Pattern determination and pattern regulation in Paramecium ... · the Paramecium system and the character of the spatial regulation of the cortical pattern remains unknown. Removal

J. Embryol. exp. Morph. 74, 47-68 (1983) 4 7Printed in Great Britain © The Company of Biologists Limited 1983

Pattern determination and pattern regulation inParamecium tetraurelia

By JANINA KACZANOWSKA1 AND BOZENA DUBIELECKA1

From the Institute of Zoology, Warsaw University

SUMMARY

Pattern regulation was investigated in the progeny of laterally fused cells of Parameciumtetraurelia. The immediate progeny of such fused cells (doublets) reveal two sets of corticalorganelles arranged roughly symmetrically. Doublets tend to transform gradually into cellswith only one set of organelles (singlets). At least two different and mutually exclusive path-ways of doublet-to-singlet transformation are reported. In intermediate stages of regulationthe cortical areas bearing different cortical landmarks may be brought into an abnormalneighbourhood. Differentiated cortical bands of cortex, bearing organellar landmarks, arefaithfully propagated even if they are improperly and asymmetrically located on the cell. Theconfrontation of such cortical bands may lead to the transient appearance of additionalduplicated organelles.

It is suggested that pattern regulation in Paramecium during doublet-to-singlet transforma-tion results from at least three factors: the regression of some part of the cortical areas, theinteraction of the juxtaposed parts remaining and the slow regulatory shift of positions of thecortical structures.

INTRODUCTION

The aim of the present paper is to investigate the mode of spatial patternregulation in a ciliate Paramecium tetraurelia.

Most ciliates show a remarkable capacity for regeneration and pattern regula-tion when pieces of the cell cortex are added or removed (Tartar, 1961; Sonne-born, 1975; Frankel, 1974). However, Paramecium represents an extrememosaic system of intracellular development (Beisson & Sonneborn, 1965) thatcontrasts with the highly regulative development of other ciliates (Tartar, 1954;Schwartz, 1963; Sonneborn, 1963). In Paramecium, the buccal primordium issituated very close to the pre-existing oral apparatus and 'de novo' formationdoes not occur after its removal (Tartar, 1954; Hanson, 1962; Hanson & Unger-leider, 1972).

The general deployment of cortical organelles over the ciliate cortex may bedescribed and quantified in terms of specific angles in a polar co-ordinate systemprojected over the cell surface (Nanney, 1966a). The cell is covered with lon-gitudinal ciliary rows marking meridians of such a system. The right ciliary rowflanking the ventral complex, comprising the oral apparatus and a site of egestion

1 Authors' address: Institute of Zoology, Warsaw University, Warsaw 00-927/1, Poland.

48 J. KACZANOWSKA AND B. DUBIELECKA

(cytoproct), is designated the ciliary row no. 1 and serves as the reference (0°meridian) to measure the longitudes of the position of any other structure interms of the angle observed on the circumferential projection of the tested cell(Fig. 1). If longitudinal ciliary rows are uniformly disposed over the cell cortex,this angle may also be expressed as a proportion of the number of ciliary rowsseparating the position of ciliary row no. 1 and tested structure to the totalnumber of longitudinal ciliary rows counted on the cell circumference. The totalnumber of longitudinal ciliary rows is referred to as the corticotype of a given cell(Nanney, 1966a, b).

Nanney (1966a,b, 1968, 1972) and Nanney, Chow & Wozencraft (1975)discovered certain cytogeometrical rules governing the positioning of thecontractile vacuole pores (CVPs) on longitudes of Tetrahymena. Positioning ofthe CVP in a given cell may be described as a consequence of specification of aninductive angle between the ciliary row involved in manufacturing the ventralcomplex and the longitude in which the CVP is found. The variability of position-ing of the CVP is described in terms of afield angle which defines the band of thecell surface which may be competent to yield a CVP. This specific cytoarchitec-ture of a given ciliate species is maintained during successive fissions, since boththe ventral complex and CVP positioning are reproduced along virtually thesame longitudinal bands of cortex in daughter cells (Frankel & Nelsen, 1981).

In experimental studies on the Tetrahymena system (Nanney, 19666; Jerka-Dziadosz & Frankel, 1979; Nanney et al. 1975; Frankel & Nelsen, 1981) theinductive and regulative character of CVP positioning on cell longitudes withrespect to the oral sector is well documented. There are no comparative data onthe Paramecium system and the character of the spatial regulation of the corticalpattern remains unknown.

Removal and grafting of longitudinal strips of cortex onto a normalParamecium cell have been performed to explore the pattern determination ofnew structures on the oral band (Hanson, 1955; Hanson & Ungerleider, 1972;Sonneborn, 1963) or the stability of anteroposterior polarity of ciliary rows(Beisson & Sonneborn, 1965; Sonneborn, 1970a). Such experiments are verylaborious and are feasible only on a limited number of cells. However, anequivalent morphological confrontation of different parts of the cortex appearsduring the spontaneous and gradual transformation of homopolar twin cells ofParamecium (comprising two sets of all cortical structures) into a single-cellcortical pattern in their progeny. Such homopolar twin cells, or doublets, maybe obtained experimentally after a complete fusion of mating cells (Hanson,1955; Sonneborn, 1963, 1970a; Butzel, 1973; Hanson & Ungerleider, 1972;Sibley, 1974; Morton & Berger, 1978). These doublets initially represent a fusionof two cortical areas with an alternate and symmetrical sequence of deploymentof cortical organelles (Fig. 1). This pattern is then reproduced during subsequentcell generations. However, it is apparent that the doublet organization of the cellcortex is somewhat unbalanced, since from time to time a spontaneous reversion

Pattern regulation in Paramecium 49

to singlet appears and this transformation is directional and irreversible (deHaller, 1965). However, data about intermediate morphological stages duringdoublet-to-singlet transformation are very confusing: while Sonneborn (1970a)reported that one of the oral apparatuses may be lost when positioned 180 ° apartfrom the second oral apparatus, Sibley (1974) described the possibility of a side-by-side configuration of both ventral sectors in doublets transforming to singlets.Hence the problem of pattern regulation during doublet-to-singlet transforma-tion is reinvestigated here.

