/ . Embryo!, exp. Morph. Vol. 21, 2, pp. 295-308, April 1969 2 9 5

Printed in Great Britain

RNA synthesis in the early embryogenesis of a fish(Misgurnus fossilis)

By C. A. KAFIANI,1 M. J. TIMOFEEVA,1 A. A. NEYFAKH,2

N. L. MELNIKOVA1 & J. A. RACHKUS1

From the Institute of Molecular Biology and Institute of DevelopmentalBiology of the Academy of Sciences of the U.S.S.R., Moscow

Synthesis of ribonucleic acids in early embryos has been extensively studiedduring recent years in a number of laboratories and has been shown to beginshortly after fertilization (Kafiani, Tatarskaya & Kanopkayte, 1958; Wilt, 1963;Brown & Littna, 1964; Decroly, Cape & Brachet, 1964; Glisin & Glisin, 1964;Kafiani & Timofeeva, 1964, 1965; Nemer & Infant, 1965). Early embryos ofXenopus (Brown & Gurdon, 1964; Brown & Littna, 1964, 1966) and of seaurchins (Wilt, 1963; Glisin & Glisin, 1964; Nemer & Infant, 1965) synthesizeup to gastrula stage predominantly or exclusively polydisperse RNA of a non-ribosomal nature usually referred to as DNA-like RNA (dRNA).

The occurrence of continuous dRNA synthesis in early embryogenesis is inapparent conflict with the periodicity of the 'morphogenetic function' of cellnuclei found by one of us (Neyfakh, 1959, 1961, 1964, 1965) in embryos of anumber of animal species. In fact, for a certain time after ferilization of eggswith a regulatory type of development, the nuclei remain morphogeneticallyinactive. Microsurgical (Harvey, 1940; Briggs & King, 1959), chemical (Gross& Cousineau, 1963; Lallier, 1963; Gross, Malkin & Moyer, 1964; Neyfakh,1965) or radiation-induced (Neyfakh, 1959, 1961; Neyfakh & Rott, 1958;Shapiro & Lander, 1960) enucleation of the egg does not visibly affect earlydevelopment up to the late blastula stage. After the initial period of inactivity,a period of 'morphogenetic activity' of the nuclei begins which assures theprocess of gastrulation. The occurrence of dRNA synthesis starting immediatelyor shortly after fertilization is hard to reconcile with the proposal of a directrelationship between the morphogenetic and the biochemical (dRNA-syn-thesizing) functions of cell nuclei. This work seeks to analyse this apparentcontraction, to further understanding of the molecular basis of earlyembryogenesis.

1 Authors' address: Institute of Molecular Biology, Academy of Sciences of USSR, VavilovStreet 32, Moscow B-312, USSR.

3 Author's address: Institute of Developmental Biology, Academy of Sciences of USSR,Vavilov Street 26, Moscow B-133, USSR.

296 C. A. KAFIANI & OTHERS

To approach this problem we used the method of quantitative evaluation ofRNA synthesis in embryos described previously (Kafiani & Timofeeva, 1964,1965). The relative rate of dRNA synthesis ('dRNA-synthesizing activity') wasestimated at different stages of development in normal embryos of loach(Misgumusfossilis) and in embryos partially or nearly wholly deprived of nucleargenetic material (haploid and 'anucleate' embryos obtained by inactivation ofone or both of the gametes with X-rays).

It was with loach embryos that morphogenetic function of nuclei was studiedfor the first time using the method of radiation-induced inactivation of nuclei;it was shown that the nuclear activity controlling the onset and the progress ofgastrulation begins at the mid-blastula stage (Neyfakh, 1959). RNA synthesiswas found to be sharply accelerated at about the same time (Kafiani &Timofeeva, 1964, 1965).

