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Stable RNAs and embryonicdifferentiation in AmphibaFiorenza De Bernardi aa Istituto di Zoologia , Università Statale di Milano , ViaCeloria 10, 20133, Milano, ItalyPublished online: 14 Sep 2009.

To cite this article: Fiorenza De Bernardi (1980) Stable RNAs and embryonic differentiation inAmphiba, Bolletino di zoologia, 47:3-4, 267-279, DOI: 10.1080/11250008009438685

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Boll. ZOO^. 47: 267-279 (1980)

Stable RNAs and embryonic differentiation in Amphibia

FIORENZA DE BERNARD1 Istituto di Zoologia, Universiti Statale di hlilano Via Celoria 10, 20133 hlilano (Italy)

ABSTRACT

Recent data on the structure and stability of mRNA in various cell lines are examined for the presence in the mRNA molecule of the polp

,adenylate segment and its possible role in main- taining high translational efficiency. Data on the synthesis and storage of maternal mRNAs in amphibian oocytes and their utilization during early embryonic development are also examined.

I n post-gastrula stages the lack of homo- geneity in the embryo leads to a separate study of the dorsal region (where differentiation is earlier). I n the dorsal regions treated with actino- mycin, active protein synthesis can be observed, clearly sustained by stable mRNAs, synthesized between gastrulation and the raising of the neural folds. An attempt to recognize some of these mRNAs led to the identification of the 26 S mRNA, codifying for ’the heavy chain of the myosin. By hybridization with ’H-poly(U), this mRNA is found to be already present in the dorsal region at the early gastrula stage.

I n bacteria the rate of protein synthesis for any specific protein is controlled by an mRNA codifying for this protein. The presence of this mRNA is determined, in turn, by the decision to promote or to inhibit the transcription of the gene cor- responding to a particular mRNA. The mechanism of mRNA production is direct, i.e. the primary transcript is the mRNA.

On the contrary, in the eukaryotes the primary transcript is not a translatable mRNA: the 5’ and 3’ ends must undergo post-transcriptional modifications requiring respectively the addition of the “cap” site and of the polyadenylated sequence. Mo- reover, this complex “processing” mecha- nism is further complicated by endonu- clear breakages of the primary transcripr, by dimensional reductions and folding of RNA (Fig. 1). At each step, several re- gulations and controls can be established. These phenomena can be collected into two main groups: transcriptional and post- transcriptional controls.

The selective amplification, about a thousandfold of ribosomal RNA genes in the oocyte at the pachytene stage (Gall, 1968), provides an example of transcrip- tional control. Observation of this me- chanism led us to try, with this approach, a general explanation of cellular differen- tiation. Much research has tried to find some amplified structural genes in those cellular systems from which a great quan- tity of purified mRNAs could be extract- ed. The best known are the cases of he- moglobin genes (Bishop et al., 1972; Gilmour et al., 1974), ovalbumin genes (Sullivan et al., 1973), and silk fibroin genes (Suzuki et al., 1972). The specia-‘ lized genes were found in one or a few copies per haploid genome, in DNA of both specialized cells and other cell types. No evidence was found for a selective gene amplification of the specific DNA sequences. On the other hand the cal- culation made by Kafatos (1972) for the cocoonase of silkmoths showed that the accumulation of proteins specific for a

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268 F. DE BERNARD1

I DNA t

I r U” Ip 3’ 5, C A P S P I SP CI

U” -xPolvlAl

1 I

CAP SP I sp cr ---- un

U A A A l U l l A A U A G

CAP 1. cr rn’G&ZGTrn

Fig. 1. - Diagram of 3 possible model of the “processing” of mRNA in the nucleus. sp = “spacer” region; 1 = ‘‘leader’’ region; cr = codi- fying region; un = untranslated region. (Modified from Darnell, 1979.)

cellular type may be explained by the available data on the transcriptional and translationa1 efficiency rate and mRNA turnover, even if the gene is present in DNA in a single copy. Nevertheless, when the relative synthesis rate of a protein becomes particularly high, an unusually long life and stability of its mRNA may be supposed. In these approaches cellular differentiation was proposed as coming from a differential gene activity. This theory found direct evidence in the reas- sociation kinetics experiments between mRNA and cDNA; mRNA from various cell-types of the same organism shows many qualitative differences, especially in the cytoplasmic form.

By measuring the content of mRNA codifying for proteins of specialized cells and the synthetic rate of these proteins, it was revealed that an increase in syn- thesis of a protein is preceded by an in- crease in synthesis of the corresponding mRNA; this fact was clearly verified for hemoglobin (Gilmour et al., 1974), oval- bumin (Palmiter, 1973), and cocoonase (Kafatos, 1972). The structural genes containing the information for the specia- lized proteins are activated when the cells are differentiating or when they are in- duced to differentiate by hormonal stimu- lus; previously the specific mRNA level was very low (Palmiter, 1973). I t seems

then that from a variation of genomic function the cellular differentiation may be understood which should be subjected to a transcriptional control.

The stabilization of mRNA has a par- ticular interest for cellular differentiation! i t is a kind of “translational amplification” allowing the production of a great num- ber of copies of the same protein mole- cule on a limited number of mRNA temp- lates. Moreover a stable mRNA, codifying for a protein typical of differentiated state, may continue to be translated even if other mRNAs for housekeeping proteins are consumed, thus leading to the terminal differentiation.

The functional stability of the mRNA seems to be related to some sequence added during the processing such as the “ cap ” or the polyadenylated segment. Most eukaryotic mRNAs, viral mRNAs and nuclear pre-mRNAs contain at the 5‘ end a m7gs’ppp5’XmYm structure in which 7-methylguanosine is bound by a 5’, 5‘- pyrophosphate bridge to the adjacent me- thylated nucleotide. This so-called “cap” structure is also present in the histone messenger in the form of heterogeneous nuclear RNA (hnRNA) and it was sug- gested that its addition precedes the poly- adenylation. The functional importance of the 7’methylguanosine 5’ terminal was demonstrated (Muthukrishnan et al., 1975) for viral mRNAs and globin mRNA. It is involved in the binding of mRNA to the ribosomal subunit 40 S; the removal of m7G reduced the translational efficien- cy; i t is recognized during the initiation and protects the mRNA from exonucleo- lytic 5’ degradation.

