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Cortical morphogenesis and synchronization in Tetrahymena pyriformis GL
Experimental
Cell Research
CORTICAL
35, 349-360
349
(1964)
MORPHOGENESIS IN TETRAHYMENA
AND SYNCHRONIZATION PYRIFORMIS GL1
J. FRANKEL Biological
Institute
of the Curlsberg
Foundation, Iowa City, Received
Copenhagen, Denmark, Iowa, U.S.A.’ Ailgust
and State University
of Iowa,
2, 1963
Tetrahymena is unique among the forms which have been artificially synchronized, in that a high degree of synchronization is attained by a prior treatment consisting of a series of environmental shocks separated by intervals of maintenance at optimal conditions [ 15, 16, 21, 23, 241. Single shock treatments produce only a limited degree of synchrony, which is not materially improved by application of one temperature cycle per generation [21, 24; cf. refs. 9, 11 for comparable studies on flagellates]. Zeuthen [22] has investigated the relation between the number of successive high temperature shocks and the degree of synchronization in T. pyriformis. He has found that in a rich medium (2 per cent proteose peptone plus 0.4 per cent liver fraction L) a second and third shock increases the proportion of cells dividing synchronously, and a four shock regime results in nearly optimal synchronization. Since the pioneering studies of Thormar [ 17, 181, it has been generally recognized that synchronization of Tetrahymena has its basis in an agedependent delay of cell division which can be induced by environmental shocks of various kinds [ 12, 13, 17, 18, 19, 211. The amount of delay increases with the age of the cell (as measured from the time of the previous division). However, the increase is not linearly related with age, and for this reason good synchronization cannot be attained by application of a single shock to an exponentially multiplying population (see [18, 191). The later shocks of the synchronizing treatment in some way alter this situation and cause a “collection” of cells into a relatively early phase of the cell cycle (see [21], p. 56). The exact phase into which cells are “collected” and the manner by which this “collection” is achieved are not immediately evident from inspection of Thormar’s [ 18: age vs. delay curves. Additional information is needed if we are to understand the dynamics of the synchronization process. 1 The doctoral Carlsberg
bulk of the work reported in this paper was performed while the author was a postfellow of the National Cancer Foundation, working at the Biological Institute of the Foundation, headed by Professor E. Zeuthen. 2 Pr&nt address: State University of Iowa. Experimentai
Cell
Research
35
350
J. Frankel
One method by which to gain further insight into the process of synchronization in Tetrahymenu is to investigate the effects of the multiple shock treatment on the stages of oral morphogenesis, which precede division in this ciliate. This was initially accomplished by Holz, Scherbaum, and 1Villiams [S], and by llrilliams and Scherhaum [‘LOO, who found that the synchronizing treatment permitted cells to develop only as far as the “anarchic field” stage of oral development. Subsequent studies [3] confirmed the earlier conclusions and also indicated that high temperature treatment applied to synchronized cells induces the resorption of oral primordia in which ciliary membranelles had commenced to form. Furthermore, interference with oral morphogenesis was found always to be correlated with prevention of cell division. Hence it appears likely that the cycle of duplication of these cortical organelles is related in some meaningful way to the cell cycle as a whole. Therefore a detailed study of oral morphogenesis during the heat shock synchronization procedure was initiated, in the expectation that it might give us some information as to how this procedure causes cells to be “collected” into some particular phase of development toward division. The object of the present study was to ascertain the effect of a single heat shock on cortical morphogenesis in exponentially growing T. pyriformis, and then to follow the further morphogenetic modifications taking place as a response to subsequent shocks. ,4n attempt was made to correlate directly the specific cortical events involved in the morphogenesis of the oral area with the attainment of synchrony in the population. METHODS
Tetrahymena pyriformis GL (an amicronucleate strain) was employed in this study. The cells were grown axenically in a medium containing 2 per cent proteose peptone (Difco), 0.4 per cent liver fraction L (Wilson), and salts as in basal medium A of Kidder and Dewey [IO] with phosphates omitted. Stocks were maintained in slanted test tube cultures kept at 28°C. Transfers were made daily. At the beginning of each experiment, about IO5 cells were inoculated into a Fernbath flask containing 150-200 ml of culture medium. The flask cultures were then grown for 16-20 hr in a temperature bath maintained at 28°C. At the end of this growth period, the population had attained a density of 2-5 x lo4 cells per ml. The cultures (or parts thereof) were then subjected to one of two types of treatment: (a) a single 20 min exposure to 34X, followed by return to 28”, or (b) exposure to the six-shock synchronization procedure described by Zeuthen and collaborators [3, 6, 131. In this procedure, 20 min periods of regulation of a water bath at 34°C are followed by 40 min intervals of regulation at 28°C. The heating and cooling of the culture, following the switches of the regulating clock, require time intervals of about 7 min each (see [3] for further details). Experimental
Cell Research
35
Morphogenesis
in T. pyriformis
351
In the course of each of these treatments, samples of the culture were fixed for silver impregnation. The sampling schedule varied with the type of experiment, and will be considered in the Results section. The Chatton-Lwoff wet silver impregnation method, as described by Corliss [I], was employed to reveal the infraciliary structures. French gelatin, kindly supplied by Dr. W. H. Furgason, was used to embed the ciliates. ; : : :
I
: :
:
; : : ;
2
: : : :
3
: :
:
4
; :
5
6
Fig. l.-Summary of the stages of oral morphogenesis in Tetrahymena. In these developing oral primordium and the region immediately surrounding it are graphical relationship of this area to the whole cell can be seen on Fig. 2). characterization of the stages, see the text. Legend: DF, division furrow; FI,, fission line; M, membranelle; GM, undulating membrane.