The present work shows that the activation of dRNA synthesis occurs notonly on a per embryo but also on a per cell base. The extent of activation dependson the quantity of genetic material in the nuclei. These facts confirm the occur-rence of the true activation, or the onset of genome transcription in the cell nucleiat the mid-blastula stage. On the other hand RNA synthesis during the precedingperiod of development (synchronous cleavage of the egg) exhibits peculiaritieswhich suggest that this 'early' synthesis of RNA is at least partly independentof the nuclei.

MATERIAL AND METHODS

Material. Mature eggs of loach were obtained 40 h after injecting the femaleswith 200 i.u. of Choriogonin (Gedeon Richter, Hungary). Eggs were fertilized,left to develop in tap water at 21 °C and staged as described previously (Neyfakh,1959).

X-ray irradiation. Eggs were irradiated with a dose of 40 kr, and sperm with80 kr using apparatus RUP-1 (190 kV, 15 mA, 5 kr/min).

Introduction of the label. Since egg membranes are poorly permeable to usualprecursors RNA synthesis was studied using [14C]carbonate. It was introducedin the eggs as described by Cohen (1954) and Flickinger (1954). Equal quantitiesof eggs at desired stages (from the same clutch of developing eggs) were incubatedin stoppered flasks in a slightly acid medium (pH 6-0-6-5) containing Na2

14CO3.In different series of experiments isotope concentration varied from 20 to40/^c/ml of final medium. Temperature (21 °C) and duration of incubation(60 or 90 min) were kept constant in each series. Penicillin and streptomycin(100 and 50 i.u./ml respectively) were added to prevent bacterial contamination.

Extraction and purification of RNA. Total RNA of embryos was extractedwith sodium dodecylsulphate (SDS) and phenol in the cold by a proceduresimilar to that used by Brown & Littna (1964). Embryos were quickly homo-genized in ice-cold tris-HCl buffer, 0-01 M (pH 6-5) containing 0-01 M-MgCl2,the homogenate made 1 % in respect to SDS and mixed for £-1 min at 10-15 °C.

RNA synthesis in a fish embryo 297The lysed material was deproteinized three times with water-saturated phenolat 3-5 °C. Nucleic acids were precipitated from the aqueous phase with ethanolafter addition of a small amount of potassium acetate.

For the determinations of RNA-synthesizing activity, the total nucleic acidprecipitate was dissolved in a small volume of cold water and made 2 M withsodium chloride. The precipitate of 'salt-insoluble RNA' (siRNA) formedovernight (—10 °C) contained ribosomal RNA (rRNA) and other kinds ofRNA (including dRNA) except transfer RNAs (tRNA). The latter, as well asDNA, polysaccharide and other contaminants, remained in the supernatant.The siRNA precipitate was freed of traces of these contaminants by three morereprecipitations and washings with 2 M-NaCl. The RNA preparations containedless than 0-3 % protein (Lowry, Rosebrough, Farr & Randall, 1951) and werefree of DNA. The label in siRNA preparations was 90-95 % sensitive to RNasetreatment. A lower proportion of counts (60-80 %) was, however, sensitive toRNase in siRNA from early cleavage eggs.

For sucrose gradient centrifugations, total RNA preparations were used. Toobtain these, initial crude nucleic acid preparations were treated for 30 min at37 °C with DNase (Worthington), 5/*g/ml, in 001 M tris-HCl buffer (pH 7-8)containing 0-0lM-MgCl2. DNase was then eliminated with three successivephenol-SDS deproteinizations, and total RNA precipitated with ethanol.

Sucrose-gradient centrifugation (Britten & Roberts, 1960). Two to 3 mg RNAdissolved in 0-5 ml of 0-1 M acetate buffer (pH 5) with 001 M - E D T A waslayered on a 5-20% sucrose gradient prepared in the same buffer. After 12 hcentrifugation at 24000 rev./min in the SW-25 rotor of the Spinco L ultra-centrifuge at 10-12 °C, the bottom of the tube was pierced and fractions collected.

