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Fission after division primordium removal in the ciliate Stentor coeruleus and comparable experiments on reorganizers
Experimental
357
Cell Research 42, 357-370 (1966)
FISSION AFTER DIVISION PRIMORDIUM REMOVAL IN THE CILIATE STENTOR COERULEUS AND COMPARABLE EXPERIMENTS ON REORGANIZERS V. TARTAR* Department
of Zoology,
University
of Washington,
Sea&e, U.S.A.
Received September 17, 1965s
and cytoplasmic division in many ciliate protozoa, including Tetrahymena and Stentor, is preceded by the formation of an oral primordium called the division primordium which produces the new set of feeding organelles for the posterior daughter cell (opisthe). Development of these organelles is completed as the fission line appears and, in constricting, separates them into the opisthe. Two methodological consequences are, first, that in larger ciliates such as Stentor incipient dividers can be recognized easily in live samples as large individuals with early division primordia; second, that a major morphogenetic process of organelle elaboration preceding and in part overlapping fission potentially may be used as an indicator of the condition of a cell before and during division. Thus in Tetrahymena both division primordium development and fission are blocked by divisionsynchronizing thermal shocks and by a variety of chemical inhibitors. Therefore it has been suggested that the substances or syntheses or processes which support division primordium development also provide the basis for formation and furrowing of the fission line, or that the primordium development triggers or may be necessary for the fission [5, 21, 22, 241. Comparable data are not yet available for Stentor but its large size permits one to excise the division primordium at any stage. What then occurs can be fruitfully compared with the performance of reorganizers and regenerators after the same operation. In the process called reorganization, as in division, an oral primordium is produced although a functional set of feeding organelles is already present, in medium-sized or even small cells as well as large ones which have completed their normal growth. No fission line appears, and the developing primordium uselessly replaces most of the original NUCLEAR
1 This work was suunorted bv research mant No. CA-03637-CB from the National Cancer Institute, 1J.S. Public &alth Sercice. a Address: U. of Wash. Field Lab., Nahcotta, Washington, U.S.A. 98637. s Revised version received January 15, 1966. Experimental
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ingestive apparatus which is selectively resorbed. Because this process usually accomplishes no apparent purpose it has remained paradoxical [ 161 but a new possible explanation was suggested by the present experiments as discussed later. In oral regeneration after excision of some or all of the feeding organelles, the oral primordium quite understandably forms a new set. The primordium and its development is the same in all three processes; and in all, the beaded macronucleus temporarily coalesces at the time of gullet formation. In dividers only, the compacted macronucleus is then pinched in two by the advancing furrow. Mitotic division of the many micronuclei occurs during regeneration [6] and reorganization [ 113 as well as division, but emicronucleation of stentors has proved that these nuclei are not essential to any of these processes [ 111. The predecessor of this investigation is to be found in the 1951 pioneering experiments of Weisz [19] who reported that S. coeruleus can go on to form a fission line and divide after removal of late-stage division primordia. I confirmed this in 1959 [ 151, adding that the macronucleus can also be removed just prior to the appearance of the fission line without precluding division. Using Glaucoma which is closely related to Tetrahymena, Frankel [3, 41 found that cytokinesis could be completed although the division primordium failed to develop, either because it was destroyed by a UV microbeam or because in certain strains it spontaneously regressed. Dissociation of division processes in Tetrahymena was obtained by Mita, Tokuzen, Fukuoka, and Nakahara [9] when treatment of division-synchronized cells with the carcinogen 4nitroquinoline-l-oxide resulted in cytokinesis without macronuclear division, yielding one anucleate daughter cell; but the fate of the division primordium was not followed. In Hanson’s [7] double Paramecium, fission could occur on the side which produced no division primordium because of UV microbeam irradiation as well as on the opposite side that did, such doublets dividing as one. In Wise’s [23] recent study of Euplotes, however, localized irradiation of the division primordia before appearance of the fission line was followed, especially in early-stage dividers, by resorption and re-formation of the primordia in delayed division, or, especially in later-stage dividers, by uninterrupted division with defective feeding organelles produced by the persisting, injured primordia, but never by division without development of division primordia. As contributing to a further analysis of cell division in ciliates, the principal object of the present experiments was to determine whether stentors could continue on division course after excision of early stage division primordia.
Experimental
Cell Research 42
Division
MATERIALS
359
in Stentor
AND
METHODS
The animals used were from several strains of S. coeruleus growing vigorously on a mixture of bacteria, flagellates, smaller ciliates, and rotifers. Dividers were abundant about 12 hr after re-nutrification of the cultures with skimmed milk and were identified as large cells with primordia visible by reflected light from an embryological lamp. Reorganizers were identified as medium-sized cells with primordia, and almost all controls did use the anlage to replace the original mouthparts instead of dividing. Some specimens were induced to begin reorganization by placing them overnight in a viscous solution of methyl cellulose [16]. This solution was also used to quiet animals for excising the primordium with a glass needle, and washed off afterwards. Each specimen was isolated into 3 drops of culture medium on a depression slide and followed with highest magnification of the dissecting microscope.
RESULTS
Excision
of the Division
Primodium.
Development of the oral primordium in Stentor has been divided stages, each marked by a distinctly visible advancement [13, 161:
into 8
(1) first appearance of the primordium as a rift in the lateral striping: (2) beginning outgrowth of cilia, producing a distinct glisten; (3) cilia half-grown, showing random beating; (4) cilia grown to final length and organized into a row of membranelles which beat metachronally; (5) slight bend at the posterior end, which presages(6) inward coiling of the posterior end of the membranellar band to form the gullet; (7) formation of a vestibulum next to the opening of the gullet as the entire primordium bends to form a ring in(8) completion of development, with circular membranellar band terminating in the mouthparts (vestibulum and gullet) (Fig. 1). Growth and increase in length occur continuously up to the end of stage 4. A stage zero may also be defined as a localized splitting or multiplication of the striping where the beginning rift will appear. Primordium development follows the same steps in dividing, regenerating, and reorganizing stentors. When applied to the division primordium and correlated events, the 8 stages mark clearly discernible stages in the course of cell division. At stage 5 the beads of the macronucleus begin coalescing within the common nuclear membrane, and a pre-divisional separation of visible carbohydrate reserve Experimental
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granules defines the locus of the fission line [ 141. Not until the middle of stage 6 is a fission line visible, as a “white” line beginning at the anterior end of the division primordium and progressing in both directions around the cell, “transecting” the pigment stripes. In stage 7 the massed macronucleus extends to a rod, and furrowing or constriction in the fission line begins and
Fig. l.-Division after removal of earliest-stage division primordium. a, Stage-l primordium, mouthparts and primordium site excised. (Membranelles shown, only in this sketch. Mouthparts at end of membranellar band. Primordium is below the mouthparts. Reserve carbohydrate granules indicated by stippling. Beaded macronucleus to left.) b, Cell heals and forms no new primordium Beaded macronucleus; granular carbohydrate reserves at posterior pole. c, Beginning fission; nuclear beads fused in one mass; carbohydrate reserves pre-divided. d, Fission completed; daughter nuclei renodulated. e, Regeneration primordia in both cells, beginning 7 hr after operation; nuclear beads coalescing. f, Oral regeneration in both cells.
