('ell Dif/>rentiation, 12 (1983) 115-120 115 Elsevier Scientific Publishers Ireland, Ltd.

The effect of transcription inhibitors on early development of the avian embryo

Bozenna Olszanska ~ and Barbara Kludkiewicz 2

~ Institute of Genetics and Animal Breeding of the Polish Academv of Sciences, Jastrzebiec, 05-551," and -" Institute q[ Biochemistry and Biop]~vsics of the Polish Academy of Science~, Wars=awa ul. Rakowiecka 36, 02-532, Poland

(Accepted 15 June 1982)

The effect of transcription [actinomycin D, 5,6-dichloro-l-fl-D-ribofuranosylbenzimidazole (DRB), a~amanitin] and translation inhibitors (cycloheximide, puromycin) on quail embryo development was investigated under in vitro conditions. The gastrulation process seemed to proceed normally in the presence of transcription inhibitors in the medium but the translation inhibitors stopped development and caused complete degeneration of the embryos.

quail embryo in vitro development a-amanitin

1. Introduct ion

Studies on the effect of transcription inhibitors upon the development of echinoderm and amphibian embryos have led to the conclusion that oocytes of these animals are furnished with maternal mRNA allowing embryogenesis to pro- ceed till gastrulation (Brachet and Malpoix, 1971: Davidson, 1976). Development of the mammalian embryo, however, has been shown to depend on de novo mRNA synthesis already after the 2 4 cell stage (Golbus et al., 1973; Manes, 1973). This difference may reflect the existence of close con- tacts of the embryo with its maternal organism.

If this assumption is true, the avian embryo which stays within the oviduct during cleavage and gastrulation but becomes separated early from the maternal organism by egg envelopes would be provided with a mRNA supply, resembling in this respect the echinoderm and amphibian embryo rather than the mammalian one.

The data concerning nucleic acid metabolism during early avian development are very scarce in contrast to detailed characterization of the mor- phological events occurring at this period. The

formation of the primitive streak is an easily ob- servable sign of the gastrulation process. We have, therefore, started to investigate this problem in quail embryos by following the morphological re- sponse to the continuous presence in the culture medium of some transcription and translation in- hibitors applied before, during or after formation of the primitive streak.

The results presented below support the hy- pothesis that mRNA stored in the avian egg meets early developmental demands including those of the gastrulation process.

2. Mater ia l s and M e t h o d s

Blastoderms of Japanese quail (Coturnix cotur- nix japonica) were obtained from eggs: a) taken out from the females about 10-15h before egg laying (uterine eggs), b) freshly laid, unincubated, c) incubated at 37.5°C for 19 h. The blastoderms were isolated and thereafter cultured in vitro on egg albumen at 37.5°C as described earlier (Olszanska and Lassota, 1980). In some cases the culture medium was supplemented with either a)

0045-6039,/83/0000-0000/$03.00 ,:, 1983 Elsevier Scientific Publishers Ireland, Ltd.

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actinomycin D, 0.05 /.tg/tnl (from Calbiochem Ltd., La Jolla, CA); 5,6-dichloro-l-,8-D-ribo- furanosylbenzimidazole (DRB), 40 ~g /ml (pro- vided by Dr. Z. Kazimierczuk, Laboratory of Bio- physics, Warsaw University, Poland); c~-amanitin (Sigma Chemical Co., St. Louis, MO), 20 p.g//ml; cycloheximide 10 5 M, i.e. 2.8 ,ug/ml (from Calbiochem AG); or b) puromycin 5 X 10 5 M, i.e. 23 / ,g /ml (from Serva, Heidelberg, F.R.G.), respectively. The blastoderms from uterine eggs were cultured in vitro for 10h in order not to overlap the post-laying period and the other ones for 19h to permit primitive streak formation. Thereafter, the blastoderms were washed with 0.9% NaCI under a binocular and the stages of develop- ment were determined according to Hamburger- Hamilton's (HH) tables (Hamburger and Hamil- ton, 1951). The effect of o~-amanitin on RNA synthesis was tested in blastoderms cultured on medium containing, along with the drug, 5',6'[-~H]uridine (spec. act. 46 Ci /mmol: The Ra- diochemical Centre, Amersham, U.K.) in the amount of 20/xCi/ml.