Radioactivity measurements. After determining RNA content in aliquots orfractions from u.v.-absorption (at 260 m/£) RNA was precipitated with cold5 % trichloroacetic acid (TCA). The precipitate was collected on nitrocellulosefilters (RUFS, Czechoslovakia, pore diameter 0-9-1-2 fi), and washed with coldTCA and ethanol. Radioactivity was counted in a liquid-scintillation spectro-meter (Lie Belin, France) with a non-polar scintillation mixture: 0-4% 2,5-diphenyloxazole and 001 % l,4-bis-2-(5-phenyloxazolyl)benzene in toluene.

RESULTS

Figure 1, A and B, show typical sedimentation patterns of total RNApreparations from loach eggs at mid-gastrula stage, obtained after 14CO2

incorporation during 1 h (A) and 5 h (B). It is seen that the bulk of RNA (u.v.-absorption, solid line) forms three peaks, two of which represent the large andsmall components of rRNA (28 S and 18 S), while the third peak (4-5 S) corre-sponds to low molecular weight RNAs, mainly tRNA.

The distribution of labelled RNA (dotted line) is quite different and revealsthe existence of a poly disperse population of newly formed RNA molecules

298 C. A. KAFIANI & OTHERS

sedimenting along the entire gradient with a few distinct peaks. The first peak,in the upper portion of the gradient, contains labelled RNAs of low molecularweight including transfer RNA. Since the latter rapidly turn over their terminalnucleotides, the incorporation of the label in this region of the gradient probablydoes not wholly correspond to the synthesis of RNA. However, a significantpart of the labelled RNA in this peak sediments faster than u.v.-absorbingmaterial. This suggests that this broad peak includes some other labelled RNAspecies besides tRNA. This is confirmed by the sedimentation pattern of asiRNA preparation obtained at the same developmental stage (Fig. 2). Here,

10

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20 30

Fraction no.40

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20 30

Fraction no.40

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Fig. 1. Sucrose-gradient centrifugation patterns of total RNA isolated from loacheggs at the mid-gastrula stage after incubation with [14C]carbonate (24 /tc/ml) forl h (A) and 5h (B). , u.v.-absorption (at 260 m/t); , TCA-precipitableradioactivity.

RNA synthesis in a fish embryo 299the 4-5 S peak of u.v.-absorption is lacking due to elimination of tRNA, whilethere is a significant amount of labelled RNA of somewhat higher molecularweight.

Most of the radioactive RNA sediments move rapidly, the 'heaviest' fractionsbeing found in greater proportions at shorter durations of 14CO2 incorporation(cf. Figs. 1A and B). But even after prolonged incorporation, the distributionof the label does not correspond to that of rRNA. This is true also for earlierstages of development (Timofeeva & Kafiani, 1965,1966).

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Fig. 2. Sucrose-gradient centrifugation pattern of a 'salt-insoluble' RNA prepara-tion from loach eggs at the mid-gastrula stage after 2 h incubation with [14C]-carbonate (20 jtic/ml).

From the sedimentation behaviour of labelled RNAs of early loach embryoswe conclude that, up to mid-gastrula stage, rRNA is not synthesized to anysignificant degree. Consequently siRNA preparations freed of tRNA containthe label predominantly, if not exclusively, in polydisperse RNA, or dRNA,which may include messenger RNA. A typical sedimentation pattern of ansiRNA sample shown in Fig. 2 illustrates the character of the RNA preparationsused throughout the present work.

In order to study the dynamics of transcription during early development inloach, we followed variations in the rate of dRNA synthesis at cleavage andblastula stages, up to the onset of gastrulation. Figure 3 summarizes the resultsof three separate experiments showing the peculiar course and reproducibilityof the curves obtained. In each experiment equal quantities of eggs from thesame batch were pulsed at different stages with 14CO2 under identical conditions,then siRNA isolated from each egg sample and specific radioactivity determined

20 J E E M 21

300 C. A. KAFIANI & OTHERS

as described in Methods. In Fig. 3 (as well as in Figs. 4 and 5) the values obtainedare plotted against hours of development, the latter representing mid-pointsbetween the beginning and the end of incubation with [14C]carbonate.

