/. Embryo!, exp. Morph. Vol. 65, pp. 57-71, 1981
Printed in Great Britain © Company of Biologists Limited 1981
57
Polymorphism and specificity of positioning
of contractile vacuole pores in a ciliate,,
Chilodonella steini
By JANINA KACZANOWSKA1
From the Institute of Zoology, Warsaw University
SUMMARY
The unicellular ciliate Chilodonella steini has a well-defined flat and ciliated ventral field.
During divisional morphogenesis two sets of new contractile vacuole pores (CVPs) are
formed on this field. During final pattern formation some of these CVP primordia and the
old parental set of CVPs are completely resorbed. Primary pattern of distribution of the
CVP primoidia and final pattern of distribution of the matured CVPs manifest an intraclonal
polymorphism.
From analysis of this polymorphism some features of mechanism(s) of CVP pattern
determination are deduced:
1. There is a strict, short-distance negative control of appearance of CVP primordia at
sites of oral morphogenesis and around the ventral field.
2. Certain indeterminacy of large-scale patterning of CVP primordia is observed over the
area competent to yield CVP formation. However, within this area three longitudinal sectors
with a high probability of occurrence of CVP primordia are alternated with sectors nearly
deprived of their occurrence.
3. Positive control of probability of occurrence and of specificity of location is found for
certain CVP primordia. An interaction of mechanism of positioning on cellular longitudes
and latitudes is proposed to account for these facts.
4. The resorption of supernumerary CVP primordia does not alter the character of the
global map of distribution of CVP primordia achieved during primary pattern formation.
The primordia located at some latitudes persist, whereas others are resorbed at random.
It is suggested that all CVP primordia which do not mature at the time of stabilization of
divisional morphogenesis are resorbed. Thus the global map of CVPs distribution would
result from the sum of the individual determinations of the fates of each CVP primordium,
superimposed on an initial spatially non-uniform distribution of CVP primordia.
INTRODUCTION
Ciliate are single-celled organisms with an ordered pattern of distribution of
their organelles over the cell cortex. There is evidence that the mechanism of
large-scale positioning of organelles is encoded in the ciliate genome (Heckmann
& Frankel, 1968; Jerka-Dziadosz & Frankel, 1979) to ensure this species-specific
order. However, in some ciliates the polymorphism exists. This may result from
modification of the global map by effects of pre-existing organization of the
ciliate cortex (Beisson & Sonneborn, 1965; Grimes, 1976; Ng & Frankel, 1977;
1
Author's address: Institute of Zoology, Warsaw University, Warsaw 00-927, Poland.
58
J. KACZANOWSKA
Final pattern
Resorption
o|
|o
Primary pattern
i
i
i
i
i
• ;
Attenuation
:° •
• o
•
Random selection
i
•!
•!
• i
|O
:• • 1
Atten uation
1
1
1
•
•
•
•
•
!
«
•
V
0
•
•
•
Random selection
0
o
'O
o
Positive control
•
•
9
0
0
0
Random selection
Fig. 1. For legend see opposite.
Ng, 1979), either from the gradual regulation of patterns in the progeny of an
abnormally patterned initial cell (Nanney, 1968), or from a probabilistic mode
of determination of the number and of the location of organelles. This latter
possibility is explored in this report on polymorphism of the disposition of
contractile vacuole pores (CVPs) on the flat ventral surface of a ciliate,
Chilodonella steini.
Any geometric description of the position of an organelle on the cell cortex
requires specification of a coordinate system. The coordinate system projected
over the cell surface allows one to measure absolute or relative distances from
chosen reference points. At least two parameters are indispensable for the exact
Contractile vacuole pores patterning in a ciliate
59
placement of the measured point on a two-dimensional surface. In a Cartesien
system these parameters correspond to latitude and longitude (Frankel, 1979).
In the polarized cortical layer of ciliates the evenly spaced meridional rows of
ciliary basal bodies form the natural meridians for measuring longitudes of the
organelle, with one of them - the stomatogenic meridian - serving as a reference. Nanney (1966a, b, 1967, 1968) discovered certain cytogeometric rules of
positioning of the CVP on longitudes in Tetrahymena. He gave a formal explanation of positioning of the CVP in a given cell as a consequence of specification
of an inductive angle between the stomatogenic meridian and the area in which
the CVP is found. The variability of positioning of the CVP between meridians
is described in terms of afield angle, which defines the sector of the cell surface
which may be competent to yield a CVP.
