p)robably reflects seasoinal variations in egg populations. No results were considered unless

Transcription

1 MITOTIC ABNORM1ALITIES IN SEA URCHIN EMBRYOS EXPOSED TO DACTINOMIYCIN* BY BARRY I. KIEFER, CHARLES F. ENTELIS, AND ANTHONY A. INFANTE DEPARTMENT OF BIOLOGY, WESLEYAN UNIVERSITY, MIDDLETOWN, CONNECTICUT Communicated by Daniel Mazia, August 20, 1969 Abstract-Dactinomycin at concentrations of 10, 20, 25, and 50 /AOg/ml causes mitotic abnormalities in sea urchin embryos. Sister chromosome separation is impaired and anaphase bridges are frequently formed. The result is an unequal distribution of the chromosome complement to daughter cells. Possible explanations are discussed. Current hypotheses regarding the relation of synthetic events during the earliest stages of embryonic development to subsequent embryonic differentiation have been largely based on experiments using Dactinomycin (Actinomycin-D) as an inhibitor of DNA-dependent RNA synthesis. In studies on sea urchin embryos, the fundamental observation is that in the presence of Dactinomycin fertilized eggs undergo delayed but regular cleavage to produce blastulae which, at the gross level, appear morphologically normal -yet fail to gastrulate.1-4 The general conclusion from this observation is that the RNA normally synthesized during cleavage is primarily concerned with events occurring after the blastula stage.4 Recent evidence suggests that some of the RNA synthesized during cleavage acts as template for the synthesis of chromosomal histones, and that this synthesis can be abolished by Dactinomycin.5 These findings taken together raise the following question: If the histones being synthesized during cleavage under the direction of newly synthesized RNA are being incorporated into and are necessary for the structure of the rapidly replicating chromosomes, how can the mitotic behavior of these chromosomes (hence, cleavage) be normal in the presence of Dactinomvcin? The evidence presented here provides a simple answer: It isn't. Materials and Methods.-Eggs of Strongylocentrotus purpuratus and Lytechinus pictus (Pacific Bio-i\Iarine Supply Co., Venice, California) were obtained, fertilized, and developed as described by Nemer and Infante.6 Each batch of eggs was divided into groups treated with 10, 20, 25, or 50 /Ag Dactinomycin (MNerck, Sharp and Dohmne) per milliliter synthetic sea water with one group serving as a control. The eggs were exposed to Dactinomycin for from 10 min to 1 hr before fertilization, or immediately after fertilization, and remained in this medium throughout the experiment. The eggs of S. purpuratus did not divide when preincubated in 25 or 50,ug/ml Dactinomvcin and in further experiments these concentrations were administered only after fertilization. The inhibition of cleavage by pre-incubation at these concentrations was not anticipated,'-3' and most p)robably reflects seasoinal variations in egg populations. No results were considered unless there was a 95% or better fertilization and normal development to at least the hatched blastula in the controls. Samples were taken at intervals of 120 to 720 mmi after fertilization, processed for microscopy using the ethanol-acetic acid method of Alazia,8 and examined with a Nikon S-Ke microscope fitted with phase contrast optics. Results.-Cleavage abnormalities were observed in all experiments with Dactinomycin. The bulk of the observations were made on the eggs of S. pur- A.57

