Experimental

POLYADENYLATION THE

Cell Research

95 (1975) 263-268

OF NASCENT

EMBRYOGENESIS

RNA

OF ZLYANASSA

DURING OBSOLETA

J. R. COLLIER Department Marine

of Biology, Brooklyn Biological Laboratory,

College, Woods

Brooklyn, NY 11210 and Hole, MA 02543, USA

SUMMARY The proportion of the total nascent RNA polyadenylated was measured during the embryogenesis of Ilyanassa. There was found a high percentage of poly(A)-RNA during early cleavage and a reduced and constant proportion at all later stages of development. It was also found that the removal of the third polar lobe at first cleavage did not significantly alter the proportion of poly(A)-RNA synthesized during the early cleavage stages by this embryo.

That the organization of egg cytoplasm and its distribution to the early blastomeres during embryogenesis plays a deciding role in the determination of the developmental fate of embryonic cells has been amply demonstrated by the observations of Crampton [l], Wilson [2], Lillie [3], and Costello [4]. More recently Clement [5, 61, Cather [7] and Atkinson [8] have made an extensive study of the effect of the polar lobe cytoplasm of the Zlyana~~a embryo on differentiation. In this prosobranch snail the removal of the third polar lobe, a cytoplasmic protrusion that occurs at the vegetal pole of the egg at first cleavage, results in a virtually complete debilitation of the differentiation of this egg. These results of experimental embryology have brought attention to the molecular biology of the Zlyanassa egg with emphasis on the molecular basis of the effect of the polar lobe as it is related to the general problem of embryonic determination. Previously, observations [9-13, 281 on IS-751814

the effect of removing the polar lobe on protein and RNA synthesis have been reported. The observations to be reported in this paper establish a set of conditions for the assay of the polyadenylation of RNA of the Zlyanassa embryo, the quantitative pattern of polyadenylation during normal development and the effect of removing the polar lobe on this pattern during early embryogenesis. The polyadenylation of RNA was selected for study because of the strong circumstantial evidence [ 141 that most messenger RNAs (mRNA) are polyadenylated, which makes the assay of this parameter one of the more effective means of quantitating the pattern of mRNA synthesis during embryogenesis. MATERIALS

AND METHODS

Snails were collected at Woods Hole, MA, and kept in a tank of recirculating sea water. Egg capsules were collected and reared at 19°C in pasteurized sea water containing 0.5 mg each of penicillin and streptomycin G . Exptl

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Res 95 (1975)

264

.I. R. C’ollicr

Table I . Bind&

of RNA to Millipore branes and Poly( U)”

teem-

% of counts A Treatment of RNA (I) Binding in 0.5 M KCI (2) Pancreatic and T, resistant counts (3) Nuclease labile counts (4) Nuclease labile counts after T, ribonuclease (5) Hybridization to Poly(U) (6) Hybridization to Poly(U) after T, ribonuclease B

Nuclease labile after sequential ofRNAat (7) pH 7.6 (8) pH 9.0

bound

10.6 2.3 8.3 0.2 8.6 0.7

counts bound extraction 8.9

8.1

(I Stage 3 embryos were pulsed for 6.5 h with 250 &i/ml of 3H-uridine and assayed as described under Methods with the variants listed in the table.

Polar lobes were isolated by gentle agitation in calcium-magnesium low sea water as previously described [ 131: In vivo labeling of nucleic acids was accomplished by incubating embryos in 250 &i/ml of 3H-uridine (27.88 Ci/mM; New England Nuclear) in the presence of antibiotics as described above

Extraction

of nucleic acids

One hundred embryos were homogenized in a4x34 mm glass tube at 4°C in 0.1 ml of homogenizing solution (HS), which was 0.1 M Tris (pH 9.0) containing 0.05 M NaCl, 0.001 M MaCl,and 0.5 % sodium dodecyl sulfate. Immediately after homogenization 0. I ml of a I : I mixture of pH 9.0 buffer saturated phenol and chloroform was added and mixed for 30 sec. The homogenate was centrifuged at I7 000 g for 15 min and the aqueous phase withdrawn. The gel and phenol phases were then extracted twice with pH 9.0 HS, and all aqueous phases were combined, diluted with KCI buffer (0.5 M KCI; 0.001 M MgC12; 0.01 M Tris, pH 7.6) and kept cold prior to binding on Millipore membranes. The gel and phenol phases were quantitatively transferred to KC1 buffer prior to acid precipitation.

