/ . Embryol. exp. Morph. Vol. 59, pp. 355-375, 1980
Printed in Great Britain © Company of Biologists Limited 1980
355
Ultrastructural aspect of size dependent
regulation of surface pattern of complex ciliary
organelle in a protozoan ciliate
By JULITA BAKOWSKA1 AND MARIA JERKA-DZIADOSZ
From the Department of Cell Biology, M. Nencki Institute of
Experimental Biology, Polish Academy of Sciences, Warsaw
SUMMARY
A regulation is shown for size and number of the elements of complex ciliary structures
forming the oral apparatus (OA) of a ciliate Paraurostyla weissei. Morphometric investigations
were performed on oral ciliature of normal and size-reduced cells. Those constituents of the
OA which exist as single structures, such as the inner and outer preoral membranelles,
are not eliminated. Both shorten and the outer membranelle becomes narrower. Within the
adornal zone of membranelles in size-reduced cells some frontal and ventral membranelles
become eliminated, whereas the respective ratio of these types remains size invariant. In
each individual adoral zone of membranelles there are membranelles of different length
specificially located along the ventral part. Membranelles from small cells are significantly
smaller than those of normal cells.
The number of kinetosomes is reduced in all four rows constructing an adoral membranelle.
The analysis showed that regardless of cell size, the number of kinetosomes in the two inner
rows of a membranelle is linearly and proportionately related. Regulation of the size of all
components of the oral ciliature in P. weissei occurs at the time when the primordia of oral
ciliature are formed.
The results are discussed in relation to recent ideas about pattern formation and size
dependent regulation of the number and size of pattern elements.
INTRODUCTION
As was recognized very early by Morgan (1901) and emphasized recently by
Wolpert (1969) the question of whether the size or position of a part is proportional to the dimensions of the whole is theoretically veiy important. The
precision of the size invariance of proportionality in patterns and the range of
sizes across which the system can regulate have seldom been quantified.
The question has been studied carefully only on a limited number of animals.
In Hydra the regulation of the internal body pattern understood as a composition
of different types of cells (ectodermal, endodermal, nervous, etc.) comprises the
number of pattern elements (Bode et ah 1973; Bode, Flick & Bode, 1977).
Similarly the number of different cell types in the fruiting body of Dictyostelium
1
Author's address: Department of Cell Biology, M. Nencki Institute of Experimental
Biology, Polish Academy of Sciences, 3, Pasteur Street, 02-093 Warsaw, Poland.
356
J. BAKOWSKA AND M. JERKA-DZIADOSZ
varies proportionately with the size (Stenhouse & Williams, 1977). In Drosophila
the bristle pattern was shown to be a function of cell number and cell size
(Held, 1979). Interestingly in higher organisms the number of somites in early
vertebrate embryos stays constant over a range of sizes, whereas the number of
cells forming each somite varies (Cooke, 19756; Flint, Ede, Wilby & Proctor,
1978; Pearson & Elsdale, 1979; Elsdale & Pearson, 1979).
These data have been generally interpreted as not fitting the general theory
whereby patterns are specified by monotonic gradients of positional information
as originally proposed by Wolpert (1969). Some such concept is nevertheless
necessary to explain the global regulation of pattern elements in relation to
overall size.
Ciliated protozoans being single-celled organisms offer special advantages
for studying pattern formation and regulation in the absence of cellular partitions. They possess highly ordered arrays of cilia and ciliary aggregates with
their fibrillar derivatives, known as the cortical pattern of the ciliature.
The object of our study is a hypotriche ciliate Paraurostyla weissei (Fig. 1) in
which the ventral ciliature is composed of multikinetosomal aggregates: oral
membranelles and cirri. Changes in the number of cortical elements take place
only through replacing the whole set with a new one. In previous studies
(Jerka-Dziadosz, 1976, 1977) it was found that the number of serially repeating
structures such as left marginal cirri and adoral membranelles is variable and
regulated proportionally to cell length. However, the effect of miniaturization
of cell dimensions on the size and composition of elements of the complex
pattern has not been studied in detail (Jerka-Dziadosz & Golinska, 1977).
Constant size of oral membranelles in Stentor with concomitant reduction in
their number was reported by Tartar (1941, 1967) whereas in starved Stylonychia cells Dembowska (1938) observed diminution of dimension of the individual
cirri.
In this paper we present the results of analysis of pattern alterations of complex ciliary structures forming the oral apparatus in Paraurostyla weissei caused
by reduction of cell dimensions. The ciliature is composed of frontal and ventral
adoral membranelles and two preoral membranelles (previously called'undulating membrane'). The details of the ultrastructure of these parts of the oral
apparatus were presented in a previous publication (Bakowska & JerkaDziadosz, 1978). Morphometric and statistical analysis was performed on
changes of the cell form and ciliary pattern. The analysis of the mode of regulation of oral ciliature in P. weissei showed that adjustment of cortical pattern to
cell dimensions comprises proportional changes not only in the number of the
elements but also in their constitution - i.e. in the number and distribution of
kinetosomes within elements.