MATERIALS AND METHODS

Materials

All stocks employed were derived from Sonneborn's Paramecium tetraureliastock 5IS. Culture and handling of paramecia followed the methods of Sonne-born (19706). They were cultured in Cerophyl, or in lettuce media, inoculatedwith Klebsiella cloacae. Results were similar in both media.

Construction of doublets

The initial doublets were derived in two independent series by recovery ofconjugating cells that failed to separate after either a brief thermal, oractinomycin D shock applied 30 min after the isolation of the tight pairs. The firstset of lines of wild-type doublets was derived from mating of complementary cellstreated for 45 min with 50 jug/ml AMD. The second set of lines was derived froma doublet created by heat shock (45 min at 37 °C) administered to conjugants ina cross of wild-type cells with homozygous twisted cells of scr6 and fna alleles.Since the scr6 and fna alleles are recessive, the resulting heterozygous doubletwas of a wild-type phenotype. The fna allele (Kung, 1971) was used only as aneasy marker for screening the completeness of sexual processes. The scr6 allele(Sonneborn, 1974) when homozygous brings about a left-handed twisting ofciliary rows around the main body axis. However, this allele has variablepenetrance and expressivity so both straight and twisted phenotypes may occurin a scr6'/scr6 clone.

A scr6, fna homozygous doublet was isolated from the heterozygous doubletline after massive autogamy and kept as subline F with straight and twistedphenotypes.

Cells were kept in test tubes at room temperature (about 22 ± 2 °C) and fedonly twice a week. Under these conditions, doublets of all sublines dividedapproximately every other day, while spontaneously derived singlets dividedabout once a day. Control singlets of corresponding genotypes and phenotypeskept at the same conditions also averaged one fission per day.

Cytological tests

Autogamy was observed using Dippell's stain (Dippell, 1955). To reveal the

50 KACZANOWSKA AND B. DUBIELECKAcortical pattern, the samples of cells were periodically fixed twice a week duringobservation periods. The Chatton-Lwoff technique of silver impregnation wasused following Frankel & Heckmann's protocol (1968).

Measurement and counting

For evaluation of the rate of appearance of singlets in samples all well-silveredspecimens from a given series were used.

To test the distribution of cortical organelles in doublets, in cells in inter-mediate stages of transformation from doublet to singlet, and in derived singlets,separate sets of camera-lucida drawings were made at the same magnification.Only well-silvered, straight cells, with no sign of divisional morphogenesis werechosen. Each of the drawings was then processed by geometrical projection ofthe angular distribution of the cortical organelles onto a circle of the diameter ofthe drawn specimen (Fig. 1). The right margin of the oral apparatus, cytoproctand CVPs were marked on this circumference. Then this circular projection was

c\ip

CVP CVP

OA'

Fig. 1. Scheme of the method of processing of a circular projection of a given ciliate,and of estimating angular values. (A) typical circular projection of a symmetricaltype I doublet. (B) typical circular projection of a symmetrical type II doublet. (C)circular projection of one of the intermediate stages in regression of a type I doublet.Black circles on the circumferences represent sites of the oral apparatus, crossesindicate sites of CVP bearing ciliary meridians. All projections are uniformly orien-ted as viewed from the anterior cells pole.

Pattern regulation in Paramecium 51verified by focusing the specimen under the microscope. The number of com-plete ciliary meridians between successive landmarks was counted. In the caseof the homozygous wild-type doublets 72 symmetrical and asymmetricaldoublets and 71 derived singlets were analysed mainly using the criteria of theprojected angles, and the numbers of ciliary rows were counted in only some ofthem (as described below). In the case of heterozygous lines, 32 completedoublets, 16 so-called incomplete doublets and 33 derived singlets were charac-terized by analysis both of projected angles and of ciliary meridian distributions.

For studies on the conservation of CVP positioning within the dorsal sector(variability of the field angle), straight and twisted cells of different genotypeswere used. The intermeridional space where the anterior CVP occurred waschosen as a reference, and the deviation of the position of the posterior CVP, ora third supernumerary CVP either to the right or to the left, was expressed as anumber of ciliary rows separating the anterior from the tested CVPs. All testswere performed on apparently non-dividing cells.

In the case of scr6] /scr6 singlet phenotypes, earnera-lucida drawings were madeon the specimens with a distinct CVP band exposed to the viewer. From thesepictures, the angle between the longitudinal cell axis and the line joining twoobliquely positioned CVPs was measured to estimate cell twisting. The cells wereclassified as 'twisted' if this angle exceeded 20 ° (136 cells). In the case of doubletsonly scr6, fna double-twisted homozygotes were used (n = 49) with twist of morethan 20°.

RESULTS

1. Characteristics of single and double Paramecium tetraurelia cells of differentphenotypes

Wild-type singlets. (Figs 2 and 3) Morphometrical and cytogeometrical studieson the cortical pattern of control (5IS) single cells revealed a virtual stability ofcortical parameters of all non-dividing cells. This pattern is consistent with thedetailed description of Sonneborn (1963) and of Kaneda & Hanson (1974). Inbrief; the oral band is marked by a complex comprising preoral suture, oralapparatus and postoral suture with a cytoproct, and is flanked to the right bynearly meridional ciliary rows. On the left side of the oral apparatus the ciliarymeridians are more densely aligned and they are bent in the form of arcs aroundthe oral apparatus. Thus the preoral and postoral sutures are flanked with slight-ly skewed right ciliary rows and with more curved left ciliary meridians. At thepostoral suture there is a long silver-stained line representing the lips of thecytoproct (Ng, 1976; Allen & Wolf, 1974). The left and right ciliary rows gradeinto pole-to-pole meridians on the dorsal surface. The position of the anteriorcontractile vacuole pore (CVP) marking the centre of the CVP band wasdeliberately chosen as the boundary for counting the right and left set ofmeridional ciliary rows (Sonneborn, 1963; Kaneda & Hanson, 1974). In a sample