proceeds relatively rapidly into stage 8 when in final separation of the filial cells the macronucleus is pinched in two [1 1. From dividing stentors in all stages the entire division primordium (or the primordium site of stage-O dividers) was excised with some surrounding ectoplasm and endoplasm. In the results diagrammed and tabulated in Table I the important cases are those in which the primordium was removed at stage zero through stage 3, long before stage 6 in which the fission line first appears; and the following summarizing statements apply to these early dividers as well as to later stages. Most stentors (100/130 -columns 2, 4, 8) continued on the course of division and divided without a division primordium. In one-third of these 100 dividers (column 4) final separation of daughter cells did not occur because, as division progressed, the operation removing the division primordium increasingly interfered, not with fission line formation or stripe cutting but with constriction or furrowing as such. Perhaps it is significant that when the mouthparts were also removed constriction was always complete (column 8). In all 32 cases of incompleted fission a fission line was formed which transected the lateral stripes and kineties into two sets, evident both in the invariable shifting of one set with reference to the other giving a double body form and in the subsequent regeneration of many as doublets (Fig, 2). Considering these cases as essentially dividing gives a Experimental
Cell Research 42
Division
361
in Stentor
truer impression of the capability for division after primordium removal. Separately, they indicate a possible dissociability of formation and constriction of the fission line. Most stentors did not replace the excised primordium with a new one (96/103). This was not because specific inhibition by the intact feeding orTABLE
I. Behavior following removal of the division primordium or both primordium and mouthparts at different stages in division.
0
0
6 3 24 15 4 -0 0 2
13 1 0 5 4 i 0 0 13
2
0
/
0 I 1 4 10 7 9 1 I 14
12
II Ill3
lo
6 2 0
6 2
2 i I
5
1 6
7
/ 0 12 3 8 2 1
.3
ganelles prevented primordium renewal [13], because when the mouthparts were also removed the majority (16/27 -column 8) also did not form a new anlage. Nor was failure to renew the division primordium because the original primordium site was necessarily removed along with the original primordium. In Stentor the normal site is not indispensable for primordium formation; a new site is provided after removal of the old one. Thus some of the operated early dividers did replace the primordium. Moreover, in a comparative test in which primordia (and original primordium sites) were removed from 7 early regenerators in stages 1 and 2, all promptly formed Experimental
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new primordia (in 1 to 1 314 hr; average: 1 213 hr), confirming a previous study [la]. In division without a division primordium the macronucleus nevertheless performed as usual, the nodes coalescing to a single large mass which later extended to a rod form, was divided about equally between the daughter
t-13 \ C
Fig. Z.-Incompleted constriction of stentor after mid-stage division primordium removed. a, Stage-4 primordium excised. (This sketch and the following show the lateral pigmented stripes between which lie the rows of body cilia.) 6, Lateral striping cut into two sets; division products characteristically shift into heteropolar orientation, indicated bv original tail-pole (t); but cells do not separate-completely. c, Regeneration-reorganization produced; doublet-next day.
cells, and then renodulated. This was observable in 5 cases following primordium removal, but most dividers were too well-fed and opaque to see nuclear changes in the living animals. When the primordium was renewed, did the specimen employ the anlage for division or for reorganizational replacement of existing mouthparts or regeneration of mouthparts if these had also been removed? A strong tendency to persist in division was evident. ,411 cells in which the original mouthparts remained used the new anlage in delayed division even though excision had reduced the cell volume (column 7). Of those in which mouthparts were also excised, at least half nevertheless divided instead of using the new anlagen for regenerating the lost mouthparts in spite of greater reduction in volume (columns 9, 10). In contrast, data already available [13] show that if the division primordium is not excised but the mouthparts or the whole head is removed, then, before stage 3, the original division primordium was used for regenerating the missing feeding organelles in the majority of cases (1 l/13). Some specimens divided unequally, always the proter having at least twice the volume of the opisthe (13-column 3). The extent of this size difference is not explainable on the basis that the part excised-the primordium and adjacent striping-was deleted from the potential opisthe which would have received it. Were this so, all divisions should have been clearly unequal and they were not. More likely, the process responsible for the migration and pre-division of the sub-ectoplasmic glycogenoid reserves Experimental
Cell Research 42
Ilivision
in Sfentor
363
was disturbed. After this migration, early in stage 6, the posterior border of the anterior portion of these reserves defines where the fission line will soon appear [ 141. Like macronuclear behavior, pre-division of this granular material did occur in the absence of a division primordium, clearly evident from typical color differences seen on the surface of the cell (Fig. 1). The
Fig. 3.-Fission in both halves of an early, stage-2 divider split longitudinally. L, Completion of division primordium development, fission line formation, and division in left half. R, Fission line formation and constriction (dorsal and ventral views) in right half without primordium; separation of quarter-cells; and their later oral regeneration next day.
cutting operation therefore in some cases may have led to irregularity of this process, indicated by insufficient migration forward of half the reserves and resulting in the fission line forming at a level considerably posterior to where it usually appeared and hence in grossly unequal division. Finally it may be noted that nearly half the minority group of cells which did not continue on the course of division nevertheless formed a new oral primordium and divided the next day, though reduced in volume by the original excision, and even after intervening regeneration (columns 5, 6, 10, 11). This may be called postponed division [13] and might be due either to maintenance of a “decision” to divide or of the basis for this “decision” in spite of the operation and even of intervening regeneration. Another way of testing what dividers can do without the division primordium is to cut them in two longitudinally, one half bearing neither the anlage nor the original mouthparts (Fig. 3). Do such halves of dividers continue on the course of division? There was one case of a stage-l divider and 35 others distributed among stages 2 through early stage 6, all before the fission line is first visible. The results can be simply stated without tabulation. None of the aboral halves without the mouth or the primordium (R in Fig. 3) formed an oral primordium promptly or within the usual time of 3 to 4 hr after oral excision in the interdivisional period; they regenerated only much later. Four halves divided completely, separating astomatous quarter-cells; one each were from stage-4 Experimental
Cell Research 42
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364
and stage-6 dividers, but the most interesting were two at stage 2: even longitudinal-half-cells without division primordia or mouthparts and cut 4 stages before the visible appearance of the fission line could continue on the division course and divide completely. Of the remaining 32 cases all but 3 formed a fission line, sectioning the kineties and pigmented stripes into two sets, with furrowing but incomplete constriction. Considering the severity of the operation and that the halves folded on themselves in healing, these specimens also may be considered as substantially dividing. Failure of final separation of division products was probably from trivial reasons involving self-adhesion in folding, and in 6 instances later regeneration with two primordia producing bistomial stentors attested the essential doubleness achieved. In only two stage-2 and one stage-4 divider was there no apparent fission line or cutting of the lateral striping. These results are substantially in agreement with those from division primordium excisions. Regarding the partner oral halves bearing the division primordium and the original mouthparts (L in Fig. 3), 8 divided completely; 24 formed a fission line and cut the striping into two sets, furrowing but not completing constriction (further evidence that self-adhesion in folding prevented final separation, not the absence of the primordium); 2 reorganized instead, the division primordium moving forward and replacing the original mouthparts which were resorbed; and 2 merely resorbed the anlage, retaining the original ingestive apparatus. Oral halves were therefore not strikingly more successful than halves without a division primordium in remaining on the division track and accomplishing fission.