Binding

Hybridization

Cell Res 95 (1975)

to poly(U)

Polyuridylic (poly(U)) cording to Kates [15]by

assay

Two aliquots of the aqueous phase and the combined gel-phenol phase were taken for acid precipitation and the determination of the total radioactivity in each fraction. Aliquots of the aqueous phase were also assayed for ribonuclease lability. Samples were chilled and precipitated in the presence of 50 pg/ml of carrier RNA by adding an equal volume of cold 10% trichloroacetic acid (TCA) and filtered onto either a MilliExptl

pore membrane or a glass fiber filter. lhe collected precipitates were washed with I5 ml of cold 5 “; TCA and counted for the assay of radioactivity. Four aliquots of the aqueous phase wet-e diluted IO-fold in cold KCI buffer and filtered slowly (with minimal suction) through a Millipore membrane (HA type; 0.45 fi pore) which had been presoaked in KCI buffer. Two of the above membranes were incubated with 20 wg pancreatic ribonuclease and 4 U of T, ribonuclease per ml in 0. I X SSC for I h at room temperature. From the radioactivity of the aliquots described above the total recovery of acid precipitable counts in the aqueous phase (92.8+0.79%), the ribonuclease stable acid precipitable counts of the aqueous phase. the total membrane bound counts, and the ribonuclease resistant membrane-bound counts were determined. From these measurements the per cent of the nascent RNA bound to the Millipore membrane was determined. All of the experiments reported below are a measure of the proportion of total nascent RNA counts that bound to the Millipore membranes. Thus, only the relative amounts of RNAs with attached poly(A) sequences (poly(A)-RNAs) were determined. Control experiments were also done in which an aliquot of the’aqueous phase was digested with 2 U/ml of T2 ribonuclease in 0. I M acetate buffer, pH 5.0, for I h at room temperature prior to binding to Millipore membranes in KCI buffer. Similarlv. DNase (electrophoretically purified from Worthington Biochemical Crop.) digestion was used as a control at concentrations ranging from 20 to 100 pg/ml in 0.1 M Tris (pH 7.0) containing 0.015 M MgCI, and 0.01 M NaCI. Preparations treated with DNase, either before or after binding onto Milhpore membranes. were incubated at 37°C for I h.

I.....“,..\ 1234567

membranes were prepared adding 150pgof poly(U)(Miles

12

ac-

J 20

1. Abscissa: pulse time (hours); or&are: % of counts bound to Millipore membranes. Polyadenylation of nascent RNA as a function of pulse time. Stage 3 embryos were pushed with ‘Huridine for the time indicated.

Fig.

Poiyadenylation I

Fig. 2. Abscissa: pg/ml of actinomycin D; ordinate: % of counts bound to Millipore membranes. The effect of actinomycin D on the per cent of nascent RNA polyadenylated. Stage 3 embryos were pre-incubated for 1 h in actinomycin D (at the concentration indicated) and then pulsed for 4 h with YH-uridine in the presence of actinomycin D.

Laboratories, Inc.) to a glass fiber filter disc (Whatman GF/A). The loaded filters were dried at 37°C and then irradiated with a IS W germicidal lamp (General Electric) for 5 min on each side at a distance of 22 cm. The filters were then air-dried and stored at 4°C in a dessicator. The poly(U) filters were washed with 50 ml of distilled water before use. The hybridization reaction was carried out by incubating a poly(U) filter for 3 h at room temperature in I ml of 0.01 M Tris (pH 7.5) containing the RNA sample. A glass filter without poly(U) was included as a control. A third poly(U) filter that was also incubated with the RNA sample was treated with pancreatic and T, ribonuclease as described above. After incubation with the RNA sample the filters were washed with 20 ml of KC1 buffer followed by 20 ml of cold TCA. The filters were then counted as described below.

