Wilhelm Roux' Archly 160, 237--242 (1968)

Effect of Chloropicrin on the Early Development of Chick Embryo*

SA~JIVA~I S. JosI~I Department of Zoology, University of Poona

Received February 1, 1967

Summary. The effects on chick embryos of treatment with the sulfhydryl inhibibor-chloropicrin have been investigated. Abnormalities of the central nervous system are described. Its action on neural induction is also discussed. All the results are compared with those obtained by other workers using inhibitory treat- ments on amphibian and chick embryos.

Zusammen/assung. Die Folgen der Behandlung von Hiihnchen-Embryonen mit den Snlfhydryl-Inhibita-Chloripikrin wurden untersucht. Abnormalit/iten des Zen- tralnervensystems wurden beschrieben. Die Wirkung des Inhibitars auf neurale Induktion wird diskutiert. Die l~esultate wurden verglichen mit Ergchnissen, die yon anderen Autoren unter Verwendung von lnhibitoren in der Amphibien- and Hfihnchenentwicklung erzielt wurden.

Introduction

B~ACH~'s (1950) strong emphasis on tile role of -SIt containing proteins in the process of induction has stimulated the Study of reaction of embryonic organism to the chemical substances like chloropicrin, which is one of the main approaches to the analysis of development. Chloropicrin has been described by earlier workers like BACQ (1941) and I~A~KI~ and BI~ACttET (1951) as an -SH inhibitor in amphibia. In the case of amphibian eggs, the effect on morphogenesis of a number of "classical" sulfhydryl reagents have been studied by a number of authors (BRAckET, 1950; BEATTY, 1949, 1951; DEVCHE1% 1957; BARTIt, 1956).

LAKS~MI (1962) working in this laboratory using chloroacetophenone has shown that there was a marked decrease in inducing capacity of node region of chick primitive streak if -SH groups were blocked. She interpreted this by explaining the possible role played by thiols in induction. Further it was shown by DIWA~ (1964) that colchicine re- sembles chloroacetophenone in its action in chick embryo. Thus the im- portance of sulfhydryl groups in living systems is increasingly recognised.

In view of this growing realisation of the importance of -StI groups it was felt desirable to study the effect of chloropicrin (B,D.H. product) on the process of morphogenesis and induction in chick embryo.

* Diese Arbeit wurde noch yon Herrn Professor I~O~EIS angenommen.

238 S.S. Jos~I:

Material and Methods Fresh, fertilised eggs of White Leghorn obtained from a local source, were

incubated to obtain the definitive primitive streak stages. Sterile precautions were observed throughout the experiments. A stock solution of 1.10 -2 M (0.01 M) Chloropicrin was prepared in fifty percent ethyl alcohol. This solution was further diluted with Pannett Compton (PC) saline to the desired strengths.

Experiment 1: Effect of Chloropicrin on chick embryos cultured in-vitro. The embryos from the incubated eggs were explanted in.vitro by NEw's tech-

nique (1955). After few preliminary experiments, a concentration of 5.10-r (0.0005 M) chloropicrin was found to be suitable. For normal (untreated) controls, fifty percent alcohol was similarly diluted with PC saline for use in place of chloro- picrin solution. Fifty embryos at the definitive streak stage were treated with chloropicrin solution and thin albumen was added around the ring. Thirty embryos were kept as controls. A second set of controls (20 embryos) was kept in order to ensure that controls treated with Mcohol-PC saline, lived normally and the alcohol percentage in control solution was therefore negligible. All the embryos were grown for 20--21 hours. They were then examined and sketched. After fixation in Bouin's fixative, they were further processed for histological study.

Experiment 2 : Effect of Chloropicri~ on the inducing capacity of Hensen's node. Some of the explanted chick embryos at the definitive streak stage were

subjected to 7.10 -a M (0.0007 M) Chloropicrin for the duration of fiteen minutes. The nodes of these donor embryos were then cut out and washed in PC saline to ensure removal of the chemical.

The host embryos at the definitive streak stage were explanted in-vitro accord- ing to the method of ~Ew (1955). The nodes of treated donor embryos were grafted into these untreated host embryos by WADDI~GTO~'S grafting technique (1952). In control experiments, the donors were treated with PC saline instead of Chloro- picrin and the grafting was performed in the same way as referred above.

In all, 35 experimental, 30 control and 15 absolute control grafts were made. All the embryos were incubated for 18--20 hours after grafting. They were then fixed in Bouin's fixative, sectioned at 8 ~ and stained in Delafield's haematoxylin for histo]ogicM examination.

Experimental results

Experiment 1: E//ect o/ Chloropicrin on chicle embryos cultured in-vitro

Chloropicrin t rea ted embryos showed a wide range of abnormali t ies , main ly of the bra in and neural tube. The abnormali t ies shown by the surviving embryos were recorded and their analysis is given in Table 1.

I n the embryo i l lustrated in Fig. 1 a, the bra in is cylindrical and open throughout . The neural folds are wavy and apar t from each other a t certain places. The closure of the neural tube thus seems to have been affected by chloropicrin t rea tment . There are 6 - -7 pairs of somites and some of them are par t ia l ly lying vent ra l to the neural folds. Hear t , fore-gut and notochord are comi)aratively normal.

I n m a n y cases, the axis of the embryo is affected to a larger extent (shortened). For comparison with the exper imental embryo, one control embryo is i l lustrated (Fig. 2).