Reorganization AjIer Removal of the Reorganization Primordium Reorganizers are like dividers in forming an oral primordium though an intact oral apparatus is present, but the new organelles then move forward and replace the original mouthparts and adjacent membranellar band which are selectively resorbed. This replacement is not because the old organelles are “worn out”, as originally and frequently supposed, for one reason because replacement of newly formed feeding organelles often occurs. For example, in the division primordium excision experiments, 5 newly formed or regenerated daughter cells promptly reorganized. Beginning reorganizers were identified as medium-sized cells with primordia. Because reorganization is normally infrequent and adventitious, 12 of the specimens were methyl-cellulose-induced reorganizers as already noted. Experimental
Cell Research 42
Division
365
in Stentor
Results are diagrammed and tabulated in Table II and summarized in the following statements. Reorganizers were like dividers in generally not replacing the excised primordium with a new one before continuing on their course, likewise contrasting with comparable regenerators which replaced the anlage promptly. TABLE
II.
Behavior
following
removal of the oral primordium
7
12
1719
3
4
s
in reorganizers.
1 6
7
* All were from specimens induced to reorganize by placing them in methyl cellulose.
In only one of the 24 cases was a new primordium promptly formed before resorption of the old mouthparts (column 7). Even when the original mouths were also excised, a new primordium appeared only after about 6 hr (column 2). Intact mouthparts were resorbed even though there was no primordium to supply a replacement (column 4). Therefore the development of new mouthparts cannot be the trigger for resorption of the old ones. Resorption occurred after about the same time -comparable to reaching stage 5 -as normally and was of the same extent. Therefore the animals continued on the course of reorganization, accomplishing what they could without a primordium. In contrast, neither experimental nor control dividers ever resorbed the original mouthparts. Experimental
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366
The only exception was one case of a stage-l reorganizer (not included in Table II) in which nothing of a morphogenetic nature happened after excising the primordium. Mouthparts were not resorbed and the specimen simply returned to the trophic phase. This case simply indicates, as did a few of the dividers, the earliest stage is transitional and therefore allows the possibility that in some specimens reorganization or division may be abolished by primordium excision. After oral resorption all specimens soon regenerated (column 5) -more promptly than astomatous products of dividers without a primordium or which were conspicuously slow without the primordium and mouthparts, in regenerating missing or excised mouthparts. In 7 operated reorganizers rapid formation and early development -but not the later development -of the regeneration primordium was notable. Following regeneration, 7 stentors which had been induced to reorganize by methyl cellulose treatment promptly divided; being medium-sized, each produced two small daughter cells (column 6). This result is intriguing because in at least 2 tests methyl cellulose did not stimulate large cells to divide or reorganize. DISCUSSION
The first of the main results to be considered is that dividers are different from regenerators in generally not forming a new primordium after the original one has been excised. Therefore the effect of the operation on dividers was in most cases not nullified by prompt primordium renewal but led to the second demonstration, that S. coeruleus can divide without normally concomitant development of a division primordium. Then it was found that reorganizers are like dividers in also generally continuing on their course (eventual resorption of the mouthparts) without replacing the excised oral primordium. In the case of regenerators, prompt re-formation of an oral primordium, sometimes with appearance of a new stage-l anlage within one hour, suggests that the cell was already prepared or activated for primordium formation and development, and therefore that much of the period of 3 to 4 hr between the stimulus to regeneration and the first appearance of the original primordium was devoted to this preparation, which then did not have to be repeated for the replacement anlage [ 121. The preparation could be at least whatever is required for the production of new kinetosomes, of which the primordium consists. Certainly an early-stage divider normally adds kinetosomes with their ciliary outgrowths to the beginning division primordium which greatly Experimental
Cell Research 42
Division
in Stentor
367
increases in length and even in width. This was demonstrated both in light and EM photomicrographs of dividing S. coeruleus by Randall and Hopkins [lo] and was clearly shown in Holtz’s [S] silver-stained Tetrahymena at Then why do early dividers bereft of their progressive stages of division. primordia usually not form new ones in replacement, and is the reason a sign of some distinctive feature of the division process? Whatever the reason for the failure in primordium renewal, one may first suppose that it is the same as for reorganizers, which replace the primordium with even less frequency than dividers. This consideration would exclude that there be some common “morphogenetic-fission substance” which, after excision of the anlage, is shunted altogether toward formation of the fission line; for reorganizers of course do not make a fission line, and besides, there was no apparent acceleration in the onset of division after removal of a “competing” division primordium. Perhaps all we can say at present is that when some other process-fission or oral resorption-follows soon after primordium development, the second process is antagonistic to a repetition of the first. That stentors and other ciliates go on to divide after removal of late-stage division primordia or other interference shortly before the appearance of the fission line is to be expected and is not very interesting because it means only that the preparation for division is already so advanced as to be irrevocable. But division of stentors after removal of even the early division primordium, unrenewed, is obviously relevant to proposing any causal sequence in their division process. It makes highly dubious Weisz’s 119, Fig. 31 hypothesis that the early primordium stimulates the macronucleus to coalesce, for even after excision of the primordium at its earliest appearance the nuclear cycle remains normal. His scheme is no longer tenable for two other reasons. First, it cannot be that micronuclear mitosis induces the fission line because these nuclei, as mentioned earlier, also divide during reorganization [ 111 and regeneration [6] and because emicronucleated stentors grow and divide normally [ll]. Second, Weisz supposed that the site of the “anarchic held” (source of kinetosomes for the oral primordium) is different in location and not identical with the early primordium itself, which has never been postulated or demonof sequential strated by anyone else for any ciliate. The one explanation events in division of Stentor is therefore out of date. It would be premature to contrive another until certain further experiments on dividers which are uniquely possible in stentors have been performed. All that need be said for the present is that the development of a division primordium is not essential Experimental
