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J. Embryol. exp. Morph., Vol. 17, 2, pp. 425-431, April 1967
With 2 plates
Printed in Great Britain
425
The effect of 2,4-dinitrophenol on the
development of early chick embryos
By PATRICIA BOWMAN 1
From the Department of Biology, Middlesex Hospital
Medical School, London
INTRODUCTION
It has been shown that when insulin is applied to chick embryos developing
in vitro it induces a syndrome of abnormalities, the main features of which are an
inhibition of brain and neural tube development at marginal concentrations
and of mesodermal derivates at higher concentrations (Barron & McKenzie,
1962). These authors found that brain and neural-tube inhibition could be
prevented by simultaneous administration of oxidized nicotinamide adenine
dinucleotide, thus lending support to the hypothesis put forward by Landauer
& Rhodes (1952) that insulin-induced anomalies are brought about by interference with oxidative phosphorylation of carbohydrates.
It has been suggested that one of the actions of insulin may be as an uncoupling agent in energy transfer (Randle & Smith, 1958 a, b) and some support
for this may be found in experiments carried out by Landauer & Clark (1964).
These authors showed that 2,4-dinitrophenol (DNP) and other uncouplers of
oxidative phosphorylation potentiate the teratogenic effects of insulin on the
chick embryo developing in ovo, although they are non-teratogenic when given
alone.
As the chick embryo developing in vitro is a much more controlled and
sensitive system for testing the effect of inhibitors than an embryo developing
in ovo, it seemed important to treat such embryos with DNP and to compare the
effects produced, if any, with insulin and other substances which interfere with
early development. Such a comparison might indicate whether insulin, in
particular, was acting in the same or in a different manner from DNP.
The effects of DNP have been tested previously on chick embryos developing
in vitro by Reporter & Ebert (1965). These authors, using the Spratt culture
technique, failed to detect any abnormalities at concentrations of DNP between
0-1 and 10-0/fcg/ml.
In the experiments reported here chick embryos cultured by the New technique have been treated with much higher concentrations of DNP than those
1
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh 9, Scotland.
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426
PATRICIA BOWMAN
used by Reporter & Ebert and abnormalities have been found which were
examined microscopically and macroscopically.
MATERIALS AND METHODS
Eggs were obtained from a White Leghorn stock supplied by a local breeder.
After an incubation period of 22-24 h at 38-5 °C they were explanted according
to the New technique (New, 1955). A five times concentrated stock solution of
DNP (British Drug Houses Ltd), was prepared in distilled water. The final
concentration of 2,4-DNP was applied both to the ventral and dorsal surfaces
of the embryos: in the former diluted with albumen and the latter with PannettCompton solution.
A modification of the New technique for the use of teratogens was used
(Billett, Collini & Hamilton, 1965). This involved storing the embryos for
approximately 16 h in a cold box at 15 °C, then replacing the solution on the
surface of the embryos with fresh solution prior to incubation. This ensures
passive diffusion of the substance to be tested before incubation begins. The
explants were then incubated for 22-24 h and examined for abnormalities.
Embryos were fixed in Bouin's fluid. Whole mount preparations were made
and stained with anthracene blue (Mahoney, 1963). Embryos to be treated
histologically were embedded in paraffin wax, sectioned at 7 /a and stained with
haematoxylin and eosin.
RESULTS
The embryos were explanted at full-streak (FS), head process (HP), head-fold
(HF) and 1-2 somite (1-2S) stages corresponding to stages 4, 5, 6 and 7 of
Hamburger & Hamilton (1951).
PLATE 1
Fixed in Bouin and stained with anthracene blue, x 13.
4
Fig. A. 10~" M DNP. Explanted at full-streak stage. Normal development.
Fig. B. 5 x 10~4 M DNP. Explanted at full-streak stage. Opening in mid-brain region.
Fig. C. 10~3 M DNP. Explanted at full-streak stage. Brain tissue extensively damaged. Somites
almost completely absent.
Fig. D. 10~4 M DNP. Explanted at head-fold stage. Normal development.
Fig. E. 5 x 10~4 M DNP. Explanted at head-fold stage. Open fore- and mid-brains.
Fig. F. 10~3 M DNP. Explanted at head-fold stage. Brain and neural tube very degenerate.
Somites absent.
Fig. G. 10~4 M DNP. Explanted at one somite stage. Normal development.
Fig. H. 5 x 10~4 M DNP. Explanted at one somite stage. Almost normal development.
Fig. I. 10~3 M DNP. Explanted at one somite stage. Neural tissue and somites severely
damaged.
PLATE 1
/. Embryo!, exp. Morph., Vol. 17, Part 2
f
A
V-.