In the internal position of many viral and eukaryotic mRNAs, several N6-methyl- adenosine residues were identified: the functional meaning of this sequence, which is not present in all the mRNAs (it is lacking in the globin mRNA), is unknown. I t does not seem to be indispensable for the translation. A possible function of a recognition site for the processing of the

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STABLE RNAS AND EhIBRYONIC DIFFERENTIATION IN AhlPHIBIA 269

mRNA was suggested (Derman & Dar- nel], 1974).

The polyadenylated segment is added to the mRNA in the nucleus and it can be lengthened or turned over in the cyto- plasm (sea urchin embryo, Slater et al., 1972; Brandhorst & Bannet, 1978; Xe- nopus oocytes, Darnbrough & Ford, 1979) at the 3’-OH end by a poly(A)polymerase, catalyzing the DNA and RNA-independent condensation of adenylic nucleotides; its length is variable (bacteriophages 1-5, fungi 50, mammals 250 AMP residues) and a course parallel to evolution was suggested (Carlin, 1978). The length of the polyadenylared segment may also be related to the stability of the mRNA mo- lecule.

Ever since the polyadenylated sequence was discovered (Edmonds & Cararnela, 1969; Lee et al., 1971), there have been speculations on its precise function. The well-known results obtained by Nude1 et al. (1976) demonstrated that the poly- adenylated segment is not indispensable for the translation of mRNA in a cell- free system (see also Sippel e f al., 19741, but its absence greatly reduces the du- ration of a good translational efficiency. The different stability of rabbit globin mRNA with polyadenylated segments of various lengths becomes evident if the translation is followed after injection into Xenopus oocytes or eggs, where, diffe- rently from a cell-free system, a high

’translation efficiency can be maintained for some days (Gurdon et al., 1974). A minimal number of 21 AMP residues seems to be needed to maintain the func- tional stability of the globin mRNA or to restore the stability when a re-adeny- lation was made (Huez et al., 1975). Observations on the shortening of the polyadenylated segment while the mRNA inolecule is translated support the idea of :I structural stabilization determined by the poly(A) segment; haemin, while it speeds up the translation of globin mRNA injected into amphibian oocytes, reduces its stability (Huez et al., 1977).

Not all the mRNAs contain the poly(A) segment; the histone mRNA is lacking and it has to be considered an “unstable” molecule, too, because in the cytoplasm of cultured somatic cells, histone mRNA was found only during S phase and after- wards it is degraded (Perry & Kelley, 1973; Melli et al., 1977). After injection into oocytes, the functional half-life of sea urchin histone mRNAs is about 3 hours (Woodland & Wilt, 1980 a), but their translation can be prolonged up to 60 hours by artificial polyadenylation (Huez et al., 1978, mammalian histone mRNA) .

Post-transcriptional addition of poIy(A) appears to be an essential step in the pro- cessing mechanism. Treatment with cor- dycepin (3’-deoxyadenosine), selectively inhibiting poly(A) synthesis, also prevents the appearance of mRNA in polysomes (Jelinek et al., 1973). The time interval required for polyadenylation was calcu- lated as 10-15 minutes, thus confirming the calculated delay of entry into the cy- toplasm in comparison with the non-poly- adenylated histone mRNA (Adesnik & Darnell, 1972).

After entering in the cytoplasm of the eukatyotic cells, the mRNA can be extract- ed from the polysomes to which it is bound during the translation or from ri- bonucloprotein particles not polysome-as- sociated. In duck erythroblasts, globin mRNA can be extracted from free cyto- plasmic RNP sedimenting 20 S. The mes- senger RNA when purified from these mRNP, containing some translational re- pressors, can direct the protein synthesis in a cell-free system (Civelli et al., 1976). Analogous mRNP particles, sedimenting 70-100 S have been identified in myoblasts before fusion (Buckingharn et al., 1974, 1976; Dym et al., .1979). From these particles it is possible to isolate the 26 S mRNA codifying for the heavy chain of myosin, while from 16-40 S particles, mRNA codifying for actin can be extracted (Bag & Sarkar, 1975). Nearly all (95%)

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270 F. DE BERSARDI

of the myosin mRNA is contained in ri- bonucleoprotein particles before the fu- sion of the myoblasts; after the fusion, on the contrary, 60% of the myosin mRNA is polysome-associated.

These results from both cellular cul- tures (Dym et al., 1979) and embryonic muscles (Bag 8: Sarkar, 1976) suggest that the ribonucleoprotein particles, iden- tifiable, to a certain extent, with Spirin’s informosomes, are involved in the accu- mulation of the mRNA not immediately translatable in a conservative form; the translation inhibitor could be a proteic one (Praeobrazhensky & Spirin, 1978). Heywood & Kennedy (1976) proposed a model of translational inhibition caused by a “translational control” RNA (tcRNA), and identified it as an oligonucleotide of 20-30 residues, with a uridine-rich se- quence, which can be extracted from the mRNP complexes; it could form double- helical, non-translatable complexes with the myosin messenger RNA.

The myosin mRNA may be synthesized in the myoblasts and stored in the ribo- nucleoprotein particles; during the fusion of the myoblasts it is transferred on the polysomes for translation. Buckingham et al. (1974) showed that the half-life of the myosin mRNA before fusion is about 10 hours; after fusion, instead, the sta- bility increases 5-6 fold.