It is already established (Kaczanowska, 1974) that CVPs in Chilodonella
cucullulus strain X form on three longitudinal sectors; the CVPs vary in number
from 5 to 11, and their location is variable. CVPs are positioned at intersections
of the longitudinal sectors with specific radii measured from a reference point,
a site of stomatogenesis. If cells are microsurgically miniaturized (Kaczanowska,
1975), the proximal radius describes the exact placement of the anterior obligatory CVPs, while posterior CVPs are drastically reduced in number.
It is believed that an analysis of the polymorphism of CVP primordia and of
CVPs distributions over the ventral field of the related species Chilodonella
steini makes possible to delineate some general characters of a large-scale
mechanism(s) operating in CVP-pattern determination. We begin by describing
the number and distribution of CVP primordia and of CVPs in different specimens of Ch. steini at the same morphogenetic stage of division, when old CVPs
Fig. 1. Theoretical models of CVP primordia and CVPs distribution within a zone
competent to yield CVP formation in Chilodonella steini. Boxes represent an entire
area of the ventral field. Black circles represent CVP primordia (left boxes), or
matured CVPs (right boxes). White circles mark resorbed CVP primordia absent in
final patterns (right boxes). Model A. Negative control, or inhibition of CVP
primordia appearance in the extreme 'forbidden' zones. Sharp boundaries (solid
lines) between CVP competent zone and CVP deprived zones. Primordia are
randomly distributed within CVP competent zone. This might lead, following
resorption of some CVP primordia, to either of two alternatives: Al -an attenuation
of the CVP competent zone (indicated by transposition of solid lines, while dashed
lines mark positioning of original boundaries), or A2 - maintenance of the original
boundaries with random selection of CVP primordia for resorption. Model B.
Probabilistic model of CVP primordia distribution along a preferred meridian
(heavy line), but with a high dispersion of placement of CVP primordia. Delineated
by dashed lines external zones mark zones of a very low probability of CVP primordia
occurrence. Following resorption there is either Bl - an attenuation of the original
dispersion (internal dashed lines), or B2 - maintenance of the original dispersion.
Model C. Positive control of placement of CVP primordia at the intersection of
two coordinates (crossing heavy lines). This might be followed by either: Cl - maintenance of CVP primordia at that intersection (dashed circle), or C2 - by a random
selection of CVPs,
60
J. KACZANOWSKA
and newly induced CVP primordia still coexist. Next we may investigate whether
there are any similarities of the test patterns. The following issues are considered:
1. Area occupied with CVP structures in all tested specimens apparently
belong to regions competent to yield CVPs. Remaining areas may be either less
competent to yield CVP formation, or are inhibited in CVP formation. If an
area occupied with CVP primordia (or CVPs) is very sharply marked off from
areas deprived of CVP primordia (or CVPs), this suggests that there are zones
'forbidden' from yielding CVP structures, i.e. they are under some form of
negative control. If however CVP-competent areas gradually fade or merge
with empty areas, this suggests that only positive controls are operating. It
means that the appearance of CVP structures is most likely along certain
meridians, with a gradual decrease in probability at more distant locations
(Fig. 1, models A and B, left boxes).
2. Grimes & L'Hernault (1979) and Frankel (1979) have established a
different character of positioning operating on the longitudes and latitudes of a
ciliate cell. Then the question arises whether or not these two supposed mechanisms act independently in determination of the position of a given organelle.
It might be ascertained whether, in Ch. steini, the probability of occurrence of
CVP structures and the degree of longitudinal dispersion remain constant within
a given sector at all of its latitudes. In the event of some cooperation between
the two putative mechanisms involved in positioning of CVP primordia, some
CVP primordia within the same sector would be spatially more precisely located
than others (Fig. 1, model C left box).