diagrams only the shown (the topoFor a brief verbal FB, fiber bundle;
A detailed consideration of the surface structures in Tetrahymena which are revealed by the silver impregnation technique can be found elsewhere [2, 3, 4, 7, 201, and will not be repeated here. However, to assist in comprehension of the Results, a brief description of the stages of oral morphogenesis, represented on Fig. 1, will be presented. The staging system follows that proposed earlier [3]. Stage 1. Loose “anarchic field”, with indefinite margins. Stage 2. Compact field, with a clearly defined outer margin; membranelles not yet visible. Stage 3. Anterior parts of one or two membranelles clearly distinguishable; third not yet visible. Stage 4. All three membranelles distinctly visible; undulating membrane, if present at all, distinct only in its anterior portion. Stage 5. Membranelles and undulating membrane complete or very nearly so; buccal cavity and fiber bundle not yet formed. Stage 6. Membranelles sunk beneath general body surface; fiber bundle in process of formation. The fission line, consisting of equatorial discontinuities in the kinetics, develops during the latter part of stage 4 and is complete by stage 5. Division furrowing takes place during stage 6, after the basic structures of the new oral area are fully formed. In one experiment observations were made on the degree of synchrony following every shock of the synchronizing regime. Samples were removed from the main culture after the end of each shock, and kept thereafter at 28°C. The samples were periodically observed through a dissecting microscope, and at the time when a maximal proportion of cells seemed to be dividing, part of the sample was fixed in 2 per cent formalin. Differential counts of dividing cells were then made, using a Sedgwick-Rafter countExperimental
Cell
Research
35
352
J. Frankel
ing chamber plus a Whipple ocular grid [5,14]. In this way it was possible precisely the percentage of cells in division at a time when this percentage to be maximal.
to ascertain was judged
RESULTS
The morphogenetic effect of is applied
to an exponentially
cells divide within but then
c~
single heat
growing
a short time interval
cell multiplication
ceases;
a single 34” shock 15 per cent of the
shock.-When
population,
about
immediately
multiplication
begins
following anew
the shock,
about
100 min
after the end of the shock (S. Nachtwep, personal communication). Observations made on silver stained preparations of cells fixed at intervals prior to, during, and after a heat shock provide information on the morphogenetic response, which is summarized in Table I. Starting 30 min after the end of the heat shock, cells with normally developing oral membranelles (stages S-3) are no longer seen and dividing cells are extremely rare. At the same time, the percentage of cells lacking oral primordia (“stage 0”) and the percentage with stage 1 primordia become somewhat higher. Most strikingly, in 7 to 10 per cent of the cells oral primordia are seen in which membranelles appear to be TABLE I. Morphogenetic events observed during and after rx 20-min heat shock. Four hundred The per cent
Time From beg. of shock 0 10 20
cells from distribution
each of the samples were classified according to their stage of development, of morphogenetic stages for each time shown on the left is indicated on the horizontal rows of the table.
(min) From end of shock
Per cent OU 61 59 59 63 68 70 61 42 27
0 10 20 30 40 60 80
1 0, No oral primordium visible. R, Oral primordium in progress ’ New primordia frequently visible old primordia. Experimental
Cell Research
35
at stage
Rb
1
2
3
4
5
7 7 10 8 6’ 4c
14 14 17 14 18 18 24 21 19
4 4 3 1
4 3 3 2 1
7 7 5 5 1
6 7 S 1
10 4
12 13
1 14
of regression. posterior (and
1 6 7 9
often
also anterior)
to regressing
6 (div.) 4 5 5 7 5 1 1 1 10
remnants
of
Morphogenesis
353
in T. pyriformis
in various stages of regression (see Figs. 4, 5, 6). In successively later samples, successively more advanced stages of membranelle regression can be seen. The structure of most of these regressing primordia indicates that they Were in fairly advanced stages of membranelle development (stages 4-5) at the time when the heat shock mas applied. Figs. 2-7 illustrate the sequence of primordium resorption vvhich can be inferred from these observations. Tmo interesting features of this resorption process warrant brief mention: (i) Although the primordia are already somewhat abnormal at the end of the heat shock (Fig. 3), resorption does not actually take place until af’fer the heat shock is over; in other vvords, the heat shock acts to trigger a resorption process (cf. 131). (ii) The resorption of advanced oral primordia is accompanied by an arrest in the development of the fission line, and a subsequent buckling and distortion of the rovvs of basal bodies (kineties) in this region (this buckling can also be observed in many synchronized cells). Stage 1 primordia appear to remain completely normal both during and after the 34” shock. The fate of the stage 2-3 oral primordia is somevvhat less clear, but it is likely that these revert to an appearance characteristic of stage 1. The distinct increase in the proportion of cells lacking oral primordia, which occurs in the first 20 min after the end of the heat shock, may be accounted for by the successful division of cells which had already begun to divide at the time vvhen the heat shock was initiated. Structurally normal cells were seen in the terminal phases of cell division both during and immediately after the heat shock. These are presumably cells which have passed a “stabilization point” at the time vvhen the heat shock was applied (cf. [3, 191). The stage at which stabilization occurs in this particular experiment is not precisely determined, but is probably during stage 5. Starting 60 min after the end of the shock, cells were seen to start membranelle formation anew; at 80 min, some cells were already seen in division. Cells in which old primordia were being resorbed frequently formed two nevv primordia, one anterior and one posterior to the resorption site (Figs. 6-7). J4orphogenetic events during synchronization.-Two experiments were performed to elucidate the morphogenetic events occurring during a six-shock synchronization regime; the results of these experiments are summarized in Fig. 8. After the first heat shock oral primordia vvith differentiating membranelles are no longer seen, and the percentage of cells bearing oral primordia declines. The second shock begins before any appreciable new development Experimental
Cell Research
35
J. Frankel of oral primordia has taken place. In the interval between the second and third shocks, most of the cells acquire stage l--2 oral primordia. Just prior to the third shock, membranelle development commences in a few cells. Subsequent to the third shock, membranelles are no longer observed, but the overall percentage of cells with oral primordia remains approximately constant. Beginning with the third shock, a distinct cyclic phenomenon becomes apparent. Essentially all the cells in the culture now have oral primordia. Between 30 and 70 per cent of these commence to form membranelles toward the end of each interval between shocks. The membranelles then disappear in the early part of the following inter-shock interval; they are apparently resorbed as a consequence of the heat shock (cf. previous section). In the middle of the intervals between shocks all the cells are in stage 1 of primordium development. In one of the two experiments, the degree of cell division synchrony was observed in samples taken from the main culture (cf. Methods section). After three heat shocks, synchrony was already fairly good (66 per cent of the population in division at the “division maximum”, 85 min after the end of the shock). It is worth noting that by the time of the third shock, most of the cells had already acquired oral primordia, which were generally in early stages of development. Subsequent to the fourth shock, the degree of synchrony remained approximately unchanged. There was, however, an unexpected decline in the degree of synchrony after the fifth shock. The poor synchrony in this case may be correlated with the unusually advanced state of membranelle differentia-
Fig. 2.-A cell fixed at the beginning of the 20 min heat shock, with a typical stage 4 primordium. The three membranelles (M) are well developed, the undulating membrane (UM) is in the process of development, and part of the original field of kinetosomes (F) is still present. The beginnings of the fission line are seen as an equatorial zone of discontinuities (D) in the longitudinal rows of kinetosomes (kineties). The oral area (OA), near the anterior end, is partly out of focus. * 1700. Fig. 3.-A cell fixed at the end of the heat shock, with a late stage 4 primordium. Its general structure is normal, but the membranelles have become somewhat ragged in outline. The discontinuities (D) marking the fission line are pronounced, and the kinety immediately to the animal’s right (viewer’s left) of the primordium shows a characteristic bending toward the primordium. x 1700. Fig. 4.-Twenty minutes after in an early phase of resorption.