The results are discussed in relation to the recent ideas about pattern formation and size dependent regulation of the number and size of pattern elements.
Pattern regulation in a ciliate
357.
MATERIAL AND METHODS
Cells used in these investigations were a clone of Paraurostyla weissei, line Z-6
isolated from single exconjugant after total conjugation (Jerka-Dziadosz &
Janus, 1975). The culture methods were as previously described (Bakowska &
Jerka-Dziadosz, 1978). Observations were made on cells from growing populations, on cells miniaturized by starvation in sterile Pringsheim solution where
they passed through repetitive physiological reorganisations (Dembowska,
1938), or on cells miniaturized by repeated cutting of normal cells into several
nucleated pieces which regenerated into small complete urostylas.
The counts of adoral membranelles were performed on preparations stained
with protargol after Jerka-Dziadosz & Frankel (1969) under light microscope
(Ortholux Leitz Wetzlar). Kinetosomes in membranelles were scored on ultrathin sections examined under an electron microscope JEM 100B. For electron
microscopy, the ciliates were prepared as described previously (Bakowska &
Jerka-Dziadosz, 1978). In some experiments cells were stained for 15-30 min in
0-1 % solution of tannic acid before dehydration. This procedure gives better
contrast to microtubular structures.
As a measure of ciliary pattern alterations the following values were utilized:
(1) for the changes of the cell form - cell width to cell length ratio,
(2) for the changes of the adoral zone structure - the ratio of the number of
frontal to ventral membranelles,
(3) for changes of architecture of membranelles - number and distribution
of kinetosomes in rows in membranelles. Length of a membranelle is understood
as the number of kinetosomes in its longest row, while the width of a membranelle is defined by the number of kinetosomal rows.
Statistical analysis of the results consisted of regression analysis and analysis
of variance. Moreover, two tests for proportionality were used: (1) the 9 5 %
confidence interval of the F-intercept was computed. If it included the value
X = 0, the relation was consistent with the conclusion of size invariance with
5 % probability of error, (2) Regression of the ratio X/Y on X was computed
and tested whether it was significantly different from zero. If not, it was concluded
that the ratio X/Y was the same at all values of X. Thus, the relation was size
invariant. These are routine techniques and descriptions can be found in Socal &
Rohlf (1969) and Finn (1974). Preparation of the statistical programme and computations were performed by the computing Centre of the Polish Academy of
Sciences.
RESULTS
I. Analysis of changes of cell dimensions caused by miniaturization
After several transections or continuous deprivation of food, Paraurostyla
cells become progressively smaller. A question arises whether the cell dimensions
change in such a way that the proportions between the cell width and length
358
J. BAKOWSKA AND M. JERKA-DZIADOSZ
70 -
60
J 50
I 40
(J
30
20
70
80
90
100
110
120
130
Cell length (jum)
140
150
160
170
180
Fig. 1. The relationship between the width (H>) and the cell length (7) for 42 of
P. weissei of different size. The line fitted by linear regression analysis is given by the
equation: w = 5-2429 + 0-33401.
remain similar to the proportions characteristic of cells of normal size. To
answer this question 41 individual cells of different size were measured. Some of
these individual cells were fixed 3-5 to 4 h after division, some were size-reduced
by operation, (i.e. they were fixed after termination of the regeneration processes
about 5-6 h after the operation) and the remainder were starved for a few days.
The results of the above measurements are plotted in Fig. 1. Statistical analysis
showed that the cell width and length are strongly correlated (r = 0-843, P =
0-01) and this relation is of linear character. Both tests for proportionality
showed that the ratio of the cell width vs. cell length is size invariant. It follows
therefore that the miniaturized cells become shorter and proportionally slender.
The question then arises whether the relation between the analysed dimensions
of the cell, and between cell size and the number of ciliary structures, is similar
in naturally small (excystant) and experimentally size-reduced (operated or
starved) cells. The results of measurements are presented in Table 1. The comparisons of linear regression lines calculated for the number of adoral membranelles against the cell length and regression of the cell width against the length
revealed that the corresponding pairs of coefficients can be replaced by a
common regression. From this comparison it follows that when the cell dimensions are similar, the regularities within the oral membranelles are the same
regardless of the origin of the cell and the cause of size reduction.