Cell Research 42
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368
to nuclear or cytoplasmic division. Frankel [3, 41 found the same in Glaucoma using different methods. Even formation or initiation of a division primordium may not be essential to division in forms like Stentor and Glaucoma. It certainly is not required in the many ciliates which divide or at least form a fission line cutting in two the longitudinal ciliary rows or kineties before stomatogenesis. Such are Balantidium [2] and Colpoda [17] and necessarily is the case with these and other forms which employ the newly-cut anterior ends of the posterior halves of the kineties in the formation of feeding organelles for the posterior daughter cell. There is no apparent reason why almost all ciliates could not do likewise, for if they simply cut and pinched themselves in two transversely the opisthe would make up its organelle deficiencies by regeneration, as in the kiinstliche Zellteilung of cutting experiments. Indeed, Uhlig [18] and I have observed rare instances in S. coeruleus of division without division primordium formation, and the astomatous posterior daughters later regenerated a normal set of feeding organelles. With the exception of Frankel’s Glaucoma, I know of no other case in which the division primordium regresses, whether from injury or spontaneously, yet the cell proceeds toward and accomplishes division. In Stentor, for example, whenever a dividing cell itself removes its primordium by resorption, as in many but by no means all cases of injured dividers [20, 131, the cell then never goes on to divide, though the division process may begin again much later. These injuries need not include cutting the primordium itself to abolish division. Hence arrest and regression affect both processes in stentors as in tetrahymenas. The questions here are whether resorption (or its milder form, arrest) is another distinct and active process or is the cessation or canceling of processes supporting division primordium development and fission line formation, and what the two processes might have in common that they should be affected alike. Reorganizers are like dividers not only in escaping head dominance or specific inhibition in beginning a second set of feeding organelles, as well as in going through the cycle of macronuclear condensation and micronuclear mitosis. They also seldom replace excised early primordia, and they usually continue on course to a second process, uiz, resorbing the old mouthparts although the primordium is no longer present to supply a replacement. This is strong resemblance: and just as there can be division without a division primordium (in the above experiments), it is possible that there might be division primordium formation and development without fission, because of Experimental
Cell Research 42
Division
in Stentor
369
the evident d&sociability of these cell processes. Therefore we should consider the possibility that reorganizers may be pseudo-dividers, i.e. cells in which formation of a division primordium is somehow triggered or released although conditions for subsequent fission are not present. That such a dissociation may occur in nature-and reorganization has been observed adventitiously in many kinds of ciliates-becomes more probable when we make a reasonable guess of when the trigger for division primordium formation in Stentor is set off. In the one type of experiment in which we can elicit an oral primordium at will, without chemical agents, viz, regeneration, in which the stimulus is oral excision, the regeneration primordium appears (stage 1) 3 to 4 hr later. If dividers require the same preparatory period, and since 1 hr is required for each stage in primordium development and the fission line appears in the middle of stage 6, the stimulus for division primordium formation may be located in time at least 8 hr before appearance of the fission line in a cell whose generation time is around 48 hr. In turn, cases of grossly unequal division sometimes occasioned by primordium removal (Table I-column 3) show that it is possible for the fission line not to be determined in respect to its location or level on the cell axis until after stage 4. If this be taken as the time of beginning fission line formation, then there would be about 6 hr between the triggering of division primordium formation sufficient difference, it would and the triggering of fission line formation, seem, to allow improper linkage of the two processes. Therefore it might happen in even small or medium-sized cells that the stimulus for primordium formation may be set off without proper conditions to elicit fissioning. Conversely, in largest stentors the latter conditions may always be present, as indicated by the fact that large cells, beheaded, form a primordium and divide instead of regenerating, producing headless proters which regenerate only after the division [16]. To regard reorganizers as pseudo-dividers could explain the otherwise entirely enigmatic occurrence of reorganization [see 161 in altogether normal ciliates with new and proportionate feeding organelles and proportionate nuclei, as well as the four mentioned resemblances between organizers and dividers.
SUMMARY
2. Following excision of division primordia even at the earliest stages, stentors can go on to complete nuclear and cytoplasmic division without the normally concomitant development of this oral primordium. 25-661804
Experimental
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2. The majority of the dividers did not replace the excised primordium by formation of a new one. 3. Reorganizers are like dividers in also continuing on this course after primordium excision, resorbing the old mouth although no new one is ready to take its place, and in not renewing the oral primordium in the majority of cases. 4. Regenerators, in contrast, promptly replace the excised oral primordium by formation of a new one, as here confirmed. The relevance of these findings to the problem of cell division in ciliates and to the interpretation of the reorganization process is indicated.
REFERENCES 1. DE TERRA, N., Exptl
Cell Res. 21, 41 (1960).
2. FAIJR&FR&&T, I;., J. Protozool..2, 54 (1955). 3. FRANKEL, J., J. Exptl Zool. 143, 175 (1960).
4. __ 5. -
J. Protozool. 8, 250 (1961). J. Exptl Zool. 155, 403 (1964). 6. GUTTES, E. and GUTTES, S., Science 129, 1483 (1959). 7. HANSON, E. D., J. Exptl Zool. 150, 45 (1962). 8. HOLTZ, G. G., Jo., B&r. Bull. 118,.84 (iSSO): 9. MITA, T., TOKUZEN, R., FUFUOKA, F. and NAKAHARA, W., Gann 56, 293 (1965). 10. RANDALL, J. and HOPKINS, J. M., Proc. Linn. Sot. London 174, 37 (1963). 11. SCHWARTZ, V., Arch. Protistenk. 85, 100 (1935). 12. TARTAR, V., J. Exptl ZooI. 139, 1 (1958).
13. __ 14. __ 15. __ 16. 17. 18. 19.
ibid. 139, 479 (1958). ibid. 140, 269 (1959). Anat. Rec. Suppl. 134, 644 (1959).
__ The Biology of Stentor. Pergamon Press, Oxford, TUFFRAU, M., Bull. biol. France-Belg. 86, 309 (1952). UHLIG, G., Arch. Protistenk. 105, 1 (1960). WEISZ, P. B., J. Expfl Zool. 116, 231 (1951).
20. --
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ibid. 131, 137 (1956).