The available data are not sufficient to clarify many aspects of the storage of mRNA in inactive form and of its mobi- lization and activation. Nevertheless, thev show the possibility that transcription and translation could take place even in sepa- rate moments of cell differentiation, and thus at different stages during embrvonic development. Monroy et al. (1965) had already demonstrated that untranslated messenger RNA was present in unferti- lized sea urchin ews: Gross et al. (1973) then identified mRNP particles containing ttc histone messenger RNA.

Furthermore, 40 S mRNP were iden- tified in Arteiizia saliita cysts (Nilsson &

Hultin, 1974; Grosfeld & Littauer, 1975), in Drosophila oocytes and earliest embryos (Lowett & Goldstein, 1977), and in Xe- iropzis oocytes (Rosbash & Ford, 1974).

I t is known that during amphibian oogenesis a great quantity of ribosomal RNA and ribosomes is synthesized; these ribosomes are sufficient to bring about the protein synthesis until the gastrula stage. Gurdon’s experiments on the anu- cleolate mutants beforehand (Brown & Gurdon, 1964), and on the translation into oocytes and eggs of exogenous mRNA injected afterwards (Gurdon et al., 1974), gave the most convincing proof that these ribosomes are functioning. It was also demonstrated that the RNA sequences synthesized in the oocytes have a great range of complexity; a lot of informational RNA transcribed in this stage can be con- served during initial embryogenesis until the mid-blastula stage (Crippa et al., 1967).

The mature amphibian oocyte contains a great variety of gene transcripts, mostly produced during the “ lampbrush” phase of diplotenic chromosomes. More recent research has criticised the traditional con- cept of the lampbrush phase as a conti- nuous period of actively transcribing ma- ternal messages which will be stored in the oocyte. It was calculated (Callan, 1963) that the “lampbrush” phase (6-8 weeks in Anura; 7 months in Urodela) wou!c! be to3 lonq, if we related it to the quantity of stored transcripts. Otherwke, Rosbash & Ford (1974), using hvbridi- zation with ’H-poly(U), found that the rate of synthesis of polyadenylated mRNA is very high in the early oogenesis staqes, before the “lampbrush” phase, and it remains quite constant, despite a greater than fourfold increase in RNA content per oocyte (it is diluted in the ribosomal one) and a-IOO-fold increase in volume. The work of Rosbash & Ford also shows that the polyadenylated RNA is mostly contained in ribonucleoprotein particles sedimenting between 25 and 80 S; less

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STABLE RNAS AND EhlBRYONIC DIFFERENTIATION IN AhlPEIIBIA 27 1 -

than 10% of this RNA cosediments with polyribosomes, it is sensitive to EDTA and may correspond to mRNA which is being actively translated. In previtelloge- nic oocytes, on the contrary (stage 1 Du- mont, 1972), about 75% of mRNA could be bound to polyribosomes (Darnbrough & Ford, 1976). The mRNA synthesized in pre-vitellogenic oocytes may have a half-life of more than two years and may be stored during entire oogenesis (Ca- bada et ul., 1977; Ford et ul., 1977). Recently Dolecki & Smith (1979) showed that polyadenylated mRNA is synthesized both at stage 3 (maximal lampbrush chro- mosome stage) and a t stage 6 (comple- tion of oocyte growth). The bulk level can be maintained by continually chang- ing rates of synthesis and degradation. It is necessary, of course, to maintain this complex steady state so that the se- quences transcribed are the same both at the stage of maximum lampbrush and in the full-grown oocyte. Cross-hybrid- ization experiments between polyade- nylated RNA from the ovary, immature oocyte and mature oocyte and the cor- responding cDNA show quite identical sequences (Perlman & Rosbash, 1978). In this may the idea that a great quantity of maternal information is contained in the cytoplasm of eggs before fertilization is confirmed for amphibian eggs as well as for those of sea urchins. In the sea urchin egg this mRNA is localized in the cytoplasm and includes RNA codifying for histones and microtubule protein.

Darnbrough & Ford (1976) found dif- ferent electrophoretic patterns for proteins synthesized “in vitro” by Xerioptis oocy- tes at different stages, from early vitello- genesis to mature oocyte. On the contrary, polyadenylated mRNA extracted from oocytes at the same stages and translated by means of a wheat germ cell-free system always gave the same patterns. This sug- gests on one hand that the mRNAs, trans- lated on the feu7 polysomes of the oocyte, are part of a selected population of stored

mRNA, on the other hand that some translational control in the oocytes may be possible.

A particular case of accumulation, which in amphibian oocytes seems to be different from in sea urchin oocytes, is the histone accumulation. During the earlier stages of Xerzopits oogenesis, a great quantity of histone is produced, non-coordinated with DNA synthesis. In the mature oocyte a histone amount about 140000 pg is present: it is sufficient for the developing egg up to the blastula stage (Adamson & Woodland, 1974, 1977). Histone synthesis increases during the oocyte maturation about 20-50 times, it is independent of the RNA synthesis (it takes place in the presence of actinomycin D or in anucleate oocytes) and its rate is higher than that of total protein syn- thesis increase. Injection experiments (Woodland & Wilt, 1980b) suggest that histone mRNAs are translated in mature eggs 1-5 times more efficiently than in oocytes, but the main factor for the in- crease of histone synthesis seems to be the mobilization of stored mRNA.

A comparison between the pattern of histone synthesis in sea urchin and am- phibian embryos can be summarized as follows. The sea urchin embryo synthe- size histones on the maternal message and on the new embryonic message (Ru- derman & Gross, 1974); the Xeiiopiis embryo, instead, utilizes histones already made on the maternal message and stored. It is perhaps likely that the accumulation of translational products is related to the greatest demand for histones during clea- vage, but an. explanation may be found also in the poor stability of histone mRNA.