£ 5

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Fig. 3. Incorporation of 14CO2 into siRNA of loach eggs at different stages ofdevelopment. The curves illustrate the results of three separate experiments inwhich equal quantities of eggs were incubated at stages indicated under followingconditions: (1) 45 min., 20/*c/ml; (2) 60min., 40/iC/ml; (3) 60min., 20/tc/ml.

Since incorporation conditions were similar for all developmental stages,since siRNA content (2 /tg per egg) remains constant over the period studied(Timofeeva & Kafiani, 1964), and since, finally, the rate of RNA labelling inconditions defined is not limited by 14CO2 incorporation into free nucleotidesof developing eggs (Kafiani & Timofeeva, 1964, 1965) we consider observedchanges in specific radioactivities of siRNA to be a measure of variations in therate of RNA synthesis on a per embryo basis. The capacity of an embryo toincorporate a given quantity of the label into siRNA per unit time (e.g. 1 h) istherefore designated as'dRNA-synthesizing activity'.

The curves in Fig. 3 have a clear-cut break at about 6 h of development. Upto this time dRNA-synthesizing activity remains very low, increasing abruptlywithin a narrow time interval between the 6th and 7th h. It was conceivablethat the increase in the over-all rate of dRNA synthesis in the embryos was dueto a corresponding increase in the number of nuclei rather than to their activation.

RNA synthesis in a fish embryo 301Figure 4 shows, however, that the rates of increase of dRNA synthesis and ofcell number are quite different. In fact, from the 3rd until the 6th h, whencleavage proceeds in a synchronous manner with a 30 min period (Neyfakh &Rott, 1958; Rott & Sheveleva, 1967) the cell number increases about 30-foldwhile dRNA-synthesizing activity increases only twofold. After the 6th h therate of dRNA synthesis increases much faster than the cell number. As a result

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Fig. 4. Changes in dRNA-synthesizing activity and in cell number in early develop-ment of loach. (1) dRNA-synthesizing activity of the embryos (specific radioactivityof siRNA at the end of 60 min pulses with 14CO2, 20 /*c/ml); (2) number of cells perembryo (from Rott & Sheveleva, 1967); (3) dRNA-synthesizing activity calculatedper cell.

the dRNA-synthesizing activity calculated per cell (see curve 3, Fig. 4) shows,at about the 7th h, a marked rise indicating a real activation of dRNA synthesis.

The left-hand, descending part of curve 3 of Fig. 4 requires special considera-tion, as it implies that RNA-synthesizing activity is very high at the time ofmorphogenetic inactivity of cell nuclei. This would seem paradoxical if thenuclei are supposed to be the sole site for RNA synthesis. This paradox mightbe resolved, however, if the 'early' RNA synthesis were partly or entirely inde-pendent of the cell nuclei.

We attempted to clarify this point by measuring the rate of RNA synthesisin embryos with a reduced quantity of nuclear genetic material. The dRNA-synthesizing activities were compared in diploid embryos, gynogenetic haploids(obtained by fertilizing normal eggs with X-ray-irradiated sperm) and 'anucle-ate' embryos (obtained either by combining X-ray-irradiated gametes or byirradiating the zygotes). Morphologically, haploid embryos develop normally

302 C. A. KAFIANI & OTHERS

at least till late gastrula, the 'haploid syndrome' appearing considerably later.In 'anucleate' embryos cleavage still occurs, resulting in a blastula-like mass ofcells virtually devoid of chromosomes (Shapiro & Lander, 1960).