II. Analysis of changes in the zone of adoral membranelles
Size-reduced cells of P. weissei show a substantial reduction in the number of
adoral membranelles. Statistical analysis showed strong correlation (r = 0-838,
15
Small excystant cells
35-6
± 4-273
33.9
± 3-390
Mean number of
membranelles
in AZM
101-24
±13-391
11111
± 14-264
Mean cell
length
31-5
± 4-740
±28-5
± 3-536
Mean cell
width
0.15g
AZM/cell length
0.236
Cell length/width
Regression coefficient b common for both
groups of analysed cells
/-test of semple means reveals no significant differences between two compared groups of cells at P = 005.
22
Size reduced cells
Type of cells
Number of
analysed
cells
Table 1. Comparison of size-reduced and small excystant cells of Paraurostyla weissei
.^
360
J. BAKOWSKA AND M. J E R K A - D Z I A D O S Z
AZM
Fig. 2. The ventral surface of P. weissei. OA, oral apparatus; AZM, adoral zone
of membranelles, hair-like appendages represent the ciliature of the ventral surface.
Protargol staining, x 600, bar represents 10 /im.
Fig. 3. The anterior part of the ventral surface of P. weissei. The AZM is divided in
two parts: frontal and ventral. The line indicates the border between them. fAZM,
frontal part of AZM; vAZM, ventral part of AZM; OPM, outer preoral membranelle; IPM, inner preoral membranelle. Protargol staining, x 1800, bar represents
10/tm.
P = 0-01) between the total number of adoral membranelles and cell length
(Jerka-Dziadosz, 1976). However, the zone of adoral membranelles is not
structurally uniform (Bakowska & Jerka-Dziadosz, 1978). The frontal and
ventral membranelles differ not only in structure but also in their location with
respect to the antero-posterior axis of the cell. Frontal membranelles ('collar'),
(Figs. 2 and 3) are arranged in the form of an arc surrounding the anterior margin
of the cell. They are composed of four rows of ciliated basal bodies (Fig. 4),
with the shortest row possessing four to five kinetosomes, and the remaining
three possessing 12-18 kinetosomes each. The cilia in the shortest row are
much shorter than the others. The distances between frontal membranelles are
larger than those between ventral membranelles.
The ventral membranelles are arranged (Fig. 7) parallel to each other and
Pattern regulation in a ciliate
361
are oriented perpendicularly to the long axis of a cell. The membranelles are
tightly packed, with the shortest anterior-most row of cilia in most cases possessing three kinetosomes. The border between frontal and ventral membranelles
is located on the left anterior margin of the body (Fig. 4).
In oider to gain some information about the relation between the two parts of
AZM, the number of frontal and ventral membranelles was counted separately
in 106 cells of different size. Statistical analysis showed that the numbers of
both kinds of membranelles are strongly correlated and they are linearly related
to each other. The regression line is superimposed in the scatter diagram in
Fig. 8. Both tests for proportionality showed that the ratio between the number
of frontal to ventral membranelles is size invariant. Hence, it can be concluded
that when the cell dimensions decrease, the number of frontal and ventral
membranelles correlates in such a way that the proportions between these
parts of AZM remain constant regardless of the cell size.
III. Analysis of size variability of ventral membranelles of
normal and size-reduced cells
It is known from the previous study (Jerka-Dziadosz, 1976), that the total
number of adoral membranelles in size-reduced cells is smaller than in the
normal ones. The question arises whether the number of kinetosomes in
membranelles is also reduced in miniature cells. In order to answer this question
two groups of cells were investigated. All cells designated to be of normal size
were from a growing population. Therefore they had an average number of
adoral membranelles of about 64 (Jerka-Dziadosz, 1976). The group of sizereduced cells was also relatively uniform in respect to the number of adoral
membranelles. They possessed from 36 to 44 adoral membranelles. For further
analysis only the ventral part of AZM was taken. The zone of the ventral
membranelles, regardless of the cell size, was arbitrarily divided into six regions
(Fig. 9). Membranelles found on sectioned material were classified to the particular region on the basis of occurrence of characteristic combinations of
structures situated in vicinity of each region of the AZM. In no case was it
possible to count kinetosomes in all membranelles from one cell. In our material
we found 67 complete membranelles from 12 cells of noimal size and 98
membianelles from 13 size-reduced cells. Therefore, the analysed membranelles
of many cells form a 'random population'.
