Brachet, J.

Denis, H.

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Work performed by Université Libre de Bruxelles 
under the Eura tom contract No. 007­61­10 BIAB 
Reprinted from 
NATURE 
Vol. 198, No. 4876 ­ 1963 m !i,fo A I fo -iiii 
Kiii 
Atomic Energy Community (EURATOM). 
under the sponsorship of the Commission of the European 
completeness, or usefulness of the information contained in this document, or that the use 
of any information, apparatus, method, or process disclosed in this document may not 
infringe privately owned rights; or 
Assume any liability with respect to the use of, or for damages resulting from the use of 
any information, apparatus, method or process disclosed in this document. 
α 
nfeaÈ! 
print is intended for restricted distribution only. If reproduces, 
by kind permission of the publisher, an article from „NATURE", 
Vol. 198, No. 4876 ­ 1963, 205­206. For further copies please apply 
to Fisher, Knight et Co. Ltd., Gainsborough Press, Lattimore Road ­
St. Albans, Hertfordshire ­ England. 
Dieser Sonderdruck ist für eine beschränkte Verteilung bestimmt. Die 
Wiedergabe des vorliegenden in „NATURE", Vol. 198, Nr. 4876 ­
1963, 205­206. erschienenen Aufsatzes erfolgt mit freundlicher Geneh­
migung des Herausgebers. Bestellungen weiterer Exemplare sind an 
Fisher, Knight et Co. Ltd., Gainsborough Press, Lattimore Road ­
St. Albans, Hertfordshire ­ England, zu richten. 
restreinte. Il Ce tiré­à­part est exclusivement destiné à une diffusion restreint 
reprend, avec l'aimable autorisation de l'éditeur, un article publié dans 
«NATURE», Vol. 198, N° 4876 ­ 1963, 205­206. Tout autre exemplaire 
Esso è stato riprodotto, per gentile concessione dell'Editore, da 
a NATURE», Voi. 198, N° 4876 ­ 1963, 205­206. Ulteriori copie 
dell'articolo debbono essere richieste a Fisher, Knight et Co. Ltd., Gains­
borough Press, Lattimore Road ­ St. Albans, Hertfordshire ­ England, 
Deze overdruk is slechts voor beperkte verspreiding bestemd. Het artikel 
is met welwillende toestemming van de uitgever overgenomen uit 
„NATURE", Vol. 198, no. 4876 ­ 1963, 205­206. Meer exemplaren 
kunnen besteld worden bij Fisher, Knight et Co. Ltd., Gainsborough 
Press, Lattimore Road ­ St. Albans, Hertfordshire ­ England. 
Dl 
wmmkÊÊOÊamÊm 
E U R I 5 6 . e 
R E P R I N T 
EFFECTS ON ACTINOMYCIN D ON MORPHOGENESIS by (in alphabetical 
order) J. BRÄCHET and H. DENIS (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 „Nature", 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 that „messenger" RNA's might play an important role in 
morphogenesis had been discussed before by 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. 
EUR 1 5 6 . e 
R E P R I N T 
EFFECTS ON ACTINOMYCIN D OX MORPHOGENESIS by (in alphabetical 
order) J. BRÄCHET and H. DENIS (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 „Nature", 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 that „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. 
EUR 1 5 6 . e 
R E P R I N T 
EFFECTS ON ACTINOMYCIN D ON MORPHOGENESIS bv (in alphabetical 
ordei) J. BRÄCHET and H. DENIS (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 „Nature", 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. 
The possibility that „messenger" RNA's might play an important role in 
morphogenesis had been discussed before by 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. 
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 tha 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, bu 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 tha 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 ha 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 tha 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 tha t actino­
mycin D obliterates the cephalo-caudal gradient of RNA svnthesis in the 
gastrula and neurula stages. 
In Acetabularia, actinomycin D completely inhibits the regeneration of 
nucleate halves, but not t ha t of anucleate halves. 
These results strongly suggest t ha t the production of a messenger — like 
RNA by the nucleus really — is a very important factor in morphogenesis in 
plants as λνβΐΐ as in animals. 
[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. In 
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 RNA does not 
begin before gastrulation. At that stage of development, 
the pre­existing polarity gradient in the distribution of 
ribosomal RNA would become activated by messenger 
RNA's produced by the nuclei : as a result, RNA 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 RNA 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 
tha 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 mm 
long), were placed in actinomycin (20­1 μg/ml.) containing 
sea­water for 8—10 days in the dark. By that 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. 