21. WHITSON, G. L. and PADILLA, G. M., ExptZ Cell Res. 36, 667 (1964). 22. WILLIAMS, N., J. Protozool. 11, 566 (1964). 23. WISE, B. N., J. Exptl Zoot. 159, 241 (1965). 24. ZEUTHEN, E. (ed.), Synchrony in Cell Division and Growth. Interscience York, 1964.
Experimental
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Publishers,
New
357
Cell Research 42, 357-370 (1966)
FISSION AFTER DIVISION PRIMORDIUM REMOVAL IN THE CILIATE STENTOR COERULEUS AND COMPARABLE EXPERIMENTS ON REORGANIZERS V. TARTAR* Department
of Zoology,
University
of Washington,
Sea&e, U.S.A.
Received September 17, 1965s
and cytoplasmic division in many ciliate protozoa, including Tetrahymena and Stentor, is preceded by the formation of an oral primordium called the division primordium which produces the new set of feeding organelles for the posterior daughter cell (opisthe). Development of these organelles is completed as the fission line appears and, in constricting, separates them into the opisthe. Two methodological consequences are, first, that in larger ciliates such as Stentor incipient dividers can be recognized easily in live samples as large individuals with early division primordia; second, that a major morphogenetic process of organelle elaboration preceding and in part overlapping fission potentially may be used as an indicator of the condition of a cell before and during division. Thus in Tetrahymena both division primordium development and fission are blocked by divisionsynchronizing thermal shocks and by a variety of chemical inhibitors. Therefore it has been suggested that the substances or syntheses or processes which support division primordium development also provide the basis for formation and furrowing of the fission line, or that the primordium development triggers or may be necessary for the fission [5, 21, 22, 241. Comparable data are not yet available for Stentor but its large size permits one to excise the division primordium at any stage. What then occurs can be fruitfully compared with the performance of reorganizers and regenerators after the same operation. In the process called reorganization, as in division, an oral primordium is produced although a functional set of feeding organelles is already present, in medium-sized or even small cells as well as large ones which have completed their normal growth. No fission line appears, and the developing primordium uselessly replaces most of the original NUCLEAR
1 This work was suunorted bv research mant No. CA-03637-CB from the National Cancer Institute, 1J.S. Public &alth Sercice. a Address: U. of Wash. Field Lab., Nahcotta, Washington, U.S.A. 98637. s Revised version received January 15, 1966. Experimental
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ingestive apparatus which is selectively resorbed. Because this process usually accomplishes no apparent purpose it has remained paradoxical [ 161 but a new possible explanation was suggested by the present experiments as discussed later. In oral regeneration after excision of some or all of the feeding organelles, the oral primordium quite understandably forms a new set. The primordium and its development is the same in all three processes; and in all, the beaded macronucleus temporarily coalesces at the time of gullet formation. In dividers only, the compacted macronucleus is then pinched in two by the advancing furrow. Mitotic division of the many micronuclei occurs during regeneration [6] and reorganization [ 113 as well as division, but emicronucleation of stentors has proved that these nuclei are not essential to any of these processes [ 111. The predecessor of this investigation is to be found in the 1951 pioneering experiments of Weisz [19] who reported that S. coeruleus can go on to form a fission line and divide after removal of late-stage division primordia. I confirmed this in 1959 [ 151, adding that the macronucleus can also be removed just prior to the appearance of the fission line without precluding division. Using Glaucoma which is closely related to Tetrahymena, Frankel [3, 41 found that cytokinesis could be completed although the division primordium failed to develop, either because it was destroyed by a UV microbeam or because in certain strains it spontaneously regressed. Dissociation of division processes in Tetrahymena was obtained by Mita, Tokuzen, Fukuoka, and Nakahara [9] when treatment of division-synchronized cells with the carcinogen 4nitroquinoline-l-oxide resulted in cytokinesis without macronuclear division, yielding one anucleate daughter cell; but the fate of the division primordium was not followed. In Hanson’s [7] double Paramecium, fission could occur on the side which produced no division primordium because of UV microbeam irradiation as well as on the opposite side that did, such doublets dividing as one. In Wise’s [23] recent study of Euplotes, however, localized irradiation of the division primordia before appearance of the fission line was followed, especially in early-stage dividers, by resorption and re-formation of the primordia in delayed division, or, especially in later-stage dividers, by uninterrupted division with defective feeding organelles produced by the persisting, injured primordia, but never by division without development of division primordia. As contributing to a further analysis of cell division in ciliates, the principal object of the present experiments was to determine whether stentors could continue on division course after excision of early stage division primordia.
Experimental
Cell Research 42
Division
MATERIALS
359
in Stentor
AND
METHODS
The animals used were from several strains of S. coeruleus growing vigorously on a mixture of bacteria, flagellates, smaller ciliates, and rotifers. Dividers were abundant about 12 hr after re-nutrification of the cultures with skimmed milk and were identified as large cells with primordia visible by reflected light from an embryological lamp. Reorganizers were identified as medium-sized cells with primordia, and almost all controls did use the anlage to replace the original mouthparts instead of dividing. Some specimens were induced to begin reorganization by placing them overnight in a viscous solution of methyl cellulose [16]. This solution was also used to quiet animals for excising the primordium with a glass needle, and washed off afterwards. Each specimen was isolated into 3 drops of culture medium on a depression slide and followed with highest magnification of the dissecting microscope.
RESULTS
Excision
of the Division
Primodium.
Development of the oral primordium in Stentor has been divided stages, each marked by a distinctly visible advancement [13, 161:
into 8
(1) first appearance of the primordium as a rift in the lateral striping: (2) beginning outgrowth of cilia, producing a distinct glisten; (3) cilia half-grown, showing random beating; (4) cilia grown to final length and organized into a row of membranelles which beat metachronally; (5) slight bend at the posterior end, which presages(6) inward coiling of the posterior end of the membranellar band to form the gullet; (7) formation of a vestibulum next to the opening of the gullet as the entire primordium bends to form a ring in(8) completion of development, with circular membranellar band terminating in the mouthparts (vestibulum and gullet) (Fig. 1). Growth and increase in length occur continuously up to the end of stage 4. A stage zero may also be defined as a localized splitting or multiplication of the striping where the beginning rift will appear. Primordium development follows the same steps in dividing, regenerating, and reorganizing stentors. When applied to the division primordium and correlated events, the 8 stages mark clearly discernible stages in the course of cell division. At stage 5 the beads of the macronucleus begin coalescing within the common nuclear membrane, and a pre-divisional separation of visible carbohydrate reserve Experimental
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granules defines the locus of the fission line [ 141. Not until the middle of stage 6 is a fission line visible, as a “white” line beginning at the anterior end of the division primordium and progressing in both directions around the cell, “transecting” the pigment stripes. In stage 7 the massed macronucleus extends to a rod, and furrowing or constriction in the fission line begins and
Fig. l.-Division after removal of earliest-stage division primordium. a, Stage-l primordium, mouthparts and primordium site excised. (Membranelles shown, only in this sketch. Mouthparts at end of membranellar band. Primordium is below the mouthparts. Reserve carbohydrate granules indicated by stippling. Beaded macronucleus to left.) b, Cell heals and forms no new primordium Beaded macronucleus; granular carbohydrate reserves at posterior pole. c, Beginning fission; nuclear beads fused in one mass; carbohydrate reserves pre-divided. d, Fission completed; daughter nuclei renodulated. e, Regeneration primordia in both cells, beginning 7 hr after operation; nuclear beads coalescing. f, Oral regeneration in both cells.
