Morphogenetic Event isn Normal and Synchronously …Morphogenetic Event isn Normal and Synchronously Dividing Tetrahymena by NORMAN E. WILLIAMS1 and OTTO H. SCHERBAUM2 From the Department

Morphogenetic Events in Normal and SynchronouslyDividing Tetrahymena

by NORMAN E. WILLIAMS1 and OTTO H. SCHERBAUM2

From the Department of Zoology, University of California, Los Angeles, and the Department ofZoology, State University of Iowa, Iowa City

WITH ONE PLATE

INTRODUCTION

SYNCHRONOUS cell-division has been induced in mass cultures of the smallciliated protozoan Tetrahymena pyriformis (Scherbaum & Zeuthen, 1954).While it is known that cells grow in a characteristic way during the synchronizingtreatment the effect on the morphogenetic events associated with the cell cycleis not clear. Studies in ciliate morphogenesis generally have established thecentral position of the ciliary basal body, or kinetosome, in developmental pro-cesses. The kinetosomes are believed to be self-duplicating structures, the kineto-somal population of a daughter cell arising directly by kinetosomal reproductionin the parent cell. The species-specific pattern of the ectoplasmic cortex is largelya matter of the distribution of kinetosomes. Further, the kinetosomes appearto function either as building blocks or 'local organizers' in most, if not all,structural syntheses occurring in the cortex, i.e. in the production cilia, cirri,membranelles, trichocysts, and other ciliate structures (see Weisz, 1954). Twoconflicting notes are available concerning the behaviour of kinetosomes andkinetosomal derivatives in synchronized cultures of Tetrahymena. Child (1957)has reported that kinetosome reproduction continues and new mouth parts areformed in cultures of T. pyriformis W subjected to the synchronizing treatment.On the other hand, Holz, Scherbaum, & Williams (1957) have reported thatformation of mouth parts and also mitosis is inhibited by the synchronizing treat-ment in T. pyriformis WH-6 (mating type I, variety 1). In the present study kine-tosomes and kinetosomal derivatives in T. pyriformis GL were studied in detailin order to elucidate the relation of the morphogenetic events of the cell cycleto the experimentally induced synchronous cell-division.

On an earlier occasion cell-growth in synchronized cultures was studied(Scherbaum, 1956). The mean cell size was found to increase from three- to four-fold as a result of the treatment. When the cells were grouped in logarithmic size

Authors' addresses: 1 Department of Zoology, State University of Iowa, Iowa City, Iowa,U.S.A. 2 Department of Zoology, University of California, Los Angeles, Calif., U.S.A.[J. Embryol. exp. Morph. Vol. 7, Part 2, pp. 241-256, June 1959]

242 N. E. WILLIAMS AND O. H. SCHERBAUM

classes and analysed with the probit method it was found that, on a percentagebasis, the small cells grew less than the large cells during treatment. Preliminaryobservations of the nuclei of synchronized cells have indicated an absoluteincrease in nuclear size (Zeuthen & Scherbaum, 1954), and detailed nucleic acidanalyses have revealed a doubling of DNA and RNA on an average cell basis(Scherbaum, 1957&). The present study was undertaken in part to determine hownuclear growth is affected by the synchronizing treatment, and the relationshipof this process to growth and differentiation of the organism as a whole.

MATERIALS AND METHODS

T. pyriformis GL was grown in 100 ml. of peptone medium in an Erlenmeyerflask as described earlier (Scherbaum & Zeuthen, 1955). At a population densityof 50,000-80,000 cells per ml. the heat treatment was started. The treatment lasted7 hours, during which the cultures were exposed to seven temperature cycles,each consisting of one half hour at 28-29° C. followed by one half hour at33-9° C. Ten-millilitre samples were removed at regular intervals for Study.

The morphological features of the cortex were studied in specimens stainedwith protargol and with silver nitrate. Protargol preparations were made asfollows: cells were fixed in 2 per cent, osmic acid for 2 minutes and passedthrough ethanol of increasing concentrations. From pure ethanol the cells weretransferred to albuminized coverslips. The coverslips with the fixed cells attachedwere transferred to a 1 per cent, solution of Winthrop-Stern pre-war protargoland allowed to remain in this solution, along with a piece of copper wire, for 24hours at 22° C. A reducing solution of 1 per cent, hydroquinone in 5 per cent,sulphite was then used. After washing the samples were transferred to a 1 percent, aqueous gold chloride solution for 5 minutes. The cells were then washed,transferred to aqueous 2 per cent, oxalic acid for 5 minutes, washed again, andtransferred to 5 per cent, sodium thiosulphate. Finally, the samples were de-hydrated in absolute alcohol, cleared in xylene, and mounted in permount. Hairswere put into the preparations to support the coverslips.