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4 5 6 7 8 9Hours of development at 21 °C

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Fig. 5. Incorporation of 14CO2 into siRNA of diploid (2/?), gynogenetic haploid (1/;)and 'anucleate' (On) embryos. The latter were obtained either by irradiating zygotesor by combining irradiated gametes. The eggs were given 90 min pulses of [14C]-carbonate (20 /*c/ml) at stages indicated.

Figure 5 and Table 1 show that until 5-6 h diploid and haploid embryosincorporate 14CO2 into siRNA at nearly the same rate while at 6-8 h diploidembryos incorporate label into siRNA about twice as fast as do haploid ones.This difference is not due to the smaller number of cells in the latter, as theycontain at this time even more cells than do diploid embryos (Rott & Sheveleva,1967). This higher rate of cell multiplication probably accounts for the com-pensation in the over-all production of dRNA shown by the reapproachmentof the In and In curves in Fig. 5 by the beginning of gastrulation.

Appearance of a direct dependence of RNA-synthesizing activity of embryoson their ploidy testifies to the onset of nucleus-directed RNA synthesis. Thelack of such correlation over the first 6 h of development suggests that RNAsynthesis is independent of the nuclei at this time. The sites for the ' early' RNA

RNA synthesis in a fish embryo 303synthesis might reside in the cytoplasm, which would account for the decreaseof the left-hand part of curve 3 in Fig. 4 as a result of division of egg cytoplasminto progressively smaller units.

This proposal is supported by experiments with 'anucleate' embryos. Thedata summarized in Fig. 5 and Table 1 show a quite definite incorporation of14CO3 into siRNA in such embryos. Early RNA synthesis rises gradually,increasing fivefold by 10 h, but fails to display the activation characteristic ofnucleated embryos. Despite the low level of incorporation most part of itappears to reflect actual RNA synthesis since it is 80 % sensitive to RNase incontrol and 60 % sensitive in anucleate embryos.

Table 1. dRNA synthesis in diploid, haploid and'anucleate' embryos

(At stages indicated, 6 ml portions of eggs were incubated with 3 ml of 0-01 Mphosphate buffer (pH 6-0-6*5) containing Na2

14CO3 (30 /*c/ml), penicillin (100 i.u./ml) and streptomycin (50 i.u./ml) for 1 -5 h as indicated in column 1. Radioactivity wascounted in aliquots of siRNA preparations (see Methods) before and after treatingthem with 'guanylic' RNase from actinomycetes. Specific radioactivities were calcu-lated by dividing RNase-sensitive cold TCA-insoluble counts by the quantity ofsiRNA present initially in aliquots.)

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87127

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obtained by irradiating:

Both gametes Zygote

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In order to get some information on the nature of the early incorporationinto siRNA we tested its sensitivity to actinomycin D. The antibiotic could beintroduced in the eggs only if applied at high concentrations (up to 100/ig/ml)immediately after fertilization (at the time of swelling). Cleavage occurred insuch eggs but development was arrested at late blastula stage. 14CO2 incorpora-tion into the RNase-sensitive portion of siRNA preparations isolated from theeggs at 6 h of development after 3 h incubation with the isotope was found tobe inhibited with actinomycin by about 50% both in normal (diploid) and'anucleate' embryos, when compared to respective untreated controls. Thissuggests that at least a part of the 14CO2 incorporation into the siRNA of normalearly cleavage eggs represents DNA-dependent RNA synthesis. Furthermorethe result supports the proposal about the cytoplasmic location of early RNA

304 C. A. KAFIANI & OTHERS

synthesis since a comparable RNase and actinomycin-sensitive incorporationcan be demonstrated in embryos virtually devoid of nuclear genetic material.The results obtained with actinomycin D should, however, be interpreted withcaution because of uncertainty of the actual concentration of the antibiotic inthe cells and its possible indirect effects.

DISCUSSION

The present paper gives some characteristics of RNA synthesis in earlyembryogenesis of a teleost fish (Misgurnus fossilis).

