J. Embryol. exp. Morph. 74, 47-68 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
47
Pattern determination and pattern regulation in
Paramecium tetraurelia
By JANINA KACZANOWSKA 1 AND BOZENA DUBIELECKA 1
From the Institute of Zoology, Warsaw University
SUMMARY
Pattern regulation was investigated in the progeny of laterally fused cells of Paramecium
tetraurelia. The immediate progeny of such fused cells (doublets) reveal two sets of cortical
organelles arranged roughly symmetrically. Doublets tend to transform gradually into cells
with only one set of organelles (singlets). At least two different and mutually exclusive pathways of doublet-to-singlet transformation are reported. In intermediate stages of regulation
the cortical areas bearing different cortical landmarks may be brought into an abnormal
neighbourhood. Differentiated cortical bands of cortex, bearing organellar landmarks, are
faithfully propagated even if they are improperly and asymmetrically located on the cell. The
confrontation of such cortical bands may lead to the transient appearance of additional
duplicated organelles.
It is suggested that pattern regulation in Paramecium during doublet-to-singlet transformation results from at least three factors: the regression of some part of the cortical areas, the
interaction of the juxtaposed parts remaining and the slow regulatory shift of positions of the
cortical structures.
INTRODUCTION
The aim of the present paper is to investigate the mode of spatial pattern
regulation in a ciliate Paramecium tetraurelia.
Most ciliates show a remarkable capacity for regeneration and pattern regulation when pieces of the cell cortex are added or removed (Tartar, 1961; Sonneborn, 1975; Frankel, 1974). However, Paramecium represents an extreme
mosaic system of intracellular development (Beisson & Sonneborn, 1965) that
contrasts with the highly regulative development of other ciliates (Tartar, 1954;
Schwartz, 1963; Sonneborn, 1963). In Paramecium, the buccal primordium is
situated very close to the pre-existing oral apparatus and 'de novo' formation
does not occur after its removal (Tartar, 1954; Hanson, 1962; Hanson & Ungerleider, 1972).
The general deployment of cortical organelles over the ciliate cortex may be
described and quantified in terms of specific angles in a polar co-ordinate system
projected over the cell surface (Nanney, 1966a). The cell is covered with longitudinal ciliary rows marking meridians of such a system. The right ciliary row
flanking 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 in
terms 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 rows
separating the position of ciliary row no. 1 and tested structure to the total
number of longitudinal ciliary rows counted on the cell circumference. The total
number 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 the
contractile vacuole pores (CVPs) on longitudes of Tetrahymena. Positioning of
the CVP in a given cell may be described as a consequence of specification of an
inductive angle between the ciliary row involved in manufacturing the ventral
complex and the longitude in which the CVP is found. The variability of positioning of the CVP is described in terms of afield angle which defines the band of the
cell surface which may be competent to yield a CVP. This specific cytoarchitecture of a given ciliate species is maintained during successive fissions, since both
the ventral complex and CVP positioning are reproduced along virtually the
same longitudinal bands of cortex in daughter cells (Frankel & Nelsen, 1981).
In experimental studies on the Tetrahymena system (Nanney, 19666; JerkaDziadosz & Frankel, 1979; Nanney et al. 1975; Frankel & Nelsen, 1981) the
inductive and regulative character of CVP positioning on cell longitudes with
respect to the oral sector is well documented. There are no comparative data on
the Paramecium system and the character of the spatial regulation of the cortical
pattern remains unknown.
Removal and grafting of longitudinal strips of cortex onto a normal
Paramecium cell have been performed to explore the pattern determination of
new 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 very
laborious and are feasible only on a limited number of cells. However, an
equivalent morphological confrontation of different parts of the cortex appears
during the spontaneous and gradual transformation of homopolar twin cells of
Paramecium (comprising two sets of all cortical structures) into a single-cell
cortical pattern in their progeny. Such homopolar twin cells, or doublets, may
be 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 fusion
of two cortical areas with an alternate and symmetrical sequence of deployment
of cortical organelles (Fig. 1). This pattern is then reproduced during subsequent
cell generations. However, it is apparent that the doublet organization of the cell
cortex 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 (de
Haller, 1965). However, data about intermediate morphological stages during
doublet-to-singlet transformation are very confusing: while Sonneborn (1970a)
reported that one of the oral apparatuses may be lost when positioned 180 ° apart
from the second oral apparatus, Sibley (1974) described the possibility of a sideby-side configuration of both ventral sectors in doublets transforming to singlets.
Hence the problem of pattern regulation during doublet-to-singlet transformation is reinvestigated here.
MATERIALS AND METHODS
Materials
All stocks employed were derived from Sonneborn's Paramecium tetraurelia
stock 5IS. Culture and handling of paramecia followed the methods of Sonneborn (19706). They were cultured in Cerophyl, or in lettuce media, inoculated
with Klebsiella cloacae. Results were similar in both media.