Ruderman et ul. (1979) demonstrated that synthesis of histones can be obtained in a cell-free system even with polyade- nylated mRNAs extracted from Xeiioptrs oocytes. With polyadenylated mRNA from eggs or embryos the histone synthesis was very poor, while non-polyadenylated mRNA is active in these stages. Remem-

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F. DE BERNARD1 272

bering earlier reports of polyadenylated mRhA codifying for histones, Ruderman & Pardue (1977) put forward the inte- resting hypothesis that the disappearance of the polyadenylated segment of histone mRNA is due to a de-adenylation during the maturation of the oocyte. If further research were to demonstrate that ade- nylation and de-adenylation take place “iii vivo”, as was shown “iii vitro” on the histone messenger (Huez et a]., 1978), the problem of correlation between ade- nylation and messenger stability could be nearing a solution (see also Slater et al., 1972).

Nevertheless, more recent calculations made by Woodland and Wilt (1980a) in- dicate that 2550% of histone mRNA in oocytes is non-polyadenylated. This mRNA must be protected from degradation. Po- lyadcnylation, indeed, seems not the only way in which mRNA can be stabilized in the oocytes. From these collected data i t is clear that the mature oocyte and un- fertilized egg represent a very complex system. A single totipotent cell is filled up with items of information such as stable maternal gene transcripts (ribonu- cleoprotein particles and polyadenylated messengers) and also translational pro- ducts (histones, DNA polymerase, RNA polymerases). The research carried out on the effects of the actinomycin D on early embryos (Giudice et al., 1968 for sea urchins; Brachet et al., 1964 for Amphi- bia) was very important in demonstrating the presence and the utilization of a pool of maternal RNA stored in the eggs. The first hypothesis of a translation of mater- nal RNA during cleavage in the sea urchin and of the existence of a post-transcrip- tional control put forward by Terman & Gross (1965) has been verified many times in Amphibia, too.

The synthesis of new embryonic RNA was studied in the classic research of Brown & Littna (1964, 1966). During cleavage, the heterogeneous RNA is syn- thesized with nuclear localization (Bach-

varova & Davidson, 1966), showing a DNA-like base composition and a rapid turnover (Brown & Gurdon, 1966; Ma- riano & Shram-Doumont, 1965); it is nonribosomal RNA (Brown & Gurdon, 1966; anucleolate mutants). The rate of synthesis of this RNA increases in the late blastula and during gastrulation, when it is stored; afterwards the rate is constant. From this research it was already clear that the increase of protein synthesis ob- served from fertilization in amphibian embryos onwards, although less dramatic that in sea urchins, had been codified by stable mRNA of maternal origin contained in the egg in sufficient amount to program the deveIopment of the embryo until the gastrula stage. It is well known from morphological research that actinomycin D cannot inhibit cleavage, but puromycin, pactamycin, cycloheximide and all inhi- bitors of translation can do this (Brachet et al., 1964). The ribosomal RNA syn- thesis (and from here the formation of embryonic ribosomes) begins only at early gastrula stage, shortly after the beginning of the synthesis of new transfer RNA at the late blastula stage.

A still open problem is the moment when the mRNAs directing the embryonic differentiation in the postgastrula stage are synthesized. They become the “li- miting factors” of protein synthesis ac- companying the differentiation, since the entire translational system seems to be ready and fully sufficient, as Gurdon’s well known experiments showed (Gurdon et al., 1974). Denis (1968) identified three forms of informational RNA during embryonic development using the compe- tition bybridization technique. 1) A mRNA which is present in the gastrula-neurula stages and cannot be found in more ad- vanced stages; 2) mRNA with rapid turnover: it IS present in all post-gastrula stages and also in the adult; 3) stable mRNA, with slow turnover, is found from the neurula stage and its appearance ac- companies the differentiation period. The

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STABLE XNAS AXD EhIBRYONIC DIFFERENTIATION IN A M P H I D I A 273

Fig. 2. - Sedimentation on sucrose gradient of RNA extracted by phenol-chloroform frorn heavy pdysomes of the dorsal region of Xemopus laevis embryos cut at early neurula stage (15 of N. and F.) and labelled with ’H-uridine up to stage 22 (dotted line). ’H-poly(U)-hybridization of RNA extracted from dorsal region of stage 22 embryos (continuous line).

presence of these stable mRNAs, stored in the cytoplasm, explains why the diffe- rentiation cannot be blocked by the acti- nomycin D or by daunomycin. In fact the inhibitors of transcription completely de- press RNA synthesis, but protein synthesis is nevertheless maintained at the same level as the control or even a little higher (De Bernardi et UZ., 1969).

The lack of homogeneity in the amphi- I bian embryo from the gastrula stage sug-

gests the existence of regional differences both in translational activity and in post- transcriptional control of the synthesis. Some results of Woodland & Gurdon (1968) indicated that the synthesis of ribo- somal RNA in the endoderm was related to the appearance of nucleoli and to the synthesis of ribosomal proteins; rRNA synthesis was starting several hours later than in the ectoderm. Moreover, a RNA called “heterogeneous” was found quickly synthesized from stage 7 (blastula) and stored in endodermal cells. This DNA-like

RNA seems to be more stable in the dorsal region (ecto-mesodermal cells) than in the endoderm (Flickinger, 1970). Brachet & Malpoix (1971), summarizing cytochemical, autoradiographic and bio- chemical data, suggested the well known diagrammatic distribution of the polyso- mes (i.e. the ribosomes actively synthe- sizing proteins) along an animal-vegetal gradient in the unfertilized egg; this gra- dient becomes dorso-ventral in the gastrula and cephalo-caudal from the tailbud stage on.

More recent research carried out by the molecular hybridization technique between nuclear RNA from Raria pipietzs neurulae and adult DNA showed that a greater number of homologous sequences are pre- sent in ecto-mesodermal than in endo- dermal cells (Flickinger & Daniel, 1972). The heterogeneous nuclear RNA from the ecto-mesodermal region shows a sedimen- tation coeficient restricted between 16 and 30 S, thus suggesting a different processing stage of primary transcripts compared with the endodermal region where the mean sedimentation coefficient is higher - if we agree, as a general rule, that the processing of most mRNAs involves a reduction of dimension (Shepherd & Flickinger, 1979).