Each membranelle regardless of the cell size is composed of four rows of
kinetosomes (Figs. 7 and 11). The two posterior rows (R-l and R-2) are the
longest and usually possess an equal number of kinetosomes. The third row
(R-3) is always shorter than the two posterior ones. The most anterior (R-4) is
the shortest and usually consists of only three kinetosomes. The number of
kinetosomes in the three posterior rows is variable and depends upon the
position of a membranelle within a zone (Table 2). The longest membranelles
are located in the central regions, i.e. Ill and IV, the shortest are located at the
362
J. BAKOWSKA AND M. JERKA-DZIADOSZ
Pattern regulation in a ciliate
363
extremities, i.e. in regions I and VI. Membranelles composed of identical number
of kinetosomes similarly arranged into four rows occur in large as well as in
small cells. However, such membranelles are differently located in vAZM of
compared groups. One of the smallest membranelles from a large cell is located
at the anterior end of region I and possesses 50 kinetosomes distributed:
17, 17, 13 and 3 in four successive rows, whereas in a small cell the membranelle
corresponding in number and distribution of kinetosomes is located in the
region III. Comparison of ranges of kinetosome number in the inner two rows
(R-2 and R-3) of ventral membranelles in normal and size-reduced cells indicated
that membranelles from small cells appear proportionately smaller.
In order to test whether this relation really occurs, two way analysis of variance
was used. Differences in number of kinetosomes in second rows of membranelles
between normal and size reduced cells, difference between region I vs. Ill and
IV, and possible interactions were simultaneously tested. Both the differences
of membranellar length (the number of kinetosomes in one of the two first
rows) between regions of AZM and between normal and size-reduced cells
proved to be highly significant (7-value of 24-9 and 12-9 respectively, D.F. = 17).
The interaction, i.e. the degree to which the differences in one factor (regions)
are not the same across the other (normal vs. size-reduced cells), was small
(2-00) and not statistically significant with a 5 % probability of error.
Two conclusions arise from the above observations: first-that in each
individual AZM band there are membranelles of different length specifically
located along the ventral part, and second - that membranelles from small cells
are significantly smaller than those from normal cells.
IV. Analysis of the number of kinetosomes and their distribution in
normal and size-reduced cells
In order to define the cytogeometrical rules governing the architecture of
ventral membranelles, the distribution of kinetosomes in the two inner rows
(R-2 and R-3) was further analysed. From Table 2 it appears that membranelles
show a rather constant relationship in the distribution of kinetosomes within
the two rows. This possibility was tested statistically by regression analysis of
F I G U R E S 4-7
Fig. 4. Section through four frontal membranelles of a size-reduced cell. Numbers
1-4 enumerate kinetosomal rows, x 10000, bar represents 1 /tm.
Fig. 5. Section through part of OPM and IPM of a normal cell. Numbers 1-5 enumerate kinetosomal rows of outer preoral membranelle. x 10 400, bar represents
1 /.im.
Fig. 6. Section through OPM and IPM of a miniature cell. Numbers 1 and 2
enumerate the kinetosomal rows of the outer preoral membranelle. x 10000, bar
represents 1 /.im.
Fig. 7. Section through the ventral part of AZM of a miniaturized cell. 1-4 enumerate
kinetosomal rows, x 4600, bar represents 1 /*m.
364
J. BAKOWSKA AND M. JERKA-DZIADOSZ
17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47
18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Ventral membranelles
Fig. 8. The relationship between the numbers of frontal (/) and ventral (v) membranelles of normal and size-reduced cells of P. weissei. The line fitted by the linear
regression analysis is given by the equation: fin = 0012 + 03820vTO.
Fig. 9. Schematic representation of the ventral part of the membranellar band
divided into six sectors numbered from anterior to posterior.
kinetosome number in inner rows of membranelles from normal and sizereduced cells (Table 3). The analysis showed that the number of kinetosomes in
the third row (R-3) is linearly related to the number of kinetosomes in the
second row (R-2), in membranelles from both normal and size-reduced cells.
R-2
R-3
R-2
R-3
R-2
III
R-3
R-2
IV
R-3
R-2
R-3
R-2
VI
R-3
Numbers in parentheses indicate the number of analysed membranelles.