Construction of doublets
The initial doublets were derived in two independent series by recovery of
conjugating cells that failed to separate after either a brief thermal, or
actinomycin D shock applied 30 min after the isolation of the tight pairs. The first
set of lines of wild-type doublets was derived from mating of complementary cells
treated for 45 min with 50 jug/ml AMD. The second set of lines was derived from
a doublet created by heat shock (45 min at 37 °C) administered to conjugants in
a 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 doublet
was of a wild-type phenotype. The fna allele (Kung, 1971) was used only as an
easy marker for screening the completeness of sexual processes. The scr6 allele
(Sonneborn, 1974) when homozygous brings about a left-handed twisting of
ciliary rows around the main body axis. However, this allele has variable
penetrance and expressivity so both straight and twisted phenotypes may occur
in a scr6'/scr6 clone.
A scr6, fna homozygous doublet was isolated from the heterozygous doublet
line after massive autogamy and kept as subline F with straight and twisted
phenotypes.
Cells were kept in test tubes at room temperature (about 22 ± 2 °C) and fed
only twice a week. Under these conditions, doublets of all sublines divided
approximately every other day, while spontaneously derived singlets divided
about once a day. Control singlets of corresponding genotypes and phenotypes
kept 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. DUBIELECKA
cortical pattern, the samples of cells were periodically fixed twice a week during
observation periods. The Chatton-Lwoff technique of silver impregnation was
used following Frankel & Heckmann's protocol (1968).
Measurement and counting
For evaluation of the rate of appearance of singlets in samples all well-silvered
specimens from a given series were used.
To test the distribution of cortical organelles in doublets, in cells in intermediate 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 were
chosen. Each of the drawings was then processed by geometrical projection of
the angular distribution of the cortical organelles onto a circle of the diameter of
the drawn specimen (Fig. 1). The right margin of the oral apparatus, cytoproct
and 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 symmetrical
type 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, crosses
indicate sites of CVP bearing ciliary meridians. All projections are uniformly oriented as viewed from the anterior cells pole.
Pattern regulation in Paramecium
51
verified by focusing the specimen under the microscope. The number of complete ciliary meridians between successive landmarks was counted. In the case
of the homozygous wild-type doublets 72 symmetrical and asymmetrical
doublets and 71 derived singlets were analysed mainly using the criteria of the
projected angles, and the numbers of ciliary rows were counted in only some of
them (as described below). In the case of heterozygous lines, 32 complete
doublets, 16 so-called incomplete doublets and 33 derived singlets were characterized 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 genotypes
were used. The intermeridional space where the anterior CVP occurred was
chosen as a reference, and the deviation of the position of the posterior CVP, or
a third supernumerary CVP either to the right or to the left, was expressed as a
number of ciliary rows separating the anterior from the tested CVPs. All tests
were performed on apparently non-dividing cells.
In the case of scr6]/scr6 singlet phenotypes, earnera-lucida drawings were made
on the specimens with a distinct CVP band exposed to the viewer. From these
pictures, the angle between the longitudinal cell axis and the line joining two
obliquely positioned CVPs was measured to estimate cell twisting. The cells were
classified as 'twisted' if this angle exceeded 20 ° (136 cells). In the case of doublets
only scr6, fna double-twisted homozygotes were used (n = 49) with twist of more
than 20°.
RESULTS
1. Characteristics of single and double Paramecium tetraurelia cells of different
phenotypes
Wild-type singlets. (Figs 2 and 3) Morphometrical and cytogeometrical studies
on the cortical pattern of control (5IS) single cells revealed a virtual stability of
cortical parameters of all non-dividing cells. This pattern is consistent with the
detailed description of Sonneborn (1963) and of Kaneda & Hanson (1974). In
brief; the oral band is marked by a complex comprising preoral suture, oral
apparatus and postoral suture with a cytoproct, and is flanked to the right by
nearly meridional ciliary rows. On the left side of the oral apparatus the ciliary
meridians are more densely aligned and they are bent in the form of arcs around
the oral apparatus. Thus the preoral and postoral sutures are flanked with slightly skewed right ciliary rows and with more curved left ciliary meridians. At the
postoral suture there is a long silver-stained line representing the lips of the
cytoproct (Ng, 1976; Allen & Wolf, 1974). The left and right ciliary rows grade
into pole-to-pole meridians on the dorsal surface. The position of the anterior
contractile vacuole pore (CVP) marking the centre of the CVP band was
deliberately chosen as the boundary for counting the right and left set of
meridional ciliary rows (Sonneborn, 1963; Kaneda & Hanson, 1974). In a sample
52
J. KACZANOWSKA AND B. DUBIELECKA
Fig. 2. Scheme of positioning of cortical organelles over: (A) ventral and (B) dorsal
surface of a single cell of Paramecium tetraurelia. Oral band is composed of the
preoral suture (PR), the oral apparatus (OA), the postoral suture (PO), with marked
longitudinal slit of the cytoproct (CYT). Ciliary rows (dashed lines) run nearly
meridionally to the right of the ventral complex. They are arched and more densely
packed to the left of the ventral complex. On the dorsal side, two contractile vacuole
pores (CVP) mark the border between the left (more dense) and the right (less
dense) system of ciliary rows. Arrows mark the CVP band of the cortex. Note that
the 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 (including the short ciliary rows arching immediately adjacent to the oral apparatus
and known as the vestibular rows). The lateral displacement of the posterior
CVP relative to the anterior one is no more than two ciliary rows in either
direction. Thus the corticotypes of control cells averaged about 74-75 and the
maximum field angle equals to about 20° (~4/75).