Radioactive

of Ilyanassa RNA

265

to poly(U) (line 5). Also, the nascent RNA hybridized to poly(U) was labile to Tz digestion (line 6). This binding behavior is consistent with the expectation that the nuclease labile counts bound to Millipore membranes because the nascent 3H-RNA contained poly(A)-rich tracts [16, 17, 18, 341. The pancreatic and T, ribonuclease resistant counts (table 1, line 3) were DNase labile, but it was found that a correction for ribonuclease resistant counts gave more consistent results than the use of DNase to remove the DNA before binding. Also, the elimination of the DNase step greatly facilitated the processing of the binding experiments when very small amounts of RNA were used. In table 1B lines 7-8 are the results of a sequential extraction of RNA from stage 3 embryos at pH 7.6 and then pH 9.0. Each of these extracts contained the same proportion of poly(A)-RNA, and while the bulk of the radioactivity was extracted at pH 7.6, good recovery (92.8+0.79%) was obtained only by a second extraction at pH 9.0. For this reason a pH 9.0 extraction was routinely used. Other experiments with older

counting

Membranes were counted either by drying in a heated vacuum oven and suspension in a toluene based scintillation solution, or wet membranes were dissolved in a dioxane based scintillation solution [33]. New England Nuclear Omnifluor was used as the scintillation fluor in either case, and the membranes were counted to 4000 counts in a scintillation spectrometer.

RESULTS From table 1A it is seen that the ribonuclease (pancreatic plus T,) labile counts of 3H-uridine labeled RNA bound to Millipore membranes (lines l-3) were eliminated by prior treatment of the RNA preparation with Tz ribonuclease (line 4) and were quantitatively the same as that hybridizable

1234

56

Fig. 3. Abscissa: days of development at 19°C; ordinote: % of counts bound to Millipore membranes. Polyadenylation of nascent RNA at different stages of development. All embryos were pulsed for 6.5 h with 3H-utidine; each point is the mean of four separate determinations. Exptl

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Res 95 (1975)

266

J. R. Collier

Table 2. Per cent of nascent RNA polyadenyluted by the I-duy normal und lobeless embryo Normal

Mean,

SE.

embryo

Lobeless

36.5 37.0 28.3 32.5

32.7 29.8 29.0 24.5

33.6k2.04

29.0?

P>O.O5.

embryo

I .68

organ anlagen; and in stages 68 there occurs the final differentiation of the veliger organs. Table 2 displays the percentages of :jHuridine labeled nascent RNAs containing poly(A)-RNA synthesized by the l-day normal and lobeless embryo. These data are from a paired experiment designed by pooling the eggs from several capsules so that the lobeless and normal eggs were randomly selected from the pooled eggs. All eggs were treated with calciummagnesium low sea water and the lobeless and normal eggs were returned to normal sea water for 23.5 h and then pulsed for 6.5 h with 3H-uridine. As seen from the P value, calculated from the F ratio obtained from an analysis of variance [20], the per cent of poly(A)-RNAs made by these embryos after 1 day of development is not significantly different from each other.

