Nucleic Acid and Histone Synthesis by Ethanol-Treated Cleavage-Arrested Sea Urchin Embryos

Differentiation

Differentiation (1982) 23 :25-28

(1, Springer-Verlag lYXZ

Nucleic Acid and Histone Synthesis by Ethanol-Treated Cleavage-Arrested Sea Urchin Embryos John W. Brookbank Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville 3261 1, USA

Abstract. It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanoltreated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20-30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.

Introduction Earlier studies have shown some degree of gene activation in sea urchin fertilized eggs prevented from normal cell division by low temperature [2, 31. These arrested embryos continued to replicate DNA. During the first 48 h of 'development', only 4 s RNA was synthesized. Later (48-96 h), the eggs began synthesis of 18, 25, and 28s RNA. These inhibited embryos also made the programmed switch in H i histone synthesis characteristic of normal development [4]. Developmental events are considerably delayed by use of low temperature, 'making temporal comparisons between control and cleavage-arrested embryos difficult. It was subsequently discovered that mixtures of isosmotic ethanol (1 M) in Millipore-filtered sea water (MSW) inhibited cytoplasmic division but allowed DNA replication to continue, albeit at a slower rate than controls. The effective ethanol concentration is species dependent. The DNA/embryo, assessed by microphotometry, is very sensitive to ethanol concentration, and the response of individual fertilized eggs in terms of amount of DNA made at a given ethanol concentration is variable. Some changes are none the less observed in ethanol-treated cultures which parallel morphologic and biochemical changes of control cultures.

Methods Sea urchins from the Gulf coast of Florida (Lytechinus variegatus = LL) and from the southern California coast (Strongylocentrotus purpuratus = S S ) were used in various phases of this study. Gametes were obtained by KCl injection. Cultures were kept at 20" C, and were covered and stirred in 1 rps. Streptomycin sulfate at 200 pg/ml was present in all cultures. Higher alcohol concentrations were required for inhibition of LL cell division (0.32 M ethanol in Millipore-filtered sea water) than for SS (0.25 M ethanol in Millipore-filtered sea water). Samples for Feulgen microphotometry were fixed in ethanol: acetic acid (3 : 1). Details of hydrolysis and microphotometry were as described previously [3]. Samples for Epon embedding were fixed in glutaraldehyde-sea water and postfixed in osmium [6]. Tritiated uridine (60Ci/mM; 10 pCi/ml) was added to the SS cultures to label RNA synthesis. RNA from these cultures was phenol extracted and analyzed by sucrose density gradient centrifugation [2]. Histones were extracted as described previously [4]. Samples were run on 13.5% acidurea gels (37.5: 1, acrylamide:bisacrylamide) and on sodium dodecyl sulfate (SDS) gels with the same acrylamide composition [8]. Gels were stained with Coomaisse brilliant blue R or with the more sensitive silver method of Merril et al. [9]. Results

Eggs of LL, fertilized and treated with 0.32 M ethanol in Millipore - filtered sea water, sometimes 'hatch' from their fertilization membranes and develop small numbers of cilia. Control embryos the same age (24 h) are hatched and beginning gastrulation. A plot of DNA/embryo as percent of control values versus ethanol concentration for inhibited embryos is shown in Fig. 1. At 0.32 M ethanol the mean DNA per fertilized egg is 25-30% of the untreated control. Feulgen-stained whole mounts (Fig. 2A and B) reveal DNA distributed in central nuclear bodies and in dispersed form throughout the cytoplasm in ethanol-arrested embryos (A). Under the conditions of hydrolysis and staining used in this and earlier studies, the cytoplasm of unfertilized eggs (as well as the sperm midpiece) are Feulgen negative, eliminating mitochondria1 DNA from the quantitative data. Controls (B) contain approximately 900 genome equivalents of DNA at this time (24 h); ethanol-treated embryos 0301-4681/82/0023/0025/$01.OO

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% 1 M ETHANOL IN SEA WATER

Fig. 1. Plot of DNA per embryo as a function of ethanol concentration in cultures of LL embryos grown at 2 0 C for 24 h postfertilization. Vertical bars indicate standard deviation from mean. Each point represents 10 determinations

Fig. 2. A Feulgen stained whole mount of LL embryos at 24 h postfertili7;ltion, 0.32 M ethanol in sea water. Dark Feulgen-positive stain in periphery as well as in more central areas. B Control culture Tor ‘A’. Early gastrulae, late blastulae, about 900 genome equivalents per embryo. In A and B, the central black bar=50 pm. C Thin (1 pm) section of embryo from culture shown in ‘ A ’ (Richardson’s stain). Peripheral nuclear body showing two nucleoli. D Control culture ‘ B ’ thin section, including archentcron only of early gastrula. Nuclei contain one or two nucleoli. In C and D. the central black bar= 12 pm