3. In Ch. steini two sets of new CVP primordia appear during divisional
morphogenesis while the old set of parental CVPs still persists. The primary
patterns of the distribution of CVP primordia is then changed by the resorption
of certain supernumerary CVP primordia. During final pattern formation, all
supernumerary CVP primordia and the old set of parental CVPs completely
disappear. The comparison of the primary and final patterns is made to test
whether resorption of some CVP primordia may modify the general character
of their distribution. Does it expand the 'forbidden' areas by eliminating CVP
primordia at the boundaries or does it occur throughout the CVP areas merely
decreasing their number but not affecting the boundaries of the CVP zone?
If it is not at random this proves that final pattern determination takes place in
two steps: first a broad outline of CVP areas, second some modification of
these areas by the process of CVP primordia resorption. The alternative possibility, of random resorption of some of CVP primordia over the ventral field,
would, if it occurred, still leave open the question of the reason of this randomness. The two different models of final pattern regulation subsequent to each of
the three types of initial pattern establishment are depicted schematically in
Fig. 1 (right boxes).
Data reported here are taken as evidence that:
1. There is a very strict negative control of appearance of CVP primordia in
Contractile vacuole pores patterning in a ciliate
61
some 'forbidden' areas. These areas are confined to the sites of stomatogenesis
and to the border of the ventral field.
2. In the remaining areas competent to yield CVP primordia, three sectors
of high probability of occurrence of CVP primordia manifest different widths.
3. Some CVP primordia are spatially much more precisely determined than
others.
4. Resorption of the supernumerary CVP primordia during final pattern
formation does not alter the character of CVP distribution over the ventral
field. However, certain CVPs, which are invariant elements of pattern, are never
resorbed.
MATERIALS AND METHODS
A clone of Chilodonella steini (Ciliata, Kinetofragmophora; Radzikowski &
Goiembiewska, 1977) line 237/10 that did not self in the immaturity period
(Kaczanowski, Radzikowski, Malejczyk & Polakowski, 1980) was isolated
from one exconjugant. Six months later, one subclone was reinitiated. All
preparations of this subclone were made during a one-month period. Other
cells tested were derived from the same subclone about 10 months later, when
at least some cells entered into permanent selfing, with retention of old macronucleus (Kaczanowski et al. 1980).
The general characteristic of this species and the methods of culturing of the
cells followed these of Radzikowski & Goiembiewska (1977). The mean generation time of the cells varied from 12-19 h when they v/ere maintained in a normal
daily photoperiod and fed every second day.
Cells from 2-day mass clonal cultures were used for silver impregnation
(method of Frankel & Heckmann, 1968). Well-silvered specimens in early
division were selected for mapping and counting of their CVPs and CVP primordia if they were properly dorsoventrally embedded in gelatin and if all ciliary
meridians were clearly distinguished. In 41 dividers of the subclone 237/10
immatured, protocols and maps were made of cortical parameters of 1842 CVP
primordia and of 587 parental CVPs. If any coordinate system was tested (Figs.
6, 7, 8 and Table 1), all of the data were grouped and then statistically analysed
(Sokal & Rohlf, 1969). In Fig. 8 three peaks were revealed in all three histograms.
The significance of these results was tested for a given coordinate system by
computing for every individual (n = 41) the difference in number of CVP
primordia between a sector selected a priori in this coordinate system as having
a high number of CVP primordia and neighbouring right and left sectors of the
same width selected as having a low number of CVP primordia. In Table 1 in
all specified sectors mean numbers and sd values of CVP primordia and of
CVPs were calculated. To estimate the rate of decrease of total number of
CVPs as compared to the total number of CVP primordia in specified sectors,
the ratio of the number of CVPs occurring in a given sector of CVP primordia
in it was calculated for every respective parental to anterior daughter, and
3
EMB 65
62
J. K A C Z A N O W S K A
Fig. 2. A ventral field of morphostatic Ch. steini. The CVPs are dispersed among
the ciliary meridians (arrows).
Fig. 3. A ventral field of early dividing Ch. steini. In addition to old parental CVPs,
new CVP primordia (arrows) are seen at the left sides of some ciliary meridians.
The R, M and L sectors are marked off by dashed lines.
Fig. 4. A ventral field of dividing Ch. steini at a stage, somewhat more advanced than
that in Fig. 3. Segment A-4 is labelled with an arrow.