^
I!
P. BOWMAN
facing p- 426
J. Embryo/, exp. Morph., Vol. 17, Part 2
PLATE 2
D
Fixed in Bouin, sectioned at 7 /t and stained with haematoxylin and eosin.
Figs. A-D, x 62; Figs. E-F, x 106.
Fig. A. Control embryo. Explanted at head-fold stage, T.S. through heart region. Normal
development.
Fig. B. 10~3 M DNP. Explanted at head-fold stage, T.S. through heart region. Knob of necrotic
cells in region of neural tube.
Fig. C. Control embryo. Explanted at full-streak stage T.S. through somite region. Normal
development.
Fig. D. 10~3 M DNP. Explanted at full-streak stage, T.S. through somite region. Absence of
neural tissue and poor development of somites.
Fig. E. Control embryo. Explanted at head-fold stage, T.S. through fore-gut region. Normal
development.
Fig. F. 10~3 M DNP. Explanted at head-fold stage, T.S. through fore-gut region. Open neural
tube with necrotic cells.
p. BOWMAN
facing p. 427
DNP and embryo development
All
Table 1. Effect of 2,4-dinitrophenol on the development of the chick embryo
Av. no.
No. showing abnormalities in:
of
\ji
Concn. of
DNP
None
10-4M
Stage
explanted
No.
somite
treated pairs
Brain
Neural
tube Somites
Heart
Blood
islands
FS
HP
HF
1-2S
FS
26
19
13
7
3
15
17
20
22
14
—
1
—
—
1
—
—
—
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
HP
HF
1-2S
FS
5
2
2
16
19
21
15
2
—
—
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
HP
HF
1-2S
FS
1
5
2
32
18
18
21
8
1
3
—
14
—
2
—
6
—
1
—
7
HP
HF
1-2S
FS
HP
HF
1-2S
12
18
4
7
9
2
1
9
7
14
2
4
3
10
8
16
4
3
6
1
1
4
11
4
2
5
1
1
2
13
4
2
3
1
1
(18-4/tg/ml)
5 x 10-4 M
(92-0/tg/ml)
10"3M
(184/fg/ml)
2X10~3M
—
•
—
—
—
—
—
—
—
4
2
i
1
1
1
1
—
—
—
—
1
—
—
The development of untreated embryos subjected to the cold delay technique
closely approximates to that of embryos subjected to immediate culture (Billett
et al. 1965).
The concentrations of DNP and their effects can be seen in Table 1. Concentrations below 10~4 M had no effect on the embryos.
Macroscopic observations
After 24 h in vitro a number of effects were observed. The regions of the
embryo particularly affected by DNP were the brain, neural tube and somites.
Typical examples of affected embryos can be seen in Plate 1. Abnormalities of
the brain included open fore- and mid-brains with lower concentrations (Plate 1,
figs. B, E), and almost complete absence of brain tissue at higher concentrations
(Plate 1,fig.F). Neural-tube closure was inhibited at low concentrations but at
higher concentrations the neural tube was often absent (Plate, 1,fig.C). Somite
development was also affected, the somites being reduced in number and very
diffuse, or at the higher concentrations completely absent.
Heart development in almost all cases was normal, as was blood island forma27-3
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PATRICIA BOWMAN
tion. The outgrowth of the blastoderms was also unaffected at all the concentrations of DNP used.
A striking feature of DNP treatment is its apparent stage-independence in
producing anomalies of neural tissue and somites, all stages being affected at
the higher concentrations used.
Histological observations
Serial sections of control and treated embryos confirmed the macroscopic
observations (Plate 2, figs. A-F). There was extensive necrosis of brain tissue
and in some cases only a knob of necrotic cells was left (Plate 2, fig. B). The
neural tube was absent in many embryos, but the notochord was resistant to
DNP and persisted even when there was no neural tissue (Plate 2, fig. D).
The somites did not show such severe degeneration as the neural tissue but
their differentiation into dermatome, myotome and sclerotome was almost
always inhibited where the neural tube was very degenerate or absent. In these
cases all that could be seen were two masses of undifferentiated cells, but
necrotic cells were only rarely observed.
Head mesenchyme was well distributed and normal. The heart and extraembryonic mesoderm did not show any cytological changes and blood island
development was comparable to controls.
DISCUSSION
The main effects of DNP on the blastoderm are progressive degeneration of
neural tissue with increasing concentration and simultaneous inhibition of
somite formation. The development of mesodermal derivatives other than the
somites is generally unaffected by DNP, as is the notochord and outgrowth of
the blastoderm.