The silver nitrate preparations were made according to the Chatton-Lwoffsilver impregnation technique as described by Corliss (1953a). In this procedurethe cells are embedded in a thin layer of gelatin on a slide prior to impregnationwith silver. French gelatin, believed by some workers to be superior to Americangelatin for this purpose, was used in the present study. All quantitative dataregarding the cortical structures were obtained from such silver nitrate prepara-tions. Kinetosomes were counted in normal and synchronized cultures. For prac-tical reasons, only the number of basal bodies in meridian n-2 was determinedand this was then used as an index of the total number of somatic kinetosomespresent. Meridian n-2 is the first meridian to the (animal's) left of the mouthwhich extends to the apex of the cell. Only those cells oriented in such a waythat the entire length of the index meridian was visible were used for counting.

MORPHOGENESIS IN SYNCHRONIZED TETRAHYMENA 243

A total of 60 cells was counted in each population considered. In additionsamples from various phases of normal and synchronized cultures were analysedfor the number of cells possessing anarchic fields of kinetosomes. In each case150 cells were examined and the number of cells with anarchic fields was ex-pressed on a percentage basis. Finally, the numbers of meridians and contractilevacuole pores were determined in various normal and heat-treated populations.In these cases a sample size of 30 was used for counting.

Macronuclear changes were studied in cells fixed with 1 per cent, osmiumtetroxide and stained with the Feulgen nuclear stain. Nuclear diameters weremeasured in these preparations. For each group under investigation the dia-meters of 100 nuclei were measured. Because of changes in cell size found inFeulgen preparations the parameters for cell volume and nuclear volume couldnot be determined on the same cells. Cell volumes were therefore determined oncells fixed in Bouin's fluid. Shrinkage was less than 10 per cent, in this fluid. Themajor and minor axes of the cells were measured on enlarged photomicrographs.The shape of the cells closely approximated a prolate spheroid, therefore theformula V = 4/3na2b was used, where a is the minor axis and b the major axis.

RESULTS AND OBSERVATIONS

Morphology of the normal organisms. The organisms used in the presentstudy agree generally with the detailed descriptions of T. pyriformis in the litera-ture (Furgason, 1940; Corliss, 19536). The essential features are the differen-tiated cortex and the macronucleus (strain GL is devoid of a micronucleus). Thedifferentiated ectoplasmic cortex consists primarily of a cytostome possessingthree membranelles and an undulating membrane, eighteen longitudinal rows ofcilia, two contractile vacuole pores, and a cytoproct. The classical technique forrevealing cortical structure and morphogenesis is silver nitrate impregnation.In the present study the application of protargol, a stain usually associated withstudies of flagellated protozoa, has made possible some new observations. Thesilver nitrate technique has the disadvantage of staining kinetosomes withoutrevealing the major kinetosomal derivatives, i.e. the cilia and ciliary membranes.Protargol was found to stain both of these elements, making possible directobservation of certain morphogenetic activities of the kinetosomes (comparePlate, figs. A and B).

In each meridian of normal cells stained with protargol 3-5 'naked' kineto-somes are usually found, i.e. basal bodies with no cilia attached (Plate, fig. A).These are often smaller than cilium-bearing kinetosomes and often lie close tothem. Short cilia have been seen regularly and presumably represent growthstages in the production of cilia by naked kinetosomes. This idea finds supportfrom the fact that cilia of all lengths from short stubs up to the full length of 7 ^have been seen. Such intermediate-length cilia were not found preferentially atany particular body-level, thus suggesting that growth of the somatic ciliature


occurs in all regions of the body. The 'fibres' and certain 'granules' seen in silvernitrate preparations are not stained by protargol (Plate, fig. A). The secondarymeridians are entirely absent in these preparations. The fibre associated with theprimary meridian of silver nitrate preparations has been found by Metz & West-fall (1954) with the electron microscope. This fibre is probably the 'kinetodesma'of Chatton & Lwoff (1935). On the other hand, the only structural feature ofTetrahymena found by these workers which corresponds to the secondary meri-

UM

STAGE 3 STAGE

TEXT-FIG. 1. Division stages of Tetrahymena. The rows of cilia are indicatedby solid lines. The system of numbering for rows of body cilia, or meridians,is indicated in the drawing of stage 1. The stomatogenic meridian is number oneand the numbers increase progressively around the animal from left to right.The four mouth membranes are indicated at the anterior region. Ml, M2, andM3 are the three membranelles while UM represents the undulating membrane.Stage 2 shows the anarchic field of basal bodies (AF). Stages 3 and 4 indicate

formation of the fully developed posterior cytostome (pc).

dians of the light microscopist is a series of pellicular rings coursing between theprimaries. It therefore appears that silver nitrate stains a variety of structuresboth on and beneath the pellicle. In contrast to this, protargol stains only thekinetosomes and their cilia. The greater specificity of protargol may presentcertain advantages for studies in ciliate morphogenesis.