Fig. 5. A ventralfieldof an advanced divider of Ch. steini. Morphogenetic movement
of the oral segments is seen (heavy arrow). The new CVP primordia are undergoing
transformation into the matured CVPs for daughter cells (arrows), while others are
gradually discarded.
parental to posterior daughter patterns of the same specimen. Then pooled data
of the mean of all these ratios for every sector were compared with a Cochran
and Cox test. The additional control cells fixed 10 months later involving only
morphostatic cells were tested for reproducibility and the maintenance of the
polymorphism of cells.
Some statistical calculations (r correlation coefficients) have been made in
the Center of Statistical Calculations of Warsaw University by Msc I. Wozniak.
RESULTS
(1) Divisional morphogenesis o/Chilodonella steini
Divisional morphogenesis of Ch. steini conforms to the general scheme
described for this genus (Radzikowski & Golembiewska, 1977).
Ch. steini is a flat asymmetric ciliate, with the ventral surface covered with
ciliary meridians and subapical oral apparatus encircled by an oral ciliature.
CVPs are distributed only over the ventral surface, but they never appear near
the oral apparatus or at the margin of the ventral surface. In silvered specimens
CVPs appear as round black circles between ciliary meridians (Fig. 2, arrows).
The first signs of approaching division are an increase of body size, differentiation of oral ciliary segments for the prospective posterior daughter cell
Contractile vacuole pores patterning in a ciliate
63
(opisthe), and differentiation of two sets of new CVP primordia for the daughter
cells as little spots or perpendicular slits near the left side of certain ciliary
meridians (Fig. 3, arrows). Oral ciliary segments and one somatic segment (the
so-called A-4 segment, Radzikowski, 1966; Kaczanowska, 1971) for the presumptive opisthe differentiate in the subequatorial region of the ventral field.
Cells in this stage were selected for further study.
In the following stage of morphogenesis (Fig. 4), all oral segments of the
presumptive opisthe and the ciliary segment A-4 begin to migrate by rotating
around the centre of the oral region. While the ciliary meridians are passively
transmitted into successive anterior daughter cells (proters), in opisthes the
enumeration of meridians of the postoral left part of the ventral field becomes
altered due to a gain of one meridian from the A-4 segment. A-4 inserted backwards between a stomatogenic meridian (no. 1) and the meridian to its right
(no. 2). When enumeration of meridians follows the rules for Tetrahymena
(Elliott & Kennedy, 1973), the former meridian 1 now becomes meridian n, the
former n becomes n-\ etc. (Figs. 4, 5). This slippage compensates for the usual
loss of the extreme left meridian, which is not represented in the equatorial zone
and is passed entirely to the proter. As a result of these migrations, the circumoral
segments turn about 120° while the preoral segment is reversed and positioned
anteriorly to them (Fig. 5). Some new CVP primordia change into long perpendicular slits, while others remain with no transformation (Fig. 4).
At a later stage of morphogenesis the old, parental oral apparatus (pharyngeal
basket) is resorbed (Fig. 5) and very quickly two new oral apparatuses for the
nascent daughters are formed: in situ for the proter and in the centre of a region
of rotating ciliary segments for the opisthe. The CVP primordia gradually are
transformed into the final round orifices in the middle of the intermeridional
space, while the parental set of CVPs and some supernumerary new CVP
primordia are resorbed.
In early dividers (Fig. 3) new CVP primordia are readily distinguished from
parental CVPs by their shape and fine positioning (slits or dots in the left side
of a ciliary meridian versus round bigger circles in the middle of the intermeridional space). In this stage, the future fission line is marked by the rupture
of stomatogenic and left meridians. The right margin of the fission line may
also be discerned as a small indentation of the dorsal surface. The distribution
of CVP primordia may, therefore, be analysed separately in prospective proters
and opisthes. In this stage the A-4 segment has not yet been added, and the old
pattern of CVPs is perfectly preserved.