Fig. 2. Scheme of positioning of cortical organelles over: (A) ventral and (B) dorsalsurface of a single cell of Paramecium tetraurelia. Oral band is composed of thepreoral suture (PR), the oral apparatus (OA), the postoral suture (PO), with markedlongitudinal slit of the cytoproct (CYT). Ciliary rows (dashed lines) run nearlymeridionally to the right of the ventral complex. They are arched and more denselypacked to the left of the ventral complex. On the dorsal side, two contractile vacuolepores (CVP) mark the border between the left (more dense) and the right (lessdense) system of ciliary rows. Arrows mark the CVP band of the cortex. Note thatthe preoral and postoral sutures terminate on the dorsal surface (Sonneborn, 1963).

of 46 cells, there were 31-8 ± 1-9 right and 43-8 ± 2-7 left ciliary meridians (in-cluding the short ciliary rows arching immediately adjacent to the oral apparatusand known as the vestibular rows). The lateral displacement of the posteriorCVP relative to the anterior one is no more than two ciliary rows in eitherdirection. Thus the corticotypes of control cells averaged about 74-75 and themaximum field angle equals to about 20° (~4/75).

Twisted singlets. Twisted singlets of genotype scr6 /scr6 expressed similarcorticotypes and left and right ciliary meridian distributions. In a sample of 20cells there were 31-5 ± 1-4 right and 44-6 ± 1-9 left ciliary meridians. The mor-phology of these cells follows the description of Whittle & Chen-Shan (1972) ofleft-handed screwy mutants. In twisted phenotypes, the ciliary rows arespiralized due to extensive local growth of the postoral right area (Kaczanowska,1977). The postoral suture with a cytoproct may be displaced to the dorsal side,and then the dorsal cortical band bearing the CVP runs obliquely from theanterior pole to the left. The anterior CVP in twisted cells remains at roughly180° to the anterior portion of the oral band. However, the dorsal ciliary rowsare no longer parallel to the main body axis. Thus, in these cells the degree ofconservation of the location of CVPs within the band of cell cortex defined bythe ciliary rows may be tested. In most cells (75 % of 136 tested specimens), thelateral displacement of CVPs was kept within the limit of variability observed inthe controls, i.e. within 20° field angle. However in the remaining cells thisdeviation was increased up to eight ciliary r6ws. The posterior CVPs were always


Fig. 3. Photomicrograph of a single control of Paramecium tetraurelia. Silver-stained preparation. On exposed ventral surface of the cell all parts of the oral bandare seen. Abbreviations as in Fig. 2.Fig. 4. Photomicrograph of the asymmetrical I type doublet with the extra cytoproctexposed. The left cytoproct (slightly out of focus) runs (arrowheads) along the con-tour of the cell. This stage corresponds to Fig. 6B. Silver-stained preparation. Allabbreviations as in Fig. 2.

shifted to the right, marking the deployment of the CVPs more parallel to themain body axis. However, even a maximum shift of positioning (extension of thefield angle to 40 °) did not fully restore the proper geometry of the CVPs relativeto the morphological axis of the twisted cell, but it did improve it.

In twisted cells an additional third CVP very often (in about 20 % of cases)appeared close to the posterior cell pole. The origin of this structure remainsunknown.

Symmetrical doublets. At least two types of regular symmetrical doublets were


Fig. 5. Scheme of the cytogeometry of (A) type I and (B) type II doublets with theirrespective circular projections. Regular symmetrical doublets. (A) Right anglesbetween oral (black circles) and CVP (crosses) landmarks on circular projection, andthe regular shape of doublet are schematically depicted. (B) Decrease of the angularvalue of the right systems of the type II doublets, and splitting of the anterior portionof the doublet are marked. All graphical conventions as in Fig. 1.

found in the sublines studied. In both, the oral and CVP band were initiallypositioned 180° apart, but subsequent regulation was very different.

(a) In symmetrical doublets, found in all sublines from the wild-type initialdoublet (Fig. 5), the two sagittal axes defined on the circumferential projectionof the cell by the oral-oral and CVP-CVP bands planes were crossed at a rightangle (Fig. 5A). This corresponds to about 28 right and 39-40 left ciliary rows(n = 10 tested cells). Such cells had corticotypes of 115 to 136 rows. These cellspossessed fused poles (Fig. 5A). They can reproduce their cytogeometry duringmore than a hundred generations, and then give rise to intermediate stages ofexclusively one type, namely of asymmetrical doublets. Derived singletsdisplayed either normal corticotypes (like control singlets) or slightly elevatedcorticotypes due to some surplus of left ciliary rows. Doublets of this type willhenceforth be called 'Type I doublets'.

(b) In symmetrical doublets from sublines derived from the progeny of the initialheterozygous doublet, the sagittal axes of oral-oral bands and CVP-CVP bandsdiameters on circumferential projections of the cells were not crossed at a rightangle but at an angle of about 70-75 ° (Fig. 5B). This corresponded to about 26-28right and 40-42 left ciliary rows (n = 11 tested cells). While the maximum cor-ticotype was also 136, some cells revealed lowered corticotypes down to 94.