Our research on the problem of the synthesis of mRNA directing the histo- logical differentiation began by cutting the embryos into two parts at various de- velopmental stages, from the gastrula until the neurula, and by blocking the RNA synthesis with actinomycin D and by la- belling the fragments with precursors of RNA and protein synthesis. The dorsal region of the embryos cut at gastrula or neural plate stage showed an incorporation reduced by 60% in comparison with the controls. On the contrary, the dorsal re- gion of the embryos cut and treated with actinomycin D from the early neurula stage showed an increase in “C-leucine incorporation in the total TCA-precipitable proteins (Leonardi Cigada et al., 1975). The‘refore further research mas carried. out

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274 F. DE BERNARD1

I n & 0

c3 E P

I 1' '= P

I

0.6

0.4

0.2

0

B

Fig. 3. - 3H-poly(U) hybridization of RNA extracted with phenol-chloroform from animal (A) or dorsal (B, C, D) regions of Xemprrs laevir embryos. A) late blastula (stage 9 of Nieuwkoop & Faber, 1956); B) gastrula (stage 10); C) neural plate (stage 12.5); D) neurula (stage 15).

to determine whether the increased incor- mycin D (De Bernardi & Bolzern, 1978). poration was to be ascribed to the whole The analysis of the incorporation in va- translational system or whether there rious polysomal classes, analyzed on su- were some ' polysomal fractions active crose gradient, demonstrated that some in translating previously formed, stable polysomal classes began to be active in mRNA even in the -presence of actino- succession in both the dorsal and the ven-

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STABLE RNAS AXD EhlBRYOSlC DIFFERENTIATION I N AhlPIIIBIA 275

20/

Fig. 4. - Percentage of ’H-labelled poly(U) hybridized in 26s RNA compared with the ’H-labelled poly(U) hybridized in total RNA at each stage of development.

tral regions of the embryos cut at early neurula stage (15 of Nieuwkoop & Faber, 1956).

To try to identify the mRNAs directing this protein synthesis and to verify when they are synthesized, w e extracted them from the heavy polysomal fractions that were also active in actinomycin-treated fragments and we performed a hybridi- zation with ’H poly(U) in order to show the presence of polyadenylated segments. The mean length of the polyadenylated segment in Xermpris mRNA, as calculated by Miller (1978), is of 125 nucleotides.

The mRNA extracted from the same polysomal fractions that me had seen en-

’ gaged in protein synthesis on evidently preformed templates and in which it was possible to show the “ superinductive ” effect of the actinomycin, can be hybri- dized with ’H poly(U) in a fraction sedi- menting at 26 S, near the ribosomal 28 S RNA (Fig. 2). This 26 S RNA has been extensively studied (Heywood & Rourke, 1974; Buckingham et nl., 1974, 1976) and identified as the messenger of the heavy chain of the myosin. Clearly the protein synthesis observed in heavy polysomal classes is greatly supported by this mes- senger, and it is probably a synthesis of

myosin. I t is to be noted that at this moment (stage 22 of N. and F.) the somites are rapidly shaping and beginning the differentiation of muscle cells.

From the data previously obtained (De Bernardi and Boliern, 1978) it was clear that this mRNA also supports protein synthesis in the presence of actinomycin D, therefore it seems to be stable and transcribed before the early neurula stage, when we began the treatment with acti- nomycin D. We tried then to identify this 26 S mRNA in earlier stages, be- ginning at the late blastula. At each stage examined the embryos were cut into two regions, leaving the presumptive ectoderm and mesoderm in the “dorsal” region and the presumptive endoderm in the “ventral” region. From each group of fragments the total RNAs were extracted and frac- tionated onto a sucrose gradient; each gradient fraction was then hybridized with labelled poly(U) (Rosbash & Ford, 1974). The RNA extracted from the animal re- gion of the blastula (stage 9 of N. and F.) hybridized in several fractions but hardly at all in 26 S (Fig. 3A); hybri- dization in this fraction slightly increased at the gastrula stage (Fig. 3B) and became more evident from the neural plate stage and at the beginning of the rising of the neural folds (stage 12.5-15; Fig. 3 C, D).

If we compare the quantity of labelled poly(U) hybridizing in 26 S RNA to the total poly(U) hybridized in each stage, me can observe that the percentage of poly(U) hybridized in myosin messenger increases about threefold from blastula to gastrula stage, and another threefold from gastrula to neural plate stage (Fig. 4). These data suggest that at this moment the myosin mRNA is actively transcribed- or that its own precursor is actively poly- adenylated. I n more advanced stages (neu- rula: stages 15-17 of N. and F.) the per- centage of hybridization of 26 S decreases, perhaps in connection with differentiation which is also in progress in other cell.ular types (nervous cells) requiring the pre-

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Adamson E.D. & Woodland H.R., 1977 - Changes in the rate of histone synthesis during oocyte maturation and very early de- velopment of Xenoprrs laevis. Develop. diol., 57: 136-149.

Adesnik hl. & Darnel! J.E., 1972 - Biogenesis and characterization of histone messenger RNA in Hela cells. J. hlol. Biol., 67: 397- 406.

Bachvarova R. & Davidson E. N., 1966 - Nuclear activation at the onset of amphibian gastru- lation. J. exp. Zool., 163: 285-296.

Bag J. & Sarkar S., 1975 - Cytoplasmic non- polysomal messenger ribonucleoprotein con- taining actin messenger RNA in chicken em- bryonic muscles. Biochemistry, 14: 3800-3807.

Bag J. & Sarkar S., 1976 - Studies on a non- polysomal ribonucleoprotein coding for myosin heavy chain from embryonic muscles. J. Biol. Chern., 251: 7600-7609.

Ballantine J. E. hl., Woodland H. R. & Sturgess E.A., 1979 - Changes in protein synthesis during the development of Xeiropzis laevis. J. Embryo]. Exp. hlorphol., 51: 137-153.