proceeds relatively rapidly into stage 8 when in final separation of the filial cells the macronucleus is pinched in two [1 1. From dividing stentors in all stages the entire division primordium (or the primordium site of stage-O dividers) was excised with some surrounding ectoplasm and endoplasm. In the results diagrammed and tabulated in Table I the important cases are those in which the primordium was removed at stage zero through stage 3, long before stage 6 in which the fission line first appears; and the following summarizing statements apply to these early dividers as well as to later stages. Most stentors (100/130 -columns 2, 4, 8) continued on the course of division and divided without a division primordium. In one-third of these 100 dividers (column 4) final separation of daughter cells did not occur because, as division progressed, the operation removing the division primordium increasingly interfered, not with fission line formation or stripe cutting but with constriction or furrowing as such. Perhaps it is significant that when the mouthparts were also removed constriction was always complete (column 8). In all 32 cases of incompleted fission a fission line was formed which transected the lateral stripes and kineties into two sets, evident both in the invariable shifting of one set with reference to the other giving a double body form and in the subsequent regeneration of many as doublets (Fig, 2). Considering these cases as essentially dividing gives a Experimental
Cell Research 42
Division
361
in Stentor
truer impression of the capability for division after primordium removal. Separately, they indicate a possible dissociability of formation and constriction of the fission line. Most stentors did not replace the excised primordium with a new one (96/103). This was not because specific inhibition by the intact feeding orTABLE
I. Behavior following removal of the division primordium or both primordium and mouthparts at different stages in division.
0
0
6 3 24 15 4 -0 0 2
13 1 0 5 4 i 0 0 13
2
0
/
0 I 1 4 10 7 9 1 I 14
12
II Ill3
lo
6 2 0
6 2
2 i I
5
1 6
7
/ 0 12 3 8 2 1
.3
ganelles prevented primordium renewal [13], because when the mouthparts were also removed the majority (16/27 -column 8) also did not form a new anlage. Nor was failure to renew the division primordium because the original primordium site was necessarily removed along with the original primordium. In Stentor the normal site is not indispensable for primordium formation; a new site is provided after removal of the old one. Thus some of the operated early dividers did replace the primordium. Moreover, in a comparative test in which primordia (and original primordium sites) were removed from 7 early regenerators in stages 1 and 2, all promptly formed Experimental
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new primordia (in 1 to 1 314 hr; average: 1 213 hr), confirming a previous study [la]. In division without a division primordium the macronucleus nevertheless performed as usual, the nodes coalescing to a single large mass which later extended to a rod form, was divided about equally between the daughter
t-13 \ C
Fig. Z.-Incompleted constriction of stentor after mid-stage division primordium removed. a, Stage-4 primordium excised. (This sketch and the following show the lateral pigmented stripes between which lie the rows of body cilia.) 6, Lateral striping cut into two sets; division products characteristically shift into heteropolar orientation, indicated bv original tail-pole (t); but cells do not separate-completely. c, Regeneration-reorganization produced; doublet-next day.
cells, and then renodulated. This was observable in 5 cases following primordium removal, but most dividers were too well-fed and opaque to see nuclear changes in the living animals. When the primordium was renewed, did the specimen employ the anlage for division or for reorganizational replacement of existing mouthparts or regeneration of mouthparts if these had also been removed? A strong tendency to persist in division was evident. ,411 cells in which the original mouthparts remained used the new anlage in delayed division even though excision had reduced the cell volume (column 7). Of those in which mouthparts were also excised, at least half nevertheless divided instead of using the new anlagen for regenerating the lost mouthparts in spite of greater reduction in volume (columns 9, 10). In contrast, data already available [13] show that if the division primordium is not excised but the mouthparts or the whole head is removed, then, before stage 3, the original division primordium was used for regenerating the missing feeding organelles in the majority of cases (1 l/13). Some specimens divided unequally, always the proter having at least twice the volume of the opisthe (13-column 3). The extent of this size difference is not explainable on the basis that the part excised-the primordium and adjacent striping-was deleted from the potential opisthe which would have received it. Were this so, all divisions should have been clearly unequal and they were not. More likely, the process responsible for the migration and pre-division of the sub-ectoplasmic glycogenoid reserves Experimental
Cell Research 42
Ilivision
in Sfentor
363
was disturbed. After this migration, early in stage 6, the posterior border of the anterior portion of these reserves defines where the fission line will soon appear [ 141. Like macronuclear behavior, pre-division of this granular material did occur in the absence of a division primordium, clearly evident from typical color differences seen on the surface of the cell (Fig. 1). The
Fig. 3.-Fission in both halves of an early, stage-2 divider split longitudinally. L, Completion of division primordium development, fission line formation, and division in left half. R, Fission line formation and constriction (dorsal and ventral views) in right half without primordium; separation of quarter-cells; and their later oral regeneration next day.