Pattern regulation in Paramecium 55These doublets have never displayed fusion of anterior poles of their com-

ponents (Fig. 5B). Thus, particularly at the anterior pole of the doublet, twosignificant acuminations representing individual single poles were separated bya notch of varying depth. Doublets of this kind can also reproduce their owncytoarchitecture for over a hundred generations. They then transform into sing-lets with intermediate stages exclusively of the character of so-called 'incompletedoublets' (Sonneborn, 1963) i.e. doublets with one or even two missing oralapparatuses but with preoral and postoral sutures and cytoprocts. Unlike theformer type of doublets, they have never generated asymmetrical completedoublets as intermediate stages. A typical asymmetrical doublet of this typepossessed one complete oral band and a second incomplete oral band comprisingthe preoral, postoral sutures and the cytoproct. Asymmetrical doublets with twoincomplete oral bands (astomous) were occasionally found. The derived 'early'singlets displayed very variable corticotypes, frequently severely reduced (toabout 52-54).

Doublets of this type will be called 'Type II doublets'. Their characteristicswere also detected in twisted doublets of the F sublines derived from theheterozygous line after a sexual process and the achievement of homozygosity.In 20 % of these cells an additional, posterior CVP was observed either in oneor in both CVP band of cortex. In 49 heavily twisted cells the rate of conservationof CVP location was tested. While in about 71 % all CVPs were located withinone or two rows of each other, in the remaining 29 % at least one, and sometimestwo, CVP bands were enlarged to up to six ciliary rows.

Both types of doublets (I and II) observed during the first month of studycontained two separate macronuclei. Only one macronucleus was found, evenin symmetrical doublets, during the last month of observations of all sublines.

2. Sequence and frequency of appearance of intermediate stages duringdoublet-to-singlet transformation in progeny of type I doublets

The data of the previous section show that the doublet-to-singlet transforma-tion involves severe reduction of the number of ciliary rows. The question iswhether in doublets transforming into singlets this decrease of the number ofciliary rows affects the angles of deployment of cortical landmarks? There is stillanother general question, namely whether this transformation is rapid, or in-volves many intermediate generations, and whether once started it requires afixed, or variable number of generations for final achievement of singlet archi-tecture.

To answer this question, the sequence and frequency of appearance of inter-mediate stages during the doublet-to-singlet transformation was studied in foursublines of cultures originating from the wild-type doublet. The sublines wereinitiated when the first derived singlet was found in test tubes of doublet progeny.This occurred after about 60 generations, with the possible intervention of twoor three autogamous cycles. At this time, four clones, A, B, C and D, were


started by isolation of apparently symmetrical doublets. The progeny of thesefour sublines were tested for the appearance of intermediate stages of trans-formation into singlets, and for the frequency of these stages, in random samplesover the next 120 days, which corresponds to about 55 generations of doublets(with one or two peaks of autogamy). For the sake of simplicity, at first all testedcells were classified into three major morphological groups: regular symmetricaldoublets, asymmetrical doublets and derived singlets. The frequencydistribution of these morphological classes is presented in Table 1.

From these data it is evident that the distribution of classes changes in a regularway: in subline B in which initially a majority of cells are symmetrical doublets,only a minor fraction of doublets are asymmetrical. Subsequently there is anincrease in the fraction of asymmetrical doublets. An increase in the proportionof asymmetrical doublets in A and C sublines coincides with an appearance ofderived singlets and in the majority of cases with a decrease in the fraction ofsymmetrical doublets. In sublines C and D, the class of symmetrical doubletscompletely disappeared and subline C eventually consisted only of single cells,while subline B did not yield singlets during the entire period of observations(although it did a few months later). It is not known if all derived singlets areviable. The slow increase in the percentage of asymmetrical doublets in thedevelopment of sublines A and B and the rarity of singlets indicate that the slowlydividing asymmetrical doublets are rather stable over a long period and probablygradually revert to the single organization. This conclusion was directly con-firmed in experiments made on isolated asymmetrical doublets reisolated aftertheir division. In a few cases singlets were found after five and seven generations,but in one case after 12 fissions all cells were asymmetric doublets.

Among all tested well-silvered cells (n = 2600) no cell was found which corre-sponded to an incomplete doublet character (i.e. lacking an oral apparatus in anoral band). All intermediate stages in all sublines corresponded exclusively toasymmetrical doublets. All stages roughly agree with the brief description givenby Sibley (1974). All symmetrical and asymmetrical doublets have functionaland normal oral apparatuses (Kaczanowska & Garlinska, 1981). Fragmentationand distortion in pattern of ciliary rows in symmetrical and slightly asymmetricaldoublets occurred exclusively in the vicinity of, or in, the CVP meridians. Insome cells one or both sets of CVPs were absent. It is not known if cells lackingosmoregulatory organelles may be viable. In any case, such cells appeared whenasymmetrical doublets occurred and were never observed among derived sin-glets.

In asymmetrical doublets the increase of asymmetry was correlated with adecrease of corticotype and cell diameter. Thus the cell circumferencediminishes mainly through a decrease of the minor side (i.e. on the side of theminor angle between oral apparatuses) of the asymmetrical doublets. Highlyasymmetrical doublets also displayed an advanced fusion of the preoral andpostoral structures (as seen in Figs 6C, D and 7). In about 30 asymmetrical

Tab

le 1

. T

he fr

eque

ncy

dist

ribu

tion

(in

%)

of c

lass

es o

f sy

mm

etri

cal

doub

lets

, as

ymm

etri

cal

doub

lets

and

sin

glet

s in

sam

ples

from

sub

line

s, A

, B

, C

and

D o

f P

aram

eciu

m t

etra

urel

ia w

hich

wer

e ob

serv

ed d

urin

g fo

ur s

ucce

ssiv

e m

onth

s

Mon

th o

fob

serv

a-ti

ons

1 2 3 4

n 48 101

240

296

Sub

line

Asy

m-

met

.d

ou

:

bs.

37-0 5-8

10-2 8-3

asym

-m

et.

dou-

bs.

63-0

94-2

87-2

87-7

sing

-le

ts

0 0 2-5

4-0

n =

the

tot

al n

umbe

r of

tes

ted

spec

imen

s.

n 61 330

286

267

Sub

line

Bsy

m-

met

.do

u-bs

.

92-0

60-6

23-8

14-8

asym

-m

et.

dou-

bs.