Developmental stages. It will be useful to divide the process of binary fissioninto a number of stages. This has been found useful in the study of ciliate mor-phogenesis by other workers and will here facilitate analysis of the morpho-


logical changes which occur in normal and synchronous division. The mostmarked and easily recognizable changes in fission occur in the cortex. For thisreason division stages are defined in terms of cortical events (Text-fig. 1).

Stage 1. From the separation of daughter cells to the beginning of anarchicfield formation by meridian number 1, the stomatogenic meridian.

Stage 2. From the beginning of anarchic field formation to the organizationof the anarchic field into bands which represent the developing mouth parts.

Stage 3. From the organization of the anarchic field into bands to the begin-ning of cytoplasmic constriction.

Stage 4. From the beginning of cytoplasmic constriction to the separation ofdaughter cells.

In cells stained with protargol the somatic ciliature at all stages of develop-ment showed naked kinetosomes and short cilia. This suggests that somatickinetosome reproduction and ciliary synthesis occurs at all stages in the cellcycle. Soon after separation the daughter cells elongate considerably. No furthermorphogenetic events are noted to occur during stage 1. Stage 2 cells stainedwith protargol indicate that there are no cilia present in the newly formed anar-chic fields. The cilia of the undulating membrane and the three membranellesgrow out immediately after the organization of the anarchic field into bands,i.e. early stage 3. The oral cilia can be seen at all stages of their growth, the youngcytostomes with short cilia and older cytostomes with longer cilia. The growingcilia of a given cytostome always appear to be uniform in length, suggesting thattheir growth is synchronous. The undulating membrane is made of a single rowof cilia while the membranelles have two or more rows each. Also during stage 3the new contractile vacuole pores arise. As protargol stains the macronuclei aswell as cortical structures it was possible to correlate nuclear-division stages withthe developmental stages defined above. These preparations showed that elon-gation of the macronucleus begins during late stage 3 of normal cell-division.Stage 4 macronuclei are seen to continue elongation and to pinch in two. Feulgenpreparations indicate that by stage 4 the chromatin granules of the macronucleihave increased in size. The coarse-granular condition is typical of the dividingmacronucleus. Other events of stage 4 are the formation of the cytopharynx andoral ribs, thus completing the formation of the new cytostome.

Morphogenesis in synchronized organisms. Subsequent to the synchronizingtreatment all cells were found to possess large anarchic fields of kinetosomes(Plate, fig. C). This was reported earlier for strain WH-6 (mating type I, variety 1)(Holz, Scherbaum, & Williams, 1957). In the present investigation the frequencyof anarchic fields in the population was determined as a function of the syn-chronizing treatment. The results are presented in Text-fig. 2. Eighteen per cent,of cells in the untreated early maximum stationary phase culture possessedanarchic fields. No increase occurred until the second heat shock. From this timeon there was a steady increase until the fifth shock, at which time all cells werefound to have anarchic fields. This means that morphologically, 100 per cent.

5584.7 R


synchrony is attained by the synchronizing treatment. Protargol preparations ofcells upon completion of the treatment showed that no cytostomal cilia werepresent, indicating the presence of normal stage 2 cytostomes. The cells werefound to remain in this condition for a period of 50-55 minutes after completionof the synchronizing treatment. No change in the ectoplasmic cortex could be

100 -

I 2 3 4 5 6 7

heat shocksTEXT-FIG. 2. Proportion of cells showing anarchic fields of kine-tosomes in normal early maximum stationary phase and heat-

treated cultures. Each point is based on 150 cells.

