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Work performed by Université Libre de Bruxelles

u n d e r the E u r a t o m contract N o . 007­61­10 BIAB

Reprinted from

N A T U R E

Vol.

198,

N o . 4876 ­ 1963

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E U R I 5 6 . e

R E P R I N T

E F F E C T S ON ACTINOMYCIN D ON M O R P H O G E N E S I S by (in alphabetical order) J. B R Ä C H E T and H. D E N I S (Université Libre de Bruxelles). Association EURATOM ­ U.L.B.

(European Atomic Energy Community and Université Libre de Bruxelles). Work performed by Université Libre de Bruxelles under the Euratom contract No. 007­01­10 BIAB.

Reprinted from „ N a t u r e " , Vol. 198, No. 4876 ­ 1963, pages 20S­206. Actinomycin Β is a strong and specific inhibitor of the enzyme ,,DNA­primed RNA polymerase". This enzyme is held responsible for the production of ,,messenger" RNA's by the genes.

The possibility t h a t „messenger" RNA's might play an important role in morphogenesis had been discussed before by one of t h e authors (J. Brächet. J. Cell. Compar. Physiol. Suppl. 1 to Vol. 60, 1­18, 1962). The possible effects of actinomycin D on developing embryos (amphibian and chick) and regenera­ ting unicellular algae (Acetabularia) have therefore been studied.

E U R 1 5 6 . e

R E P R I N T

E F F E C T S ON ACTINOMYCIN D OX M O R P H O G E N E S I S by (in alphabetical order) J. B R Ä C H E T and H. D E N I S (Université Libre de Bruxelles). Association EURATOM ­ U.L.B.

(European Atomic Energy Community and Université Libre de Bruxelles). Work performed by Université Libre de Bruxelles under the Euratom contract No. 007­01­10 BIAB.

Reprinted from „ N a t u r e " , Vol. 198, No. 4876 ­ 1963, pages 205­206. Actinomycin D is a strong and specific inhibitor of the enzyme ,,DNA­primed RNA polymerase". This enzyme is held responsible for the production of „messenger" RNA's by the genes.

The possibility t h a t „messenger" RNA's might play an important role in morphogenesis had been discussed before bv one of the authors (J. Brächet. J. Cell. Compar. Physiol. Suppl. 1 to Vol. 60, 1­18, 1962). The possible effects of actinomycin D on developing embryos (amphibian and chick) and regenera­ ting unicellular algae (Acetabularia) have therefore been studied.

E U R 1 5 6 . e

R E P R I N T

E F F E C T S ON ACTINOMYCIN D ON M O R P H O G E N E S I S bv (in alphabetical ordei) J. B R Ä C H E T and H. D E N I S (Université Libre de Bruxelles). Association EURATOM ­ U.L.B.

(European Atomic Energy Community and Université Libre de Bruxelles). Werk performed by Université Libre de Bruxelles under the Euratom contract No. 007­61­10 BIAB.

Reprinted from „ N a t u r e " , Vol. 198, No. 4876 ­ 1963, pages 205­206. Actinomycin D is a strong and specific inhibitor of the enzyme „DXA­primed RNA polymerase". This enzyme is held responsible for the production of „messenger" RNA's by the genes.

I t was found t h a t actinomycin D very strongly inhibits the differentiation of the nervous system, especially the brain : strongly microcephalic embryos can easily be obtained in this way. Cytological studies indicate t h a t actino­ mycin D obliterates the cephalo-caudal gradient of RNA synthesis in t h e gastrula and neurula stages.

In Acetabularia, actinomycin D completely inhibits the regeneration of nucleate halves, b u t not t h a t of anucleate halves.

These results strongly suggest t h a t the production of a messenger — like RNA by the nucleus really — is a very important factor in morphogenesis in plants as well as in animals.

I t was found t h a t actinomycin D very strongly inhibits the differentiation of the nervous system, especially the brain : strongly microcephalic embryos can easily be obtained in this way. Cytological studies indicate t h a t actino­ mycin D obliterates the cephalo-caudal gradient of RNA synthesis in the gastrula and neurula stages.

In Acetabularia, actinomycin D completely inhibits the regeneration of nucleate halves, but not t h a t of anucleate halves.

These results strongly suggest t h a t the production of a messenger — like RNA by the nucleus really — is a very important factor in morphogenesis in plants as well as in animals.

I t was found t h a t actinomycin D very strongly inhibits the differentiation of the nervous system, especially the brain : strongly microcephalic embryos can easily be obtained in this way. Cytological studies indicate t h a t actino­ mycin D obliterates the cephalo-caudal gradient of RNA svnthesis in t h e gastrula and neurula stages.

In Acetabularia, actinomycin D completely inhibits the regeneration of nucleate halves, but not t h a t of anucleate halves.