2 8fi8 ZOOLOGY: KIEFER ET AL. PROC. N. A. S. puratus. A second species of s4ai urchin, L. pictus, was utilized primarily to be certain that our results were not due to species or population specific phenomena. The results were essentially the same for both species although the degree of visible effect was less in eggs of L. pictus than those of S. purpuratus. The influence of Dactinomycin on cleavage is twofold. First, there is either a delay or a complete inhibition of cleavage depending on the concentration and length of exposure. This is a common observation'-4, and will not be dwelt on here. Secondly, there are mitotic abnormalities, the most obvious of which is the abnormal separation of the chromosomes during anaphase. In this respect, the results reported here are essentially identical to those obtained by Karnovsky and Simmel and by Mazia and Gontcharoff in BUdR-treated sand dollar8 9 and sea urchin10 embryos. The disturbance of sister chromosome separation can be described as a tendency toward "stickiness" which is expressed in the form of anaphase bridges (Fig. 1). In some cases the bridges appear to consist simply of extremely stretched chromosomes. The more common situation is that in addition to the stretched chromosomes, large portions of chromosomes apparently fail to' separate and remain at the equator. In either case, the bridge may break before or during cytokinesis (Fig. 2), or remain intact after the cleavage furrow has divided the blastomeres. In the latter case, it appears that the furrow constricts the unseparated chromosome mass but cannot pass through it (Fig. 3), resulting in bridged nuclei (Fig. 4). These nuclei proceed through the next mitotic cycle, resulting in a severely disturbed anaphase. It is quite obvious that the chromosome complement is not being equally distributed to daughter cells, and in late-cleavage-stage embryos, large amounts of chromatin "debris" are often seen (Fig. 5). The frequency with which these chromosome abnormalities are observed is dependent upon the concentration of Dactinomycin and the length of exposure and ranges from roughly 5 per cent (one mitotic figure out of 20 observed is abnormal) at 10,ug/ml to over 50 per cent at 50 Ag/ml for 4 to 8 cell embryos. The latter class shows about 90 per cent abnormal cells by the late cleavage stages. These values vary from one batch of eggs to the next. Additionally, there are cleavage irregularities of a more general nature (Fig. 6). These are most likely a direct result of the mitotic disturbance. Among the treated embryos there is a comparatively high incidence of odd-numbered cell stages. This is most obvious during the second and third divisions. Also observed are blastomeres of unequal size, nuclei of unequal size, and two nuclei in one cell. Discussion.-The evidence reported here indicates that the cleavage of sea urchin embryos in the presence of Dactinomycin can no longer be considered normal. The premise that one can inhibit most of differentiation with Dactinomycin without inhibiting cell division4 must at least be modified. In attempting to explain our results we make the assumption that the Dactinomycin is, in fact, getting into the cells, and that the observed abnormalities are related to the binding of the Dactinomycin to DNA', 12 This assumption is contrary to the report of Thaler, Cox, and Villee. 13 On the basis of studies on the rate of uptake of Dactinomycin-14C, these authors conclude that the antibiotic is

3 ~~~~~~~~~~~~~~~~~~tro *W _ Q ' :: ' s K W VOT.. 64, 1969 ZOOLOGY: KIEFER ET AL aa C I 40 kr4o *'...A~~~~~~~~~~~~~~ib.:.A PI..-.. *sikw ' > *s~re~ 5 -ik r = r_= _e;tri....'. x;;.w ' - : y.*.,x, ~~4 - ~ ~ ~.t C as i ses i M ;: 0t; 2.; s "A Xs e.:. \~~~~~~~~~s ETshb 4; l'ik r~ju:u a t 1 U E W I X 0f L' t; ; '? "; $ Ct 4. FIG. 1.-Examples of anaphase bridges. (a) Anaphase of second cleavage, S. purpuratus. In addition to stretched chromosomes, a mass of chromatin is being left at the equator (arrow), X 1302; (b) S. purpuratus, X 3255; (c) L. pictus, X FIG. 2.-Anaphase bridges broken either before or during furrow formation. The cells pictured are entering prophase of the division following the formation of the bridge. In all cases, the material which comprised the bridge remains extended; S. purpuratus, X excluded from eggs and embryos of Arbacia punctulata until the blastula hatches. The validity of this conclusion in light of considerable evidence of the inhibitory effect of Dactinomycin on RNA synthesis in cleaving sea urchin embryos'-4 remains unclear. The concentration and exposure effects observed in the present study are consistent with the findings of Gross and Cousineaul, 2 and others

4 860 ZOOLOGY: KIEFER ET AL. PROC. N. A. S. that the degree of inhibition of RNA synthesis by Dactinomycin is increased with increased dosage. It has been suggested that these data probably reflect a very low permeability of the cell to Dactinomycin. I Although it was the possibility of the absence of histone synthesis' which essentially led to this study, the abnormal chromosome behavior reported here t ml '' AM div.. _ FIG. 3.-Anaphase bridges which remain intact after furrow formation. In the region of the furrow, the mass of chromatin is gieatly constricted and arranged symmetrically on either side of the furrow. (a) and (c) The region of the furrow (riot visible) is indicated by the arrow, S. purpuratus; (a) X 1302; (c) X3255. (b) An extiemely thin strand connects the chiomatin mass on each side of the furrow, S. piirpiiratfus, X FIG. 4.-Bridged nuclei, S. purpuratuis. (a) Interphase; (b) Prophase. In both figures the other pair of nuclei have separated normally, X 1302.