detected. We refer to this period, therefore, as a period of arrested development.The only change noted to occur during this period was perhaps an increase in thesize of the chromatin granules within the macronucleus. Cells examined 1 hourafter treatment had resumed development. They possessed normal early stage 3cytostomes, characterized by the short, stubby, growing cilia arising from thefour bands of kinetosomes which are the basal plates of the incipient mouthmembranes (Plate, fig. E). A few cells 1 hour after treatment still had anarchicfields present. Since prior to this all cells were in the same stage of developmentthis represents an increase in the morphological variability. Other morpho-genetic events occurring after the period of arrested development and prior tocytoplasmic constriction were the production of contractile vacuole pores andthe beginning of macronuclear elongation. Cytoplasmic constriction began 1hour and 20 minutes after treatment. Silver preparations of cells at this timeshowed a continuing increase in the morphological variability, i.e. while mostcells were beginning constriction many others either had stage 3 cytostomes andwere not constricted, or else were well along in constriction and had fully formedcytostomes. The silver preparations also showed that the oral ribs and cyto-pharynx are formed after cytoplasmic constriction is well under way in syn-chronously dividing cells. Thus, aside from the 50-55-minute period of arrested


development following the synchronizing treatment, the developmental pro-cesses in synchronously dividing cells appear to be fundamentally the same asthose in normally dividing cells.

Counts were made of the number of meridians and the number of contractilevacuole pores in untreated cells, in cells immediately after the synchronizingtreatment, and in cells just prior to the synchronous division. The results arereported in Table 1. It is seen that no change in either meridian number or con-tractile vacuole pore number results from synchronization. In all cases themodal meridian number was eighteen and the modal contractile vacuole porenumber was two.

TABLE 1

Number of meridians and contractile vacuole pores in normal andsynchronously dividing organisms

MeanMode

Untreated cells

Meridians

17-6818

C.V.P.

2162

After treatment

Meridians

17-5218

C.V.P.

2-22

After induced lag

Meridians

17-6018

C.V.P.

2-262

TABLE 2

Number of kinetosomes in meridian n-2

Sample number

1Con t ro l , m a x i m u m s ta t ionary

phase . . . .

2During fourth heat shock .

3After seventh heat shock .

4Prior to synchronous division

5After synchronous division .

Mean

40-14

4606

57-64

59-86

34-34

Range

29-60

33-60

49-68

48-69

28^3

Standarddeviation

9-88

7-14

4-84

4-67

3-28

Coefficient ofvariation

0-246

0155

0084

0078

0095

The meridians of synchronized cells were also morphologically similar to themeridians of untreated cells. The distance between the meridians, however, wasclearly greater in synchronized cells and is to be expected on the basis of thegreater width of synchronized cells reported below. A second meridianal pecu-liarity of the heat-treated cells is to be noted in the subequatorial regions ofmeridians 2,3, and sometimes 4 and 5 (Plate, fig. C). These meridians are archedto the right in this region in association with the 'bulge' or 'notch' previouslyreported in mating type I, variety 1. Protargol preparations revealed that cells atall periods throughout treatment and the subsequent synchronous cell-division


possessed naked kinetosomes and short cilia. Although no quantitative datawere obtained it can be stated that no obvious alteration of the frequencies ofthese two elements was noted.

oc

cr0)

h—i

1

• A

3

-n

15

28 36 44 52 60 68

kinetosomes per m e r i d i a n

TEXT-FIG. 3. Number of kinetosomes in meridian n-2 in normal and syn-chronized cultures (excluding cells in the process of cytoplasmic constriction).The following populations are represented: early maximum stationary phasecells (1), during the fourth heat shock (2), after the last heat shock (3), prior tosynchronous division (4), and after the synchronous division (5). The syn-chronizing treatment increases the mean number of kinetosomes while reducing

the coefficient of variation. Each histogram is based on 60 cells.

The number of kinetosomes in meridian n-2 was determined for individualsin populations at various times during and after treatment. These data are pre-sented in Text-fig. 3. It should be emphasized that it was not possible to countthe number of kinetosomes in the meridians of cells in the process of cytoplasmicconstriction. The bias thus introduced will be considered in the discussion. It isseen from Table 2 that while the mean number of kinetosomes in meridian n-2gradually increased from 40 to 57 during the synchronizing treatment, thevariance progressively decreased. The coefficient of variation decreased from


0-246 in the untreated cells to 0-084 after the seventh heat shock. There is verylittle change in the mean and variance during the long induced lag between theend of treatment and the resumption of development. The synchronous fissionwhich follows reduces the mean to 34 kinetosomes. The standard deviation is

EXPONENT. MULTIPLIC.

MAX. STAT. PHASE

JTBETW. 3 d - 4 f t HEAT SHOCK

AFTER 7th HEAT SHOCK

PRIOR TO SYNCHR. 01V.

DURING SYNCHR. DIV.

rlr-r-t

AFTER SYNCHR. OIV.

NUCLEAR DIAMETERS

TEXT-FIG. 4. Nuclear diameters at differentstages of normal and synchronized cultures.Each histogram is based on the measurement

of 100 nuclei.

also reduced but the coefficient of variation is slightly increased. An interpreta-tion of these data will be presented in the discussion.