8-0

39-4

76-2

85-2

sing

-le

ts 0 0 0 0

n 71 294

194

200

Sub

line

Csy

m-

met

.do

u-bs

.

40-3

13-5 0 0

asym

-m

et.

dou-

bs.

58-7

72-3 9-0

0

sing

-le

ts 1-0

14-2

91-0

100-

0

n 49 264

Sub

line

Dsy

m-

asym

-m

et.

met

.do

u-

dou-

bs.

bs.

0 38

-50

27-8

Not

tes

ted

Not

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ted

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ts

61-5

72-2

<S K ^̂ <»» O s s 1 o c


Fig. 6. Sequence of doublet-to-singlet transformation of type I doublets. (A) Slight-ly asymmetrical doublet with two oral bands, and variable (see Table 2) number ofCVP bands. (B) Asymmetrical doublet with the extra cytoproct positioned betweentwo oral bands; variable number (Table 2) of CVP bands. (C) More asymmetricaldoublet; beginning of the fusion of postoral sutures marked by the V or Y shape offused cytoprocts; none or one CVP band. (D) Very asymmetric doublet with two oralapparatuses positioned side-by-side and with only one cytoproct; none or one CVPband. All graphical conventions as Fig. 1.

doublets, taken from the sample with a majority of derived singlets, the numberof ciliary rows on the major side of doublets is fairly stable with 28-30 right andabout 39-40 left ciliary rows. Thus the major side of the doublets retains anumber of ciliary rows in its right and left parts that is low but still within thenormal range. It is the minor side between two oral apparatuses which is ap-parently regressing.

The various stages of regression of the minor side of doublets and the fusionstages of preoral and postoral structures within the asymmetrical doublets mayeasily be classified on the basis of the angle between two oral apparatuses andviewed on circular projections of individual cells (Table 2 and Figs 4, 6 and 7):

(a) nearly symmetrical doublets (180-160° oral apparatuses apart) alwaysrevealed two separate cytoprocts. In some of these cells one, or both CVP setsdisappeared (Table 2).

(b) slightly asymmetrical doublets (160-120 ° oral apparatuses apart) includedcells with a strong tendency towards fragmentation of the ciliary rows in thevicinity of CVP meridians. There are two separate cytoprocts (Fig. 6A).

(c) asymmetrical doublets (120-65 ° oral apparatuses apart) comprised cellswith only one CVP sector or none. The CVPs, if present, were always localizedmidway between two oral apparatuses on the major side of doublets. The post-oral and preoral sutures were partially fused, but there were still two separate

Pattern regulation in Paramecium 59cytoprocts. In five cases such side-by-side configurations of ventral complexesbrought about the appearance of an extra third cytoproct midway between thetwo normal cytoprocts (Figs 4 and 6B). These additional cytoprocts wereseparated from the normal ones by about 8-16 ciliary rows. The additionalcytoproct does not possess a central dot characteristic of the silvered image of thefunctional organelle (Ng, 1976). In some cells belonging to this group, there areonly two cytoprocts fused into V or Y configuration (Fig. 6C). In some cells, onlyone cytoproct is observed lying on the fused postoral suture.

•V:

8

Fig. 7. Photomicrograph of the very asymmetrical I type doublet. This stage corre-sponds to Fig. 6D. Silver-stained specimen. All abbreviations as in Fig. 2.Fig. 8. Photomicrograph of the incomplete symmetrical II type doublet with ex-posed an incomplete oral band. The preoral (PR), postoral (PO) sutures and thecytoproct (CYT) are shown, whereas there is no even remnant of the oral apparatus.Arrows indicate the ciliary row joining the preoral and postoral sutures which marksthe position of the incomplete oral band on a cell's circumference projection. Thisstage corresponds to Fig. 9B. Silver-stained specimen.

Wi;


Table 2. Frequency distribution of specimens {in %) of four classes specified bythe angular values of disposition of two oral apparatuses on the circumference oftype I doublets in relation to (a) the number of existing CVP bands and (b) the

number of cytoprocts

Angular values of disposition of oral apparatuses% % % %

180°-160° 160°-120° 120°-65° 65°-30°Number of cortical structures n (N-16) (N-28) (N-12) (N-16)

(a) the number of CVP bands0 (lethal?)12

(b) the number of cytoprocts123

33327

17505

37-537-525-0

0100-0

0

n = total number of tested specimens in each class.N = number of specimens of a given class.

46-450-0

3-6

0100-0

0

25-075-0

0

16-641-641-6

68-731-2

0

93-76-30

(d) very asymmetric doublets (Figs 6D and 7) include cells with oral apparatusesat an angle of 65-30 °. In these cases, two oral apparatuses assume a side-by-sideposition, with very few wrinkled ciliary rows between them. In most cases thesecells possessed only one cytoproct. Even in this group many cells lacked both setsof CVPs.

(e) derived singlets. Among the observed cells, only two cases of singlets withbranched preoral and postoral sutures were noted. Apparently the left oralapparatus was totally missing (including even vestibular ciliary rows) in both ofthese cases. Both branches were separated by 12-16 ciliary rows and they in-cluded a surplus of the left ciliary rows.

Table 2 summarises the occurrence of CVPs and cytoprocts in very well silver-stained specimens in which these structures could not have been overlooked.

These data indicate that:(1) Regression of cortical areas may occur on both sides of doublets close to

dorsal cortical strips. However, in most viable cells this regression is restrictedto only one cortical side, which brings about an increasing asymmetry in thelocation of the oral apparatuses.

(2) Oral apparatuses are morphologically normal even when in a side-by-sideconfiguration.

(3) Confrontation of oral bands of cortex at an angle\of 120-65 ° may bringabout the appearance of a third cytoproct. This structure s&ems to be transient,or selected against, since no such case is reported in other groups of asymmetricaldoublets.


(4) No case of an incomplete symmetrical doublet was found (Sonneborn,1970a).