Three independent experiments have shown that actino­
mycin inhibits growth and morphogenesis much more 
strongly in the nucleate algae than in the others. This 
effect is more easily obtained at the highest concentration 
used (20­10 μg/ml.) than at the lowest ones. In anucleate 
halves, cap formation is usually slightly delayed ; but in 
the experiments made at the 10 μg/ml. level, all anucleate 
fragments formed caps which grew at a slower pace than 
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 s tated 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). In both species, 
cleavage is completely unaffected ; t ha t this negative 
result is not due to lack of penetration of actinomycin was 
shown by experiments in which the antibiotic was micro­
injected in fertilized eggs : again, cleavage proceeded 
normally. Cleavage of fertilized sea urchin eggs also 
remained unimpaired by the addition of actinomycin. In 
Xenopus, t reatment of the eggs with actinomycin a t the 
highest concentrations (10 μg/ml.) used always produced 
exogastrulation, while gastrulation was usually normal in 
Pleuroäeles. If, during gastrulation, the vitelline mem­
brane oí Pleuroäeles embryos is removed, very remarkable 
results can be obtained in the presence of actinomycin : 
at 10 μg/ml., neural induction quickly stops, although the 
embryos survive for several days and elongate somewhat. 
The nervous system is practically absent, even in sections, 
in 80 per cent of the cases (Fig. 1). The head can scarcely 
be recognized from the tail and its basophilia is abnormally 
low. Chorda and somites are generally present, but they 
are very poorly differentiated. At lower concentrations 
(5­2­5 μg/ml.), the nervous system is present ; but 
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 that of the brain. 
These results stand in excellent agreement with the 
hypotheses which have been presented earlier in this 
communication : they suggest that 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 thank 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 
Euratom (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). 
Primed in Great Britain by Fisher, Knight & Co., Ltd., St. Albans. 

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EFFECTS OF ACTINOMYCIN D ON MORPHOGENESIS. EUR 156.e

f«OTT» ¡f-4 gnu ■ra UR I S * REPRINT m BW.. Ï'W . S !lî*t|l>WlVl rhFriSRi Hfl] Mi ; t*tt ASSOCIATION EURATOM ­ U.L.B. (European Atomic Energy Community and Université Libre de Bruxelles) m •"Vi >m 'äw Wi m SS: SK* » f f l »Λ!; !¡íí> •FFECTS OF ACTINOMYCIN D ι ΐ ' ί ν (Université Libre de Bruxelles) 4r *'l!'^Jii«!il£!ìùL3cnX$i!Ki!i! < 'ur i !*i*¡í fUft­r ; · : HI uWfr.JU.'Jf. 'iìM'i fôfc œs t"«Hr;;^3.­!i ».mü tua pHi HKl« ¡M ' . f i Sí &*'<<! l'tlthb LÌ 1 " ' · · ' · ' . ' l'Hall m fina i 1963 ü li ι*, ii i * * j titt« W: m m lia* íjli}" M IjiSI íiAfciW! m mm 'm Frl i! ifMií [¡tå IP Work performed by Université Libre de Bruxelles under the Eura tom contract No. 007­61­10 BIAB Reprinted from NATURE Vol. 198, No. 4876 ­ 1963 m !i,fo A I fo -iiii Kiii Atomic Energy Community (EURATOM). under the sponsorship of the Commission of the European completeness, or usefulness of the information contained in this document, or that the use of any information, apparatus, method, or process disclosed in this document may not infringe privately owned rights; or Assume any liability with respect to the use of, or for damages resulting from the use of any information, apparatus, method or process disclosed in this document. α nfeaÈ! print is intended for restricted distribution only. If reproduces, by kind permission of the publisher, an article from „NATURE", Vol. 198, No. 4876 ­ 1963, 205­206. For further copies please apply to Fisher, Knight et Co. Ltd., Gainsborough Press, Lattimore Road ­St. Albans, Hertfordshire ­ England. Dieser Sonderdruck ist für eine beschränkte Verteilung bestimmt. Die Wiedergabe des vorliegenden in „NATURE", Vol. 198, Nr. 4876 ­1963, 205­206. erschienenen Aufsatzes erfolgt mit freundlicher Geneh­migung des Herausgebers. Bestellungen weiterer Exemplare sind an Fisher, Knight et Co. Ltd., Gainsborough Press, Lattimore Road ­St. Albans, Hertfordshire ­ England, zu richten. restreinte. Il Ce tiré­à­part est exclusivement destiné à une diffusion restreint reprend, avec l'aimable autorisation de l'éditeur, un article publié dans «NATURE», Vol. 198, N° 4876 ­ 1963, 205­206. Tout autre exemplaire Esso è stato riprodotto, per gentile concessione dell'Editore, da a NATURE», Voi. 198, N° 4876 ­ 1963, 205­206. Ulteriori copie dell'articolo debbono essere richieste a Fisher, Knight et Co. Ltd., Gains­borough Press, Lattimore Road ­ St. Albans, Hertfordshire ­ England, Deze overdruk is slechts voor beperkte verspreiding bestemd. Het artikel is met welwillende toestemming van de uitgever overgenomen uit „NATURE", Vol. 198, no. 4876 ­ 1963, 205­206. Meer exemplaren kunnen besteld worden bij Fisher, Knight et Co. Ltd., Gainsborough Press, Lattimore Road ­ St. Albans, Hertfordshire ­ England. Dl wmmkÊÊOÊamÊm E U R I 5 6 . e R E P R I N T EFFECTS ON ACTINOMYCIN D ON MORPHOGENESIS by (in alphabetical order) J. BRÄCHET and H. DENIS (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 „Nature", 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 that „messenger" RNA's might play an important role in morphogenesis had been discussed