17-26 13-17 26-32 18-19 29^0 19-30 35-41 25-30 24-26 16-17 20-23 12-8
(17)
(9)
(15)
(20)
(3)
(3)
14-22 10-16 21-25 14-18 25-33 16-24 31-33 22-24 23-28 16-21 17-18 9-10
Size-reduced cells
(24)
(24)
(11)
(5)
(9)
(2)
—
13-15 11-12 17-20 13-16 18-21 14-17 16-18 10-12
12
3
U
Minute cells
(2)
(5)
(5)
(2)
(1)
11
8
13
9-10 14-19 10-13
—
—
—
S
(1)
(2)
(5)
Total number of membranelles
42
37
36
30
14
6
Normal cells
Group of cells
II
AZM region
8
165
15
75
67
67
vTotal number of
membranelles
Table 2. Range of distribution of kinetosome number in ventral adoral membranelles of different AZM region
ON
a
^
r3 = - 3 1 3 9 + 0-956 r2
15
10
Cell U and S
Cell U
r3 = - 4 0 9 3 +1016 r2
r3 = -9-566 + 0-989 r2
rz = -8-772 + 0-995 r2
35
16
rz = l-814 + 0-734r2
rz = -3-834 + 0-846 r2
rz = -0-489 +0-724 r2
75
31
40
r3 = -2-238 + 0-778 r2
67
Regression equation
Normal cells
Size reduced cells
Membranelles from region
III and IV:
Normal cells
Size-reduced cells
Membranelles possessing
from 24 to 33 kinetosomes
in row no 2
Normal cells
Size-reduced cells without
cell U and S
All membranelles from:
The analysed group of
membranelles
Number
of analysed
membranelles
P
P
P
P
yes
= 0001
yes
= 0001
yes
= 0001
yes
= 0001
yes
P = 0001
yes
P = 0001
yes
P = 0001
yes
P = 0001
P
no P
yes
P
yes
P
no
no
no
the slope
the elevation
A
c/a
o
w
F0.01 = 4 0 0
D.F. = 1/67
F= 8-2
yes
/Voi = 2-74
D.F. = 3/71
yes
F = 80148
F0.01 = 4 0 0
D.F. = 1/68
yes
F= 230
N
o
(—|
CN
u
i—i
>
m
no
no
>
a =-1073
b = 0-743
F = 2-68
F = 2-48
Fo.ni = 3-92
>ooi = 3-92
z;
D.F. = 1/138 D.F. = 1/139 M
f
Significant differences between regression coefficients
no
yes A
= 0001 b = 0-988
yes
F = 0074
F0.05 = 2-75
= 001
D.F. = 3/68
no
= 005
no
= 005 1
—
no
P = 005
no
yes P = 005
no
Does the
Is the slope 95% consignificantly fidence
Does the
diffeinterval
include ratio rz/r2
rent
change
the value
from
with r2
X=0
zero
Table 3. The statistical analysis of the internal distribution of kinetosomes within membranelles of normal and
size-reduced cells o/Paraurostyla weissei
366
Pattern regulation in a ciliate
367
40
30
S 20
10
10
20
30
Number of kinelosomes in row no. 2
40
50
Fig. 10. The relationship between the number of kinetosomes in the two inner rows of
kinetosomes of ventral membranelles. The common line for membranelles of normal
and miniature cells fitted by linear regression analysis is given by the equation
r3 = 1-0731+0-7434 r2.
The tests for proportionality showed that the relationship between the number
of kinetosomes in the two inner rows is proportional. The comparison of
regression equations showed that the two groups do not differ significantly.
Relation between the number of kinetosomes in R-3 and R-2 in membranelles of
normal and size-reduced cells can be described by a common linear regression
(Table 3, Fig. 10). This relation is proportional.
In two exceptionally small cells the fourth (R-4) row in several membranelles
has only two kinetosomes. The total number of ventral membranelles was
lower than 20 in both cells. The cell denned as S had 14 ventral membranelles,
so an extrapolation based on the results of Fig. 8 gives a total number of all
membranelles equal to 19. This indicates a three-fold diminution in the size of
the oral apparatus of this cell as compared to an average normal cell. The
fourth row in all but the first anterior ventral membranelles was two-kinetosomal. The number of kinetosomes in rows R-l, R-2 and R-3 was also drastically
reduced. The cell defined as U was slightly larger than the cell S. It possessed
about 19 ventral membranelles, implying a total of about 27 adoral membranelles. The R-4 in ventral membranelles of this cell is either three- or twokinetosomal with no specific pattern of arrangement of these membranelles
within the zone. The above facts indicate that when cells of P. weissei undergo
a decrease in the size of the membranelle band of more than 2-5 fold, the number
24-2
368
J. BAKOWSKA AND M. JERKA-DZIADOSZ
of kinetosomes in the anterior rows of each membranelle is also reduced from
three to two. At sizes near threshold, the transition between the two patterns is
gradual rather than sudden.
V. Relation between the length and position of membranelles within
the AZM and the internal distribution of kinetosomes in
membranelles
From the analysis described in previous sections it follows that some basic
rules of patterning of the ventral band of membranelles should be the same in
normal and size-reduced cells, i.e., they should be size-independent.
There are two possibilities regarding the architectural regularities in the
structure of membranelles of normal and size-reduced cells:
(1) the distribution of kinetosomes in the two inner rows is the same within
the membranelles of the same length (the membranellar length is understood as
the number of kinetosomes in R-l) regardless of the cell size,
(2) membranelles, regardless of their length with similar position in the adoral
zone show similar relationship in distribution of kinetosomes i n the two inner rows.
In order to check the first hypothesis, a statistical analysis was performed on
membranelles of size-reduced and normal cells possessing from 24 to 33
kinetosomes in each of the first two rows. Since the membranelles in VI region
are different from the other, membranelles having less than 24 kinetosomes in
the first row were excluded in this analysis. The results are presented in Table 3.