Twisted singlets. Twisted singlets of genotype scr6 /scr6 expressed similar
corticotypes and left and right ciliary meridian distributions. In a sample of 20
cells there were 31-5 ± 1-4 right and 44-6 ± 1-9 left ciliary meridians. The morphology of these cells follows the description of Whittle & Chen-Shan (1972) of
left-handed screwy mutants. In twisted phenotypes, the ciliary rows are
spiralized 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 the
anterior pole to the left. The anterior CVP in twisted cells remains at roughly
180° to the anterior portion of the oral band. However, the dorsal ciliary rows
are no longer parallel to the main body axis. Thus, in these cells the degree of
conservation of the location of CVPs within the band of cell cortex defined by
the ciliary rows may be tested. In most cells (75 % of 136 tested specimens), the
lateral displacement of CVPs was kept within the limit of variability observed in
the controls, i.e. within 20° field angle. However in the remaining cells this
deviation was increased up to eight ciliary r6ws. The posterior CVPs were always
Pattern regulation in Paramecium
53
Fig. 3. Photomicrograph of a single control of Paramecium tetraurelia. Silverstained preparation. On exposed ventral surface of the cell all parts of the oral band
are seen. Abbreviations as in Fig. 2.
Fig. 4. Photomicrograph of the asymmetrical I type doublet with the extra cytoproct
exposed. The left cytoproct (slightly out of focus) runs (arrowheads) along the contour of the cell. This stage corresponds to Fig. 6B. Silver-stained preparation. All
abbreviations as in Fig. 2.
shifted to the right, marking the deployment of the CVPs more parallel to the
main body axis. However, even a maximum shift of positioning (extension of the
field angle to 40 °) did not fully restore the proper geometry of the CVPs relative
to 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 remains
unknown.
Symmetrical doublets. At least two types of regular symmetrical doublets were
54
J. KACZANOWSKA AND B. DUBIELECKA
Fig. 5. Scheme of the cytogeometry of (A) type I and (B) type II doublets with their
respective circular projections. Regular symmetrical doublets. (A) Right angles
between oral (black circles) and CVP (crosses) landmarks on circular projection, and
the regular shape of doublet are schematically depicted. (B) Decrease of the angular
value of the right systems of the type II doublets, and splitting of the anterior portion
of the doublet are marked. All graphical conventions as in Fig. 1.
found in the sublines studied. In both, the oral and CVP band were initially
positioned 180° apart, but subsequent regulation was very different.
(a) In symmetrical doublets, found in all sublines from the wild-type initial
doublet (Fig. 5), the two sagittal axes defined on the circumferential projection
of the cell by the oral-oral and CVP-CVP bands planes were crossed at a right
angle (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 cells
possessed fused poles (Fig. 5A). They can reproduce their cytogeometry during
more than a hundred generations, and then give rise to intermediate stages of
exclusively one type, namely of asymmetrical doublets. Derived singlets
displayed either normal corticotypes (like control singlets) or slightly elevated
corticotypes due to some surplus of left ciliary rows. Doublets of this type will
henceforth be called 'Type I doublets'.
(b) In symmetrical doublets from sublines derived from the progeny of the initial
heterozygous doublet, the sagittal axes of oral-oral bands and CVP-CVP bands
diameters on circumferential projections of the cells were not crossed at a right
angle but at an angle of about 70-75 ° (Fig. 5B). This corresponded to about 26-28
right and 40-42 left ciliary rows (n = 11 tested cells). While the maximum corticotype was also 136, some cells revealed lowered corticotypes down to 94.
Pattern regulation in Paramecium
55
These doublets have never displayed fusion of anterior poles of their components (Fig. 5B). Thus, particularly at the anterior pole of the doublet, two
significant acuminations representing individual single poles were separated by
a notch of varying depth. Doublets of this kind can also reproduce their own
cytoarchitecture for over a hundred generations. They then transform into singlets with intermediate stages exclusively of the character of so-called 'incomplete
doublets' (Sonneborn, 1963) i.e. doublets with one or even two missing oral
apparatuses but with preoral and postoral sutures and cytoprocts. Unlike the
former type of doublets, they have never generated asymmetrical complete
doublets as intermediate stages. A typical asymmetrical doublet of this type
possessed one complete oral band and a second incomplete oral band comprising
the preoral, postoral sutures and the cytoproct. Asymmetrical doublets with two
incomplete oral bands (astomous) were occasionally found. The derived 'early'
singlets displayed very variable corticotypes, frequently severely reduced (to
about 52-54).