the end of the heat shock. x 1700.
A stage 4 or 5 oral primordium
is shown
Fig. 5.-Thirty minutes after the end of the heat shock. A stage 4 or 5 primordium is seen in a late phase of resorption; the membranelles are much diminished in size and the undulating membrane has practically disappeared. Note the extreme buckling of the kinety to the right of the primordium, posterior to the original fission line discontinuity (D). x 1700. Esperimental
Cell Research
35
Morphogenesis
in T. pyriformis
355
Experimenfal
Cell Research
35
J. Frankel
Fig. B.-Sixty minutes after the end of the heat shock. Hemnants of an almost completely resorbed oral primordium (RP) are still visible. There are two new primordia, both in stage 1. One of these (NP 1) is anterior and the other (NP 2) is posterior to the remnants of the original primordium. Note the distortion of the fission line. The anterior oral area (OA membranelles out of focus) remains completely normal. x 1400. Fig. 7.-Eighty minutes after the the resorption site (R) being clearly (cf. Figs. 5, 6). Two new primordia
end of the heat shock. Primordium resorption marked as a focus of extensive local buckling (NP 1, NP 2) are both in stage 4. x 1400.
is completed, of the kineties
tion attained by many cells at the beginning of the fifth shock--50 per cent of the cells possessed stage 4 primordia. At the beginning of the sixth shock this excessively advanced oral primordium development did not occur, and synchrony was good after this shock (83 per cent of the population in division, 85 min after the end of the shock).
DISCUSSION
The results of the present study indicate that the effect of a heat shock on oral morphogenesis is the same in normal exponentially growing Tetrahymena as it is in the synchronized organism (cf. [3]). In both cases, there is clear Experimental
Cell Research
35
Morphogenesis
in T. pyriformis
357
evidence that high temperature induces resorption of developing membranelles. This is most apparent in stage 4 primordia, but also appears to occur in stage 3 primordia (cf. [3, 191). On the other hand, “anarchic field” primordia (stage 1) are not visibly affected by a heat shock. Stage 2 primordia probably revert to stage 1. There is a considerable delay between the end of a single heat shock and the resumption of morphogenetic activity (see Table I). Thus oral primordium formation is not observed between the first and second heat shocks of the synchronizing treatment (see Fig. 8). However, between the second and third heat shocks the majority of cells develop oral primordia. Clearly, the second heat shock is not hindering development as severely as the first. One might ,lOO-
Fig. 8.-Morphogenesis and division synchrony during the synchronization procedure. The schedule of administration of heat shocks serves as the abscissa; the shocks begin at hourly intervals. The cortical morphogenetic events are summarized by two curves: curve A indicates the percentage of cells which have oral primordia, in any stage of development; curve B indicates the percentage with stage 3 or 4 oral primordia, i.e. those in which developing membranelles are visible. The percentage of cells with stage l-2 oral primordia can be obtained by subtracting B from A. Each point is based on a tally of 100 to 200 cells. The uppermost curve indicates the degree of division synchrony, as observed following each heat shock. Two experiments were performed, under conditions as near as possible to being identical. The results of one are indicated by open circles, those of the other by filled circles. The degree of division synchrony was observed only in the latter experiment. Experimental
Cell
Research
35
358
J. Frankel
therefore postulate that in addition to the specific stage-dependent effects, an initial heat shock also induces a non-specific delay in development. Following this initial shock the cells adjust so that after a second shock the non-specific delay is either reduced or absent. Recent experiments, in which individual Tetrahymena of known ages were given either one heat shock or two successive shocks, revealed that a second shock generally has considerably less effect in delaying cell division than does the first shock; these results thus provide independent evidence which supports the above postulate.1 Division synchrony following two shocks is not much better than after a single shock. Following the third shock, however, synchrony is strikingly improved (cf. [22], Fig. 4~). It is at this time that the majority of cells have come to acquire oral primordia (see Fig. 8). By a slight extrapolation, one may correlate the time at which good synchrony is established in the population with the time at which all the cells in the population reach the phase of oral primordium development. Toward the end of the intervals between shocks, membranelle development starts in a considerable fraction of the cells; following the shocks these membranelles are resorbed. This phenomenon is clearly dependent on the duration of the inter-shock intervals: if these intervals had been appreciably longer, cell division would have occurred during the synchronizing treatment, and effective synchronization would have been impossible; if the intervals had been shorter, membranelles would not have had a chance to form. Williams and Scherbaum [‘LO], using a synchronizing treatment in which the intervals between shocks were only 30 min, found that during the synchronization procedure there is a steady increase in the proportion of cells with “anarchic field” primordia (eventually reaching 100 per cent), but there was no development of membranelles. Clearly, the transitory membranelle development observed in the present experiments is not an obligatory feature of the synchronization process. Indeed, when membranelle development goes too far in a significant proportion of the cells, synchronization is intrrfered with. The more advanced oral primordia (those in stage 4) undergo total resorption and replacement as a consequence of the heat shock (Figs. 2-7, cf. also [3]). This sequence of events requires a greater amount of time than does the more limited replacement or recovery process of cells affected at earlier stages (see [ 191 for direct confirmation of this conclusion). Cells in stage 4 at the time of the heat treatment arrive at cell division at a 1 These experiments were performed the aid of an N.I.H. grant (GM-08777-02) ance of Howard E. Buhse. Experimental