Growth in heat-treated organisms. Nuclear size was determined in logarith-mic phase cells, stationary phase cells, and at various times during and after thesynchronizing treatment. These data are presented in arbitrary units represent-ing nuclear diameters in Text-fig. 4. It is seen from this histogram that stationaryphase cells had nuclei considerably smaller than cells in logarithmic phase ofgrowth. A similar observation was reported previously by Summers, Bernstein,& James (1957). After three heat shocks in the synchronizing treatment the


nuclear size was increased. This increase continued until the nuclei after treat-ment were approximately three times as large as normal logarithmic phase cells(Table 3). Interestingly, some of the already greatly oversized nuclei continuedto increase in size after treatment and prior to the synchronous cytoplasmic con-

1.5MEAN LENGTH

TEXT-FIG. 5. Mean cell width and mean cell length as a function of the tempera-ture treatment. The mean width and mean length of the average normal cell(marked O m t n e figure) are arbitrarily set equal to 1 for comparison with themean cell length and width at various growth phases. Designation of the othersymbols: + between third and fourth heat shock, x after seventh heat shock,• prior to division, A after synchronous division, V maximum stationary phase.The broken line indicates a constant width /length ratio. Each point is the average

of measurement of 300 cells.

striction. It is not known whether this growth takes place during the period ofarrested development, subsequent to this, or during both periods since thesample was taken immediately prior to cytoplasmic constriction. During thesimultaneous division the nuclear volumes become remarkably uniform. Sixty-four per cent, of the measured nuclei fall into one size class. The mean volumeis also decreased during the synchronous division more than the expected one-half. This implies a change in density of the nuclear material or a loss of nuclear

M O R P H O G E N E S I S IN S Y N C H R O N I Z E D TETRAHYMENA 251

material into the cytoplasm. The Feulgen preparations indicate such a loss ofmacronuclear chromatin to the cytoplasm during the synchronous division.Quantitative consideration of this extranuclear chromatin will appear in aseparate study. After division the relative size of the average macronucleusreadjusts itself to a value close to one-half that found just prior to the syn1-chronous division.

TABLE 3

Nuclear volumes in normal and synchronized cultures(The mean volume is given in arbitrary units at the left. In the right column under 'Meanvolume' the volume of untreated cells is taken as 1 and the other populations compared to this)

Sample

Control, exponential multiplication

Between third and fourth heat shock .

After seventh shock . . . .

Prior to synchronous division

During synchronous division

After synchronous division .

Maximum stationary phase .

Mean

265

397

816

1,063

430

571

122

volume

100

1-50

308

401

1-62

216

0-46

Range

149-458

246-898

553-1,554

553-3,328

308-769

380-898

82-246

Standarddeviation

80

118

166

460

101

142

26

Coefficient ofvariation

0-30

0-30

0-20

0-43

0-24

0-25

0-22

TABLE 4

Cell volumes in normal and synchronized cultures(To facilitate comparison the values for length, width, and volume of the normal

average cell is also taken arbitrarily as 1)

Sample

1Control, exponential multiplication

2Between third and fourth heat shock

3After the seventh shock

4Prior to synchronous division

6After synchronous division .

7Maximum stationary phase .

Mean length

511

58-8

66-4

690

64-3

50-3

100

115

1-30

1-35

1-26

0-98

Mean

30-8

360

51-6

511

42-6

27-1

width

100

1-17

1-67

1-66

1-38

0-88

Mean volume

25,380

39,900

92,590

94,370

61,360

19,340

100

1-57

3-65

3-72

2-42

0-76

Cell size was determined in normal and heat-treated cultures and the nuclearsize data related to this. Stationary phase cells show a decreased width-lengthratio while heat-treated cells show an increased width-length ratio when com-pared with cells in the logarithmic phase of growth (Text-fig. 5). The data on cellvolumes is reported in Table 4. While the nuclei of stationary phase cells are


reduced to approximately one-half the volume of logarithmic phase cells, thetotal cell volume is reduced by less than this amount. The average stationaryphase nucleus, therefore, is smaller in relation to the total cell volume than theaverage logarithmic phase nucleus (Text-fig. 6). In the course of the heat treat-ment the mean cell volume increased more on a percentage basis than the meanmacronuclear volume. During the time subsequent to the treatment and prior to

TEXT-FIG. 6. Changes of nuclear versus cellvolume at different growth stages in arbi-trary units. Normal cell volume and nuclearvolume (marked Q in the figure) are bothtaken as one. Designation of the other sym-bols: + between the third and fourth heatshock, x after the seventh heat shock, Qprior to division, & after the synchronousdivision, V maximum stationary phase ofgrowth in untreated cells. The broken lineindicates a constant nucleo-cytoplasmicratio. Each point is based on the measure-

cell volume ment of 300 cells and 100 nuclei.

the synchronous cytoplasmic constriction the cell volume increased only slightly,while the macronuclear volume increased 30 per cent. These shifts in nucleo-cytoplasmic relations can be visualized from Text-fig. 6. After the synchronouscell-division the nuclear size was found to be reduced in relation to the cell size.The nuclear volume was reduced by approximately half by the synchronousdivision but the cell volume was reduced by less than this proportion.