3. Intermediate stages during doublet-to-singlet transformation in progeny oftype II doublets

Two sublines were studied in detail. These were subline M, heterozygous forscr6, (but homozygous wild-type at/ha locus, genetical data not shown) of thewild-type phenotype (straight cells), and a subline of twisted scr^fnahomozygotes. Both of these sublines were derived from doublets after thesecond massive burst of autogamy. Despite the different phenotype (due todifferent genotype) the sequence of appearance and the morphology of theintermediate stages were similar in both sublines. They are in sharp contrast tothose observed in the type I doublets described above.

Intermediate stages in transformation to singlets consisted exclusively of dif-ferent kinds of incomplete doublets. These incomplete doublets, with one or twooral apparatuses lacking, correspond to the description and definition given bySonneborn (1963,19706). These cells manifest the doublet sets of cortical organ-elles, with the exception that one or even two oral apparatuses are missing.However any incomplete oral band, even if the oral apparatus is completelyabsent, may be readily recognized due to the preserved landmarks of the preoraland postoral sutures with the cytoproct (Fig. 8). Thus the location of the ciliaryrows joining preoral and postoral sutures of the incomplete ventral complex (asmarked by arrows at Fig. 8) stands as the position of this incomplete oral bandon the circular projection of cells (labelled as the empty circles on circularprojection schemes in Fig. 9B, C and D).

The loss of the oral apparatus was observed in perfectly symmetrical doubletsand in all asymmetrical doublets (Table 3 and Fig. 9B, C and D). At the timewhen the first incomplete symmetrical doublets were found in samples of the Mand F sublines, many other doublets displayed apparent malformation, partialregression, or displacements of oral apparatuses within the oral bands of one orboth sides. In some cases totally astomous doublet cells were found: with twonormal dorsal CVP sets, with two preoral and postoral sutures, with twocytoprocts, but with both oral apparatuses absent. It is known (Tartar, 1954) thatsuch cells are in viable. The stages of regression of oral apparatuses correspondto the description of these events in Paramecium doublets with locally irradiatedoral structures (Hanson, 1955, 1962).

Morphology, symmetry and corticotypes were analysed in 81 perfectly silvereddoublets and derived singlets and 41 control singlets. Data are represented inTable 3 and Figs 8 and 9. In completely symmetrical doublets with two normaloral apparatuses, the number of ciliary rows in particular right and left systemsmay be variable (Table 3; first, second and third rows), even though geometricalproportions tend to remain similar (Fig. 9A). There is some variability in theangular values for CVPs placements among cells belonging to this group (the

EMB74


Fig. 9. Sequence of doublet-to-singlet transformation of type II doublets. Circularprojections of intermediate stages in a corresponding sequence of Table 3 are presen-ted. Variability of angular values for right ciliary rows systems are marked by thedotted areas. These dotted areas correspond to CVP field angle (Nanney, 1966a),i.e. limiting the maximal and minimal angles between the respective oral band andits right positioned CVP band. (A) Symmetrical complete doublet with two oral, andtwo CVP sets of landmarks. This circular projection corresponds to cells depicted inFig. IB and 5B. The right systems of ciliary rows (extending to the right from the oralapparatus to the location of the dorsal CVPs bearing strip) may occupy from 60-90 °.(B) Symmetrical incomplete doublet. The symmetry of positioning of the complete(black circle) and of the incomplete (empty circle) oral bands is shown. There is alsoa symmetrical disposition of the CVP bands (crosses). Variability of the angularvalues of both right systems ranges from 30-60°. (C) Asymmetrical incompletedoublet with the 160-120° angle between the complete and the incomplete oralbands. These angles are always manifested to the left from the complete ventralcomplex. This configuration brings about an increase of the angular values for theright system on an opposite major side of a doublet to 90-120°. On the minor sideof a doublet the side-by-side location of the incomplete oral band and of the CVPband is marked with an arrow. (D) Very asymmetrical incomplete doublet with anangle of 120-90 ° between the complete and the incomplete oral bands. On the minorside of a doublet the markers of the CVP band completely disappeared. On the majorside of a doublet a further increase of the angular values of the right system isobserved. (E) Derived singlets. All landmarks of the incomplete oral band disap-peared. An angle for the remaining right ciliary system increased to 160-180°.Nearly normal singlet's disposition of cortical landmarks is manifested both in thenormal and tiny derived singlets.

variability of these angular values is marked as dotted areas in Fig. 9), but thereis a strong tendency to maintain the 180 ° apart locations of the CVP sets in eachof the individual specimens.

Incomplete doublets may appear even among cells with high corticotypes, e.g.,115 (Table 3, fourth row). However there is no case of a normal doublet, with twooral apparatuses with a corticotype below 95 (Table 3, third row). Thus there is noinvariant relation between the appearance of incomplete doublets and cor-ticotypes , though this phenomenon becomes a rule in low corticotypes. The loss ofthe oral apparatus is always related to some disturbance in the cortex in its vicinity.Incomplete doublets of corticotypes below 93 were all asymmetrical (Table 3, sixthand seventh rows; and Fig. 9C and D). Thetotal number of ciliary rows between anincomplete oral band (without an oral apparatus) and a remaining complete one(Fig. 9D and Table 3, seventh row) may be as low as 22 ciliary rows.

Tab

le 3

. Mea

n nu

mbe

r an

d S.

D.

of c

ilia

ry r

ows

appe

arin

g in

seq

uent

ial

left

1,

righ

t 1 a

nd le

ft 2

and

rig

ht 2

sys

tem

s of

typ

e II

doub

lets

of

spec

ifie

d st

ages

(F

ig.

9) o

f do

uble

t-to

-sin

glet

tra

nsfo

rmat

ion

No.

of

row

and

refe

renc

e to

sta

ges

in F

ig.

