effect of colchicine on morphogenesis
253
It has been shown in exp. 1 of this part that increase in the concentration of
colchicine caused a decrease in the number of grafts differentiating. Apart from
this fact it has also been shown that colchicine has no effect on ectodermal
competence. This suggests a direct action of colchicine on the mesoderm of the
graft.
Table 4
Experimental
grafts
Control grafts (colchicine treated
(treated with
grafts treated
00025 g/1.
subsequently with
colchicine) 00025 g/1.cysteine)
Quality of induction
Strong inductions
Moderate inductions
Absence of inductions
/o
(%)
10
101
550
150
300
88-9
DISCUSSION
Several authors have emphasized the importance of sulfhydryl groups in
morphogenesis (Deuchar, 1957; Brachet, 1960; Lakshmi, 1962a). Abnormalities
caused by blocking sulfhydryl groups are shown to be consistent with their
distribution in Amphibia (Brachet, 1960). The present investigation has shown
that colchicine (0-05 mg/1.) at the primitive streak stage produces abnormalities
mainly in the brain region (Figs. 1 A, C, D). An identical treatment at the head
process stage does not cause any abnormalities. Using chloroacetophenone,
similar results have been obtained by Lakshmi (1962 a). Rapkine & Brachet
(1951) also observed inhibition of formation of nervous system in dorsal explants using oxidized glutathione or alloxane to oxidize the sulfhydryl groups.
From the results presented here, it is possible to assume that colchicine like
chloroacetophenone affects the activity of the organizer, and once the inducing
stimulus is given it becomes ineffective. The assumption is further supported
by the fact that Hensen's node loses much of its inducing capacity as a result of
treatment with colchicine (Fig. 3D, E). A similar effect on the inducing capacity
of the node has been shown by using chloroacetophenone by Lakshmi (1962&).
Further, it has also been shown that the sulfhydryl content of Hensen's node
decreases considerably after treatment with chloroacetophenone (Lakshmi &
Mulherkar, 1963).
In considering the effects of colchicine on the process of induction it was felt
significant to study its effects on the competence of the host ectoderm. The
results of Exp. 2 (Part II) indicated that the chemical does not affect the competence of the ectoderm of the treated hosts, since the normal grafts gave rise
to good inductions in these cases (Fig. 3G, H). Obviously, therefore, colchicine
254
B. A. DIWAN
affected only the inducing capacity of the node and has no effect on the reacting
tissue of the host.
Often the mechanism of drug activity has been revealed when antagonist is
used. Since colchicine-produced abnormalities ('Colchicine syndrome') are
INT
0-2 mm
I
HNT
f
,INT GR .•
HNT
:,
"
B
INT
,1
0-2 mm
I
I
INT
GR
0-2 mm*'*! E
Fig. 4. A, T.S. through control induction produced by graft treated with 0-0025 g/1. colchichine. B, T.S. through a strong induction produced by an experimental graft. C, T.S.
through a control induction produced by graft treated with 00025 g/1. colchicine showing
absence of induction. D, T.S. through a case showing strong induction reaction to an experimental graft. E, T.S. through a case showing absence of induction reaction to a control
node graft. F, T.S. through a strong induction produced by an experimental graft.
probably mediated by blocking sulfhydryl groups as mentioned above, an attempt has been made to nullify its action by supplying the embryos with a compound which contains sulfhydryl groups. From the results of Exp. 2 (Part I) it is
clear that cysteine hydrochloride can reverse the various effects produced by
Effect of colchicine on morphogenesis
255
colchicine (Fig. 2B, D). Further it has also been shown that the inducing capacity
of the colchicine-treated nodes is restored by a subsequent treatment with
cysteine hydrochloride (Fig. 4B, D, F).
It is therefore reasonable to assume that the loss in the inducing capacity of
the node treated with colchicine is due to its effect on sulfhydryl groups since the
effect is reversed by cysteine hydrochloride. This reasoning is also supported
by other work in our laboratory. It was found (Waheed, 1965) that the postnodal grafts treated with sulfhydryl group-containing substances (cysteine and
glutathione) differentiated into axial tissues such as neural tube and somites and
elicited differentiation of neural tissue in the host ectoderm. The differentiation
into somites seems to be promoted by cysteine (Fraser, 1960).