From the labelled blastoderms total RNA was isolated by hot phenol-SDS extraction at pH 5 (Olszanska et al., 1974) and digested twice with RNase-free DNase, added each time in the amount of 10/~g/ml (Knowland, 1970).

Polyadenylated RNA was separated by affinity chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The radioactivity of RNA samples was measured in a Beckman scintillation counter with PPO-POPOP- to luene scintillant.

3. Results and Discussion

In blastoderms from uterine eggs the primitive streak (PS) did not start forming during the 10 h of culture. After 19 h of in vitro culture, the embryos from the freshly laid eggs reached stages 3-4, undergoing gastrulation as indicated by the forma- tion of the primitive streak (Fig. la). In embryos from the incubated eggs the formation of the PS was already Well advanced in ovo and during 19 h of in vitro culture these embryos reached stages 6 10 (Fig. lg).

During in vitro culture 25-30% of control

blastoderms degenerated (Table l). The morpho- logical differences between the normally devel- oping embryos and the degenerating ones were clearly visible already before PS formation, since a dense, irregular clump arose usually in the centre of the latter. Degeneration during PS formation was manifested similarly and any traces of PS structure were absent (Fig. lb). Degeneration after PS formation resulted in a strong constriction or even disappearance of area opaca and pellucida, formation of dense clumpy structures at the border of these areas, fusion or absence of somites, defor- mation of head structures and strong constriction of the whole embryo.

The presence of transcription and translation inhibitors in culture medium reduced the per- centage of normally developing embryo (Table I).

The morphological symptoms of degeneration induced by transcription inhibitors were similar to those observed in degenerating control embryos of the same age. Actinomycin D in concentration known to inhibit rRNA synthesis (Penman et al., 1968; Perry and Kelley, 1970) caused degeneration of all blastoderms when applied after PS forma- tion, while about one-third of embryos treated during PS formation continued to develop and formed a normal primitive streak (Fig. lc). Degen- eration of all embryos tested during PS formation occurred when the concentration of actinomycin D in the medium was increased to 0.08 i~g/ml resulting in a more general inhibition of RNA synthesis (Fig. ld).

The lower sensitivity of embryos during PS formation than after PS formation was also evi- dent when another drug, DRB, reported to inhibit preferentially mRNA synthesis (Sehgall et al., 1976), was present in the culture medium at these two periods.

a-Amanitin is known to act in vitro as a specific inhibitor of nucleoplasmic RNA polymerase I1 responsible for mRNA synthesis (Jacob et al., 1970); and the embryos treated after PS formation were more susceptible to the drug than during this process (see Table I and Fig. le, ~a). The respon- siveness to o~-amanitin of blastoderms tested be- fore PS formation was also higher than during PS formation (all 12 tested blastoderms from uterine eggs degenerated in the presence of o~-amanitin

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Fig. 1. Quail embryos after 19 h of in vitro culture on egg albumen supplemented with inhibitors of transcription and translation. Embryos from freshly laid eggs cultured on: a) standard medium, normal embryo; b) standard medium, embryo degenerated: c) med ium+ac t inomyc in D (0.05 ~g /ml ) : d) med ium+ac t inomyc in D (0.08 p.g/ml): e) m e d i u m + a - a m a n i t i n (20 btg/ml); f) medium + puromycin (2.8/~g/ml). Embryos from incubated eggs (19 h, 37.5°C) cultured on: g) standard medium, normal embryo; h) m e d i u m + a-amanit in (20 #g /ml ) ; i) medium+cycloheximide (2.8 ,ag/ml) arrested at stage 3: j) medium + cycloheximide (2.8/~g/ml) degenerated.

while 24 out of 32 controls developed normally). In embryos from freshly laid eggs the primitive streak formed in vitro even when the concentra- tion of a-amanitin in the medium amounted to 50 /~g/ml. The penetration rate of a-amanitin into embryos seems to influence its effect since prein- cubation at suboptimal temperature (20-22°C) for 2h with 20 /~g/ml of this drug resulted in a degeneration of 100% of embryos treated after PS formation. However, similar preincubation with c~-amanitin of embryos during PS formation did

not intensify the effect of inhibitor. The apparent resistance of the embryos during PS formation did not concern translation inhibitors. In the presence of cycloheximide all tested embryos were affected: the embryos during PS formation degenerated as described above and those treated after PS forma- tion either degenerated or ceased to develop re- maining at stage 3-5 (Fig. l i, j). In embryos arrested at stage3, the posterior part of the PS disappeared (Fig. li), revealing injury due to the inhibitor. Puromycin caused degeneration in 60%