Cell
Research
35
by the author in the laboratory to Dr. Williams, and with
of Dr. N. E. Williams, the valuable technical
with assist-
Morphogenesis
in T. pyriformis
359
later time than cells in stages 1 to 3. Good synchronization can therefore only be obtained if the majority of cells are in stages 1 to 3 of primordium development at the time of the final shock. From the above considerations, we may conclude that an essential element in the synchronization process is the “collection” of cells in a phase of development which has the beginning of oral primordium formation as its salient structural feature. This phase occurs at a time which is somewhat less than halfway through the normal division cycle of Tefrahymena ([3], p. 1%). \Ve may interpret this to mean that synchronized Tefrahymena are actually “collected” into a phase which corresponds more closely to the middle than to the beginning of the normal division cycle. It is possible to characterize to some degree the stage-specific responses to a sequence of heat shocks, on which the “collection” of cells is based. Cells in the earliest phases of the cell cycle (stage O-lacking oral primordia) are considerably delayed in development by the first heat shock (cf. [18]), but adapt and form oral primordia in the succeeding inter-shock intervals. Cells with stage 1 (“anarchic field”) oral primordia retain their primordia through the heat shock treatment; however, in these cells some underlying system responsible for further development is affected, since there is a period development” [20] between the end of the last synchronizing of “arrested shock and the beginning of membranelle formation. Finally, cells which have started forming membranelles just prior to or during the synchronizing treatment or during the inter-shock intervals are thrown back by the heat shocks to the beginning of stage 1, and thus must start primordium development over again. The “collection” process can thus to a large extent be described in terms of morphogenetic responses, although observation of these responses does not tell us what the significant underlying processes are. Investigation into the physical and chemical bases of cortical morphogenesis might, however, yield significant information concerning the processes by which synchronization in Tefrahymena comes about.
SUMMARY
A study was made of the effects of a single heat shock, and of a multiple heat shock synchronizing treatment, on oral morphogenesis in Tefrahymena pyriformis GL. Heat shocks were found to have different effects, depending on the stage of cortical development. Cells with “anarchic field” primordia (stage 1) were not visibly affected by the shocks, while cells with primordia Experimental
Cell Research
35
360
J. Frankel
which had started to form membranelles (stages 3-4) resorbed their developing structures as a consequence of a heat shock and thus were returned to an earlier phase of development. Cells initially lacking oral primordia (“stage 0”) were able to develop primordia in the intervals between heat shocks. As a consequence of these stage-specific effects, there was a rapid increase, during the early part of the synchronizing treatment, in the proportion of cells possessing oral primordia; the primordia remained in (or were returned to) the “anarchic field” phase of development. Fairly good synchronization was achieved already by the end of the third shock, at which time the majority of the cells had begun oral primordium development. From these and earlier findings, it was concluded that the synchronizing treatment “collects” cells in a phase of development whose salient structural feature is the onset of oral primordium development. This phase may be considered to correspond roughly to the middle of the normal cell generation in Tetrahymena pyriformis.
The author would like to thank Drs. Earl D. Hanson, George G. Holz, D. Stuart Nachtwey, Norman E. Williams, and Erik Zeuthen for carefully reading this paper in manuscript
form,
and for supplying
constructive
comments
and suggestions.
REFERENCES 1. CORLISS, J. O., Stain Tech&. 28, 97 (1953). 2. __ Parasitology 43, 49 (1953). 3. FRANKEL, J., Compt. Rend. Trav. Lab. Curlsberg 33, 1 (1962). 4. FURGASON, W. H., Arch. Protistenk. 94, 224 (1940). 5. HALL, R. P., JOHNSON, D. F. and LOEFER, J. B., Trans. Am. Microscop. Sot. 54, 298 (1935). 6. HA~IBURGER, K., PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in Synchrony in Cell Division and Growth. E. ZEUTHEN (ed.) Interscience, New York, In press. 7. HOLZ, G. G., Bio[. Bull. 118, 84 (1960). 8. HOLZ, G. G., SCHERBAUM, 0. H. and WILLIAMS, N. E., Exptl Cell Res. 13, 618 (1957). 9. JAMES, T. W., Ann. N.Y. Acad. Sci. 90, 550 (1960). IO. KIDDER, G. W. and DEWEY, V. C., in Biochemistry and Physiology of the Protozoa, p. 323. A. LWOFF ted.) Academic Press, New York, 1951. 11. PADILLA, G. M. and JAMES, T. W. Exptt Cett Res. 20, 401 (1960). 12. RASMUSSEN, L., Compt. Rend. Trav. Lab. Carlsberg 33, 53 (1962). 13. RASMUSSEN, L. and ZEUTHEN, E., ibid. 32, 333 (1962). 14. SCHERBAUM, O., Acta Pathol. Microbial. &and. 40, 7 (1957). 15. ~ Ann. Rev. Microbial. 14, 283 (1960). 16. SCHERBAUM, 0. and ZEUTHEN, E., Exptl Cell Res. 6, 221 (1954). 17. THORMAR, H., Danish Thesis, Univ. of Copenhagen, 1956.
18. ~
19. 20. 21. 22. 23.
Compt.
Rend.
Trav.
Lab.
Carlsberg
31, 207 (1959).
WILLIAMS, N. E., EzptZ Cell Res., submitted for publication. WILLIAMS, N. E. and SCHERBAUM, O., J. Embryol. Exptt Morphot. 7, 241 (1959). ZEUTHEN, E., Adv. Biol. Med. Phys. 6, 37 (1958). ~ in Growth in Living Systems, p. 135. M. X. ZARROW (ed.) Basic Books, New York, -in Synchrony in Cell Division and Growth, E. ZEUTHEN (ed.) Interscience, New
1961. York.
In press. 24.
ZEUTHEN, E. and SCHERBAUM, KITCHING (ed.) Butterworths,
Experimental
Cell Research
35
O., in Recent London,
Developments 1954.
in
Cell
Physiology,
p. 141.
