Effect of colchicine on mitosis in the neural tube of the forty-eight hour chick embryo.код для вставкиСкачать
EFE'ECZ' O F CO1,CIIICINE ON MlTOSIS IX THE NEURAI, TUBE O F THE E'ORTY-EIGHT HOUR CHICK EMBRYO' T. M. WOODARD, JR. AND SARAH B. ESTES Department of Biology, Vanderbilt University, iVashville, Tenn. INTRODUCTION OIW of t l i ~d f w t s of colchicine upon mitotic nuclei is to produce inetaphase block. A t adequate concentrations and as time goes 011 more aud I ~ O I ' C nuclei a r e thus affected, so that in counts the number. of mitotic iiuclei appears to illcrease. This increase was first interpreted by many to mean that mitosis had been stimulated. It was then realized that if all mitosis in progress at a giveii time is arrested at the metaphase ail increase might be the result simply of a n accumulation of nuclei in this phase (Ludford, ' 3 6 ) . The latter interpretation is now geiierdly accepted, but even yeceiitly certain results with colchicine on animal material have been attributed to stiniulatiou. This is the case, for example, in the chick i i i which PafY ( '39) found that one result of treating 48-hour' chick cnibryos with (.olehicine was to pi-oduee local regions of overgrowth in the neural tube. These he attributed to mitotic stimulation. It seeins desirable, therefore, to determine in a quantitative way, if possible, whether or not mitosis is stimulated by colchicine. This is especially important in view of the frequency with which colchicine is being used to arrest mitosis as an experimental method. MATERIAL AND METHODS The window tecluiic of introducing substances to the embryo chick leaves much to be desii*edin speed and in the assurance that the embryo is uniformly surrounded by the substances introduced. Although we tried this method and various niodifications of it in preliminary esperiments, it was soon abandoned in favor of the method described below which we found to be simpler and to give more uniform results. Eggs were incubated slightly over 48 hours. Each egg was then opened, and most of the contained albumen was poured into a 100-cc. beaker containing 10 cc. of a solution of colchicine in a warmed, physiThe senior author is indebted t o the Division of Natural Sciences, Vanderbilt University, for financial aid in this work. 51 52 T. M. WOODARD, JH. A N D SARAH B. ESTES ological salt solution. The albumen and colchiciiie solution were then mixed by agitation and the intact yolk and remaining albumen were added. A strip of gauze was wrapped around each beaker so a s to project over the rim, and a glass plate was rested on the edges of the cloth as a cover. Each beaker with contents was placed in a n incubator where it remained for some measured time. Embryos so treated and controls were removed from the yolks in the usual way. They were fixed in warmed B-15 fluid, dehydrated very gradually, cleared and imbedded. Sections were prepared and stained with iron alum hematoxylin. Counts were made of the number of nuclei in each of the phases of mitosis and of the number of resting nuclei. A modified Howard micrometer disc ruled into nine squares was used in counting. Counts were recorded for each micrometer area, and these were combined at random until a total number of nuclei near 2000 was reached. These totals were then used as samples in determining the standard deviations and were further combined for presentation. Special care was taken to make counts here and there over the neural tube, since it is known (Derrick, '37) that the mitotic index varies widely in different regions. The method described above of maintaining embryos during the experimental period must furnish conditions which are essentially normal well beyond the duration of the experiment ( 3 hours), for controls (10 cc. of salt solution plus albumen) were normally developed after 24 hours. Incidentally several embryos were carried successfully well beyond this period, even up to 2 and 3 days. Concentrations of colchicine ranging from 0.0078 mg., to 1.0 mg., per embryo were tried