DISCUSSION

Analysis of cortical differentiation in cultures of T. pyriformis GL subjectedto the synchronizing treatment has indicated that the cells are temporarilyarrested in their development, but are otherwise structurally normal. The normalsequence of morphogenetic events associated with binary fission has beenblocked at a characteristic point in these cells, i.e. at late stage 2. The specificdevelopmental processes which occur in treated cells prior to the block andsubsequent to the continuation of development in the synchronous division arefundamentally the same as for normal binary fission. Treated cells have the samenumber of meridians, the same number of contractile vacuole pores, and thesame cortical anatomy as cells from normal cultures that are in the same stage ofthe cell cycle. The number of kinetosomes should be discussed in this connexion.

The larger number of kinetosomes found in cells subjected to the synchroniz-ing treatment does not appear to be the result of an abnormal increase. Therange for the number of kinetosomes in meridian n-2 in untreated cells was from


29 to 60. It was observed that the low end of the distribution represents youngcells and the high end represents cells just prior to cytoplasmic constriction(kinetosome counts could not be carried out on cells undergoing constriction).This means that the number of kinetosomes in meridian n-2 in late stage 3 un-treated cells is in the neighbourhood of 60. The mean number of kinetosomes inmeridian n-2 for cells in late stage 3 of the synchronous division was 59-86. Thissuggests that the number of kinetosomes in cells after the synchronizing treat-ment is the same as the number of kinetosomes in untreated cells that are at acomparable stage in the cell cycle. The greater mean number of kinetosomes inmeridian n-2 in synchronized cells is therefore a result of the fact that all cellsare brought into synchrony at late stage 2, a period in the cell cycle characterizedby a large number of somatic kinetosomes. A second feature of the frequencydistribution of the number of kinetosomes in meridian n-2 of heat-treated cellsis the reduced coefficient of variation. Since the number of kinetosomes increaseas the cells approach the division stage, much of the variability in kinetosomenumbers in a normally growing population will be due to the distribution of cellsat various points in the cell reproductive cycle. A reduction in the coefficient ofvariability is thus to be expected when any degree of synchrony is induced in thepopulation.

The mean number of kinetosomes in meridian n-2 was 57-64 after the syn-chronizing treatment. The cells resumed development in synchrony and werefound to possess a mean number of 59-86 kinetosomes in meridian n-2 just priorto cytoplasmic constriction, i.e. at late stage 3. As it was not possible to count kine-tosomes in cells undergoing cytoplasmic constriction there is no direct evidencethat there is a kinetosome increase during this stage. However, the mean numberof 3434 kinetosomes found in meridian n-2 in cells just after the synchronousdivision suggests that during stage 4 there may have been an increase from 59-86to twice the number found in the daughter cells, i.e. 68-68. The data from syn-chronously dividing T. pyriformis, therefore, suggest that the growth of thesomatic ciliature may be most rapid during cytoplasmic constriction and after,with a reduction in the rate of increase occurring prior to constriction. Interest-ingly, this type of increase with respect to the cell cycle of Tetrahymena has beenreported for respiration by Zeuthen (1953).

The period of arrested development was found to occupy about 50-55minutes. With the exception of an increase in the size of the chromatin granulesof the macronucleus, no changes were noted to occur during this period. Previousinvestigations have led to the general conclusion that the blockade to cell-division in heat-treated cells may be due to a reversible denaturation of a singleenzyme system (Scherbaum, 1957a). From the present study it would appear thatthe enzyme system in question interferes with morphogenesis in a non-specificway, affecting the developmental sequence as a whole but not selectively inhibit-ing any constituent process. This does not mean, however, that a single develop-mental event cannot be the point of action of the synchronizing treatment. This


could actually be the case if all events normally occurring after stage 2 weredependent upon the integrity of this step. Biochemical investigations of arresteddevelopment in Tetrahymena might be rewarding.