J. A.
Cell Research
CORTICAL
35, 349-360
349
(1964)
MORPHOGENESIS IN TETRAHYMENA
AND SYNCHRONIZATION PYRIFORMIS GL1
J. FRANKEL Biological
Institute
of the Curlsberg
Foundation, Iowa City, Received
Copenhagen, Denmark, Iowa, U.S.A.’ Ailgust
and State University
of Iowa,
2, 1963
Tetrahymena is unique among the forms which have been artificially synchronized, in that a high degree of synchronization is attained by a prior treatment consisting of a series of environmental shocks separated by intervals of maintenance at optimal conditions [ 15, 16, 21, 23, 241. Single shock treatments produce only a limited degree of synchrony, which is not materially improved by application of one temperature cycle per generation [21, 24; cf. refs. 9, 11 for comparable studies on flagellates]. Zeuthen [22] has investigated the relation between the number of successive high temperature shocks and the degree of synchronization in T. pyriformis. He has found that in a rich medium (2 per cent proteose peptone plus 0.4 per cent liver fraction L) a second and third shock increases the proportion of cells dividing synchronously, and a four shock regime results in nearly optimal synchronization. Since the pioneering studies of Thormar [ 17, 181, it has been generally recognized that synchronization of Tetrahymena has its basis in an agedependent delay of cell division which can be induced by environmental shocks of various kinds [ 12, 13, 17, 18, 19, 211. The amount of delay increases with the age of the cell (as measured from the time of the previous division). However, the increase is not linearly related with age, and for this reason good synchronization cannot be attained by application of a single shock to an exponentially multiplying population (see [18, 191). The later shocks of the synchronizing treatment in some way alter this situation and cause a “collection” of cells into a relatively early phase of the cell cycle (see [21], p. 56). The exact phase into which cells are “collected” and the manner by which this “collection” is achieved are not immediately evident from inspection of Thormar’s [ 18: age vs. delay curves. Additional information is needed if we are to understand the dynamics of the synchronization process. 1 The doctoral Carlsberg
bulk of the work reported in this paper was performed while the author was a postfellow of the National Cancer Foundation, working at the Biological Institute of the Foundation, headed by Professor E. Zeuthen. 2 Pr&nt address: State University of Iowa. Experimentai
Cell
Research
35
350
J. Frankel
One method by which to gain further insight into the process of synchronization in Tetrahymenu is to investigate the effects of the multiple shock treatment on the stages of oral morphogenesis, which precede division in this ciliate. This was initially accomplished by Holz, Scherbaum, and 1Villiams [S], and by llrilliams and Scherhaum [‘LOO, who found that the synchronizing treatment permitted cells to develop only as far as the “anarchic field” stage of oral development. Subsequent studies [3] confirmed the earlier conclusions and also indicated that high temperature treatment applied to synchronized cells induces the resorption of oral primordia in which ciliary membranelles had commenced to form. Furthermore, interference with oral morphogenesis was found always to be correlated with prevention of cell division. Hence it appears likely that the cycle of duplication of these cortical organelles is related in some meaningful way to the cell cycle as a whole. Therefore a detailed study of oral morphogenesis during the heat shock synchronization procedure was initiated, in the expectation that it might give us some information as to how this procedure causes cells to be “collected” into some particular phase of development toward division. The object of the present study was to ascertain the effect of a single heat shock on cortical morphogenesis in exponentially growing T. pyriformis, and then to follow the further morphogenetic modifications taking place as a response to subsequent shocks. ,4n attempt was made to correlate directly the specific cortical events involved in the morphogenesis of the oral area with the attainment of synchrony in the population. METHODS
Tetrahymena pyriformis GL (an amicronucleate strain) was employed in this study. The cells were grown axenically in a medium containing 2 per cent proteose peptone (Difco), 0.4 per cent liver fraction L (Wilson), and salts as in basal medium A of Kidder and Dewey [IO] with phosphates omitted. Stocks were maintained in slanted test tube cultures kept at 28°C. Transfers were made daily. At the beginning of each experiment, about IO5 cells were inoculated into a Fernbath flask containing 150-200 ml of culture medium. The flask cultures were then grown for 16-20 hr in a temperature bath maintained at 28°C. At the end of this growth period, the population had attained a density of 2-5 x lo4 cells per ml. The cultures (or parts thereof) were then subjected to one of two types of treatment: (a) a single 20 min exposure to 34X, followed by return to 28”, or (b) exposure to the six-shock synchronization procedure described by Zeuthen and collaborators [3, 6, 131. In this procedure, 20 min periods of regulation of a water bath at 34°C are followed by 40 min intervals of regulation at 28°C. The heating and cooling of the culture, following the switches of the regulating clock, require time intervals of about 7 min each (see [3] for further details). Experimental
Cell Research
35
Morphogenesis
in T. pyriformis
351
In the course of each of these treatments, samples of the culture were fixed for silver impregnation. The sampling schedule varied with the type of experiment, and will be considered in the Results section. The Chatton-Lwoff wet silver impregnation method, as described by Corliss [I], was employed to reveal the infraciliary structures. French gelatin, kindly supplied by Dr. W. H. Furgason, was used to embed the ciliates. ; : : :
I
: :
:
; : : ;
2
: : : :
3
: :
:
4
; :
5
6
Fig. l.-Summary of the stages of oral morphogenesis in Tetrahymena. In these developing oral primordium and the region immediately surrounding it are graphical relationship of this area to the whole cell can be seen on Fig. 2). characterization of the stages, see the text. Legend: DF, division furrow; FI,, fission line; M, membranelle; GM, undulating membrane.