embryos gave a slightly higher partitioning of counts in the pH 9.0 extract, but there was in no case the extreme partitioning of poly(A)-RNA in the pH 9.0 extract reported by Lee et al. [18] for mouse sarcoma 180 cells. Fig. 1 illustrates the proportion of poly(A)-RNAs as a function of pulse time for the 3-day embryo. As expected, a shorter pulse time, which minimizes the counts accumulated in rRNA, shows a DISCUSSION higher percentage of poly(A)-RNA. Fig. 2 presents the results of inhibiting rRNA by The results presented in this paper show low concentrations of actinomycin D; it was that the binding of 3H-uridine labeled Zlyufound that 3.1 pg of actinomycin D/ml was ~~SSU RNA to Millipore membranes is a measure of the proportion of nascent RNA suffkient for maximal repression of rRNA accumulation, which gave a maximum of that is polyadenylated. Nuclear and cytoplasmic fractions were not prepared from 16 % poly(A)-RNA for the 3-day embryo. The percentage of poly(A)-RNA after a these embryos, but from the relatively long pulse times used (4-6.5 h), and the finding 6.5 h pulse at several stages of development is displayed in fig. 3. Each point is the of Darnell et al. [21] that 80% of the p(A)mean of at least four separate determinaRNA accumulates in the cytoplasm after a tions; there is no significant difference be- 150 min pulse, indicates that most of the tween the values for stages 2 through 6. p(A)-RNAs measured were probably All stages of embryos are days of developpresent in the cytoplasm. That 16% of the nascent RNA polyadenylated when rRNA ment at 19°C. The earliest stage measured was the 4-cell embryo, followed by a stage 1 synthesis is differentially repressed by low of actinomycin D agrees embryo that contained 29 to 30 cells [13]. concentrations The developmental features of each stage very well with the binding of 17% of the have been previously described [13, 191: nascent RNA after a short pulse. Both of stage 2 is a gastrula; during stages 3-5 there these conditions minimize the contribution is cell proliferation and organization of of rRNA to the labeled RNA and provide Exptl

Cell

Res 95 (1975)

Polyadenylation

an estimate of the proportion of nonribosomal RNA that is adenylated. The percent of nascent RNA during a 6.5 h pulse that was adenylated was determined for all stages of embryogenesis. The earliest stage studied (fig. 3) was the 4-cell embryo, in which 40.3 % of the RNA made during the next 6.5 h was p(A)-RNA. Similarly, at the end of the first day of development, when the embryo consists of 29 to 30 cells, 33 % of the nascent RNA was p(A)RNA. These two stages are particularly important in the development of this spiralian egg, because it is during this period of early cleavage that the developmental fate of many of these early blastomeres is established [6, 231. Thus, these data show that mRNA is synthesized during early development and that it is a dominant species of RNA being accumulated at this time. The synthesis of mRNA during early cleavage by the Zlyanassa embryo is similar to the pattern of RNA synthesis during early cleavage of the sea urchin embryo [24] and appears to be significantly greater than the per cent of p(A)-RNA made during early rabbit development [25]. When the data of Wilt [34] for the distribution of p(A)-RNA during early cleavage of the sea urchin egg is corrected for a 10% contribution of radioactivity in the poly(A)-rich tracts [27], the 40.1% p(A)-RNA in Zlyanassa is quite comparable to the corresponding stage of the sea urchin embryo. During gastrulation, morphogenesis and organogenesis the per cent of p(A)-RNA was decreased to about 10%. This is considerably lower than corresponding values for the sea urchin gastrula as estimated by Wilt [34]. However this difference is expected, because the Zlyanassa embryo synthesizes and accumulates much more rRNA [13] than does the sea urchin em-

of Ilyanassa RNA

267

bryo. This would substantially decrease the proportion of total nascent RNA appearing as p(A)-RNA. This constant accumulation ratio in Zlyanassa suggests that there is a transcriptional linkage for the transcription of p(A)-RNA, rRNA and tRNA that begins at gastrulation and lasts throughout the remainder of development. It has previously been shown that the extirpation of the polar lobe of this embryo alters the rate of DNA synthesis by the lobeless embryo [13], the pattern of protein synthesis [9, lo] and the incorporation of uridine into RNA [l 1, 121. The finding that the normal and lobeless embryo synthesize the same relative proportions of poly(A)RNAs, confirm the observations of Koser [12] for early embryogenesis and extend the criteria of analysis to a parameter other than the molecular size of the RNA. While my experiments did not measure the amount of poly(A) synthesized by these two classes of embryos, the similarity in the amount of nascent RNA bound by attached poly(A)-rich tracts suggests that the polar lobe cytoplasm does not play a limiting role at this stage of development in the minimal cytoplasmic polyadenylation of RNAs. This does not, however, eliminate the possibility that the polar lobe cytoplasm may have a role in the transposition of poly(A)RNAs, perhaps partially adenylated RNAs, from one cytoplasmic compartment to another, e.g. the transfer from a subribosomal to a ribosomal fraction as demonstrated in sea urchin embryogenesis by Slater et al. [22]. Nor do they eliminate a contribution by the polar lobe cytoplasm to the polyadenylation of oogenic transcripts, which would not be labeled in the experiments reported here. The possibility also remains that there is a differential synthesis of individual mRNAs and/or that there is an absolute reduction of the amount of RNA synExpri