(2) Variability of the cortical patterns and of the total number of
CVPs and CVP primordia
Dividing specimens of Ch. steini manifested an array of corticotypes (i.e.
total number of ciliary meridians) from 24 to 30 with corticotypes 27 and 28
in the majority of cells. In cells with corticotypes 26, 27 and 28 of the group
3-2
64
J. KACZANOWSKA
tested for CVPs and CVP primordia, the distribution of the total dimensions
of the ventral field were nearly identical. If the first (furthest right) stomatogenic
meridian is designated as no. 1, it divides the whole set of ciliary meridians into
right (nos. 2, 3, 4 etc.) and left ciliary meridians (nos. n, n-\, «-2, etc.). Cells of
the same corticotypes often have different patterns of the total number of right
and left meridians.
The total number of parental CVPs varies from 8 to 25 with a mean number
of 14-7 ± 3-9 (n = 41). The newly formed sets of the CVP primordia included
respectively 13 to 35 (mean 22-1 ± 5-0) for proters and 10 to 38 (mean 22-8 ± 6-2;
n = 41) for opisthes. There is a significant (P = 0-01) difference between the
total number of parental CVPs and the number of CVP primordia in descendants.
It is deduced that some of the CVP primordia are discarded during formation
of final pattern (about 34-5 % of the total number of CVP primordia).
There is no statistically significant correlation between the total number of
parental CVPs and the number of CVP primordia in proters (r = 0-14) or in
opisthes (r = 0-18). There is a slight positive correlation of the total number
of the CVP primordia in proter and opisthe (r = 0-55 with P = 0-05).
The total number of the CVP primordia observed in particular intermeridional space is variable (from 0 to 10). Among 41 tested dividers no two cells
had identical patterns of distribution of CVPs or CVP primordia.
Hence in Ch. steini there is a polymorphism of corticotypes, of right/left
pattern of ciliary meridians, and of a number and disposition of CVP primordia
and CVPs.
(3) Localization of the CVP primordia in early dividers, primary patterns
The maps of CVP primordia show that in all specimens there are zones strictly
devoid of CVP primordia. These 'forbidden' zones include the margin of the
ventral field, the regions of the parental oral apparatus and of the preoral ciliary
segment, the area of the future oral primordium for the opisthe, and the region
of the forming fission line.
The remaining surface of the ventral field is more or less competent to yield
CVP primordia. However, these competent zones include longitudinal sectors
of relatively high frequency of occurrence of CVP primordia. The right (R) sector
follows the curvature of the right margin of the ventral field, but at some distance
from it. The median (M) sector appears in the right half of the ventral field
parallel to the main longitudinal axis of cells. This sector is roughly composed
of two separate groups of CVP primordia for every daughter cell: one just
posterior to the oral areas, which corresponds to the sites of CVP-1 described
in the related species, Ch. cucullulus (Kaczanowska, 1974), and the second at
the rear end of the ventral surface of the prospective daughter cell, corresponding
to the posterior CVP-4 site of Ch. cucullulus. Finally, the left (L) sector appears
in the middle portion of the left part of the ventral field. All of these sectors are
diagramatically marked in Fig. 3.
Contractile vacuole pores patterning in a ciliate
65
4i
3-
OJ ~^r14
12
10
8
_2 - 4 - 6
- 8 -10 -12
Fig. 6. Diagram of the mean number of CVP primordia per given intermeridional
space counted from the stomatogenic axis for prospective proters of three different
corticotypes. Corticotype 28 (n = 8) - dashed line, corticotype 27 (n = 11) - heavy
line, and corticotype 26 (n = 6) - dashed and dotted line.
0J
14
12
10
8
-2 -4 -6
-8 -10 -12
Fig. 7. Diagram of the mean number of CVP primordia per given intermeridional
space counted from the stomatogenic axis for prospective opisthes of three different
corticotypes. Illustrative conventions and number of studied patterns as in Fig. 6.