diagrams only the shown (the topoFor a brief verbal FB, fiber bundle;
A detailed consideration of the surface structures in Tetrahymena which are revealed by the silver impregnation technique can be found elsewhere [2, 3, 4, 7, 201, and will not be repeated here. However, to assist in comprehension of the Results, a brief description of the stages of oral morphogenesis, represented on Fig. 1, will be presented. The staging system follows that proposed earlier [3]. Stage 1. Loose “anarchic field”, with indefinite margins. Stage 2. Compact field, with a clearly defined outer margin; membranelles not yet visible. Stage 3. Anterior parts of one or two membranelles clearly distinguishable; third not yet visible. Stage 4. All three membranelles distinctly visible; undulating membrane, if present at all, distinct only in its anterior portion. Stage 5. Membranelles and undulating membrane complete or very nearly so; buccal cavity and fiber bundle not yet formed. Stage 6. Membranelles sunk beneath general body surface; fiber bundle in process of formation. The fission line, consisting of equatorial discontinuities in the kinetics, develops during the latter part of stage 4 and is complete by stage 5. Division furrowing takes place during stage 6, after the basic structures of the new oral area are fully formed. In one experiment observations were made on the degree of synchrony following every shock of the synchronizing regime. Samples were removed from the main culture after the end of each shock, and kept thereafter at 28°C. The samples were periodically observed through a dissecting microscope, and at the time when a maximal proportion of cells seemed to be dividing, part of the sample was fixed in 2 per cent formalin. Differential counts of dividing cells were then made, using a Sedgwick-Rafter countExperimental
Cell
Research
35
352
J. Frankel
ing chamber plus a Whipple ocular grid [5,14]. In this way it was possible precisely the percentage of cells in division at a time when this percentage to be maximal.
to ascertain was judged
RESULTS
The morphogenetic effect of is applied
to an exponentially
cells divide within but then
c~
single heat
growing
a short time interval
cell multiplication
ceases;
a single 34” shock 15 per cent of the
shock.-When
population,
about
immediately
multiplication
begins
following anew
the shock,
about
100 min
after the end of the shock (S. Nachtwep, personal communication). Observations made on silver stained preparations of cells fixed at intervals prior to, during, and after a heat shock provide information on the morphogenetic response, which is summarized in Table I. Starting 30 min after the end of the heat shock, cells with normally developing oral membranelles (stages S-3) are no longer seen and dividing cells are extremely rare. At the same time, the percentage of cells lacking oral primordia (“stage 0”) and the percentage with stage 1 primordia become somewhat higher. Most strikingly, in 7 to 10 per cent of the cells oral primordia are seen in which membranelles appear to be TABLE I. Morphogenetic events observed during and after rx 20-min heat shock. Four hundred The per cent
Time From beg. of shock 0 10 20
cells from distribution
each of the samples were classified according to their stage of development, of morphogenetic stages for each time shown on the left is indicated on the horizontal rows of the table.
(min) From end of shock
Per cent OU 61 59 59 63 68 70 61 42 27
0 10 20 30 40 60 80
1 0, No oral primordium visible. R, Oral primordium in progress ’ New primordia frequently visible old primordia. Experimental
Cell Research
35
at stage
Rb
1
2
3
4
5
7 7 10 8 6’ 4c
14 14 17 14 18 18 24 21 19
4 4 3 1
4 3 3 2 1
7 7 5 5 1
6 7 S 1
10 4
12 13
1 14
of regression. posterior (and
1 6 7 9
often
also anterior)
to regressing
6 (div.) 4 5 5 7 5 1 1 1 10
remnants
of
Morphogenesis
353
in T. pyriformis
in various stages of regression (see Figs. 4, 5, 6). In successively later samples, successively more advanced stages of membranelle regression can be seen. The structure of most of these regressing primordia indicates that they Were in fairly advanced stages of membranelle development (stages 4-5) at the time when the heat shock mas applied. Figs. 2-7 illustrate the sequence of primordium resorption vvhich can be inferred from these observations. Tmo interesting features of this resorption process warrant brief mention: (i) Although the primordia are already somewhat abnormal at the end of the heat shock (Fig. 3), resorption does not actually take place until af’fer the heat shock is over; in other vvords, the heat shock acts to trigger a resorption process (cf. 131). (ii) The resorption of advanced oral primordia is accompanied by an arrest in the development of the fission line, and a subsequent buckling and distortion of the rovvs of basal bodies (kineties) in this region (this buckling can also be observed in many synchronized cells). Stage 1 primordia appear to remain completely normal both during and after the 34” shock. The fate of the stage 2-3 oral primordia is somevvhat less clear, but it is likely that these revert to an appearance characteristic of stage 1. The distinct increase in the proportion of cells lacking oral primordia, which occurs in the first 20 min after the end of the heat shock, may be accounted for by the successful division of cells which had already begun to divide at the time vvhen the heat shock was initiated. Structurally normal cells were seen in the terminal phases of cell division both during and immediately after the heat shock. These are presumably cells which have passed a “stabilization point” at the time vvhen the heat shock was applied (cf. [3, 191). The stage at which stabilization occurs in this particular experiment is not precisely determined, but is probably during stage 5. Starting 60 min after the end of the shock, cells were seen to start membranelle formation anew; at 80 min, some cells were already seen in division. Cells in which old primordia were being resorbed frequently formed two nevv primordia, one anterior and one posterior to the resorption site (Figs. 6-7). J4orphogenetic events during synchronization.-Two experiments were performed to elucidate the morphogenetic events occurring during a six-shock synchronization regime; the results of these experiments are summarized in Fig. 8. After the first heat shock oral primordia vvith differentiating membranelles are no longer seen, and the percentage of cells bearing oral primordia declines. The second shock begins before any appreciable new development Experimental
Cell Research
35
J. Frankel of oral primordia has taken place. In the interval between the second and third shocks, most of the cells acquire stage l--2 oral primordia. Just prior to the third shock, membranelle development commences in a few cells. Subsequent to the third shock, membranelles are no longer observed, but the overall percentage of cells with oral primordia remains approximately constant. Beginning with the third shock, a distinct cyclic phenomenon becomes apparent. Essentially all the cells in the culture now have oral primordia. Between 30 and 70 per cent of these commence to form membranelles toward the end of each interval between shocks. The membranelles then disappear in the early part of the following inter-shock interval; they are apparently resorbed as a consequence of the heat shock (cf. previous section). In the middle of the intervals between shocks all the cells are in stage 1 of primordium development. In one of the two experiments, the degree of cell division synchrony was observed in samples taken from the main culture (cf. Methods section). After three heat shocks, synchrony was already fairly good (66 per cent of the population in division at the “division maximum”, 85 min after the end of the shock). It is worth noting that by the time of the third shock, most of the cells had already acquired oral primordia, which were generally in early stages of development. Subsequent to the fourth shock, the degree of synchrony remained approximately unchanged. There was, however, an unexpected decline in the degree of synchrony after the fifth shock. The poor synchrony in this case may be correlated with the unusually advanced state of membranelle differentia-
Fig. 2.-A cell fixed at the beginning of the 20 min heat shock, with a typical stage 4 primordium. The three membranelles (M) are well developed, the undulating membrane (UM) is in the process of development, and part of the original field of kinetosomes (F) is still present. The beginnings of the fission line are seen as an equatorial zone of discontinuities (D) in the longitudinal rows of kinetosomes (kineties). The oral area (OA), near the anterior end, is partly out of focus. * 1700. Fig. 3.-A cell fixed at the end of the heat shock, with a late stage 4 primordium. Its general structure is normal, but the membranelles have become somewhat ragged in outline. The discontinuities (D) marking the fission line are pronounced, and the kinety immediately to the animal’s right (viewer’s left) of the primordium shows a characteristic bending toward the primordium. x 1700. Fig. 4.-Twenty minutes after in an early phase of resorption.