[Reprinted from Nature, Vol. 198, No. 4876, pp. 205­206,

April 13, 1963)

Effects of Actinomycin D on Morphogenesis

T H E possible role in morphogenesis of ribonucleic acids (RNA) produced by the cell nuclei (presumably 'messenger' RNA's) has been discussed recently by one of us1. I n short, it was suggested that, in a relatively simple system such as the regenerating unicellular alga Acetabularia, the 'morphogenetic substances' of Hämmerling2 could be equated to messenger (informational) RNA ; however, these substances might as well be proteins, the synthesis of which is controlled by a specific RNA of nuclear origin. The situation is obviously much more complex in the case of embryonic differentiation ; we have suggested1 that, in amphibian eggs, synthesis of messenger R N A does not begin before gastrulation. At t h a t stage of development, the pre­existing polarity gradient in the distribution of ribosomal R N A would become activated b y messenger RNA's produced by the nuclei : as a result, R N A and protein synthesis would proceed further along the well­ known cephalocaudal and dorsoventral morphogenetic gradients.

Since actinomycin D strongly and selectively inhibits the production of nuclear R N A by combining with the DNA template3'4, it was of interest to test the possible effects of this antibiotic on morphogenesis in both Acetabu­ laria and developing eggs. This communication sum­ marizes the results so far obtained.

(1) Acetabularia mediterranea. Since it is known5'8 t h a t the production of the 'morphogenetic substances' still proceeds in the dark during 1­2 weeks and since actinomycin solutions are fairly sensitive to light, the following experimental conditions were adopted : nucleate and anucleate fragments of large algae almost ready for cap formation, as well as young whole algae (10—15 m m long), were placed in actinomycin (20­1 μg/ml.) containing sea­water for 8—10 days in the dark. By t h a t time, growth and whorls formation had stopped in the controls (dark­ ness) as well as in the actinomycin­treated algae. The algae, which looked exactly alike, were then placed in the light (actinomycin being renewed every 5 days) and cultivated during 2­3 weeks.

Fig. 1. Pleuroäeles eggs cultivated for 4 days. Left, actinomycin Ώ 10 μβ/πιΐ. ; right, control

in the controls. The results of these experiments are practically identical with those obtained in this laboratory by de Vitry' when she treated Acetabularia halves with fluorodeoxyuridine. This analogue has now been found to inhibit strongly the transfer of nuclear RNA to the cyto­ plasm of the algae (de Vitry, unpublished). While the results of our experiments stand in agreement with the hypothesis stated here, the reasons for the unexpected inhibition of growth in the caps formed by the anucleate fragments in the presence of actinomycin remain to be studied.

(2) Embryonic development. Most of the experiments have been performed on amphibian eggs (Pleuroäeles and

Xenopus), using concentrations of actinomycin ranging between 10 and 1 μg/ml. (in the dark). I n both species, cleavage is completely unaffected ; t h a t this negative result is not due to lack of penetration of actinomycin was shown b y experiments in which t h e antibiotic was micro­ injected in fertilized eggs : again, cleavage proceeded normally. Cleavage of fertilized sea urchin eggs also remained unimpaired b y t h e addition of actinomycin. I n

Xenopus, t r e a t m e n t of the eggs with actinomycin a t t h e highest concentrations (10 μg/ml.) used always produced exogastrulation, while gastrulation was usually normal in

low. Chorda and somites are generally present, b u t they are very poorly differentiated. At lower concentrations (5­2­5 μg/ml.), the nervous system is present ; b u t microcephaly, microphthalmy and even anophthalmy are the rule. The cephalocaudal RNA gradient is no longer recognizable cytochemically ; it can even be reversed. The development and basophilia of the nucleoli are highly variable. Many nuclei undergo pycnotic degeneration.

Preliminary experiments on explanted chick embryos have given results which are in substantial agreement with those obtained with the amphibians : again, actinomycin strongly inhibits the differentiation of the nervous system, especially t h a t of the brain.

These results stand in excellent agreement with the hypotheses which have been presented earlier in this communication : they suggest t h a t the production of nuclear (presumably messenger) RNA must be of little importance until cleavage ends ; inhibition of its synthesis with actinomycin results in the abolishment or consider­ able reduction of cephalocaudal and dorsoventral differen­ tiation. But extensive biochemical studies are obviously required before the RNA fraction, the synthesis of which is sensitive to actinomycin, can be definitely identified as messenger RNA.

We t h a n k Misses V. Pohl, J. Quertier and E . Van Stevens for help in the experiments, Messrs. Merck, Sharpe and Dohme for a generous gift of actinomycin and E u r a t o m (contract 016­61­10 ABIB) for financial help.

J . BRÄCHET H . D E N I S (Charge do Recherches au F.N.R.S.) Laboratoire de Morphologie animale,

Université libre de Bruxelles.

1 Brächet, J., J. Cell. Comp. Physiol., 60, Suppl. 1,1 (1962).

* Hãmmerling, J., Roux Ârch. Enlicsmech. Organ., 131,1 (1934).

3 lì eich, E . , Goldberg, I. H., and Rabinowitz, M., Sature, 186, 743 (1962). 4 Shatkin, A. J., Bioch. Biophys. Acta, 61, 310 (1962).

* Brächet, J., Chantrenne, Η., and Vanderhaeghe, F., Biochim. Biophys. Acta, 18, 544(1955).

« Hãmmerling, J . , and Hãmmerling, Cli., Planta, 52, 516 (1959). ' de Vitry, F . , Exp. Cell. Res., 25, 697 (1962).

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