Doublets of this type will be called 'Type II doublets'. Their characteristics
were also detected in twisted doublets of the F sublines derived from the
heterozygous line after a sexual process and the achievement of homozygosity.
In 20 % of these cells an additional, posterior CVP was observed either in one
or in both CVP band of cortex. In 49 heavily twisted cells the rate of conservation
of CVP location was tested. While in about 71 % all CVPs were located within
one or two rows of each other, in the remaining 29 % at least one, and sometimes
two, CVP bands were enlarged to up to six ciliary rows.
Both types of doublets (I and II) observed during the first month of study
contained two separate macronuclei. Only one macronucleus was found, even
in symmetrical doublets, during the last month of observations of all sublines.
2. Sequence and frequency of appearance of intermediate stages during
doublet-to-singlet transformation in progeny of type I doublets
The data of the previous section show that the doublet-to-singlet transformation involves severe reduction of the number of ciliary rows. The question is
whether in doublets transforming into singlets this decrease of the number of
ciliary rows affects the angles of deployment of cortical landmarks? There is still
another general question, namely whether this transformation is rapid, or involves many intermediate generations, and whether once started it requires a
fixed, or variable number of generations for final achievement of singlet architecture.
To answer this question, the sequence and frequency of appearance of intermediate stages during the doublet-to-singlet transformation was studied in four
sublines of cultures originating from the wild-type doublet. The sublines were
initiated when the first derived singlet was found in test tubes of doublet progeny.
This occurred after about 60 generations, with the possible intervention of two
or three autogamous cycles. At this time, four clones, A, B, C and D, were
56
J. KACZANOWSKA AND B. DUBIELECKA
started by isolation of apparently symmetrical doublets. The progeny of these
four sublines were tested for the appearance of intermediate stages of transformation into singlets, and for the frequency of these stages, in random samples
over 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 tested
cells were classified into three major morphological groups: regular symmetrical
doublets, asymmetrical doublets and derived singlets. The frequency
distribution of these morphological classes is presented in Table 1.
From these data it is evident that the distribution of classes changes in a regular
way: in subline B in which initially a majority of cells are symmetrical doublets,
only a minor fraction of doublets are asymmetrical. Subsequently there is an
increase in the fraction of asymmetrical doublets. An increase in the proportion
of asymmetrical doublets in A and C sublines coincides with an appearance of
derived singlets and in the majority of cases with a decrease in the fraction of
symmetrical doublets. In sublines C and D, the class of symmetrical doublets
completely 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 are
viable. The slow increase in the percentage of asymmetrical doublets in the
development of sublines A and B and the rarity of singlets indicate that the slowly
dividing asymmetrical doublets are rather stable over a long period and probably
gradually revert to the single organization. This conclusion was directly confirmed in experiments made on isolated asymmetrical doublets reisolated after
their 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 corresponded to an incomplete doublet character (i.e. lacking an oral apparatus in an
oral band). All intermediate stages in all sublines corresponded exclusively to
asymmetrical doublets. All stages roughly agree with the brief description given
by Sibley (1974). All symmetrical and asymmetrical doublets have functional
and normal oral apparatuses (Kaczanowska & Garlinska, 1981). Fragmentation
and distortion in pattern of ciliary rows in symmetrical and slightly asymmetrical
doublets occurred exclusively in the vicinity of, or in, the CVP meridians. In
some cells one or both sets of CVPs were absent. It is not known if cells lacking
osmoregulatory organelles may be viable. In any case, such cells appeared when
asymmetrical doublets occurred and were never observed among derived singlets.
In asymmetrical doublets the increase of asymmetry was correlated with a
decrease of corticotype and cell diameter. Thus the cell circumference
diminishes mainly through a decrease of the minor side (i.e. on the side of the
minor angle between oral apparatuses) of the asymmetrical doublets. Highly
asymmetrical doublets also displayed an advanced fusion of the preoral and
postoral structures (as seen in Figs 6C, D and 7). In about 30 asymmetrical
48
101
240
296
1
2
3
4
37-0
5-8
10-2
8-3
63-0
94-2
87-2
87-7
0
0
2-5
4-0
singlets
n = the total number of tested specimens.
n
Month of
observations
Subline A
sym- asymmet. met.
dou : doubs.
bs.
61
330
286
267
n
92-0
60-6
23-8
14-8
8-0
39-4
76-2
85-2
Subline B
sym- asymmet. met.
dou- doubs.
bs.
0
0
0
0
singlets
71
294
194
200
n
40-3
13-5
0
0
58-7
72-3
9-0
0
Subline C
sym- asymmet. met.
dou- doubs.
bs.