contain about 200-300 genome equivalents. At higher magnification of 1 micron sections (Fig. 2C and D), the arrested embryo shows a nuclear body with two nucleolar structures located in the periphery. The control earyl gastrula (Fig. 2D) has nuclei of uniform size containing one or two nucleoli. The nucleoli have been described previously [7] and make their appearance at the beginning of gastrulation (22-24 h after fertilization). Bodies resembling nucleoli have been observed during early cleavage [q.These structures differ from later nucleoli in that the latter possess more of a granular component in addition to amorphous fibrous material prevalent in the nucleoli of cleavage stages. Electron micrographs of normal (A) and ethanol-arrested LL embryos (B) are shown in Fig. 3. The normal gastrula nucleolus is granular and compact, whereas the nucleolus of the arrested embryo is somewhat more diffuse, but still has the granular elements characteristic of nuclcoli of later stages. The ‘cell’ in Fig. 3B is about the same size as the nucleus of Fig. 3A, and probably arose by a process of blistering of the plasma membrane rather than by a normal mitosis. Mitochondria of the arrested cell are about the same diameter as those visible in cross-section in Fig. 3 A. The interior of the cytoplasm of the arrested ‘cell’ is filled with membranated vacuoles which might have originated as inward tunnels from the plasma membrane. Nuclear pores are evident in the nuclear membrane of the ethanoltreated embryo, but appear less frequently in the section of the control nucleus. Feulgen-stained whole mounts of arrested embryos show localization of DNA in the periphery as well as in the central nuclear bodies, and autoradiographs of 3H-uridine pulsed cold-inhibited embryos reveal an intense incorporation of uridine in the peripheral (perhaps cortical) area of older cold-arrested embryos [2]. A similar pattern of uridine incorporation has been observed in ethanol-treated SS embryos (see below). The localization of labeled uridine follows the same pattern as the Feulgen stain shown in Fig. 2A. Sucrose density gradient centrifugation analysis of RNA synthesis was performed on cultures of SS embryos. These embryos were grown at 15-17” C, with some cultures exposed to 0.25 M ethanol in Millipore filtered sea water to inhibit cleavage. Control and ethanol cultures were pulsed for 2 h prior to sampling with 10pCi/ml 3H-uridine. Samples were taken at 17 h (controls are unhatched blastulae) and at 41 h (controls are early gastrulae), phenol extracted, and the RNA layered over 5-20% linear sucrose gradients. At 41 h, the DNA per embryo of ethanol-treated embryos was estimated by microphotometry to be 20% of the control values (at 41 h, controls range between 400-500 genome equivalents per embryo). The results of sucrose gradient experiments are shown in Table 1. Based on the A,,, ratios of the 28S/18S rRNA peaks, little degradation took place during the isolation of RNA. The specific activity of the 28s peak is lower than that of the 18s peak by a factor of about 5. The departure from a 1 : 1 ratio may be related to the 2 h labeling time, which may not have allowed for complete incorporation of the uridine label into the 28s rRNA. Changes in synthesis of H1 histone are presented in Fig. 4. In normal development of LL embryos, there is a shift from a single slow component to two faster components at the late blastula stage as resolved on acid-urea gels. The earlier H1 is conserved during later development. On SDS gels, the earlier H1 is the smaller, more rapidly

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Fig. 3. A Electronmicrograph of LL late blastula nucleus (24 h). Central granular nucleolus and scattered chromatin; mitochondria in cross-section outside nuclear membrane. Bfack bar =0.5 pm. Arrow indicates nuclear pore. B Electronmicrograph of ethanol-arrested egg. Single peripheral ‘cell’ showing central nucleus with membrane and nuclear pores; central nucleolus. Vacuolated cytoplasm containing longitudinal sections of mitochondria. Black bar = 0.5 pm. Arrow indicates nuclear pore

Ethanol (17 h)

(41 h)

migrating component, followed at the late blastula stage by a single, slower H1. These Hl’s have been termed H l m (early) and H l g (late) by other workers [lo]. As can been seen in Fig. 4, the ethanol-treated embryos do make the shift from H l m to Hlg. In contrast with controls, the Hlg is present as an unresolved single band on acid urea gels, which seems to migrate with the faster H l g present in controls. The SDS gels show a single H l g band in both elhanoltreated and control embryos.

32 5

16 26

Discussion

5 1

51

Table 1. Specific activity (cpm/A,,,) x Size analysis of total RNA extracted from control and ethanol-arrested cultures of SS embryos. Results expressed as specific activity (cpm/A,,, x of fractions collected from linear 5 2 0 % sucrose gradients. In the absence of an internal standard, the rRNA peaks are assumed to fall at 18 and 28S, as found in eucaryotic systems. Values represent mean of three experiments RNA

peak 4s 18s 25s 28s

Control (17h)

23 14 16 3

Control (41 h) 32 60 111 9

Ethanol

6

In normal sea urchin development, there is an early period of dependence on stored maternal messages, with some contribution from the zygote genome as regards histone synthesis. Later development at the mesenchyme blastula and early gastrula stages is characterized by increased synthesis