9

Cla

ss o

f sp

ecim

ens

oftr

ansf

orm

ing

type

II

doub

lets

Tot

alnu

mbe

r of

cili

ary

row

son

who

lece

lls

num

ber

of c

ilia

ry r

ows

syst

ems:

(mea

ns a

nd S

.D.)

left

lri

ght

1le

ft 2

righ

t 2

Obs

erve

d lo

cali

zati

on o

ffr

agm

ente

d ci

liar

y ro

ws

onth

e ci

rcum

fere

nce

of w

hole

cells

1. F

ig.

9A

2. F

ig.

9A

3. F

ig.

9A

4. F

ig.

9B

5. o

nly

on

e ca

se

6. F

ig.

9C

7. F

ig.

9D

8. F

ig.

9E9.

Con

trol

sym

met

ric

(180

-160

°)

18

135-

115

39-2

±2

-5

23-4

±1

-5

37-2

±3

-0

23-4

±1

-0co

mpl

ete

doub

lets

9 11

4-11

0 35

-5 ±

2-4

20

-7 ±

1-3

34

-9 ±

2-0

20

-2 ±

2-8

sym

met

ric

(180

-160

°)co

mpl

ete

doub

lets

sym

met

ric

(180

-160

°)co

mpl

ete

doub

lets

sym

met

ric

(180

-160

°)in

com

plet

e do

uble

tsas

ymm

etri

c co

mpl

ete

doub

let

wit

h o

ne

CV

P s

etas

ymm

etri

c (1

60-1

20°)

inco

mpl

ete

doub

lets

asym

met

ric

(120

-90°

)in

com

plet

e do

uble

tsde

rive

d si

ngle

tssi

ngle

ts (

cont

rol)

4 6 1 4 6 33 41

99-95

115-93

84

92-74

94-79

73-52

68-75

28-2

±2

-4

19-7

±1

-1

28-2

±1

-9

17-5

±1

-7

36-3

±3

-4

18-2

±2

-9

33-0

±2

-4

21-8

±4

-4

27

24

33

28-7

±4

-9

24-0

±2

-6

23-7

±1

-7

9-3

±1

-5

34-0

±6

-4

25-3

±3

-0

29-3

±2

-9

38-7

±2

-7

26-5

±2

-643

-8 ±

2-7

31

-8 ±

1-9

syst

ems

righ

t 1

and

righ

t 2

clos

e to

the

ora

l be

nds

disp

erse

d

clos

e to

the

ora

l ba

nds

not

know

n

syst

ems

righ

t 2

and

left

2sy

stem

sri

ght

2 an

d le

ft 2

not

obse

rved

not

obse

rved

Lef

t 1

and

righ

t 1

syst

ems

alw

ays

refe

rs t

o th

e co

mpo

nent

of

the

doub

let

with

the

hig

her

num

ber

of t

he s

um o

f th

e le

ft a

nd r

ight

cil

iary

row

s.A

ngul

ar v

alue

s in

par

enth

eses

in

the

seco

nd c

olum

n de

scri

be t

he l

imit

s of

var

iabi

lity

of

rela

tive

dis

posi

tion

of

two

oral

ban

ds o

n th

e ci

rcum

fere

nce

of d

oubl

ets.

n =

tot

al n

umbe

r of

obs

erve

d ce

lls

belo

ngin

g to

a g

iven

spe

cifi

ed c

lass

.D

ata

in 1

, 2

and

3 ro

ws

refe

r to

the

sam

e cy

toge

omet

rica

l st

age-

Fig

. 9A

; su

bdiv

isio

n w

as b

ased

on

diff

eren

t lo

cali

zati

on o

f fr

agm

ente

d ci

liar

yro

ws

and

coin

cide

s w

ith

thre

e di

ffer

ent

grou

ps o

f co

rtic

otyp

es.


Some incomplete asymmetrical doublets (160-120° Table 3, sixth row) main-tained a CVP band on their minor sides as well as on their major sides (Fig. 9C).The minimal distance between landmarks of two differently differentiated bandswas 8-11 rows (marked with an arrow on Fig. 9C).

On the major side of incomplete doublets two different tendencies are obser-ved:

(1) although the total corticotype of doublets decreased sharply with an in-crease in the asymmetry of cells, the number of ciliary rows in the right system(right 1 Table 3) on the major side of the cells actually increased; in thesymmetric incomplete doublets (Table 3, fourth row) of this system it averagesabout 18 ciliary rows, while in the asymmetric incomplete doublets (Table 3,sixth and seventh rows) it increased to about 24-25 ciliary rows.

(2) The angle between the oral band and CVP band on the major side ofdoublets tends to increase from about 60° (Fig. 9B) to about 120° (Fig. 9D).

In derived singlets, corticotypes may fall below the average of the controls(Table 3, eighth row vs. ninth row). The corticotype of some tiny singlets maybe as low as 52. But even very tiny derived singlets still manifested about 60-52ciliary rows.

Only two exceptional cases of duplication of cortical landmarks in a side-by-side position were observed among many type II doublets (M subline). In oneslightly asymmetrical doublet (oral apparatuses 160 ° apart) of a high corticotypeof 133, a third extra CVP band occurred midway between the oral apparatuseson the major side of the doublet. Thus two side-by-side positioned CVP bandswere separated by 15 ciliary rows.

A second doublet of the corticotype of 111 was symmetrical as indicated in Fig.5B projection. However one left system of ciliary rows represented only 28ciliary rows, while the other left system was crowded with 43 ciliary rows. Withinthe latter system an extra third oral apparatus appeared. This third oral ap-paratus was inverted (but not right-left reversed) and was not topographicallyrelated to any preoral or postoral sutures. This oral apparatus was positioned 16ciliary rows to the left of the normal oral apparatus.

DISCUSSION

The doublet system in Paramecium tetraurelia is inherently unstable over along term and tends to regulate to the singlet state (Sonneborn, 1963; de Haller,1965). However, some doublets may be maintained over more than 100 genera-tions. The transformation from doublet to singlet is not a single event, but aseries involving changes of corticotypes, loss of cortical organelles and theassumption of only one nuclear apparatus.