The comparison of the regression lines for membranelles of the same length of
normal and size-reduced cells showed statistically significant differences between
them. The value of the regression coefficient is higher for membranelles from
miniature cells.
From this analysis it follows that membranelles of the same length but of
different locations within the AZM of cells of different size have different
internal patterns.
In order to test the second hypothesis, which postulates that membranelles of
different length but of similar locations within adoral zones show a similar
relationship between numbers of kinetosomes in the two inner rows, membranelles
from region III and IV (from normal and size-reduced cells) were chosen for
statistical analysis and comparison.
The analysis of membranelles possessing 29-41 kinetosomes in the second
row, derived from normal cells, showed linear relation (Table 3). This relation
is not proportional.
In small cells, the membranelles located centrally in the adoral zone have
25 to 33 kinetosomes in the second row (Table 2). As in the previous cases
numbers of kinetosomes in the second and third row are linearly related, but
the relation is not proportional (Table 3).
Similar analysis of kinetosomal distribution performed separately for cells
U and S and cell U alone showed also that there exists a linear relation. How-
Pattern regulation in a ciliate
369
ever, the proportions between the number of kinetosomes in third vs second row
do not depend upon the membranellar length (Table 3).
The value of regression coefficients calculated for analysed groups of centrally
located membranelles are similar. In order to find out whether the differences
between them are significant or not the test for equality of slopes of several
regression lines was used. This showed that all of regression coefficients b can be
replaced bya common coefficient which is equal to 0-98794. In other words, when
the number of kinetosomes in the second row (and the first as well) of centrally
located membranelles decreases by one kinetosome then the third row shortens
also by one kinetosome regardless of membranellar length. This indicates that
the relationship in kinetosomal distribution between the inner row of these
membranelles is relatively constant. It follows therefore that the position of a
membranelle has a decisive effect on the value of the regression coefficient
regardless of the length of that membranelle.
Summarizing the results obtained from comparison of the data concerning
membranelles from normal and size-reduced cells, it can be stated that membranelles of the same length vary with respect to their inner pattern according
to their position within AZMs during regulation of the size of oral apparatus in
relation to overall cell dimensions. In other words, at least for central AZM
locations, membranelles of similar position in the AZM are identical with
respect to their inner pattern regardless of their length.
Another aspect of the proportions of membranelles concerns their total
architecture, i.e the ratio between the number of kinetosomal rows in a membranelle and the length of the rows of kinetosomes. Since each membranelle is
always composed of four rows of kinetosomes and the length of kinetosomal
rows can vary, the shape of membranelles depends on membranellar size. This
means that membranelles from small cells corresponding in position with
membranelles from normal cells are relatively shorter and thicker (Fig. 11).
The comparison of characteristics of membranelles from normal and sizereduced cells reveals regularities common for both groups of data (Table 2):
(1) The length range of membranelles of a given AZM is the larger the more
membranelles it contains. Therefore, this range depends on the cell size.
(2) The value of the regression coefficients b close to 1 is a constant feature
of kinetosomal distribution between the inner rows of centrally located membranelles in all kinds of cells.
(3) The smaller the total number of membranelles, the shorter are the
membranelles located centrally.
(4) The number of ventral membranelles is highly correlated with the number
of frontal membranelles and the total number of the adoral membranelles is in
turn correlated with the length of a cell (Jerka-Dziadosz, 1976). It follows from
the above that the number of ventral membranelles is also correlated with the
cell length. This allows one to conclude that the distribution of kinetosomes in
ventral membranelles depends on the overall dimension of the cell.
370
J. BAKOWSKA AND M. J E R K A - D Z I A D O S Z
Fig. 11. Membranelles of the same relative position in the ventral band (sector
posterior IV) from a normal cell {A) and a miniature cell {B). x 11500, bar represents 1 /*m.
VI. Analysis of ultrastructural variability in preoral membranelles of
normal and size-reduced cells
The right side of the oral opening in P. weissei is bounded by two longitudinal
ciliary aggregates called collectively the 'membrana undulans' UM, (Figs. 2, 3).
These are two separate structures: inner and outer ciliary rows (de Puytorac,
Grain & Rodrigues de Santa Rosa, 1976; Bakowska & Jerka-Dziadosz, 1978).
The inner preoral membranelle (IPM) formed by a single row of ciliated
kinetosomes originates on the ventral side of the oral opening at the same level
as the most posterior AZM membranelles and runs anteriorly reaching a level
of the border between II and III region of the ventral part of the AZM (Fig. 3).