Comparison of means of occurrence of CVP primordia in particular intermeridional spaces in cells of the same corticotypes (Fig. 6 for proters and Fig. 7
for opisthes) revealed that histograms for the proters and for the opisthes are
very similar. The R, M and L sectors of frequent occurrence of CVP primordia
alternate with four sectors of infrequent occurrence. The sectors at the left and
right edges of the histograms belong to the 'forbidden' zones, while the sectors
located between the R, M and L sectors, are characterized by a relative
absence of the CVP primordia. The different corticotypes have a very similar
variability of the mean frequency of CVP primordia. The longitudes of the CVP
primary pattern might be computed for the whole ventral field by combining
data for the prospective proters and opisthes for all 41 specimens. To test
whether the observed R, M and L sectors might be also specified with reference
66
J. KACZANOWSKA
1
3
5
7
29 27 25 23
14 12 10
8
9
21
6
11
19
4
13
17
2
15 17 19 21 23 25 27 29
15 13 11 9
7
5 3
1
n -2 - 4 - 6 - 8 - 1 0 - 1 2 -14
Fig. 8. Diagram of the mean number of CVP primordia per given intermeridional
space occurring both in prospective proter and opisthe in 41 specimens using the
following reference coordinates: (a) the right extreme meridian, solid line; (b) the
left extreme meridian, dashed and dotted line; (c) the stomatogenic axis, dotted line.
Note that three different conventions of counting of ciliary meridians were used for
ordering data about the CVP primordia deployment: convention (a), the most
right ciliary meridian is designated as a ciliary meridian 1 and followed by 2, 3, etc.;
convention (b), the most left ciliary meridian is designated as a ciliary meridian
number 1 and followed in reverse direction by 2, 3, etc.; convention (c), furthest
right postoral meridian is designated as a ciliary meridian number 1, to the right
this meridian is followed with ciliary meridians 2, 3, etc., to the left this meridian is
followed with ciliary meridians n, n-l,n-2, etc. This convention corresponds to that in
Figs. 6 and 7. Three conventions of enumerations indicated on three abscissae.
Ordinate: mean number of CVP primordia per intermeridional space.
to the boundaries of the ventral field, the positioning of all CVP primordia was
then assessed in all specimens in terms:
(1) of the number of meridians from the right margin of the ventral field,
(2) or in terms of the number of meridians from the left margin of the ventral
field,
(3) or in terms of the number of meridians from the stomatogenic axis.
In three histograms (Fig. 8) the R, M and L peaks are visualized in all three
coordinate systems. Statistical tests were carried out to assess the significance
of the peaks appearing within the three coordinate systems with respect to the
neighbouring valleys. Particular intermeridional spaces were selected on an a
priori basis as having either a high or low probability of appearance of CVP
primordia. It was found that statistically significant results were obtained for
the R and L sectors only if these were constituted of three intermeridional
spaces, not one or two.
The three intermeridional spaces of the R peak had a significantly greater
average number of CVP primordia than did the neighbouring valleys when these
Contractile vacuole pores patterning in a ciliate
67
spaces were counted from the right margin of the ventral field; similar results
were obtained for the left L peak with reference to the left margin. When the
positions of the right and left peaks were enumerated with respect to the stomatogenic axis (Fig. 8, dotted line), these peaks were significant when compared to
the proximal valleys (i.e. those between the stomatogenic axis and the peak)
but, surprisingly, not with respect to the distal zones situated near the
margins.
The M peak differed from the two other peaks in that it was made up of only
a- single intermeridional space, and that it was significantly specified only with
respect to the stomatogenic axis. This can be clearly appreciated by noting in
Fig. 8 how much sharper this peak is in the spatial system keyed to the stomatogenic axis (dotted line) than in that keyed to the right (solid line) or left (dashed
line) margins.
The simplest interpretation consistent with this analysis is that the M sector
consists of only a single intermeridional space while the R and L sectors consist
of three or, in the case of the L sector, probably more such spaces. The stomatogenic axis probably serves as the primary reference point for the M sector and
may participate in specifying the R and L sectors; however, the 'forbidden'
sectors at the two margins are significantly specified only in relation to the
nearby cell margins.
Although, as mentioned earlier, the distribution of CVP primordia is probabilistic, and no two cells have an identical distribution, certain specific CVP
primordia can be followed more reliably than others. We will here consider the
two CVP primordia that may appear in the M sector, one of them (CVP-1)
located a short distance posterior to the oral region, the other (CVP-4) situated
not far from the posterior end of the nascent cells (Figs. 3, 5), as well as CVP-5,
the anterior preoral CVP primordium in the R sector (these CVP primordia are
numbered as in Ch. cucullulus stock X; Kaczanowska, 1974, 1975). The CVP-1
primordium was found in all but one of the 82 proter and opisthe examined
patterns, and was always placed in the same intermeridional space with reference
to the stomatogenic axis. Thus it occurred that CVP-1 primordium was a stable
element of primary pattern of Ch. steini.