the end of the heat shock. x 1700.
A stage 4 or 5 oral primordium
is shown
Fig. 5.-Thirty minutes after the end of the heat shock. A stage 4 or 5 primordium is seen in a late phase of resorption; the membranelles are much diminished in size and the undulating membrane has practically disappeared. Note the extreme buckling of the kinety to the right of the primordium, posterior to the original fission line discontinuity (D). x 1700. Esperimental
Cell Research
35
Morphogenesis
in T. pyriformis
355
Experimenfal
Cell Research
35
J. Frankel
Fig. B.-Sixty minutes after the end of the heat shock. Hemnants of an almost completely resorbed oral primordium (RP) are still visible. There are two new primordia, both in stage 1. One of these (NP 1) is anterior and the other (NP 2) is posterior to the remnants of the original primordium. Note the distortion of the fission line. The anterior oral area (OA membranelles out of focus) remains completely normal. x 1400. Fig. 7.-Eighty minutes after the the resorption site (R) being clearly (cf. Figs. 5, 6). Two new primordia
end of the heat shock. Primordium resorption marked as a focus of extensive local buckling (NP 1, NP 2) are both in stage 4. x 1400.
is completed, of the kineties
tion attained by many cells at the beginning of the fifth shock--50 per cent of the cells possessed stage 4 primordia. At the beginning of the sixth shock this excessively advanced oral primordium development did not occur, and synchrony was good after this shock (83 per cent of the population in division, 85 min after the end of the shock).
DISCUSSION
The results of the present study indicate that the effect of a heat shock on oral morphogenesis is the same in normal exponentially growing Tetrahymena as it is in the synchronized organism (cf. [3]). In both cases, there is clear Experimental
Cell Research
35
Morphogenesis
in T. pyriformis
357
evidence that high temperature induces resorption of developing membranelles. This is most apparent in stage 4 primordia, but also appears to occur in stage 3 primordia (cf. [3, 191). On the other hand, “anarchic field” primordia (stage 1) are not visibly affected by a heat shock. Stage 2 primordia probably revert to stage 1. There is a considerable delay between the end of a single heat shock and the resumption of morphogenetic activity (see Table I). Thus oral primordium formation is not observed between the first and second heat shocks of the synchronizing treatment (see Fig. 8). However, between the second and third heat shocks the majority of cells develop oral primordia. Clearly, the second heat shock is not hindering development as severely as the first. One might ,lOO-
Fig. 8.-Morphogenesis and division synchrony during the synchronization procedure. The schedule of administration of heat shocks serves as the abscissa; the shocks begin at hourly intervals. The cortical morphogenetic events are summarized by two curves: curve A indicates the percentage of cells which have oral primordia, in any stage of development; curve B indicates the percentage with stage 3 or 4 oral primordia, i.e. those in which developing membranelles are visible. The percentage of cells with stage l-2 oral primordia can be obtained by subtracting B from A. Each point is based on a tally of 100 to 200 cells. The uppermost curve indicates the degree of division synchrony, as observed following each heat shock. Two experiments were performed, under conditions as near as possible to being identical. The results of one are indicated by open circles, those of the other by filled circles. The degree of division synchrony was observed only in the latter experiment. Experimental
Cell
Research
35
358
J. Frankel
therefore postulate that in addition to the specific stage-dependent effects, an initial heat shock also induces a non-specific delay in development. Following this initial shock the cells adjust so that after a second shock the non-specific delay is either reduced or absent. Recent experiments, in which individual Tetrahymena of known ages were given either one heat shock or two successive shocks, revealed that a second shock generally has considerably less effect in delaying cell division than does the first shock; these results thus provide independent evidence which supports the above postulate.1 Division synchrony following two shocks is not much better than after a single shock. Following the third shock, however, synchrony is strikingly improved (cf. [22], Fig. 4~). It is at this time that the majority of cells have come to acquire oral primordia (see Fig. 8). By a slight extrapolation, one may correlate the time at which good synchrony is established in the population with the time at which all the cells in the population reach the phase of oral primordium development. Toward the end of the intervals between shocks, membranelle development starts in a considerable fraction of the cells; following the shocks these membranelles are resorbed. This phenomenon is clearly dependent on the duration of the inter-shock intervals: if these intervals had been appreciably longer, cell division would have occurred during the synchronizing treatment, and effective synchronization would have been impossible; if the intervals had been shorter, membranelles would not have had a chance to form. Williams and Scherbaum [‘LO], using a synchronizing treatment in which the intervals between shocks were only 30 min, found that during the synchronization procedure there is a steady increase in the proportion of cells with “anarchic field” primordia (eventually reaching 100 per cent), but there was no development of membranelles. Clearly, the transitory membranelle development observed in the present experiments is not an obligatory feature of the synchronization process. Indeed, when membranelle development goes too far in a significant proportion of the cells, synchronization is intrrfered with. The more advanced oral primordia (those in stage 4) undergo total resorption and replacement as a consequence of the heat shock (Figs. 2-7, cf. also [3]). This sequence of events requires a greater amount of time than does the more limited replacement or recovery process of cells affected at earlier stages (see [ 191 for direct confirmation of this conclusion). Cells in stage 4 at the time of the heat treatment arrive at cell division at a 1 These experiments were performed the aid of an N.I.H. grant (GM-08777-02) ance of Howard E. Buhse. Experimental