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Fig. 4. Polyacrylamide gel electrophoretic patterns of histones from control and cleavage-arrested cultures (LL). Upper Acid-urea gel, 13.5% acrylamide. Lanes 1-3, early blastula histones; lanes 4, 5, gastrula histones; lanes 68,pluleus histones; lanes 9. 10, histones from 24 h culture in 0.32% ethanol; lanc 11. control cultures at 24 h. H l m and Hlg indicated by ‘m’ and ‘g’ (Coomaisse stain). Lower SDS gel, 13.5% acrylamide. Lanes 1-3, early blastula histones; lancs 4 4 , late blastula histones; lanes 7-9, gastrula histones; lanes 10-12, pluteus histones; lanes 13, 14 histones from 24 h, 0.32 M ethanol-treated cultures; lanes 15, 16, histones from 24 h control cultures. Hlm and g indicated (silver stain)

of high molecular weight heterogeneous RNA and by the synthesis of rRNA associated with newly formed nucleoli. The Hlg histone is translated from newly synthesized message derived from the zygote genome [l, 51. It appears that the ethanol-arrested embryos are ‘attempting’ to follow some of the normal developmental program. Hatching of cleavage-arrested embryos may or may not indicate translation of mRNA for the hatching enzyme. The culture medium and stirring procedure during the experimental period could have caused softening of the membranes and subsequent mechanical removal. The appearance of small numbers of cilia over the surface of the arrested embryos is an indication of microtubule assembly. Table 1 provides evidence that ethanol-treated embryos increase their transcription of higher molecular weight RNA, as do sea water controls. These RNA species represent mainly rRNA. That other species of RNA are also transcribed in older cleavage-arrested embryos is shown by the appearance of the H l g histone characteristic of late blastula-early gastrula control embryos. H l g has been shown to be under transcriptional control. The increased specific activity in the 25s region of the sucrose gradients is not understood at the time of writing, but has been observed by others (Whitely AH, personal communication). Ultrastructural observations show that nucleoli make their appearance in ethanol-arrested embryos at about the same time as they appear in control embryos. Since these organelles are associated with rRNA synthesis, their appearance correlates with the data presented on RNA synthesis. The results reported here indicate that these pro-

grammed changes in transcription occur in a precise temporal sequence regardless of whether or not normal morphogenesis is taking place. Further, this report indicates that the total amount of DNA/embryo does not correlate with programmed changes in RNA and histone synthesis. Previous results [5] do show that near complete inhibition of DNA synthesis by cordycepin does result in the failure of those embryos to make the shift in H1 histone synthesis. Earlier experiments with cold-inhibited embryos [4] are in agreement with results reported here, but because of the protracted time required for changes to occur in cold-inhibited embryos, correlations with time between events occurring in control and arrested cultures could not be made. In experiments with cold-arrested embryos, DNA/embryo was the developmental parameter used to compare cultures growing at the control temperature of 20°C with those at 10-11” C. To facilitate comparisons, cold cultures were allowed to develop until they had reached the DNA/embryo level displayed by controls, regardless of time required. The observed changes in RNA and histone synthesis taking place in cold-arrested embryos were thus automatically correlated with DNA/embryo values by experimental design. In the present experiments with ethanol-inhibited embryos, time was used as the correlating parameter. After equivalent development times at the same temperature, it was observed that even though the inhibited embryos lagged behind controls in DNA/embryo values, changes in the pattern of transcription still took place at the appropriate time set by control cultures. Acknowledgements. This research was supported in part by grant GM 22290 from the National Institutes of Health. The technical assistance of Ms Susan DeCostanza is gratefully noted. Contribution no. 3460 from the Agricultural Experiment Station, Institute of Food and Agricultural Sciences, University of Florida.

References 1.Arceci RJ, Senger DR, Gross PR (1976) The programmed switch in lysine-rich histone synthesis at gastrulation. Cell 9: 171-178 2. Brookbank JW (1976) DNA and RNA synthesis by fertilized, cleavage-arrested sea urchin eggs. Differentiation 6: 33-39 3.Brookbank JW (1976) DNA synthesis by hybrid echinoid

embryos produced from parent species of low and high temperature tolerance. Differentiation 5 :9-1 3 4. Brookbank JW (1 978) Histone synthesis by cleavage-arrested sea urchin eggs. Cell Differentiation 7: 153-1 58 5. Brookbank JW (1980) Effects of cordycepin and cell dissociation on the synthesis of H1 histone by sea urchin embryos. Cell Differentiation 9 :3 1S 3 2 1 6. Conway CM, Metz CB (1974) In vitro maturation of Arbuciu punctulatu oocytes and initiation of heavy body formation. Cell Tissue Res 150 :271-279 7. Karasaki S (1968) The ultrastructure and RNA metabolism of nucleoli in early sea urchin embryos. Exptl Cell Res 52: 13-26 8. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680-685 9. Merril CR, Boldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerbrospinal fluid proteins. Science 21 1 :1437-1438 10. Ruderman JV, Gross PR (1974) Histones and histone synthesis in sea urchin development. Develop Biol 36: 286-298

Received December 1981 / Accepted in revised form July 1982