A number of other agents affect the development of neural tissue and somites
in explanted chick embryos. Insulin inhibits brain differentiation and neuraltube closure and frequently causes extensive necrosis of cells in the neural
tissue (Barron & McKenzie, 1962). Increasing concentrations of insulin affect
somite development, but there is no effect on the heart or notochord.
Aminopterin also inhibits closure of the neural tube, but the most persistent
effect is the failure of blood channel formation (O'Dell & McKenzie, 1963).
Chloroacetophenone, an —SH inhibitor, causes irregularities in brain and
neural-tube formation, but there is no necrosis (Lakshmi, 1962). Another
substance which inhibits neural tube closure is D-threo-chloramphenicol
(Billett et al. 1965). This agent affects closure in the posterior part of the neural
tube in contrast to the substances mentioned previously, where the anterior
part of the tube is particularly affected. D-threo-chloramphenicol also inhibits
blood island formation, its teratogenic action being attributed to an impairment
of protein synthesis.
DNP and embryo development
429
The effects produced by DNP most closely approximate to those produced by
nitrogen-mustard derivatives (Jurand, 1960), where there is extensive necrosis
of neural tissue and degeneration of somites, but not complete absence of
neural tissue as with DNP.
The effective concentrations of DNP which were used in the present experiments were very much greater than those which uncouple oxidative phosphorylation in isolated mitochondria (Racker, 1961) and also much greater than
those used by Reporter & Ebert (1965) on the chick embryo. These authors
found no abnormalities within the range 0-1-10-0 /*g/ml DNP. They also found
that other uncouplers (oligomycin, amytal, dicumarol and thyroxine) produced
some distortion of the nervous system, but the primary effect was on formation
of heart, which was completely absent.
If the DNP is acting as an uncoupler in the present experiments, then the
anomalies observed may be the result of depletion of ATP reserves in the affected
tissues, neural tissue being particularly sensitive. However the effective concentrations of DNP are rather high and it may in addition be inhibiting some
enzyme system. The chick embryo, and in particular brain tissue, has been
shown to be more dependent on the pentose phosphate pathway relative to the
citric acid cycle and glycolysis (Burt & Wenger, 1961; Newburgh, Buckingham
& Herrmann, 1962). This pathway may act as a source of reduced nicotine
adenine dinucleotide phosphate (NADPH) for synthetic processes and as a
source of pentose for RNA synthesis. Burt & Wenger (1961) found peaks of
activity of this pathway in the early chick brain which they interpreted as
alternating periods of proliferation and differentiation. If the DNP is interfering
with some point in this metabolic pathway this might explain the stage independence found in these experiments, DNP differing from most other inhibitors of
early chick development in this respect. However, without biochemical analyses
of the affected tissues, one cannot go any further in suggesting the mode of
action of dinitrophenol.
The effects produced by insulin and by DNP are in some respects similar,
both agents damaging neural tissue with extensive necrosis of cells, but DNP
having a more drastic effect which results in almost complete absence of neural
tissue.
SUMMARY
1. The effect of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, has been studied on the chick embryo developing in vitro.
2. 2,4-dinitrophenol in concentrations ranging between 10~4 M and 2 x 10~3 M
causes a syndrome of abnormalities, the main features of which are degeneration
and sometimes complete absence of neural tissue accompanied by reduction in
number of and inhibition of the somites. Heart, notochord and blood islands
are unaffected.
3. A comparison is drawn between the effects of 2,4-dinitrophenol and other
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PATRICIA BOWMAN
substances which affect the development of neural tissue and somites in explanted
chick embryos.
RESUME
Action du 2,4-dinitrophenol sur le developpement de jeunes
embryons de poulet
1. On a etudie l'action du 2,4-dinitrophenol, un inhibiteur de la phosphorylation oxydative, sur le developpement de l'embryon de poulet in vitro.
2. A des concentrations variant de 1 0 ~ 4 M a 2-10~ 3 M, le 2,4-dinitrophenol
provoque un syndrome d'anomalies dont les principaux caracteres sont la
degenerescence et parfois l'absence complete de tissu nerveux, accompagnees
d'une reduction en nombre et de l'inhibition des somites. Le cceur, la notochorde
et les ilots sanguins ne sont pas atteints.
3. On etablit une comparaison entre les effets du 2,4-dinitrophenol et
d'autres substances qui affectent le developpement du tissu neural et des
somites dans les embryons de poulet explant.es.
I wish to thank Dr F. S. Billett for helpful advice and for reading the manuscript. I also
wish to thank Professor D. R. Newth for providing me with facilities in his department.
I am grateful to Mr B. Hind for taking the photographs and to Miss A. Hornbruch for
technical assistance. I acknowledge the support of the Medical Research Council.
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