1-0
14-2
91-0
100-0
singlets
49
264
n
0
38-5
0
27-8
Not tested
Not tested
Subline D
sym- asymmet.
met.
dou- doubs.
bs.
61-5
72-2
singlets
Table 1. The frequency distribution (in %) of classes of symmetrical doublets, asymmetrical doublets and singlets in samples
from sublines, A, B, C and D of Paramecium tetraurelia which were observed during four successive months
c
o
1
ss
K
^^
<»»
O
<S
58
J. KACZANOWSKA AND B. DUBIELECKA
Fig. 6. Sequence of doublet-to-singlet transformation of type I doublets. (A) Slightly asymmetrical doublet with two oral bands, and variable (see Table 2) number of
CVP bands. (B) Asymmetrical doublet with the extra cytoproct positioned between
two oral bands; variable number (Table 2) of CVP bands. (C) More asymmetrical
doublet; beginning of the fusion of postoral sutures marked by the V or Y shape of
fused cytoprocts; none or one CVP band. (D) Very asymmetric doublet with two oral
apparatuses positioned side-by-side and with only one cytoproct; none or one CVP
band. All graphical conventions as Fig. 1.
doublets, taken from the sample with a majority of derived singlets, the number
of ciliary rows on the major side of doublets is fairly stable with 28-30 right and
about 39-40 left ciliary rows. Thus the major side of the doublets retains a
number of ciliary rows in its right and left parts that is low but still within the
normal range. It is the minor side between two oral apparatuses which is apparently regressing.
The various stages of regression of the minor side of doublets and the fusion
stages of preoral and postoral structures within the asymmetrical doublets may
easily be classified on the basis of the angle between two oral apparatuses and
viewed on circular projections of individual cells (Table 2 and Figs 4, 6 and 7):
(a) nearly symmetrical doublets (180-160° oral apparatuses apart) always
revealed two separate cytoprocts. In some of these cells one, or both CVP sets
disappeared (Table 2).
(b) slightly asymmetrical doublets (160-120 ° oral apparatuses apart) included
cells with a strong tendency towards fragmentation of the ciliary rows in the
vicinity of CVP meridians. There are two separate cytoprocts (Fig. 6A).
(c) asymmetrical doublets (120-65 ° oral apparatuses apart) comprised cells
with only one CVP sector or none. The CVPs, if present, were always localized
midway between two oral apparatuses on the major side of doublets. The postoral and preoral sutures were partially fused, but there were still two separate
Pattern regulation in Paramecium
59
cytoprocts. In five cases such side-by-side configurations of ventral complexes
brought about the appearance of an extra third cytoproct midway between the
two normal cytoprocts (Figs 4 and 6B). These additional cytoprocts were
separated from the normal ones by about 8-16 ciliary rows. The additional
cytoproct does not possess a central dot characteristic of the silvered image of the
functional organelle (Ng, 1976). In some cells belonging to this group, there are
only two cytoprocts fused into V or Y configuration (Fig. 6C). In some cells, only
one cytoproct is observed lying on the fused postoral suture.
•V:
8
Fig. 7. Photomicrograph of the very asymmetrical I type doublet. This stage corresponds to Fig. 6D. Silver-stained specimen. All abbreviations as in Fig. 2.
Fig. 8. Photomicrograph of the incomplete symmetrical II type doublet with exposed an incomplete oral band. The preoral (PR), postoral (PO) sutures and the
cytoproct (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 marks
the position of the incomplete oral band on a cell's circumference projection. This
stage corresponds to Fig. 9B. Silver-stained specimen.
60
J. KACZANOWSKA AND B. DUBIELECKA
Table 2. Frequency distribution of specimens {in %) of four classes specified by
the angular values of disposition of two oral apparatuses on the circumference of
type I doublets in relation to (a) the number of existing CVP bands and (b) the
number of cytoprocts
Number of cortical structures n
(a) the number of CVP bands
0 (lethal?)
1
2
(b) the number of cytoprocts
1
2
3
Angular values of disposition of oral apparatuses
%
%
%
%
180°-160° 160°-120° 120°-65°
65°-30°
(N-16)
(N-28)
(N-12)
(N-16)
33
32
7
37-5
37-5
25-0
46-4
50-0
3-6
25-0
75-0
0
68-7
31-2
0
17
50
5
0
100-0
0
0
100-0
0
16-6
41-6
41-6
93-7
6-3
0
n = total number of tested specimens in each class.
N = number of specimens of a given class.
(d) very asymmetric doublets (Figs 6D and 7) include cells with oral apparatuses
at an angle of 65-30 °. In these cases, two oral apparatuses assume a side-by-side
position, with very few wrinkled ciliary rows between them. In most cases these
cells possessed only one cytoproct. Even in this group many cells lacked both sets
of CVPs.
(e) derived singlets. Among the observed cells, only two cases of singlets with
branched preoral and postoral sutures were noted. Apparently the left oral
apparatus was totally missing (including even vestibular ciliary rows) in both of
these cases. Both branches were separated by 12-16 ciliary rows and they included a surplus of the left ciliary rows.