The outer preoral (OPM) membranelle is formed by several longitudinal
rows of alternatively distributed ciliated kinetosomes (Fig. 5) and is slightly
shorter than the inner membranelle. The length and spacing of both membranelles is correspondingly smaller in size-reduced cells. Kinetosomes in both membranelles were not counted because it was impossible to obtain sections through
all cilia even on serial sections.
If one accepts that the distance between kinetosomes of the UM's are the
same in normal and size-reduced cells and the length of the whole membranelle
is correspondingly smaller in miniature cells it could be concluded that the
number of kinetosomes in the inner preoral membranelle is proportionally
coordinated with the size of the oral apparatus - that is with the size of the cell.
This conclusion - concerning the length of the membranelle - is true also for
the outer preoral membranelles. In addition to this the outer membranelle is
regulated also in its width. In normal cells the outer membranelle is usually
formed by 4-5 longitudinal rows of kinetosomes (Fig. 5). The two rows on the
Pattern regulation in a ciliate
371
AZM side are the longest and are usually equally long. The subsequent rows to
the right of the preceeding ones do not reach both ends of the membranelle.
This means that a membranelle is widest in its middle part.
In cells reduced in size by starvation or transection, outer preoral membranelles possessing only three or two rows of kinetosomes in the widest part were
observed (Fig. 6). However, we have never observed an outer membranelle
having only one row of kinetosomes. In our material, therefore, the maximal
number of kinetosomal rows in normal cells is 6-7 and the minimal number of
them in size-reduced cells is 2.
DISCUSSION
Explanation of how spatial regulation of pattern is accomplished is not
possible without knowledge about regularities by which size of the regulating
structure and its inner proportions are defined and the precision and limitations
of these regularities. If our understanding of pattern formation is ever to progress
beyond a conceptual framework it seems essential that regulative responses of
patterns be analysed exactly.
From studies performed on the ciliate Paraurostyla it is evident that experimental alterations in the cell dimensions or deletion of the ciliary pattern cause
replacement of the whole ciliature by a new set. The positions of ciliary porn ordia as well as the formed structures are regulated in such a way that their
final dimensions are adjusted to the size of the whole.
The oral apparatus of P. weissei responds to overall dimensions in the following way: all components, that is frontal and ventral membranelles, inner and
outer preoral membranelles, show reduction in the numbers of their constituent
kinetosomes. This already indicates that during early stages of formation of the
ciliary primordia, smaller numbers of kinetosomes are produced.
Reduction in overall number of kinetosomes is not the only response to size
diminution - the other is the reduction of the number of serially repeated
membranelles (AZM). Frontal as well as ventral membranelles are eliminated
in such a way that their respective quantitative ratios remain size invariant.
Those constituents of the oral ciliature which exist as single structures (inner
and outer preoral membranelles) are never eliminated, they become shorter and
the OPM becomes narrower.
All kinetosomes, even from very tiny cells, show normal fine structure - they
are composed of 9 triplets of microtubules. The question of whether a kinetosome is the smallest unit of regulation, or whether the number of microtubular
protofilaments (Burton, Hinkley & Pierson, 1975) can also vary is not resolved.
Interestingly the cytogeometry within individual adoral membranelles also
turned out to be regulated. Within a large range of sizes the internal architecture
of membranelles shows size invariant proportionality (Table 3). All these
observations taken together suggest that there must be some global control
which monitors the overall cell size and the 'bigger' parts of the oral ciliature
372
J. BAKOWSKA AND M. JERKA-DZIADOSZ
(frontal vs. ventral membranelles and UM's) and also a local control which
monitors the inner proportions of each membranelle in concert with the whole
oral apparatus dimensions.
As to the mechanism for achieving such regulation, where everything can
change except for the relative proportions, we have no suggestions to present.
We can ask the question: when during morphogenesis does the size adjustment take place? From studies on the formation of the oral ciliature (OP)
in P. weissei (Jerka-Dziadosz & Frankel, 1969; Jerka-Dziadosz, 1980 b) and
other hypotrich ciliates as well (Grimes, 1972; Bohatier, 1979) we know that
the OP originates as a longitudinal field of randomly but densely packed kinetosomes. The length of that field seems to be related to the size of a fragment.
This field then segments into promembranelles composed of two equally long
kinetosomal rows (R-l and R-2). This segmentation procedes in a wave-front
mode from anterior to posterior, similarly to segmentation of the mesodermal
streak into somites in vertebrate embryos (Flint et al. 1978; Pearson & Elsdale,
1979). During that process the final number of all membranelles (frontal plus
ventral) is established. After that, again in antero-posterior sequence, new
rounds of kinetosome proliferation occur anteriorly to each promembranelle and
the third (R-3) and then fourth (R-4) rows of kinetosomes are induced at
appropriate locations. The process of differentiation of membranelles is accompanied by a shift of the whole developing primordium anteriorly, and then the
anterior 1/4 of the original OP forms the frontal membranelles, oriented at right
angles to the long axis of the cell. When the decision concerning differentiation
toward frontal-type membranelles is taken is not clear. It seems that the final
length of R-l and R-2 of frontal membranelles is set before the membranelles
start to move to the right. The proliferation of R-3 and R-4 of each membranelle
also starts before shifting of these membranelles. All details of these morphogenetic phenomena will be given in the next publication of this series (JerkaDziadosz, 19806).