The CVP-4 primordium, on the other hand, was absent in 17 of the 82 patterns,
preferentially in opisthes (but see also Fig. 4), and its location with respect to
the stomatogenic axis was not absolutely fixed; in fact, all of the variation in
the M sector is accounted for by variation in the placement of the CVP-4
primordium.
The preoral CVP-5 primordium failed to appear in only 2 out of the 82
daughter patterns. Its position, though not completely invariant, is mainly
restricted to the interior intermeridional space within three ones constituting
the R peak.
It thus appears that the level of indeterminacy in both the occurrence and the
positioning of specific CVP primordia differs, both for primordia located at
68
J. KACZANOWSKA
Table 1
Mean number of CVP primordia (n = 82), of CVPs (n = 41), and ratios of the
total number of CVPs to the total number of CVP primordia in respective parental
and daughter patterns in selected sectors specified in a coordinate system with the
stomatogenic axis as a reference (explained in text).
Sector
R
R, M
M
M, L
Intermeridional spaces
9,8,7
6,5,4
2
1, n, n-\
CVP primordia
6-90±3-40 1-23 ±1-41 1-94±108 0-58 ±0-75
(mean number and sd)
CVPs
4-35±20O O-75±O-8O l-54±0-98 0-22±0-52
(mean number and sd)
Ratio:
0-94±0-93 036±0-62 100±0-85 016±0-38
number of CVPs
number of CVP primordia
(mean number and sd)
Number of tested pairs
82
72
79
36
n-5,n-6,n-7
4-85 ±2-22
3-58±l-43
0-93±0-81
81
different latitudinal levels in the same sector (CVP-1 and CVP-4) and in different
sectors at a somewhat similar latitude (CVP-1 and CVP-5).
(4) Comparison of the primary andfinalpatterns of CVP distribution
In Ch. steini, only about 65-5 % of the CVP primordia persist in the final
patterns. The remainder are resorbed. The question arises whether the probability of resorption is uniform over the whole competent area of occurrance of
CVPs, or whether it is specifically confined to certain sectors (Fig. 1, right
boxes).
Some decrease in the mean number of CVPs is observed in all of the specified
sectors, both in the peaks of preferential occurrence of CVP primordia and in
the valleys of relative absence of these primordia (Table 1). Further, the ratios
of CVPs to CVP primordia did not differ significantly among sectors, as evaluated by the Cochran and Cox test (P = 0-05). This result strongly suggests the
uniform resorption of CVP primordia over the whole CVP competent zone
(corresponding to models of the alternatives nos. 2 among right boxes in Fig. 1).
But on the other hand, when specific CVPs (matured CVP-1, CVP-4 and
CVP-5) were considered, CVP-1 was found to be absent at its expected site only
in 1-6% of the specimens, CVP-5 was absent in 8 % of the specimens, while
the posterior CVP-4 was absent in 52-8 % of the cells. Thus the CVP-1 primordium
tends to persist at a non-random fashion (Fig. 1 Cl model), while the CVP-4
primordia are much more readily resorbed.
These different data are taken as evidence for a generally uniform resorption
of the total number of CVP primordia that is superimposed upon, and independent of, the spatially non-uniform probability of formation of CVP primordia
and of their persistence.
Contractile vacuole pores patterning in a ciliate
69
DISCUSSION
In Chilodonella steini, CVP primordia may occur near any ciliary meridians
except those in certain 'forbidden' areas. The borders of the ventral field and
the site of stomatogenesis were used here as a priori reference points for CVP
positioning on longitudes. Absolute absence of CVP primordia in these areas
is consistent with a hypothesis of a short-distance inhibitory effect of the site
of stomatogenesis and of the boundaries of the ventral field on the competence
to yield CVP primordium formation.