An interesting feature of synchronized cultures of Tetrahymena is the disso-ciation of nuclear growth, cell growth, and morphogenesis. 'Growth' as usedhere refers to increases in volume. During the synchronizing treatment the cellsapparently proceed to developmental stage 2 and stop, while nuclear and cellvolumes continue to increase. During the period subsequent to the synchronizingtreatment and prior to the onset of cytoplasmic constriction there is an appre-ciable increase in nuclear volume with no concurrent increase in cell volume. Itis not known whether this nuclear growth takes place during the period ofarrested development, after this period, or at both times, since the measuredsample was taken just prior to cytoplasmic constriction. However, a 27 per cent,increase in DNA has been reported to occur during a one-hour interval subse-quent to the synchronizing treatment (Scherbaum, \951b), suggesting that mostof the nuclear volume increase and the DNA synthesis may occur during arresteddevelopment. If this is correct we may conclude that the extensive morpho-genesis which occurs in all cells synchronously after the period of arresteddevelopment takes place in the absence of any appreciable increase in nuclearand cell volume.

SUMMARY

1. A study of differentiation in the ectoplasmic cortex of T. pyriformis GLsubjected to the standard temperature cycling for the induction of synchronousbinary fission has demonstrated an arrest in development which occurs in allcells at a characteristic point in the cell cycle. After treatment all cells werefound to possess anarchic fields of kinetosomes in the stomatogenic region,indicating 100 per cent, synchrony. The cells remain in this condition for aperiod of 50-55 minutes after the last heat shock. During this time no changesother than in increase in the size of the macronuclear chromatin granules couldbe detected. At the end of the period of arrested development morphogenesisresumes and binary fission continues in synchrony. The degree of synchrony issomewhat reduced as development proceeds, resulting in about 85 per cent,synchrony at the time of cytoplasmic constriction.

2. In order to facilitate analysis of morphogenesis in synchronously dividingTetrahymena, a series of four developmental stages has been defined. Based onwork which has confirmed available descriptions of stomatogenesis and macro-nuclear fission, the sequence includes new data on development of the somaticciliature. All observations, including quantitative consideration of the somatickinetosomes, ciliary meridians, and contractile vacuole pores has indicated that,aside from synchrony and the period of arrested development, the morpho-genetic events in synchronized organisms are fundamentally no different fromthese processes in untreated cultures.

M O R P H O G E N E S I S IN S Y N C H R O N I Z E D TETRAHYMENA 255

3. Growth of the organism, growth of the macronucleus, and morphogenesisare to some extent dissociated in synchronized cultures of Tetrahymena. Duringthe heat treatment morphogenesis is blocked while cell and macronucleargrowth continues. After treatment and prior to the synchronous cytoplasmicconstriction there is a significant increase in mean nuclear volume with no signi-ficant increase in total cell volume. After resumption of development in syn-chronized cultures morphogenesis proceeds unaccompanied by any increase incellular volume. In addition, it is believed that the nuclear increase occurs duringthe period of arrested development and not during the subsequent period ofdevelopment.

4. The use of protargol staining promises to be of value in studies of ciliatemorphogenesis. Of those structures in and on the ciliate cortex, protargolappears to be highly specific for kinetosomes. In addition to this the cilia andciliary membranes are revealed by this technique.

ACKNOWLEDGEMENTS

We would like to thank Dr. W. H. Furgason, Department of Zoology, Univer-sity of California at Los Angeles, for reading the manuscript and for suggestingimprovements. We also wish to thank Dr. Furgason for providing the Frenchgelatin made available to him through the kindness of Dr. A. Lwoff of thePasteur Institute. We are indebted to Dr. W. Balamuth, Department of Zoology,University of California, Berkeley, for making available samples of protargol.We are also indebted to Mr. A. Louderback for the protargol preparations andother technical assistance. This work was supported in part by grant 2490 fromthe National Science Foundation.

REFERENCES

CHATTON, E., & LWOFF, A. (1935). Les Ciltes apostomes. I. Apercu historique et ge'ne'ral; e"tudemonographique des genres et des especes. Arch. zool. exp. gen. 77, 1—453.

CHILD, F. M. (1957). Morphogenetic changes during heat-shock synchronization of Tetrahymena.J. Protozool. 4 (suppl.), 12.