Sucrose-gradient analysis showed that synthesis of ribosomal RNAs doesnot occur to any significant extent at least until mid-gastrula stage, and theembryos synthesize predominantly or exclusively polydisperse RNAs of a non-ribosomal nature designated as dRNA. Selective inhibition of expression ofribosomal cistrons seems to be a general phenomenon in early embryonic devel-opment since it has been demonstrated also in pre-gastrula stages of Xenopuslaevis (Brown & Gurdon, 1964; Brown & Littna, 1964, 1966) and sea urchins(Glisin & Glisin, 1964; Nemer & Infant, 1965; Guidice & Mutolo, 1967).

A quantitative approach (Kafiani & Timofeeva, 1964) was applied here forevaluating changes in the activity of the embryonic genome in transcription ofgenetic information during pregastrular development. It consists of comparingspecific radioactivities of RNA of the eggs after pulsing them, at different stages,under standard conditions, with a precursor (14CO2) which is rapidly incor-porated into the free nucleotide pool. In view of the non-specific nature of theprecursor and possible terminal incorporation into tRNA, thoroughly purifiedRNA preparations were used freed of tRNA and other contaminants byrepeated precipitations with sodium chloride. Such RNA preparations containedrRNA and dRNA of which only the latter was labelled. Specific radioactivitiesof the RNA preparations corresponded to the approximate rate of dRNAsynthesis and were considered a measure of 'dRNA-synthesizing activity' ofthe embryos.

dRNA-synthesizing activity changes strikingly during the early developmentof loach. It remains very low for the first 6 h of cleavage and increases abruptlyat 6-7 h of development (mid-blastula), suggesting operation of a triggermechanism. The large extent of the activation suggests, furthermore, that thetriggering involves a large part of the cell population.

The sharp activation of nuclear RNA synthesis in loach at the mid-blastulastage has been shown previously (Kafiani & Timofeeva, 1964, 1965). In thepresent paper additional evidence is given that this activation really involvesnuclear genetical material: (a) the increase in dRNA-synthesizing activity calcu-lated per cell, and (b) the appearance of a correlation between the rate of dRNAsynthesis and ploidy of embryos.

Similar activation of RNA synthesis has been found in Rana pipiens

RNA synthesis in a fish embryo 305(Bachvarova, Davidson, Allfrey & Mirsky, 1966) and in X. laevis (Brown &Littna, 1966) at the late blastula stage, which correlates with the onset of morpho-genetic activity (Neyfakh, 1964). In starfish, transcription of templates sup-porting gastrulation is reported to begin at early mid-blastula stage (Barros,Hand & Monroy, 1966). An abrupt activation of RNA synthesis was found inmouse embryos at the morula-early blastocyst stage (Monesi & Salfi, 1967).Stimulation of genome transcription appears therefore to be a characteristicevent in early embryogenesis.

In loach, the activation of RNA synthesis coincides with important cytologicalchanges: the beginning of fusion of caryomeres (Pankova, 1963), and a dropin the mitotic index and lengthening of mitotic cycle due to lengthening ofinterphase (Rott & Sheveleva, 1967). Since RNA synthesis occurs mainly duringinterphase our data are in accord with the cytological observations.

By the 7th h of development an important change in the developmentalpotency of loach embryos occurs, the blastoderms becoming able to differentiatein artificial media (Kostomarova & Neyfakh, 1964).

Finally, at the 6th h of development morphogenetic function of the cellnuclei starts (Neyfakh, 1959). Concomitant activation of dRNA synthesis inthe nuclei is unlikely to be a coincidence and suggests that the beginning ofactive gene transcription forms a biochemical basis for nuclear function indirecting gastrulation.

The correlation between the dRNA-synthesizing and morphogenetic activityof the nuclei seems, however, to be confined to the time of their commencement.Morphogenetic activity exhibits periodicity, the first period lasting for 2-^h(from 6 to 8^ h) followed by an 'interruption of morphogenetic function' whichlasts until the 14th h. This is not so, however, with RNA synthesis, which goeson with increasing speed after being switched in at the 6th h. The reasons forthe discrepancy between biological and biochemical function remain obscure.