cutting operation therefore in some cases may have led to irregularity of this process, indicated by insufficient migration forward of half the reserves and resulting in the fission line forming at a level considerably posterior to where it usually appeared and hence in grossly unequal division. Finally it may be noted that nearly half the minority group of cells which did not continue on the course of division nevertheless formed a new oral primordium and divided the next day, though reduced in volume by the original excision, and even after intervening regeneration (columns 5, 6, 10, 11). This may be called postponed division [13] and might be due either to maintenance of a “decision” to divide or of the basis for this “decision” in spite of the operation and even of intervening regeneration. Another way of testing what dividers can do without the division primordium is to cut them in two longitudinally, one half bearing neither the anlage nor the original mouthparts (Fig. 3). Do such halves of dividers continue on the course of division? There was one case of a stage-l divider and 35 others distributed among stages 2 through early stage 6, all before the fission line is first visible. The results can be simply stated without tabulation. None of the aboral halves without the mouth or the primordium (R in Fig. 3) formed an oral primordium promptly or within the usual time of 3 to 4 hr after oral excision in the interdivisional period; they regenerated only much later. Four halves divided completely, separating astomatous quarter-cells; one each were from stage-4 Experimental
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and stage-6 dividers, but the most interesting were two at stage 2: even longitudinal-half-cells without division primordia or mouthparts and cut 4 stages before the visible appearance of the fission line could continue on the division course and divide completely. Of the remaining 32 cases all but 3 formed a fission line, sectioning the kineties and pigmented stripes into two sets, with furrowing but incomplete constriction. Considering the severity of the operation and that the halves folded on themselves in healing, these specimens also may be considered as substantially dividing. Failure of final separation of division products was probably from trivial reasons involving self-adhesion in folding, and in 6 instances later regeneration with two primordia producing bistomial stentors attested the essential doubleness achieved. In only two stage-2 and one stage-4 divider was there no apparent fission line or cutting of the lateral striping. These results are substantially in agreement with those from division primordium excisions. Regarding the partner oral halves bearing the division primordium and the original mouthparts (L in Fig. 3), 8 divided completely; 24 formed a fission line and cut the striping into two sets, furrowing but not completing constriction (further evidence that self-adhesion in folding prevented final separation, not the absence of the primordium); 2 reorganized instead, the division primordium moving forward and replacing the original mouthparts which were resorbed; and 2 merely resorbed the anlage, retaining the original ingestive apparatus. Oral halves were therefore not strikingly more successful than halves without a division primordium in remaining on the division track and accomplishing fission.
Reorganization AjIer Removal of the Reorganization Primordium Reorganizers are like dividers in forming an oral primordium though an intact oral apparatus is present, but the new organelles then move forward and replace the original mouthparts and adjacent membranellar band which are selectively resorbed. This replacement is not because the old organelles are “worn out”, as originally and frequently supposed, for one reason because replacement of newly formed feeding organelles often occurs. For example, in the division primordium excision experiments, 5 newly formed or regenerated daughter cells promptly reorganized. Beginning reorganizers were identified as medium-sized cells with primordia. Because reorganization is normally infrequent and adventitious, 12 of the specimens were methyl-cellulose-induced reorganizers as already noted. Experimental
Cell Research 42
Division
365
in Stentor
Results are diagrammed and tabulated in Table II and summarized in the following statements. Reorganizers were like dividers in generally not replacing the excised primordium with a new one before continuing on their course, likewise contrasting with comparable regenerators which replaced the anlage promptly. TABLE
II.
Behavior
following
removal of the oral primordium
7
12
1719
3
4
s
in reorganizers.
1 6
7
* All were from specimens induced to reorganize by placing them in methyl cellulose.
In only one of the 24 cases was a new primordium promptly formed before resorption of the old mouthparts (column 7). Even when the original mouths were also excised, a new primordium appeared only after about 6 hr (column 2). Intact mouthparts were resorbed even though there was no primordium to supply a replacement (column 4). Therefore the development of new mouthparts cannot be the trigger for resorption of the old ones. Resorption occurred after about the same time -comparable to reaching stage 5 -as normally and was of the same extent. Therefore the animals continued on the course of reorganization, accomplishing what they could without a primordium. In contrast, neither experimental nor control dividers ever resorbed the original mouthparts. Experimental
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The only exception was one case of a stage-l reorganizer (not included in Table II) in which nothing of a morphogenetic nature happened after excising the primordium. Mouthparts were not resorbed and the specimen simply returned to the trophic phase. This case simply indicates, as did a few of the dividers, the earliest stage is transitional and therefore allows the possibility that in some specimens reorganization or division may be abolished by primordium excision. After oral resorption all specimens soon regenerated (column 5) -more promptly than astomatous products of dividers without a primordium or which were conspicuously slow without the primordium and mouthparts, in regenerating missing or excised mouthparts. In 7 operated reorganizers rapid formation and early development -but not the later development -of the regeneration primordium was notable. Following regeneration, 7 stentors which had been induced to reorganize by methyl cellulose treatment promptly divided; being medium-sized, each produced two small daughter cells (column 6). This result is intriguing because in at least 2 tests methyl cellulose did not stimulate large cells to divide or reorganize. DISCUSSION
The first of the main results to be considered is that dividers are different from regenerators in generally not forming a new primordium after the original one has been excised. Therefore the effect of the operation on dividers was in most cases not nullified by prompt primordium renewal but led to the second demonstration, that S. coeruleus can divide without normally concomitant development of a division primordium. Then it was found that reorganizers are like dividers in also generally continuing on their course (eventual resorption of the mouthparts) without replacing the excised oral primordium. In the case of regenerators, prompt re-formation of an oral primordium, sometimes with appearance of a new stage-l anlage within one hour, suggests that the cell was already prepared or activated for primordium formation and development, and therefore that much of the period of 3 to 4 hr between the stimulus to regeneration and the first appearance of the original primordium was devoted to this preparation, which then did not have to be repeated for the replacement anlage [ 121. The preparation could be at least whatever is required for the production of new kinetosomes, of which the primordium consists. Certainly an early-stage divider normally adds kinetosomes with their ciliary outgrowths to the beginning division primordium which greatly Experimental
Cell Research 42
Division
in Stentor
367