CORLISS, J. O. (1953a). Silver impregnation of ciliated protozoa by the Chatton-Lwoff technique.Stain Tech. 28, 97-100.(19536). Comparative studies on holotrichous ciliates in the Colpidium-Glaucoma-

Leucophrys-Tetrahymena group. Parasitology, 43, 49-87.FURGASON, W. H. (1940). The significant cytostomal pattern of the 'Glaucoma-ColpidiunC group,

and a proposed new genus and species, Tetrahymena geleii. Arch. Protistenk. 94, 224-66.HOLZ, G. G., SCHERBAUM, O. H., & WILLIAMS, N. E. (1957). The arrest of mitosis and stomato-

genesis during temperature-induction of synchronous division in Tetrahymena pyriformis,mating type I, variety 1. Exp. Cell Res. 13, 618-21.

METZ, C. B., & WESTFALL, J. A. (1954). The fibrillar systems of ciliates as revealed by the elec-tron microscope. II. Tetrahymena. Biol. Bull. Wood's Hole, 107, 106-22.

SCHERBAUM, O. (1956). Cell growth in normal and synchronously dividing mass cultures ofTetrahymena pyriformis. Exp. Cell Res. 11, 464-76.(1957a). Studies on the mechanism of synchronous cell division in Tetrahymena pyriformis.

Exp. Cell Res. 13, 11-23.


SCHERBAUM, O. (19576). The content and composition of nucleic acids in normal and syn-chronously dividing mass cultures of Tetrahymena pyrifonnis. Exp. Cell Res. 13, 24-30.& ZEUTHEN, E. (1954). Induction of synchronous cell division in mass cultures of Tetra-

hymena pyriformis. Exp. Cell Res. 6, 221-7.(1955). Temperature-induced synchronous divisions in the ciliate protozoon Tetra-

hymena pyriformis growing in synthetic and proteose-peptone media. Exp. Cell Res. Suppl.3, 312-25.

SUMMERS, L., BERNSTEIN, E., & JAMES, T. W. (1957). A correlation between nuclear activity andthe growth phase in cultures of protozoan cells. Exp. Cell Res. 13, 436-7.

WEISZ, P. P. (1954). Morphogenesis in protozoa. Quart. Rev. Biol. 29, 207-29.ZEUTHEN, E. (1953). Growth as related to the cell cycle in single-cell cultures of Tetrahymena

pyriformis. J. Embryol. exp. Morph. 1, 239-49.& SCHERBAUM, O. (1954). Synchronous divisions in mass cultures of the ciliate protozoonTetrahymena pyriformis, as induced by temperature changes. Colston Papers, 7, 141-55.

E X P L A N A T I O N OF PLATE

FIG. A. Photomicrograph of a normal individual stained with protargol, showing cytostome(c) and ciliary rows. Both the cilia and their basal bodies (kinetosomes) are revealed by this tech-nique. Note kinetosomes without cilia found in various regions of the body (NK). These mayrepresent newly formed kinetosomes prior to ciliary synthesis.

FIG. B. Photomicrograph of a normal individual stained according to the Chatton-Lwoffsilver impregnation technique. Kinetosomes and fibrils are revealed but not the cilia or ciliarymembranes. Meridian n-2 was used for kinetosome counts and is designated in this photomicro-graph. This particular cell shows 32 kinetosomes in meridian n-2, indicating that the organism isin the early part of the cell cycle.

FIG. C. Photomicrograph of a cell impregnated with silver immediately after the synchronizingtreatment. Note the anarchic field of kinetosomes (AF) present in the equatorial region and thelarge numbers of kinetosomes in the meridians. This is the condition in all cells subsequent totreatment, i.e. 100 per cent, synchrony has been obtained. For purposes of orientation it shouldbe noted that the ventral surface has been photographed from the dorsal side of this organism.Thus, in this case, the animal's right is on the viewer's right.

FlG. D. Photomicrograph of a silver-impregnated cell 1 hour and 10 minutes after the syn-chronization treatment. The kinetosomes of the anarchic field have organized into the patterncharacteristic of the fully developed cytostome, indicating that development has been resumed. Themeridianal pattern in the region of the developing cytostome foreshadows the beginning of con-striction which began 10 minutes later in the population from which this individual was taken.Nearly all cells in the population were at this stage of development, although synchrony was nolonger 100 per cent.

FIG. E. Photomicrograph of a protargol-stained cell at approximately the same stage in thesynchronous division as the above. At this time the growing cilia of the developing undulatingmembrane (DC) and membranelles are still shorter than the neighbouring somatic cilia.

(Manuscript received 13: xi: 58)

J. Embryol. exp. Morph. Vol. 7, Part 2

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N. E. WILLIAMS «n^ O. H. SCHERBAUM

Documents

Morphogenetic Event isn Normal and Synchronously …Morphogenetic Event isn Normal and Synchronously Dividing Tetrahymena by NORMAN E. WILLIAMS1 and OTTO H. SCHERBAUM2 From the Department