Cell

Res 95 (1975)

268

J. R. Collier

thesized by the lobeless embryo. The latter possibility remains because all of the available evidence is based on the proportional measurement of the various RNAs synthesized. The determination of the absolute amounts of RNA synthesized by these two types of embryos is now in progress in this laboratory. This work was supported in part by the NSF (grant number GB- 15290). I thank Miss Louise Garone for excellent technical assistance.

REFERENCES I. Crampton, H E, Wilhelm Roux Arch Entwicklungsmech Organ 3 (18%) 1. 2. Wilson, E B, J exp zool I (1904) I. 3. Lillie, F R, J exp zool 3 (1906) 153. 4. Costello, D P, J exp zoo1 100 (1945) 19. 5. Clement, A C, J exp zoo] 121 (1952) 593. 6. -Ibid 149 (1962) 193. 7. Cather, J N, J exp zoo1 166 (1967) 205. 8. Atkinson, J W, J morphol 133 (1968) 339. 9. Collier, J R, Actaembryol morpholexp4 (1961)70. 10. Teitelman, G, J embryo1 exp morphol 29 (1973) 267. 1 I. Davidson, E H, Haslett, G W, Finney, R J, Allfrey, V G & Mirskv. A E. Proc natl acad sci US 54 (1965) 696. . 12. Koser, R B, Doctoral thesis. City Univ of New York (1974). 13. Collier, J R, Exp cell res 95 (1975). 14. Weinberg, R A, Ann rev biochem 42 (1973) 329.

Expti

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15. Kates. J, Methods in cell brol (ed D M Prescott) p. 53. Academic Press, New York (1973). 16. Mendecki, J. Se Yong, L & Brawer-man. G, Biochemistry 1I (1972) 792. 17. Gillespie, S, Marshall. S & Gallo, K C‘. Nature new biol 236 (1972) 227. 18. Lee, S Y, Mendecki. J & Brawerman. Ci. Proc natl acad sci US 68 (1971) 1331. 19. Collier, J R, The biochemistry of animal development (ed R Weber) p. 203. Academic Press. New York (1965). 20. Sokal, R R & Rohlf, J, Biometry. W H Freeman and Co, San Francisco, CA (1969). 21. Darnell, J E, Philipson, L, Wall, R & Adesnik. M, Science I74 (1971) 507. 22. Slater, I, Gillespie, D & Slater, D W. Proc natl acad sci US 70 (1973) 406. ‘3. Clement, A C, Experimental embryology of marine and fresh-water invertebrates (ed G Reverberi) p. 188. North-Holland, London (1971). 24. Emerson, C P & Humphreys. T, Dev biol23 (1970) 86. 25. Schultz, G, Manes. C & Hahn, W E. Dev biol 30 (1973) 418. 26. Wilt, F H, Proc natl acad sci US 70 (1973) 2345. 27. Slater, I & Slater, D W, Proc natl acad sci US 71 (1974) 1103. 28. Berg, W E & Kato, Y, Acta embryo1 morphol exp 2 (1959) 227. 29. Collier, J R, EXD cell res 21 (1960) 126. 30. Donohoo, P& Kafatos, FC, Dev biol32( 1973)224. 31. Freeman, S B. J embrvol 26 (1971) . exn. morphol 339. 32. Newrock, K M & Raff, R A. Personal communication (1974). 33. Bray, G A, Anal biochem I (1960) 279. 34. Wu, R S & Wilt, F H, Biochem biophys res commun 54 ( 1973) 704. Received December 30, 1974 Revised version received March 25, 1975