Bishop J. O., Pernberton R. & Baglioni C., 1972 - Reiteration frequency of haemoglobin genes in the duck. Nature N.B., 235: 231.

Brachet J., Denis H. & de Vitry F., 1961 - The effects of actinomycin D and puromycin on morphogenesis in amphibian eggs and Acefa- bidaria mediierratzea. Develop. Biol., 9: 398- 434.

Brachet J. & hlalpoix P., 1971 - hlacromolecular synthesis and nucleocytoplasmic interactions in early development. Adv. Morphog., 9: 263- 316.

Brandhorst B. P. & Bannet hl., 1978 - Terminal completion of poly(A) synthesis in sea urchin embryos. Develop. Biol., 63: 421-431.

Brown D.D. & Gurdon J.B., 1963 - Absence of ribosomal RNA synthesis in the anucleolate

sence of a wide range of messengers. At stage 22 (the neural tube closed), when the differentiation of muscle cells (and therefore the synthesis of myosin) is in progress, the higher proportion of radio- activity bound to the polyadenylate seg- ment of 26 S mRNA is restored.

The results obtained by us, compared with indications from the research cited above, suggest that protein synthesis in the dorsal region of Xenopus laevis em- bryos during the closing of the neural tube proceeds on the template of stable mRNA, transcribed and polyadenylated before the raising of the neural folds (stage 15 of N. and F.). The myosin mRNA may be an example of this me- chanism.

It may be remembered that at the late gastrula stage in Ram the presence of myosin can be detected by an immunolo- gical method (Ranzi & Citterio, 1955). Recent work (Ballantine et al., 1979) strengthens our findings by showing that another main protein of muscles, the a-actin, and its mRNA can be found from the late blastula stage, in the dorsal region, while Q and y actin (non-muscu- lar) are already synthesized in the oocyte.

From the research carried out on am- phibian oocytes and embryos the folloming conclusions can be drawn: stable mRNAs, codifying for proteins specific for diffe- rentiated cells, first identified by research on cell cultures, are in many cases trans- lated quite a long time after the transcrip- tion; a sort of specific control (translatio- nal control) of the expression of these mRNAs must exist, switching on and off their activity; this translational control may be committed to an inhibitor, pro- bably of protein nature, which is present in ribonucleoprotein particles, whatever their structure may be.

REFERENCES Adamson E.D. & Woodland H.R., 1973 - Histone

synthesis in early amphibian development: histone and DNA synthesis are not coor- dinated. 1. hlol. Biol., 88: 263-285.

mutant of Xet iopk laevis. Proc. Nat. Acad. Sci. USA, 51: 139-146.

Brown D.D. & Littna E., 1961 - RNA synthesis during the development of Xetzoprrs laevis, the South African clawed toad. J. hlol. Biol., 8: 669-687.

Brown D.D. & Littna E., 1966 - Synthesis and accumulation of DNA-like RNA during em- bryogenesis of Xetzoprrs laevis. J. hlol. Biol., 20: Xl-93 -_, ..

Brown D.D. & Gurdon J.B.. 1966 - Size distri- bution and stability of DNA-like RNA syn- thesized during development of anucleolate embryos of Xetzopiis laevis. J. hlol .Biol., 19:

Buckinfiham hl. E., Caput D., Cohen A,, Whalen R.G. & Gros F., 1974 - The synthesis and stability of cytoplasmic messenger RNA dur- ing myobIast differentiation in culture. Proc.

.Nat. Acad. Sci. USA, 71: 1466-1470.

3991122. .

Dow

nloa

ded

by [

Cas

e W

este

rn R

eser

ve U

nive

rsity

] at

18:

38 0

4 N

ovem

ber

2014

STABLE RNAS AND EMBRYONIC DIFFERENTIATION IN AhiPIilBIA 277

Buckingham hi. E., Cohen A. & Gross F., 1976 - Cytoplasmic distribution of pulse-labeled poly (A):containing RNA, particularly 26 S RNA, during myoblast growth and differentiation. J. hiol. Biol., 103: 611-626.

Cabada hi.O., Darnbrough C. & Ford P. J., 1977 - Differential accumulation of two size classes of poly(A) associated with mRNA dur- ing oogenesis in Xerzopirs laevis. Develop. Biol., 57: 427-439.

Callan H. G., 1963 - The nature of lampbrush chromosomes. Int. Rev. Cytol., 15: 1-34.

Carlin R.K., 1978 - The poly(A) segment of mRNA: 1) evolution and function and 2) the evolution of viruses. J. Theor. Biol., 71: 323- 338.

Civelli O., Vincent A., Buri J.F. & Scherrer K., 1976 - Evidence for a translational inhibitor linked to globin mRNA in untranslated free cytoplasmic messenger ribonucleoprotein com- plexes. FEBS Lett., 72: 71-76.

Crippa hf., Davidson E.H. & hlirsky A.E., 1967 - Persistence in early amphibian embryos of informational RNAs from the lampbrush chromosome stage of oogenesis. Proc. Natl. Acad. Sci. USA, 57: 885-892.

Darnbrough C. & Ford P. J., 1976 - Cell-free translation of messenger RNA from oocytes of Xenoprrs laevis. Develop. Blol., 50: 285- 301.

Darnbrough C. 8~ Ford P. J., 1979 - Turnover and processing of poly(A) in full-grown oocytes and during progesterone induced oocyte maturation in Xenoprrs laevis. Develop. Biol., 71: 323-340.

Darnel1 J.E., 1979 - Transcription units for mRNA production in eukaryotic cells and their DNA viruses. Progr. Nucl. Ac. Res.,

De Bernardi F. & Bolzern A.M., 1978 - O n protein synthesis in Xenoplrr laevis embryos. Rend. Acc. Naz. Lincei (Sc. Fis.) (E), 64: 302- 310.