The results of morphometric and statistical analysis presented in this paper,
as well as the analysis of the mode of differentiation of oral structures in P.
weissei, seem to confirm the notion about the dual control of pattern formation
in ciliated protozoa (reviewed in Frankel, 1979; Jerka-Dziadosz, 1979). This
dual control involves so called short-range, or nearest neighbour interaction
and a long-range, gradient-field type interaction. The short-range control
(basically non-regulatable) is based on restricted self-assembly (Tucker, 1977)
where the local environment provides all spatial information required for ordered
assembly of kinetosomes and other associated differentiations (Sonneborn,
1975). This mechanism then may partially be responsible for patterning of each
individual membranelle.
The long-range forces responsible for asymmetry of structures (Jerka-Dziadosz
& Frankel, 1979), positioning of primordia (Jerka-Dziadosz, 1974) are in
many respects similar to the forces presumed to act in a morphogenetic field
Pattern regulation in a ciliate
373
(Frankel, 1974). The simplest and most popular type of model based on Wolpert's (1969) theory of positional information is that of chemical diffusible
components forming a monotonic gradient, whose local concentrations are
'read off' and 'interpreted' into differentiating structure. These concepts however, possess their own shortcomings because they cannot be adopted either
to cases where patterns depend on cell number (Held, 1979), or to those where
serially repeating structures such as the vertebrate somites are formed
(Cooke, 1975 a, b). A modification of the model which partially accounts for
generation and regulation of serially repeated structures was given by Flint
et al. (1978) who studied somitogenesis in normal and amputated mutants of
mouse. They proposed a wave-form gradient of morphogen which accounts for
the definition of somite boundaries and the capacity for regulation in adjusting
constant somite number to overall axial length.
Cooke & Zeeman (1976) formulated a theoretical model of pattern formation
(though see criticisms of Pearson & McLaren, 1977, and Zahler & Sussman,
1977) which shows how a wave of change passing through a field of coordinate
cells can function like an escapement mechanism in a clock to provide for a
repetitive series of discontinuities events. This 'clock and wave-front' model
postulated that all the presomite cells become physiologically phase-linked with
respect to some oscillator whose rhythm interacts with a wave-front of rapid cell
change, causing spatially periodic delays and advances in the overt expression of
that wave as cell behaviour. This underlies the spatially regular causes of fissures
of cellular de-adhesion segregating successive somites. This model induced new
experiments (Cooke, 1978; Elsdale & Pearson, 1979; Pearson & Elsdale, 1979)
which showed that a wave is indeed involved in the specification and regulation
of segmental pattern.
Since the formation of adoral membranelles from oral primordium in Paraurostyla is essentially a segmentation of uniform longitudinal kinetosomal field
into successive subpopulations of kinetosomes (so is the formation of the somatic
cirri - Jerka-Dziadosz, 1980a; Bakowska & Jerka-Dziadosz, 1979) in spatial and
temporal sequence down the body axis, it is possible that a determination wave
precedes the visible pattern formation. This wave-like change induces the
kinetosomes to assemble and aggregate into orderly patterned membranelles.
The difficulty with adopting the above mentioned models to pattern specification and regulation in serially repeated structures in ciliates is that the models
require discrete subunits of the field (the cells) to act autonomously but coordinately (cellular processing). If, however, one considers the kinetosomes as units
of regulation (in which timing is encoded), and the cell surface structures as a
carrier of the positional information variables (Kaczanowska, 1974; Hewitt,
1978), then the general idea of the two-step pattern determination where, first,
a presumptive primordial region is programmed and later undergoes segmentation, could be adopted and further tested, This problem will be considered with
more detail in the next publication of this series.
374
J. BAKOWSKA AND M. J E R K A - D Z I A D O S Z
The authors would like to thank Dr Joseph Frankel for suggesting some of the statistical
tests. We also wish to express our appreciation for valuable comments and discussions concerning earlier drafts of the manuscript by Drs Krystyna Golinska, Janina Kaczanowska and
Joseph Frankel. Dr Jonathon Cooke kindly helped us with the English. Excellent technical
assistance of Mrs Wiernicka is kindly acknowledged. The work was supported by grant
MR.II from Polish Academy of Sciences.
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