5 VOL. 64, 1969 ZOOLOGY: KIEFER ET AL. 861 FIG. 5. Chromatin masses in late cleavage stage of Dactinomycin-treated S. purpuratus embiyos. (a) X319; (b) enlarged chiomatin mass seen in (a) (arrow), X FIG. 6.-Examples of irregular cleavage planes. The upper embryo contains cells of unequal size. The lower embryo has been partitioned into three cells but contains at least five nuclei, three of which are grouped together (arrow), S. purpuratus, X319. could be the result of any one (or all) of three functionally distinguishable effects of Dactinomycin:'1' 12 interference with RNA and concomitant protein synthesis, interference with DNA synthesis, and a physical alteration- of DNA (chromosome) structure. A consideration of any of these possibilities is hampered by the scantiness of information on the role of the constituents of chromosomes in their organization and behavior. However, it is obvious that chromosomal proteins must play an important part and that these proteins increase during cleavage. This suggests that the observed abnormalities may be due to the inhibition of the mrna necessary for the synthesis of chromosomal proteins (e.g., histones5). If this were the case, it would mean that both new mrna and its protein product are needed for normal development as early as the first cleavage division. However, if these new proteins are chromosomal proteins, this cannot be taken as an event of differentiation (as carefully defined by Gross4) unless the synthesis of these proteins is qualitatively different from one blastomere to the next-an intriguing, but unlikely, possibility. Therefore, these findings alone would not significantly alter the generalizations that the events up to the blastula stage are primarily under the control of maternal molecules, and that the mrna synthesized during cleavage is primarily concerned with events after the blastula stage. While the second possible explanation of the observed abnormalities, that of the interference with DNA synthesis by Dactinomiycin, seems least likely in view of the apparently negligible effect of the antibiotic on DNA polymerase, 11' 12 such an explanation cannot be eliminated until more detailed information is available for this system. Lastly, the fact that Dactinomycin and BUdR'0 cause the same chromosomal abnormalities in sea urchin embryos suggests that these abnormalities may be due to a common effect of the two agents. The possibility that the alteration of the

6 862 ZOOLOGY: KIEFER ET AL. PRoc. N. A. S. physical properties of DNA by BUdR could be responsible for an impairment of chromosome behavior has been proposed by Mazia and Gontcharoff.8 It follows that the results reported in the present communication may be due to a direct effect on chromosome structure by the physical binding of Dactinomycin to DNA rather than an indirect effect on RNA or DNA synthesis. The relevance of these studies to those using Dactinomycin on other cell systems remains to be determined. * This work was supported in part by U.S. Public Health Service grants GM (B.I.K.) and HD (A.A.I.), and grant E-537 (A.A.I.) from the American Cancer Society. 1 Gross, P., and G. H. Cousineau, Biochem. Biophys. Res. Commun., 10, 321 (1963). 2 Gross, P., and G. H. Cousineau, Exp. Cell Res., 33, 368 (1964). 3 Giudice, G., V. Mutolo, and G. Donatuti, Wilhelm Roux' Archiv, 161, 118 (1968). 4Gross, P., in Current Topics in Developmental Biology, eds. A. A. Moscona and A. Monroy (New York: Academic Press, Inc., 1968). vol. 2, p Nemer, M., and D. T. Lindsay, Biochem. Biophys. Res. Commun., 35, 156 (1969). 6 Nemer, M., and A. A. Infante, Science, 150, 217 (1965). 7 Infante, A. A., and M. Nemer, these PROCEEDINGS, 58, 681 (1967). 8 Mazia, D., and M. Gontcharoff, Exp. Cell Res., 35, 14 (1964). 9 Karnovsky, D., and E. Simmel, Progr. Exp. Tumor Res., 3, 254 (1963). 10 Gontcharoff, M., and D. Mazia in preparation. 11 Reich, E., and I. H. Goldberg, in Progr. Nucl. Acid Res. Mol. Biol., ed. J. N. Davidson and W. E. Cohn (New York: Academic Press, 1964), Vol. 3, p Waring, M. J., Nature, 219, 1320 (1968). 13 Thaler, M. M, M. C. L. Cox, and C. A. Villee, Science, 164, 832 (1969).