As for the first period of morphogenetic inactivity of the nuclei (up to 6 hpostfertilization), the observed limited RNA synthesis seems to be at least partlynuclear-independent. This view is supported by the fact that the rate of RNAsynthesis in early cleavage eggs correlates neither with cell number nor withthe ploidy of embryos. The 'early' RNA synthesis is presumed to occur in somecytoplasmic DNA-containing structures. Such a possibility has been suggestedfor anucleate halves of sea-urchin eggs (Baltus, Quertier, Ficq & Brachet, 1965).Loach-egg cytoplasm contains sufficient DNA to support measurable RNAsynthesis, since as shown previously the DNA content of an egg is 6-10 x 10~3 figwhile that of a sperm cell is 2-4 x 10~6/*g, the 'extra' DNA corresponding tothe nuclear DNA of about 1000 diploid cells (Timofeeva & Kafiani, 1964). Wehave, however, no direct data concerning the exact localization of nuclear-independent 'early' RNA synthesis nor are we able to answer the questionwhether cell nuclei are totally inactive during early cleavage.

In conclusion, the data presented show that there are two clearly distin-

306 C. A. KAFIANI & OTHERS

guishable functional states of cell nuclei during early embryonic developmentof loach. The nuclei are virtually inactive in transcription of genetic informationduring early cleavage, and at this time they are also inactive morphogenetically.At the mid-blastula stage the nuclei progress into another state characterizedby a high rate of gene transcription and morphogenetic activity.

SUMMARY

1. RNA synthesis during early embryogenesis of loach (Misgurnus fossilis)was studied using 14CO2 incorporation into the 2 M-NaCl-precipitated fractionof total embryonic RNA.

2. From fertilization until mid-gastrula stage the RNA preparations containlabel in non-ribosomal RNAs (dRNA) which have a high degree of sizeheterogeneity.

3. A quantitative method was used for evaluating relative dRNA-synthesizingactivities of embryos at different stages of development by measuring specificradioactivities of NaCl-precipitated RNA preparations isolated from eggspulsed with 14CO2 under standard conditions.

4. The dRNA-synthesizing activity is very low for early (synchronous) cleavagestages and increases slowly compared to cell division. At this time the rate ofRNA synthesis is nearly equal in diploid and haploid embryos. A detectableRNA synthesis occurs also in 'anucleate' embryos. It is concluded tentativelythat at least a part of the 'early' RNA synthesis occurs independently of thecell nuclei, in some DNA-containing cytoplasmic structures.

5. At the mid-blastula stage, dRNA synthesis is sharply accelerated perembryo as well as per cell, the rate of synthesis becoming directly dependent oncell ploidy. It is concluded that after an initial period of cleavage an activation,or onset of gene transcription, occurs in the cell nuclei which may be the basisof their morphogenetic function.

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of RNA synthesis associated with gastrulation. Proc. natn. Acad. Sci. U.S.A. 55, 358-65.BALTUS, E., QUERTIER, J., FICQ, A. & BRACHET, J. (1965). Biochemical studies of nucleate

and anucleate fragments isolated from sea-urchin eggs. A comparison between fertilizationand parthenogenetic activation. Biochim. biophys. Ada 95, 408-17.

BARROS, C, HAND, G. S. & MONROY, A. (1966). Control of gastrulation in the starfish,Asterias forbesii. Expl Cell Res. 43, 167-82.

BRIGGS, R. & KING, T. J. (1959). Nucleocytoplasmic interactions in eggs and embryos. InThe Cell (ed. J. Brachet and A. Mirsky), vol. i, pp. 537-618. New York and London:Academic Press.

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(Manuscript received 14 September 1967, revised 23 August 1968)