It is suggested by Brachet (1964) and Mulherkar, Rao & Joshi (1965) that the
biological activity and the molecular structure of the proteins which take part
in the process of induction depend upon the integrity of sulfhydryl groups and
disulfide groups. Colchicine, like chloroacetophenone, is expected to react with
sulfhydryl groups of these proteins, thus rendering them unable to give the
inducing stimulus.
SUMMARY
1. Chick embryos at the primitive streak and head process stage were treated
with 5 x 10~3 g/1. colchicine. Various types of abnormalities were observed,
particularly in the brain region and neural system, when treated at definitive
primitive streak stage. Embryos treated at the head process stage did not develop
any abnormalities.
2. The effects of colchicine on morphogenesis could be nullified by cysteine
hydrochloride applied after treatment with colchicine.
3. The effect of colchicine on the inducing capacity of Hensen's node at the
definitive streak stage has been studied. Increasing concentrations of colchicine
progressively affect the differentiation of the graft.
4. The quality of induction was also affected by the colchicine treatment. At
higher concentrations of colchicine (0-002 g/1.) there was a decrease in the percentage of strong induction, and in the case of nodes treated with 0-0025 g/1.
colchicine no case of strong induction was seen.
5. Colchicine was found to have no effect on ectodermal competence.
6. The inhibitory effects of colchicine on the inducing capacity of Hensen's
node are reversed by a subsequent treatment with cysteine hydrochloride. A
qualitative improvement of induction is seen as evidenced by an increase in the
number of strong inductions and decrease in the cases of absence of induction
after subsequent treatment with cysteine. The differentiation of the grafts also
improves after treatment with cysteine hydrochloride following colchicine.
256
B. A. DIWAN
RESUME
Etude des effets de la colchicine sur la morphogenese et
Vinduction chez Vembryon de Poulet
1. Des embryons de Poulet aux stades de la ligne primitive et du prolongement cephalique ont ete traites par la colchicine a la dose de 5 x 10~5 g/1. Divers
types d'anomalie ont ete observes, particulierement dans la region cerebrale et le
systeme nerveux, lorsque le traitement avait ete applique au stade de la ligne
primitive constitute. Les embryons traites au stade du prolongement cephalique
ne developpent aucune anomalie.
2. Les effets de la colchicine sur la morphogenese peuvent etre annules par le
chlorhydrate de cysteine agissant apres le traitement colchicinique.
3. L'effet de la colchicine sur les capacites inductrices du nceud de Hensen
a ete etudie au stade de la ligne primitive definitive. Des concentrations de plus
en plus elevees affectent graduellement la differentiation de la greffe.
4. La qualite de l'induction est egalement affectee par la colchicine. Pour des
concentrations elevees de colchicine (0,002 g/1.), on note une diminution du
pourcentage d'induction 'forte', et, lorsque la concentration atteint 0,0025 g/1.,
plus aucune induction forte ne se manifeste.
5. Les effets inhibiteurs de la colchicine sur les capacites inductrices du nceud
de Hensen sont reversibilises par un traitement ulterieur au chlorhydrate de
cysteine. Une amelioration qualitative de l'induction, se manifestant par
l'augmentation de nombre des inductions fortes et la diminution des cas d'absence
d'induction, est constatee lorsqu'un traitement a la cysteine est instaure apres la
colchinisation, traitement qui ameliore egalement la differentiation des greffes.
The author is grateful to Professor Leela Mulherkar, M.Sc, Ph.D. (Edin.), for her invaluable guidance in the present work. Grateful thanks are due to Professor C. H. Waddington
and Professor D. R. Newth for reading the manuscript and making helpful suggestions.
I also thank the Government of India for awarding me a scholarship under the Research
Training Scheme.
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{Manuscript received 1 March 1966)