As in Tetrahymena (Nanney et al. 1975; Frankel & Nelsen, pers. comm.) inParamecium pathways of regulation from doublet to singlet always involve un-stable intermediate configurations of cortical landmarks. At least two different


pathways are reported here. These pathways conform to both contradictorydescriptions respectively reported by Sonneborn (1970a) and by Sibley (1974).The sequence of intermediate stages is deduced from the sequence of theirappearance in samples and in some cases were confirmed experimentally. Wesuggest that the pathway of regulation and the nature of the intermediary stagesdoes not depend on genotype, but on the initial disturbance of doublet symmetry.If one supposes that during initial fusion, accompanied by dedifferentiation ofciliary rows in the area of the cytoplasmic bridge between mates (Watanabe,1978), this area of fusion is shifted somewhat to the right or to the left, the initialdoublet can acquire a different shape and the line of confrontation of cortexesof different origin may be differently positioned. In Fig. 10A it is suggested thatin type I doublets, the area of fusion might have been slightly further from theoral apparatuses, leading cells to join firmly along the right ciliary meridianwhich extends straight from pole to pole. However, such union would bring somemechanical pressure on the dorsal sides. Thus such doublets would tend toregress their dorsal, and not ventral, cortical areas. In contrast, in type IIdoublets the fusion area in the right ciliary system would be localized close to theoral apparatuses, along the ciliary rows extending from the preoral to the post-oral sutures. Thus the areas close to the oral apparatuses and sutures would bedistorted and may become a site of mechanical stresses. Thus such doubletswould tend to regress their ventral, and not dorsal, cortical areas (Fig. 10B). Thishypothesis is consistent with observations that areas of regression may involveeither one, or even two sets of corresponding cortical structures in a givendoublet. It is assumed that selection is the mechanism bringing about the survivalof cells, which maintained a complete set of indispensable organelles and thecortical area of corticotype of at least about 52 ciliary rows (the lowest cortico-type found in derived singlets, Table 3, eighth row).

From these it is clear that Paramecium is able to form oral and CVP structureseven if they are improperly located with respect to each other. Regression of thecortical area may bring about very different configurations of the remainingcortical landmarks. From comparison of patterns of deployment of cortical land-marks in cells at intermediate stages of doublet-to-singlet transformation at leastthree developmental processes are deduced:

(1) Side-by-side location of the separate functional oral bands may bring aboutthe appearance of an additional structure along the line of confrontation of thecortex of an apparently different origin. Such cases of induction of the extracytoproct have been previously reported in Paramecium in grafting experiments(Sonneborn, 1963). The appearance of the additional structures in general termsis reminiscent of cases of duplication of structures after specific confrontation ofparts of developing systems with different positional values (French, Bryant &Bryant, 1976; Tartar, 1961).

(2) The maintenance of a given set of landmarks in two cortical bandspositioned side by side requires a minimal distance between them. Thus we


.CVP

CVP

CVP

CVI

CVP

Fig. 10. Hypothesis of the origin of diversity of two different developmental path-ways of doublet-to-singlet regulation in Paramecium tetraurelia. (A) Type I doubletprogeny originates from the initial fusant with the seams joining cortexes of differentorigins (dashed outline vs solid outline of mating cells) running along the right ciliaryrow. This kind of fusion put the mechanical stresses onto the dorsal surfaces. Hencethe oral bands are more stable than the CVP bands. Resulting doublet manifests onlyone fused anterior pole. (B) Type II doublet progeny originates from the initialfusant with the seams confronting cortexes of different origins (dashed outline vssolid outline of mating cells) running along the right ciliary, systems very close to theoral bands i.e. in the vicinity of the preoral and postoral sutures. This kind of fusionbrings about an instability of the oral bands themselves. Splitting of the anterior polesreleases the mechanical stresses on the dorsal surfaces, which become more stablethan those in I type of doublet. Disposition of the area of the fusion of cortexes ofdifferent origins is marked by a heavy line on the schemes of the exposed to viewersurfaces of both types of doublets (far right schemes of A and B sets of drawings).

suggest that a strip of cortex of some width is required to manifest 'identity ofdifferently determined states' (Meinhardt & Gierer, 1980). If this distancedecreases either fusion of both bands occurs (as observed in fusion of preoral andpostoral sutures in type I doublets), or one set of structures disappears (left oralapparatus in the same cases).

(3) Both in twisted cells, and in very asymmetrical incomplete doublets of typeII some regulatory shift of positioning of CVPs was observed. It is deduced thatan increase of field angle (Nanney, 1966a) permits step-by-step restoration of theproper symmetry and the numbers of ciliary rows between cortical landmarks.


Thus it is suggested that pattern regulation in Paramecium during doublet-to-singlet transformation is an interplay of at least three factors: the regression ofcortical areas caused by mechanical stresses, an interaction of confronted frag-ments of cortical pattern and regulatory shift of localization of cortical struc-tures. If the symmetry of deployment of the cortical organelles, the maintenanceof one set of indispensable set of cortical organelles and minimum of corticalareas between them is achieved, the whole organism falls into a stablecytogeometrical balance and this pattern may then be faithfully reproducedwithout further change. This idea is consistent with a mode of 'stability sink'proved in Tetrahymena (Nanney, 1968).

We acknowledge with deep gratitude the valuable advice of Dr Joseph Frankel and his helpin preparing the manuscript. We thank Dr David Nanney for comments and corrections ofmanuscript. We wish to thank Dr Mario Nelsen and Dr Andrzej Kaczanowski for manyhelpful suggestions.

This work is partially supported by research grant of the Polish Academy of Sciences.

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{Accepted 9 November 1982)

Documents

Pattern determination and pattern regulation in Paramecium ... · the Paramecium system and the character of the spatial regulation of the cortical pattern remains unknown. Removal