before by 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 regenera­ting unicellular algae (Acetabularia) have therefore been studied. EUR 1 5 6 . e R E P R I N T EFFECTS ON ACTINOMYCIN D OX MORPHOGENESIS by (in alphabetical order) J. BRÄCHET and H. DENIS (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 „Nature", 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 that „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 regenera­ting unicellular algae (Acetabularia) have therefore been studied. EUR 1 5 6 . e R E P R I N T EFFECTS ON ACTINOMYCIN D ON MORPHOGENESIS bv (in alphabetical ordei) J. BRÄCHET and H. DENIS (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 „Nature", 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. The possibility that „messenger" RNA's might play an important role in morphogenesis had been discussed before by 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 regenera­ting unicellular algae (Acetabularia) "have therefore been studied. 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 tha t actino­mycin 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, bu 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 tha t actino­mycin 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 ha 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 tha 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 tha t actino­mycin D obliterates the cephalo-caudal gradient of RNA svnthesis in the gastrula and neurula stages. In Acetabularia, actinomycin D completely inhibits the regeneration of nucleate halves, but not t ha t of anucleate halves. These results strongly suggest t ha t the production of a messenger — like RNA by the nucleus really — is a very important factor in morphogenesis in plants as λνβΐΐ as in animals. [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. In 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 RNA does not begin before gastrulation. At that stage of development, the pre­existing polarity gradient in the distribution of ribosomal RNA would become activated by messenger RNA's produced by the nuclei : as a result, RNA and protein synthesis would proceed further along the well­known cephalocaudal and dorsoventral morphogenetic gradients. Since actinomycin D strongly and selectively inhibits the production of nuclear RNA by combining with the DNA template3 '4, it was of interest to test the possible effects of this antibiotic on morphogenesis in both Acetabu­laria and developing eggs. This communication sum­marizes the results so far obtained. (1) Acetabularia mediterranea. Since it is known5 '8 tha 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 mm long), were placed in actinomycin (20­1 μg/ml.) containing sea­water for 8—10 days in the dark. By that time, growth and whorls formation had stopped in the controls (dark­ness) 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. Three independent experiments have shown that actino­mycin inhibits growth and morphogenesis much more strongly in the nucleate algae than in the others. This effect is more easily obtained at the highest concentration used (20­10 μg/ml.) than at the lowest ones. In anucleate halves, cap formation is usually slightly delayed ; but in the experiments made at the 10 μg/ml. level, all anucleate fragments formed caps which grew at a slower pace than 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 cyto­plasm of the algae (de Vitry, unpublished). While the results of our experiments stand in agreement with the hypothesis s tated 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). In both species, cleavage is completely unaffected ; t ha t this negative result is not due to lack of penetration of actinomycin was shown by experiments in which the antibiotic was micro­injected in fertilized eggs : again, cleavage proceeded normally. Cleavage of fertilized sea urchin eggs also remained unimpaired by the addition of actinomycin. In Xenopus, t reatment of the eggs with actinomycin a t the highest concentrations (10 μg/ml.) used always produced exogastrulation, while gastrulation was usually normal in Pleuroäeles. If, during gastrulation, the vitelline mem­brane oí Pleuroäeles embryos is removed, very remarkable results can be obtained in the presence of actinomycin : at 10 μg/ml., neural induction quickly stops, although the embryos survive for several days and elongate somewhat. The nervous system is practically absent, even in sections, in 80 per cent of the cases (Fig. 1). The head can scarcely be recognized from the tail and its basophilia is abnormally low. Chorda and somites are generally present, but they are very poorly differentiated. At lower concentrations (5­2­5 μg/ml.), the nervous system is present ; but 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 that of the brain. These results stand in excellent agreement with the hypotheses which have been presented earlier in this communication : they suggest that 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 consider­able reduction of cephalocaudal and dorsoventral differen­tiation. 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 thank 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 Euratom (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). Primed in Great Britain by Fisher, Knight & Co., Ltd., St. Albans. 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