Table 2 summarises the occurrence of CVPs and cytoprocts in very well silverstained 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 restricted
to only one cortical side, which brings about an increasing asymmetry in the
location of the oral apparatuses.
(2) Oral apparatuses are morphologically normal even when in a side-by-side
configuration.
(3) Confrontation of oral bands of cortex at an angle\of 120-65 ° may bring
about 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 asymmetrical
doublets.
Pattern regulation in Paramecium
61
(4) No case of an incomplete symmetrical doublet was found (Sonneborn,
1970a).
3. Intermediate stages during doublet-to-singlet transformation in progeny of
type II doublets
Two sublines were studied in detail. These were subline M, heterozygous for
scr6, (but homozygous wild-type at/ha locus, genetical data not shown) of the
wild-type phenotype (straight cells), and a subline of twisted scr^fna
homozygotes. Both of these sublines were derived from doublets after the
second massive burst of autogamy. Despite the different phenotype (due to
different genotype) the sequence of appearance and the morphology of the
intermediate stages were similar in both sublines. They are in sharp contrast to
those observed in the type I doublets described above.
Intermediate stages in transformation to singlets consisted exclusively of different kinds of incomplete doublets. These incomplete doublets, with one or two
oral apparatuses lacking, correspond to the description and definition given by
Sonneborn (1963,19706). These cells manifest the doublet sets of cortical organelles, with the exception that one or even two oral apparatuses are missing.
However any incomplete oral band, even if the oral apparatus is completely
absent, may be readily recognized due to the preserved landmarks of the preoral
and postoral sutures with the cytoproct (Fig. 8). Thus the location of the ciliary
rows joining preoral and postoral sutures of the incomplete ventral complex (as
marked by arrows at Fig. 8) stands as the position of this incomplete oral band
on the circular projection of cells (labelled as the empty circles on circular
projection schemes in Fig. 9B, C and D).
The loss of the oral apparatus was observed in perfectly symmetrical doublets
and in all asymmetrical doublets (Table 3 and Fig. 9B, C and D). At the time
when the first incomplete symmetrical doublets were found in samples of the M
and F sublines, many other doublets displayed apparent malformation, partial
regression, or displacements of oral apparatuses within the oral bands of one or
both sides. In some cases totally astomous doublet cells were found: with two
normal dorsal CVP sets, with two preoral and postoral sutures, with two
cytoprocts, but with both oral apparatuses absent. It is known (Tartar, 1954) that
such cells are in viable. The stages of regression of oral apparatuses correspond
to the description of these events in Paramecium doublets with locally irradiated
oral structures (Hanson, 1955, 1962).
Morphology, symmetry and corticotypes were analysed in 81 perfectly silvered
doublets and derived singlets and 41 control singlets. Data are represented in
Table 3 and Figs 8 and 9. In completely symmetrical doublets with two normal
oral apparatuses, the number of ciliary rows in particular right and left systems
may be variable (Table 3; first, second and third rows), even though geometrical
proportions tend to remain similar (Fig. 9A). There is some variability in the
angular values for CVPs placements among cells belonging to this group (the
EMB74
62
J. KACZANOWSKA AND B. DUBIELECKA
Fig. 9. Sequence of doublet-to-singlet transformation of type II doublets. Circular
projections of intermediate stages in a corresponding sequence of Table 3 are presented. Variability of angular values for right ciliary rows systems are marked by the
dotted areas. These dotted areas correspond to CVP field angle (Nanney, 1966a),
i.e. limiting the maximal and minimal angles between the respective oral band and
its right positioned CVP band. (A) Symmetrical complete doublet with two oral, and
two CVP sets of landmarks. This circular projection corresponds to cells depicted in
Fig. IB and 5B. The right systems of ciliary rows (extending to the right from the oral
apparatus 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 also
a symmetrical disposition of the CVP bands (crosses). Variability of the angular
values of both right systems ranges from 30-60°. (C) Asymmetrical incomplete
doublet with the 160-120° angle between the complete and the incomplete oral
bands. These angles are always manifested to the left from the complete ventral
complex. This configuration brings about an increase of the angular values for the
right system on an opposite major side of a doublet to 90-120°. On the minor side
of a doublet the side-by-side location of the incomplete oral band and of the CVP
band is marked with an arrow. (D) Very asymmetrical incomplete doublet with an
angle of 120-90 ° between the complete and the incomplete oral bands. On the minor
side of a doublet the markers of the CVP band completely disappeared. On the major
side of a doublet a further increase of the angular values of the right system is
observed. (E) Derived singlets. All landmarks of the incomplete oral band disappeared. An angle for the remaining right ciliary system increased to 160-180°.