De Bernardi F., Leonardi Cigada hi., hlaci R. I ' & Ranzi S., 1969 - O n protein synthesis

during the development of lithium-treated embryos. Experientia, 25: 211-213.

Denis H., 1968 - Role of messenger ribonucleic acid in embryonic development. Adv. hior-

Derman E. & Darnell J.E., 1974 - Relationship of chain transcription to poly(A) addition and processing of hnRNA in HeLa cells. Cell, 3:

Dolecki G.J. & Dennis Smith L., 1979 - Poly- (A)+ RNA metabolism during oogenesis in X. Zaevis. Develop. Biol., 69: 217-236.

Dumont J. N., 1972 - Oogenesis in Xenopus laevis Daudin. 1 Stages of oocyte development in laboratory maintained animals. J. hiorphol., 136: 153.

22: 327-353.

phog., 7: 115-150.

255-261.

Dym H. P., Kennedy D. S. & Heywood S. hl., 1979 - Sub-cellular distribution of the cyto- plasmic myosin heav chain mRNA during myogenesis. Differentiation, 12: 145-155.

Edmonds hi. & Caramela hl.G., 1969 - The isolation and characterization of adenosine monophosphate-rich polynucleotides synthesi- zed by Ehrlich ascites Cells. J. Biol. Chem.,

Flickinger R.A., 1970 - Effect of Actinomycin D on KNA and protein synthesis in region of developing frog embryos. Experientia, 26:

Flickinger R.A. & Daniel J.C., 1972 - Nuclear DNA-like RNA of regions of developing frog embryos. \Vilhelm Roux' Archiv., 169: 350- 352.

Ford P. J., hlathieson T. & Rosbash M., 1977 - Very long-lived messenger RNA in ovaries of Xetmpirs laevis. Develop. Biol., 57: 417-426.

Gall J-G., 1968 - Differential Synthesis of genes of ribosomal RNA during amphibian ooge- nesis. Proc. Nat. Acad. Sci. USA, 60: 553- 560.

Gilmour R.S., Harrison P. R., Windass J.D.., Affara N.A. & Paul J., 1974 - Globin mes- senger RNA synthesis and processing during haemoglobin induction in friend cells. I. Evi- dence for transcriptional control in clone h12. Cell Differ., 3: 9-21.

Giudice G., hlutolo V. & Donatuti G., 1968 - Gene expression in sea urchin development. Wilhelm Roux' Arch., 161: 118-128.

Gross K. IV., Jacobson-Lorena M., Baglioni C. & Gross P. R., 1973 - Cell-free translation of maternal messenger RNA from sea urchin eggs. Proc. Nat. Sci. USA, 70: 2614-2618.

Grosfeld H. & Littauer U.Z., 1975 - Cryptic form of mRNA in dormant Artetizia saliria cysts. Biochem. Biophys. Res. Commun., 67: 176-181.

Gurdon J. B., Woodland H. R. & Lingrel J. B., 1974 - The translation of mammalian globin mRNA injected into fertilized eggs of Xetzo- prts laevis. I. Message stability in develop- ment. Develop. Biol., 39, 125-133.

Heywood S . hl. & Rourke A. W., 1974 - Cell- free synthesis of myosin. hieth. in Enzymol., 30: 669-674.

Heywood S. hl. & Kennedy D. S., 1976 - Trans- lational control in embryonic muscle. Progr. Nucl. Acid. Res., 19: 477484.

Huez G., hlarbaix G., Hubert E., Cleuter Y., Leclercq hi., Chantrenne H., Devos R., Soreq H., Nude1 U. & Littauer U. Z., 1975 - Reade- nylation of polyadenylate-free globin mes- senger RNA restores its stability in vivo. Eur. J. Biochem., 59: 589-592.

Huez G., hlarbaix G., Burny A,, Hubert E., Leclercq hl., Cleuter Y., Chantrenne H., Soreq H. & Littauer U.Z., 1977 - Degra-

244: 1314-1324.

778-779.

Dow

nloa

ded

by [

Cas

e W

este

rn R

eser

ve U

nive

rsity

] at

18:

38 0

4 N

ovem

ber

2014

-~ I;. DE BERNARD1 278

dation of deadenylated rabbit a-globin mRNA in Xenoprrs oocytes is associated with its translation. Nature, 266: 473-474.

Huez G., hlarbaix G., Gallmitz D., Weinberg E., Devos R., Hubert E. & Cleuter Y., 1978 - Functional stabilization of HeLa cell histone messenger RNA injected into Xerropris oocytes by 3‘-OH polyadenylation. Nature, 271: 572- 573.

Jelinek \XI., Adesnik hl., Salditt hl., Sheiness D., Wall R., hlolloy G. ,Philipson L. & Darnel1 J. E., 1973 - Further evidence on the nuclear origin and transfer to the cytoplasm of poly- adenylic acid sequences in mammalian cell RNA. J. hlol. Biol., 75: 515-532.

Kafatos F.C., 1972 - The cocoonase zymogen cells of silk moths: a mode! of terminal cell differentiation for specific protein synthesis. Curr. Top. Develop. Biol., 7: 125-191.

Kaumeyer J.F., Jenkins N.A. & Raff R.A., 1978 - hfessenger ribonucleoprotein particles in unfertilized sea urchin eggs. Develop. Biol., 63: 266-278.

Lee S. Y., hlendecki J. & Brawerman G., 1971 - A polynucleotide segment rich in adenylic acid in H n rapidly-labeled polyribosomal RNA component of mouse sarcoma 180 ascites cells. Proc. Nat. Acad. Sci. USA, 68: 1331-1335.