increases in length and even in width. This was demonstrated both in light and EM photomicrographs of dividing S. coeruleus by Randall and Hopkins [lo] and was clearly shown in Holtz’s [S] silver-stained Tetrahymena at Then why do early dividers bereft of their progressive stages of division. primordia usually not form new ones in replacement, and is the reason a sign of some distinctive feature of the division process? Whatever the reason for the failure in primordium renewal, one may first suppose that it is the same as for reorganizers, which replace the primordium with even less frequency than dividers. This consideration would exclude that there be some common “morphogenetic-fission substance” which, after excision of the anlage, is shunted altogether toward formation of the fission line; for reorganizers of course do not make a fission line, and besides, there was no apparent acceleration in the onset of division after removal of a “competing” division primordium. Perhaps all we can say at present is that when some other process-fission or oral resorption-follows soon after primordium development, the second process is antagonistic to a repetition of the first. That stentors and other ciliates go on to divide after removal of late-stage division primordia or other interference shortly before the appearance of the fission line is to be expected and is not very interesting because it means only that the preparation for division is already so advanced as to be irrevocable. But division of stentors after removal of even the early division primordium, unrenewed, is obviously relevant to proposing any causal sequence in their division process. It makes highly dubious Weisz’s 119, Fig. 31 hypothesis that the early primordium stimulates the macronucleus to coalesce, for even after excision of the primordium at its earliest appearance the nuclear cycle remains normal. His scheme is no longer tenable for two other reasons. First, it cannot be that micronuclear mitosis induces the fission line because these nuclei, as mentioned earlier, also divide during reorganization [ 111 and regeneration [6] and because emicronucleated stentors grow and divide normally [ll]. Second, Weisz supposed that the site of the “anarchic held” (source of kinetosomes for the oral primordium) is different in location and not identical with the early primordium itself, which has never been postulated or demonof sequential strated by anyone else for any ciliate. The one explanation events in division of Stentor is therefore out of date. It would be premature to contrive another until certain further experiments on dividers which are uniquely possible in stentors have been performed. All that need be said for the present is that the development of a division primordium is not essential Experimental
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to nuclear or cytoplasmic division. Frankel [3, 41 found the same in Glaucoma using different methods. Even formation or initiation of a division primordium may not be essential to division in forms like Stentor and Glaucoma. It certainly is not required in the many ciliates which divide or at least form a fission line cutting in two the longitudinal ciliary rows or kineties before stomatogenesis. Such are Balantidium [2] and Colpoda [17] and necessarily is the case with these and other forms which employ the newly-cut anterior ends of the posterior halves of the kineties in the formation of feeding organelles for the posterior daughter cell. There is no apparent reason why almost all ciliates could not do likewise, for if they simply cut and pinched themselves in two transversely the opisthe would make up its organelle deficiencies by regeneration, as in the kiinstliche Zellteilung of cutting experiments. Indeed, Uhlig [18] and I have observed rare instances in S. coeruleus of division without division primordium formation, and the astomatous posterior daughters later regenerated a normal set of feeding organelles. With the exception of Frankel’s Glaucoma, I know of no other case in which the division primordium regresses, whether from injury or spontaneously, yet the cell proceeds toward and accomplishes division. In Stentor, for example, whenever a dividing cell itself removes its primordium by resorption, as in many but by no means all cases of injured dividers [20, 131, the cell then never goes on to divide, though the division process may begin again much later. These injuries need not include cutting the primordium itself to abolish division. Hence arrest and regression affect both processes in stentors as in tetrahymenas. The questions here are whether resorption (or its milder form, arrest) is another distinct and active process or is the cessation or canceling of processes supporting division primordium development and fission line formation, and what the two processes might have in common that they should be affected alike. Reorganizers are like dividers not only in escaping head dominance or specific inhibition in beginning a second set of feeding organelles, as well as in going through the cycle of macronuclear condensation and micronuclear mitosis. They also seldom replace excised early primordia, and they usually continue on course to a second process, uiz, resorbing the old mouthparts although the primordium is no longer present to supply a replacement. This is strong resemblance: and just as there can be division without a division primordium (in the above experiments), it is possible that there might be division primordium formation and development without fission, because of Experimental
Cell Research 42
Division
in Stentor
369
the evident d&sociability of these cell processes. Therefore we should consider the possibility that reorganizers may be pseudo-dividers, i.e. cells in which formation of a division primordium is somehow triggered or released although conditions for subsequent fission are not present. That such a dissociation may occur in nature-and reorganization has been observed adventitiously in many kinds of ciliates-becomes more probable when we make a reasonable guess of when the trigger for division primordium formation in Stentor is set off. In the one type of experiment in which we can elicit an oral primordium at will, without chemical agents, viz, regeneration, in which the stimulus is oral excision, the regeneration primordium appears (stage 1) 3 to 4 hr later. If dividers require the same preparatory period, and since 1 hr is required for each stage in primordium development and the fission line appears in the middle of stage 6, the stimulus for division primordium formation may be located in time at least 8 hr before appearance of the fission line in a cell whose generation time is around 48 hr. In turn, cases of grossly unequal division sometimes occasioned by primordium removal (Table I-column 3) show that it is possible for the fission line not to be determined in respect to its location or level on the cell axis until after stage 4. If this be taken as the time of beginning fission line formation, then there would be about 6 hr between the triggering of division primordium formation sufficient difference, it would and the triggering of fission line formation, seem, to allow improper linkage of the two processes. Therefore it might happen in even small or medium-sized cells that the stimulus for primordium formation may be set off without proper conditions to elicit fissioning. Conversely, in largest stentors the latter conditions may always be present, as indicated by the fact that large cells, beheaded, form a primordium and divide instead of regenerating, producing headless proters which regenerate only after the division [16]. To regard reorganizers as pseudo-dividers could explain the otherwise entirely enigmatic occurrence of reorganization [see 161 in altogether normal ciliates with new and proportionate feeding organelles and proportionate nuclei, as well as the four mentioned resemblances between organizers and dividers.
SUMMARY
2. Following excision of division primordia even at the earliest stages, stentors can go on to complete nuclear and cytoplasmic division without the normally concomitant development of this oral primordium. 25-661804
Experimental
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2. The majority of the dividers did not replace the excised primordium by formation of a new one. 3. Reorganizers are like dividers in also continuing on this course after primordium excision, resorbing the old mouth although no new one is ready to take its place, and in not renewing the oral primordium in the majority of cases. 4. Regenerators, in contrast, promptly replace the excised oral primordium by formation of a new one, as here confirmed. The relevance of these findings to the problem of cell division in ciliates and to the interpretation of the reorganization process is indicated.
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Cell Res. 21, 41 (1960).
2. FAIJR&FR&&T, I;., J. Protozool..2, 54 (1955). 3. FRANKEL, J., J. Exptl Zool. 143, 175 (1960).
4. __ 5. -
J. Protozool. 8, 250 (1961). J. Exptl Zool. 155, 403 (1964). 6. GUTTES, E. and GUTTES, S., Science 129, 1483 (1959). 7. HANSON, E. D., J. Exptl Zool. 150, 45 (1962). 8. HOLTZ, G. G., Jo., B&r. Bull. 118,.84 (iSSO): 9. MITA, T., TOKUZEN, R., FUFUOKA, F. and NAKAHARA, W., Gann 56, 293 (1965). 10. RANDALL, J. and HOPKINS, J. M., Proc. Linn. Sot. London 174, 37 (1963). 11. SCHWARTZ, V., Arch. Protistenk. 85, 100 (1935). 12. TARTAR, V., J. Exptl ZooI. 139, 1 (1958).
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__ The Biology of Stentor. Pergamon Press, Oxford, TUFFRAU, M., Bull. biol. France-Belg. 86, 309 (1952). UHLIG, G., Arch. Protistenk. 105, 1 (1960). WEISZ, P. B., J. Expfl Zool. 116, 231 (1951).
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21. WHITSON, G. L. and PADILLA, G. M., ExptZ Cell Res. 36, 667 (1964). 22. WILLIAMS, N., J. Protozool. 11, 566 (1964). 23. WISE, B. N., J. Exptl Zoot. 159, 241 (1965). 24. ZEUTHEN, E. (ed.), Synchrony in Cell Division and Growth. Interscience York, 1964.
Experimental
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Publishers,
New