in preliminary experiments. At all of these concentrations effects on mitosis appeared as early as 20 to 30 minutes after treatment had begun. Since it was not desired to carry these experiments beyond a few hours, that dose was selected (1.0 mg.) which produced complete block of most nuclei within 3 hours. All of the data here recorded are based upon embryos treated with this amount for. periods of time ranging from 40 minutes to a maximum of 3 hours. OBSERVATIONS A N D DISCUSSION The results of our experiments are shown iit table 1. Considering the percentage of each phase of mitosis referred to the total number of iiuclei counted, the data show clearly that there is no increase in the percentage of any phase except metaphase. If colchicine acted as a stimulant there would have to be an increase in all phases of mitosis. The increase in nictaphase can be entirely accounted for in terms of 53 EFFECT OF COLCHICINE O N lllITOSIS accumulation of arrested cells. Thus, anaphases 2nd telophases have almost completely disappeared after 40 minutes, while at this time theper cent of prophases is still almost unchanged, indicating that cells are still coming through prophase to metaphase at the normal rate. The increase in metaphase during these 40 minutes, from 2.4% to 10.6%, represents the nuclei which during this time have entered metaphase. TABLE 1 Effect of 1.0 wig., of colcliicine on mitosis in the neural tube of the 48-holrr chick. The percentage of each phase of ~tittosisis based on the total n z c ~ n b ~ofr nnclei c o w t e d . I n some C U S P S only prophases were counted an certain samples, so that the s u m of the percentages o f each p?&uscmay not always eqical the nzitntic index. TIME TREAThn Control 40 minutes 1hour lthour 2hours Bhours NUMBS11 OF NUCLF,I COUNTXD NUMRHR OF MITOTIC NUCLEI 6363 14534 3928 12816 19074 19579 598 1989 627 1670 3240 3977 MITOTIC INDEX ST A NDAH n DSVIATION 9.3 13.6 15.9 r 1.1 13:O 17.1 20.2 -t 3.8 & 3.1 & 4.2 * 1.1 PER CUNT PKOPHAEE 2.8 2.4 3.0 2.3 0.6 1.1 PER,C E N T P E R C E N T P S K C B N 3 MEFAPHARE 2.4 10.6 8.4 9.2 15.8 20.5 ANAPHASE TELOPHASE 0.2 0.0 0.0 0.0 0.0 0.0 4.6 0.3 0.0 1.4 0.1 0.0 In view of tile results of these experiments the rcgious of excessive overgrowth ill the neural tube described by Paff ('39) a i d observed in our material also must be attributed to some other cause tliaii colr11ic:ine stimulation. Sauer ( ' 3 5 ) has shown that in the neural tube epithelium nuclei migrate from the peripheiy into the germinal layer where they then undergo division, returning then after completing division. Since colchicine arrests the division at metaphase, it may be that the return of resting nuclei is also prevented but their migration into the germinal layer is not. I f this be true, nuclei would then tend to accumulate in the germinal layer producing the characteristic swellings or regions of apparent overgrowth. SUMMARY 1. Chick embryos of 48 hours iiicubatioii were treated with 1.0 mg., of colchicine and allowed to continue development for different lengths of time. Counts were then made of the number of resting and mitotic nuclei in the neural tube. 2. The mitotic index rises to a maximum of 20.5% in 3 hours. The percentage of each phase of mitosis, except that of metaphase, eventu-ally declines. The rise in metaphases can be accounted for in terms of the accumulation of nuclei i n this phase. It is concluded that colchicine at this concentration acts only in this way and not as a mitotic stimulant. 54 T. M. WOODARD, JB. AND SARAH B. ESTES LITERATURE CITED 1937 An analysis of the early development of the chick by means of the mitatic index. J. Morpli., vol. 61, pp. 257-284. LUDFORD, R. J. 1936 The actioii of toxic substances upon division of normal aiid malignaiit cells. Arch. exp. Zellf., vol. 18, pp. 411-441. PAFF,G. H. 1939 Action of eolehieine upon the forty-eight hour chick embryo. Am. J. Anat., V O ~64, . pp. 331-348. SAUER, F. C. 1935 Mitosis in the neural tube. J. Comp. Neur., vol. 62, pp. 377-405. DERRICK, G. E.