Cell
Research
35
by the author in the laboratory to Dr. Williams, and with
of Dr. N. E. Williams, the valuable technical
with assist-
Morphogenesis
in T. pyriformis
359
later time than cells in stages 1 to 3. Good synchronization can therefore only be obtained if the majority of cells are in stages 1 to 3 of primordium development at the time of the final shock. From the above considerations, we may conclude that an essential element in the synchronization process is the “collection” of cells in a phase of development which has the beginning of oral primordium formation as its salient structural feature. This phase occurs at a time which is somewhat less than halfway through the normal division cycle of Tefrahymena ([3], p. 1%). \Ve may interpret this to mean that synchronized Tefrahymena are actually “collected” into a phase which corresponds more closely to the middle than to the beginning of the normal division cycle. It is possible to characterize to some degree the stage-specific responses to a sequence of heat shocks, on which the “collection” of cells is based. Cells in the earliest phases of the cell cycle (stage O-lacking oral primordia) are considerably delayed in development by the first heat shock (cf. [18]), but adapt and form oral primordia in the succeeding inter-shock intervals. Cells with stage 1 (“anarchic field”) oral primordia retain their primordia through the heat shock treatment; however, in these cells some underlying system responsible for further development is affected, since there is a period development” [20] between the end of the last synchronizing of “arrested shock and the beginning of membranelle formation. Finally, cells which have started forming membranelles just prior to or during the synchronizing treatment or during the inter-shock intervals are thrown back by the heat shocks to the beginning of stage 1, and thus must start primordium development over again. The “collection” process can thus to a large extent be described in terms of morphogenetic responses, although observation of these responses does not tell us what the significant underlying processes are. Investigation into the physical and chemical bases of cortical morphogenesis might, however, yield significant information concerning the processes by which synchronization in Tefrahymena comes about.
SUMMARY
A study was made of the effects of a single heat shock, and of a multiple heat shock synchronizing treatment, on oral morphogenesis in Tefrahymena pyriformis GL. Heat shocks were found to have different effects, depending on the stage of cortical development. Cells with “anarchic field” primordia (stage 1) were not visibly affected by the shocks, while cells with primordia Experimental
Cell Research
35
360
J. Frankel
which had started to form membranelles (stages 3-4) resorbed their developing structures as a consequence of a heat shock and thus were returned to an earlier phase of development. Cells initially lacking oral primordia (“stage 0”) were able to develop primordia in the intervals between heat shocks. As a consequence of these stage-specific effects, there was a rapid increase, during the early part of the synchronizing treatment, in the proportion of cells possessing oral primordia; the primordia remained in (or were returned to) the “anarchic field” phase of development. Fairly good synchronization was achieved already by the end of the third shock, at which time the majority of the cells had begun oral primordium development. From these and earlier findings, it was concluded that the synchronizing treatment “collects” cells in a phase of development whose salient structural feature is the onset of oral primordium development. This phase may be considered to correspond roughly to the middle of the normal cell generation in Tetrahymena pyriformis.
The author would like to thank Drs. Earl D. Hanson, George G. Holz, D. Stuart Nachtwey, Norman E. Williams, and Erik Zeuthen for carefully reading this paper in manuscript
form,
and for supplying
constructive
comments
and suggestions.
REFERENCES 1. CORLISS, J. O., Stain Tech&. 28, 97 (1953). 2. __ Parasitology 43, 49 (1953). 3. FRANKEL, J., Compt. Rend. Trav. Lab. Curlsberg 33, 1 (1962). 4. FURGASON, W. H., Arch. Protistenk. 94, 224 (1940). 5. HALL, R. P., JOHNSON, D. F. and LOEFER, J. B., Trans. Am. Microscop. Sot. 54, 298 (1935). 6. HA~IBURGER, K., PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in Synchrony in Cell Division and Growth. E. ZEUTHEN (ed.) Interscience, New York, In press. 7. HOLZ, G. G., Bio[. Bull. 118, 84 (1960). 8. HOLZ, G. G., SCHERBAUM, 0. H. and WILLIAMS, N. E., Exptl Cell Res. 13, 618 (1957). 9. JAMES, T. W., Ann. N.Y. Acad. Sci. 90, 550 (1960). IO. KIDDER, G. W. and DEWEY, V. C., in Biochemistry and Physiology of the Protozoa, p. 323. A. LWOFF ted.) Academic Press, New York, 1951. 11. PADILLA, G. M. and JAMES, T. W. Exptt Cett Res. 20, 401 (1960). 12. RASMUSSEN, L., Compt. Rend. Trav. Lab. Carlsberg 33, 53 (1962). 13. RASMUSSEN, L. and ZEUTHEN, E., ibid. 32, 333 (1962). 14. SCHERBAUM, O., Acta Pathol. Microbial. &and. 40, 7 (1957). 15. ~ Ann. Rev. Microbial. 14, 283 (1960). 16. SCHERBAUM, 0. and ZEUTHEN, E., Exptl Cell Res. 6, 221 (1954). 17. THORMAR, H., Danish Thesis, Univ. of Copenhagen, 1956.
18. ~
19. 20. 21. 22. 23.
Compt.
Rend.
Trav.
Lab.
Carlsberg
31, 207 (1959).
WILLIAMS, N. E., EzptZ Cell Res., submitted for publication. WILLIAMS, N. E. and SCHERBAUM, O., J. Embryol. Exptt Morphot. 7, 241 (1959). ZEUTHEN, E., Adv. Biol. Med. Phys. 6, 37 (1958). ~ in Growth in Living Systems, p. 135. M. X. ZARROW (ed.) Basic Books, New York, -in Synchrony in Cell Division and Growth, E. ZEUTHEN (ed.) Interscience, New
1961. York.
In press. 24.
ZEUTHEN, E. and SCHERBAUM, KITCHING (ed.) Butterworths,
Experimental
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35
O., in Recent London,
Developments 1954.
in
Cell
Physiology,
p. 141.
J. A.