Nearly normal singlet's disposition of cortical landmarks is manifested both in the
normal and tiny derived singlets.
variability of these angular values is marked as dotted areas in Fig. 9), but there
is a strong tendency to maintain the 180 ° apart locations of the CVP sets in each
of 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 two
oral apparatuses with a corticotype below 95 (Table 3, third row). Thus there is no
invariant relation between the appearance of incomplete doublets and corticotypes , though this phenomenon becomes a rule in low corticotypes. The loss of
the 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, sixth
and seventh rows; and Fig. 9C and D). Thetotal number of ciliary rows between an
incomplete 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.
symmetric (180-160°)
18 135-115
complete doublets
9 114-110
symmetric (180-160°)
complete doublets
symmetric (180-160°)
4 99-95
complete doublets
symmetric (180-160°)
6 115-93
incomplete doublets
1
asymmetric complete
84
doublet with one CVP set
asymmetric (160-120°)
4 92-74
incomplete doublets
asymmetric (120-90°)
6 94-79
incomplete doublets
33 73-52
derived singlets
41 68-75
singlets (control)
Class of specimens of
transforming type II
doublets
23-4 ±1-5
20-7 ±1-3
19-7 ±1-1
18-2 ±2-9
24
24-0 ±2-6
25-3 ±3-0
26-5 ±2-6
31-8 ±1-9
35-5 ±2-4
28-2 ±2-4
36-3 ±3-4
27
28-7 ±4-9
34-0 ±6-4
38-7 ±2-7
43-8 ±2-7
right 1
39-2 ±2-5
leftl
33
9-3 ±1-5
21-8 ±4-4
17-5 ±1-7
20-2 ±2-8
23-4 ±1-0
right 2
29-3 ±2-9
23-7 ±1-7
33-0 ±2-4
28-2 ±1-9
34-9 ±2-0
37-2 ±3-0
left 2
number of ciliary rows systems:
(means and S.D.)
systems
right 2 and left 2
systems
right 2 and left 2
not observed
not observed
not known
close to the oral bands
dispersed
systems
right 1 and right 2
close to the oral bends
Observed localization of
fragmented ciliary rows on
the circumference of whole
cells
Left 1 and right 1 systems always refers to the component of the doublet with the higher number of the sum of the left and right ciliary rows.
Angular values in parentheses in the second column describe the limits of variability of relative disposition of two oral bands on the circumference
of doublets.
n = total number of observed cells belonging to a given specified class.
Data in 1, 2 and 3 rows refer to the same cytogeometrical stage-Fig. 9A; subdivision was based on different localization of fragmented ciliary
rows and coincides with three different groups of corticotypes.
8. Fig. 9E
9. Control
7. Fig. 9D
6. Fig. 9C
5. only one case
4. Fig. 9B
3. Fig. 9A
2. Fig. 9A
1. Fig. 9A
No. of row and
reference to stages
in Fig. 9
Total
number of
ciliary rows
on whole
cells
Table 3. Mean number and S.D. of ciliary rows appearing in sequential left 1, right 1 and left 2 and right 2 systems of type II
doublets of specified stages (Fig. 9) of doublet-to-singlet transformation
64
J. KACZANOWSKA AND B. DUBIELECKA
Some incomplete asymmetrical doublets (160-120° Table 3, sixth row) maintained 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 bands
was 8-11 rows (marked with an arrow on Fig. 9C).
On the major side of incomplete doublets two different tendencies are observed:
(1) although the total corticotype of doublets decreased sharply with an increase 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 the
symmetric incomplete doublets (Table 3, fourth row) of this system it averages
about 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 of
doublets 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 may
be as low as 52. But even very tiny derived singlets still manifested about 60-52
ciliary rows.
Only two exceptional cases of duplication of cortical landmarks in a side-byside position were observed among many type II doublets (M subline). In one
slightly asymmetrical doublet (oral apparatuses 160 ° apart) of a high corticotype
of 133, a third extra CVP band occurred midway between the oral apparatuses
on the major side of the doublet. Thus two side-by-side positioned CVP bands
were 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 28
ciliary rows, while the other left system was crowded with 43 ciliary rows. Within
the latter system an extra third oral apparatus appeared. This third oral apparatus was inverted (but not right-left reversed) and was not topographically
related to any preoral or postoral sutures. This oral apparatus was positioned 16
ciliary rows to the left of the normal oral apparatus.
DISCUSSION
The doublet system in Paramecium tetraurelia is inherently unstable over a
long term and tends to regulate to the singlet state (Sonneborn, 1963; de Haller,
1965). However, some doublets may be maintained over more than 100 generations. The transformation from doublet to singlet is not a single event, but a
series involving changes of corticotypes, loss of cortical organelles and the
assumption of only one nuclear apparatus.
As in Tetrahymena (Nanney et al. 1975; Frankel & Nelsen, pers. comm.) in
Paramecium pathways of regulation from doublet to singlet always involve unstable intermediate configurations of cortical landmarks. At least two different
Pattern regulation in Paramecium
65
pathways are reported here. These pathways conform to both contradictory
descriptions respectively reported by Sonneborn (1970a) and by Sibley (1974).