TABEE I

Influence of RNA and protein synthesis inhibitors on the development of quail embryos in vitro

Inhibitor Concentration used in medium

( # g / m l )

Embryos grown in vitro in the presence of inhibitor

During PS formation After PS formation

No. tested % Normal No. tested % Normal

None 0 340 70 460 75 Actinomycin D 0.05 66 35 30 0 DRB 40 52 65 83 38 a-Amanitin 20 95 62 151 12 Cycloheximide 2.8 47 0 79 0 "

(10 5 M)

Puromycin 23.5 67 0 ~ 47 8 (5X 10 ~ M)

" Development of 52% of embryos arrested at stage 3 5. b In 39% of embryos the primitive streak formed, but was abnormal.

of embryos during PS formation and in the re- maining 40% the development was affected since primitive streak was swollen and abnormally dense in the region of Hensen's node (Fig. lf).

In the embryos treated with puromycin after PS formation, degeneration symptoms appeared in more than 90% of the tested individuals.

These results indicate that undisturbed protein synthesis is essential for normal embryonal devel- opment during and after PS formation. By con- trast, embryos tested during PS formation seem to withstand the temporary inhibition of mRNA synthesis. This may be interpreted in terms of a pre-existing mRNA stock supplying the process of gastrulation, but probably synthesized during cleavage or blastulation rather than in the oocyte, since the uterine eggs did degenerate in the pres- ence of a-amanitin. However, it cannot be pre- cluded that during gastrulation the sites of tran- scription become inaccessible to inhibitors. In order to check this possibility the effects of ~- amanitin, which were the most evident morpho- logically, were tested at the molecular level. The results (Table II) testify against the above assump- tion. The amount of polyadenylated and total RNA synthesized during, as well as after, PS for- mation in c~-amanitin-treated embryos was invaria- bly lower than in untreated controls, indicating that the drug penetrated into embryonic cells. The inhibitory effect of c~-amanitin in vivo is known to concern the synthesis of mRNA as well as that of

119

rRNA (Niessing et al., 1970; Tata et al., 1972; Hadjiolov et al., 1974). It is interesting that inhibi- tion of poly(A+) RNA synthesis due to c~-amani- tin occurred in both abnormal and apparently normal embryos. The inhibition should probably reach some quantitative a n d / o r qualitative threshold to result in morphological abnormalities. Up to the end of gastrulation this threshold may be high owing to the pre-existing stock of mRNA.

The possibility that RNA polymerase responsi- ble for mRNA synthesis during gastrulation is resistant to c~-amanitin remains open. Such poly- merases were reported to occur in Eucaryota (Wulf and Bautz, 1976; Johnson and Preston, 1980), however; their switching on and off with the start and the end of the gastrulation process seems rather improbable.

In conclusion, by comparison to the situation in echinoderm and amphibia, we suppose that mRNA synthesized during oogenesis is not sufficient to carry out cleavage and blastulation, but that this mRNA together with mRNA synthesized during the latter stages permits gastrulation to proceed normally. After gastrulation de novo synthesis be- comes again necessary.

Acknowledgements

Our thanks are due to Dr. T. Rogulska, In- stitute of Zoology of the Warsaw University, Po-

TABLE I1

RNA synthesis in quail embryos grown 19 h in vitro in the presence of c~-amanitin (20 ~tg/ml of medium)

Stages covered during in vitro development (HH)

Treatment [3H]Uridine label incorporated ~ (cpm/embryo)

Total RNA Poly(A + ) RNA

Normal embryos Degenerated Normal embryos embryos

Degenerated embryos

I - 4 None 46,015 35,953 957 Amanit in 44,567 25,930 741

4 -10 None 118,836 75,269 2,913 Amanit in 88,760 45,014 1,796

662 359

991 634

" Results of two to three experiments comprising l0 20 embryos each.

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land, for critical reading of the manuscript. This work was supported by the Polish Academy

of Sciences within the project 09.'7.1.5.

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