Every meridian on the remainder of the ventral field is able to support CVP
formation, although the probability of this event is much higher in three longitudinal sectors. From this result three conclusions may be drawn: (a) CVP
organellogenesis is not restricted to particular meridians but rather to certain
territories, (b) there is some indeterminacy in the large-scale mechanism of
specification of these territories, as CVP primordia sometimes form outside of
the territories and since even within the territories the disposition of CVP
primordia is variable, and (c) there is some periodicity of longitudes of high
probability of the occurrence of CVP primordia in a form of the R, M and L
sectors alternated with sectors of low probability of CVP primordia occurrence.
The right and left sectors of a high probability of occurrence of CVP structures
cover more than one intermeridional space. In terms of Nanney's formalism
(19666) they represent a broad field angle. The median sector, however, is
limited to one intermeridional space.
A virtual stability of occurrence and of localization of the CVP-1 primordium
with respect to the stomatogenic meridians, which undergo cortical slippage in
every generation of opisthes, indicates that the stomatogenic axis is not inherited
by the structural identity of a particular meridian, but as a territory in which
stomatogenesis takes place. The position of the M sector, and especially the
CVP-1 primordium is determined in relation to this territory.
At least CVP-1 primordium placement is determined much more specifically
than the placement of the other CVP primordia. This suggests that along a given
sector of high probability of appearance of CVP primordia, there exists, at some
latitudes a spatial constraint on the CVP placement along longitudes (Fig. 1,
model C). Thus a cytogeometric model of CVP distribution in Chilodonella
(Kaczanowska, 1974) may result from some cooperation of mechanisms of
positioning on longitudes and on latitudes, perhaps in a form of a mosaic of
nodes of increased specificity of CVP positioning.
Ultrastructural investigations (Kaczanowska & Moraczewski, in preparation)
indicate that during late division stages some CVP primordia are very advanced
in their differentiation, while other neighbouring ones are still in an early stage
of development. This asynchronous development of individual CVP primordia
and next resorption of part of them, while others persist evidence a very local
character of completion of CVP organellogenesis, which is different from the
70
J. KACZANOWSKA
global character of assessment of CVP-competent territories observed at the
cellular level of organization. This statement is consistent with a distinction made
between large-scale and short-range mechanisms of patterning in ciliates
(Frankel, 1979).
The final pattern of CVPs results from the spatially uniform resorption of
about 35 % of the total number of CVP primordia. Resorption of the supernumerary CVP primordia does not modify the global map of CVP distribution
over the ventral field. However, CVP primordia occurring at particular sites
(e.g. CVP-1) are positively selected to persist. This juxtaposition of positive
selection of certain of the CVP primordia and a randomness of the global fates
of CVPs observed at the cellular level of organization might be understood by
assuming an early structural maturation of the CVP primordia positioned at
sites of increased specificity of CVP positioning. They might be sufficiently
developed at the critical period of divisional morphogenesis (Kaczanowska &
Kiersnowska, 1976) of Chilodonella to escape resorption.
Thus the global map of CVPs distribution in Ch. steini would result from the
sum of the individual determinations of the fates of each CVP primordium,
superimposed on an initially spatially non-uniform distribution of CVP
primordia.
In terms of the set of theoretical models of CVPs distribution on the ventral
surface of Ch. steini (Fig. 1) presented here data are consistent with model A
applied to dissect a CVP competent zone out of 'forbidden' zones at the sites
of stomatogenesis and around the border of the ventral field. On remaining
zone there are three preferred sectors of CVP primordia occurrence with certain
dispersion of their placement (Model B). However, along these sectors the
positive control of placement of certain CVP primordia is also established
(perhaps consistent with Model C). Resorption, though globally random,
involves a positive selection of at least CVP-1 primordium (Model Cl).
I am most grateful to Dr Joseph Frankel for extensive discussions, helpful suggestions and
criticisms in the development and final shaping of this manuscript. The author would like
to thank Dr Maria Jerka-Dziadosz, Dr Krystyna Golinska and Dr Andrzej Kaczanowski
for critical reading of the draft of this manuscript.
This work is partially supported by a research grant of the Polish Academy of Sciences
P.A.N.-II. 1.3.7.
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{Received 6 May 1980, revised 10 April 1981)