Chloropicrin on Chick Embryos 239

Experiment 2: E/[ect o/Chloropicrin on the inducing capacity o/ Hensen ' s node

Chlorop ic r in seems to h a v e a t e l l ing ef fec t on t h e i nduc ing c a p a c i t y

of t h e node . I t is seen f r o m Table 2, t h a t t h e p e r c e n t a g e of i n d u c t i o n

�9 : : . , ~ : ,

( "!f~ �9 . : . .

Fig. 1 a. ~Iacroscopic view of chick embryo treated in-vitro with 5.10 -~ M ehloropicrin, • 24

~'~,~;. .~ .~ " . ~ . ~ " ~ . ..... .~;,.;+~- .,~:" ..:... y . ,:i"," . g :~ ' , ~

, ,~,~, =::, :,1,~:~::~:4.:,:~ . . . . . :::~-~ ,:-!.'-?~.: '~;~:: ,~ .~,~,,M~:~'~'..,:6~5~,,:~.i:'"" % "''":~=~"..'~.~-" ~o ' . .~ "

Fig. I b. Transverse section through the mid-brain of embryo in I a, showing open brain, and absence of gut cavity

has fa l l en a f t e r ch lo rop ic r in t r e a t m e n t a n d t he r e also exis ts a qual i -

t a t i v e change .

The various induction cases were classified into two main categories,

namely strong inductions and moderate inductions. In the case

of strong induction the graft induced a neural tube of considerable

240 S.S. Josm:

Table 1. Analysis o/mal/ormations shown by chloropicrin treated chick embryos

Organs showing malformations Percentage of abnormalities

Fore brain 75 % Posterior portion of brain 79% Neural tube, behind brain 60%

Optic vesicles: a)Both absent 21% b) Both present and abnormal 65%

Heart: a) Vitelline veins not fused 15% b) Vitelline veins fused 42%

Notochord 10 %

Somites: a) Abnormal 39 % b) Not formed 16%

Subnormal length of embryonic axis 72%

dimensions; wilhe in the case of moderate induc t ion the graft caused the format ion of only a medul la ry plate or thick palisade. I n some cases, the graft had differentiated into neural tube bu t i t did no t cause any neural isa t ion of the overlying ectoderm. There were also

few cases, where the grafts had not differentiated and had not caused induc t ion though the cells had

Table 2. Analysis o/the induction capacity o/ Hensen' s node after chloropicrin treatment

Concentration of cMoropicrin 7.10 -4 M No. of grafts made 35 No. of grafts taken for analysis 30 % of strong induction 53 % % of moderate induction 22 % Absence of induction 15% Over all % of induction 75 % Over all % of induction by corresponding 95%

control grafts

i#. .:$?.~ ~.: ~:.: :'..:% ~ ~ : i ~i'"::. :~;:~

=~2.-:i . :i~U ' ~:i .,~'

remained heal thy. Here, the loss of inducing capaci ty may not be t aken as due to the non-dif ferent ia t ion of the graft.

Fig. 2. Macroscopic view of control chick embryo for the experimental embryo in I a, • 24

Chloropicrin on Chick Embryos 241

The experimental cases were also studied from the point of view of differentiation of the graft, which seems to have been affected by Chloropicrin treatment.

Thus the non-differentiation of the graits and the loss of inducing capacity they incur seems to be the consequence of ehloropierin treatment.

Discussion

In the case of amphibian eggs, a number of workers (BI~AC~IET, 1950 ; BEATTY, 1949, 1951; BARTH, 1956; D~UCnER, 1957; TE~ CATE, 1957) have obtained inhibition of nervous system formation by removing -SH groups through oxidation with monoiodoacetic acid, monoiodoacetamide, chloroacetophenone, oxidized gluthatione, alloxane etc. All these agents produce similar effects. Similar observations were recorded by BACQ (1941) in amphibia using chloropicrin. The embryos showed very defective nervous systems (thick, open plate), while the differentia- tion of adjacent chorda and somites was relatively normal. Thus the results of the present investigation find support from the work of BACQ (1941).

The extremely low percentage of any mesoderm abnormalities, noted in the present results, was a feature of RAP~:I~]~ and BRACKET'S findings (1951) in Rana embryos which had been treated with -SH inhibitors like iodoacetamide and chloropicrin.

I t was further shown by BAcQ (1941), that chloropicrin shows a great affinity for -SH groups. I t also rapidly stops mitosis in the eggs of amphibia and as in sea-urchin egg; there is a tendency for blasto- meres to separate. The fact that chioropicrin and iodoacetamide produce rapid blocking of development, leads one to suspect that -SH groups are indispensible for the development.

Chloropicrin like chloroacetophenone (LA~s~I , 1962b), and colchi- cine (DIwA?r 1966) affects the activity of the organizer by reacting with suifhydryl groups of proteins, thus rendering them unable to give the inducing stimulus. This assumption is further supported by the fact that Hensen's node loses much of its inducing capacity as a result of treatment with colchieine (DIwA~, 1966) and chloroacetophenone (LAKs~3II, 1962b). Further, it has also been shown that the sulfhydryl content of IIensen's node decreases considerably after t reatment with chloroacetophenone (LA~:S~MI and M~YLI~E~J~AI~, 1963).

The results of the present experiments in chick confirm these earlier findings in amphibia and chick thus supporting BnAc~T ' s hypothesis assigning an important role to -SH groups in morphogenesis and em- bryonic induction.

242 S.S. J o s m : Chloropicrin on Chick Embryos

Acknowledgements. The author wishes to thank Prof. LEELA IV[ULtIERKAt~, Head of the Zoology Department, for providing necessary facilities. Grateful thanks are due to the Indian Council of MedicM Research, for awarding a post- doctoral fellowship.

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Dr. SANJIVANI S. J o s m Department of Zoology University of Poona Poona 7, Indien