The sequence of intermediate stages is deduced from the sequence of their
appearance in samples and in some cases were confirmed experimentally. We
suggest that the pathway of regulation and the nature of the intermediary stages
does not depend on genotype, but on the initial disturbance of doublet symmetry.
If one supposes that during initial fusion, accompanied by dedifferentiation of
ciliary 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 initial
doublet can acquire a different shape and the line of confrontation of cortexes
of different origin may be differently positioned. In Fig. 10A it is suggested that
in type I doublets, the area of fusion might have been slightly further from the
oral apparatuses, leading cells to join firmly along the right ciliary meridian
which extends straight from pole to pole. However, such union would bring some
mechanical pressure on the dorsal sides. Thus such doublets would tend to
regress their dorsal, and not ventral, cortical areas. In contrast, in type II
doublets the fusion area in the right ciliary system would be localized close to the
oral apparatuses, along the ciliary rows extending from the preoral to the postoral sutures. Thus the areas close to the oral apparatuses and sutures would be
distorted and may become a site of mechanical stresses. Thus such doublets
would tend to regress their ventral, and not dorsal, cortical areas (Fig. 10B). This
hypothesis is consistent with observations that areas of regression may involve
either one, or even two sets of corresponding cortical structures in a given
doublet. It is assumed that selection is the mechanism bringing about the survival
of cells, which maintained a complete set of indispensable organelles and the
cortical area of corticotype of at least about 52 ciliary rows (the lowest corticotype found in derived singlets, Table 3, eighth row).
From these it is clear that Paramecium is able to form oral and CVP structures
even if they are improperly located with respect to each other. Regression of the
cortical area may bring about very different configurations of the remaining
cortical landmarks. From comparison of patterns of deployment of cortical landmarks in cells at intermediate stages of doublet-to-singlet transformation at least
three developmental processes are deduced:
(1) Side-by-side location of the separate functional oral bands may bring about
the appearance of an additional structure along the line of confrontation of the
cortex of an apparently different origin. Such cases of induction of the extra
cytoproct have been previously reported in Paramecium in grafting experiments
(Sonneborn, 1963). The appearance of the additional structures in general terms
is reminiscent of cases of duplication of structures after specific confrontation of
parts 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 bands
positioned side by side requires a minimal distance between them. Thus we
66
J. KACZANOWSKA AND B. DUBIELECKA
.CVP
CVP
CVP
CVI
CVP
Fig. 10. Hypothesis of the origin of diversity of two different developmental pathways of doublet-to-singlet regulation in Paramecium tetraurelia. (A) Type I doublet
progeny originates from the initial fusant with the seams joining cortexes of different
origins (dashed outline vs solid outline of mating cells) running along the right ciliary
row. This kind of fusion put the mechanical stresses onto the dorsal surfaces. Hence
the oral bands are more stable than the CVP bands. Resulting doublet manifests only
one fused anterior pole. (B) Type II doublet progeny originates from the initial
fusant with the seams confronting cortexes of different origins (dashed outline vs
solid outline of mating cells) running along the right ciliary, systems very close to the
oral bands i.e. in the vicinity of the preoral and postoral sutures. This kind of fusion
brings about an instability of the oral bands themselves. Splitting of the anterior poles
releases the mechanical stresses on the dorsal surfaces, which become more stable
than those in I type of doublet. Disposition of the area of the fusion of cortexes of
different origins is marked by a heavy line on the schemes of the exposed to viewer
surfaces 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 of
differently determined states' (Meinhardt & Gierer, 1980). If this distance
decreases either fusion of both bands occurs (as observed in fusion of preoral and
postoral sutures in type I doublets), or one set of structures disappears (left oral
apparatus in the same cases).
(3) Both in twisted cells, and in very asymmetrical incomplete doublets of type
II some regulatory shift of positioning of CVPs was observed. It is deduced that
an increase of field angle (Nanney, 1966a) permits step-by-step restoration of the
proper symmetry and the numbers of ciliary rows between cortical landmarks.
Pattern regulation in Paramecium
67
Thus it is suggested that pattern regulation in Paramecium during doublet-tosinglet transformation is an interplay of at least three factors: the regression of
cortical areas caused by mechanical stresses, an interaction of confronted fragments of cortical pattern and regulatory shift of localization of cortical structures. If the symmetry of deployment of the cortical organelles, the maintenance
of one set of indispensable set of cortical organelles and minimum of cortical
areas between them is achieved, the whole organism falls into a stable
cytogeometrical balance and this pattern may then be faithfully reproduced
without 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 help
in preparing the manuscript. We thank Dr David Nanney for comments and corrections of
manuscript. We wish to thank Dr Mario Nelsen and Dr Andrzej Kaczanowski for many
helpful suggestions.
This work is partially supported by research grant of the Polish Academy of Sciences.
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{Accepted 9 November 1982)