Leonardi Cigada hl., Laria De Bernardi F. & Bolzern Scarpetti A. hl., 1975 - Sintesi di acidi ribonucleici e di proteine nelle diverse regioni dell’embrione di Xeriopzrs laeuis. Rend. Acc. Naz. Lincei (Sc. fis.) (8), 59:

Lowett J.A. & Goldstein E. S., 1977 - The cytoplasmatic distribution and characterization of poly(A)+ RNA in oxytes and embryos of Drosophila. Develop. Biol., 61: 70-78.

hlariano E.E. & Schram-Doumont A.. 1965 - Rapidly labeled ribonucleic acid in Xeriopris laevis embryonic cells. Biochim. Biophys. Acta, 103: 610-622.

hlcCarthy B. J. & Hoyer B.H., 1964 - Identity of DNA and diversity of messenger RNA molecules in normal mouse. Proc. Nat. Acad. Sci. USA, 52: 915-922.

hlelli hl. L., Spinelli H. & Arnold E., 1977 - Synthesis of histone messenger RNA of HeLa cells during the cell cycle. Cell, 12: 167-174.

Miller L., 1978 - Re!ative amounts of newly synthesized poly(A)+ and poly( A)- messenger RNA during development of Xeiiopus laeuis Develop. Biol., 64: 118-129.

hlonroy A., hlaggio R. & Rinaldi A. hf., 1965 - Experimentally induced activation of the ribo- somes of the unfertilized sea urchin egg. Proc. Nat. Acad. Sci. USA, 54: 107-111.

hluthukrishnan S., Both G.W., Furuichi Y. & Shatkin A. J., 1975 - 5’-terminal -/-methyl- guanosine in eukaryotic mRNA is required for translation. Nature, 255: 33-37.

152-163.

Nieuwkoop P. D. 8: Paber J., 1956 - Normal table of Xenopzrs laevis. North Holland Publ. Co. Amsterdam.

Nilsson hi. 0. & Hultin T., 1974 - Characteristic and intracellular distribution of messenger-like RNA in encysted embryos of Arletlria sali?a. Develop. Biol., 38: 138-149. I

Nude1 U., Soreq H., Littauer U. Z., hlarbaix G., Huez G., Leclercq hl., Hubert E. 8: Chan- trenne H., 1976 - Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xeriopris oocytes. Eur. J. Biochem., 64: 115-121.

Yalmiter R.D., 1973 - Rate of ovalbumin mes- senger ribonucleic acid synthesis in the oviduct of ihe estrogen-primed cells. J. Biol. Chem., 248: 8260-8270.

Perlman S. & Rosbash hi., 1978 - Analysis of Xeriopris Iueuis ovary and somatic cell poly- adenylated RNA by molecular hybridization. Develop. Biol., 63: 197-212.

Perry R.P. & Kelley D.E., 1973 - Messenger RNA turnover in mouse L cells. J. hlol. Biol., 79: 681-696.

Praeobrazhensky A.A. & Spirin A.S., 1978 - Informosomes and their protein components: the present state of knowledge. Progr. Nucl. Ac. Res., 21: 1-38.

Ranzi S. & Citterio P., 1955 - Le comportement des differentes fractions protiiques au cows du developpement embryonnaire de Raria esrrden!a. Rev. Suisse Zool., 62: 275-281.

Rosbash hi. & Ford P. J., 1974 - Polyadenylic acid - containing RNA in Xenopus Iaeuis oxytes. J. hlol. Biol., 85: 87-101.

Rudermm J.V. & Pardue ML., 1977 - Cell- free translation anaIysis of messenger RNA in echinoderm and amphibian early development. Develop. Biol., 60: 48-68.

Ruderman J. V. & Gross P. R., 1974 - Histones and histone synthesis in sea urchin develop ment. Develop. Biol., 36: 286-298.

Ruderman J.V., Woodland H.R. & Sturgess F. A., 1979 - hfodulation of histone messenger RNA during the early development of Xe- nopus laevis. Develop. Biol., 71: 71-82.

Shepherd G .W. & Flickinger R., 1979 - Hetero- geneous nuclear RNA (HnRNA) size in deve- loping frog embryos. Exp. Cell. Res., 120: 73-77.

Sippel A. E., Stavrianopuolos J. G., Schultz G. & Feigelson P., 1974 - Translation properties of rabbit globin mRNA after specific removal of poly(A) with ribonuclease H. Proc. Nat. Acad. Sci. USA, 71: 4635-1639.

Slater D. W., Slater J. & Gillespie D., 1972 - Post-fertilization synthesis of polyadenylic acid in sea urchin embryos. Nature, 240: 333-337.

Dow

nloa

ded

by [

Cas

e W

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ve U

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rsity

] at

18:

38 0

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STABLE RNAS AND EMBRYONIC DIFFERENTIATION IN AhlPHIBIA 279

Sullivan S., Palacios R., Stavnezer J., Taylor J. hI., Faras A. J., Kiely hl.L., Summers N. hl., Bishop J. hl. & Schimke R. T., 1973 - Syn- thesis of a deoxyribonucleic acid sequence complementary to ovalbumin messenger rib* nucleic acid and quantification of ovalbumin genes. J. Biol. Chem., 248: 7530-7539.

Suzuki Y., Gage L.P. & Brown D.D., 1972 - The genes for silk fibroin in Bonzbyx moti. J. hiol. Biol., 70: 637-649.

Terman S.A. & Gross P.R., 1965 - Translation level control of protein synthesis during early development. Biochem. Biophys. Res. Com- mum, 21: 595.

Woodland H.R. & Gurdon J.B., 1968 - The relative rates of synthesis of DNA, sRNA and rRNA in the endodermal region and other parts of Xerroprrs Iaevis embryos. J. Embryol. Exp. hlorph., 19: 363-385.

Woodland H.R. & Wilt F.H., 1980a - The functional stability of sea urchin histone mRNA injected into oocytes of Xerropris Iaevis. Develop. Biol., 75: 199-213.

Woodland H.R. & Wilt F.H., 198Ob - The stability and translation of sea urchin histone messenger RNA molecules injected into Xc- iropris laevis eggs and developing embryos. Develop. Biol., 75: 214-221.

Dow

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