/ . Embryol. exp. Morph. Vol. 33, 3, pp. 553-580, 1975
Printed in Great Britain
553
An analysis of the spatial distribution of ciliary
units in a ciliate, Euplotes minuta
By JOSEPH FRANKEL 1
From the Department of Zoology, University of Iowa
SUMMARY
1. Six to eleven longitudinal ciliary rows are arrayed over the dorsal surface of the ciliate
Euplotes minuta. Forty-two to 129 ciliary units are distributed among these rows. The number
of rows depends on genotype, clonal age, and vegetative ancestry, while the total number of
units is controlled partly by the number of rows and also by the separate action of genetic
and environmental factors.
2. The pattern of distribution of units among different rows of non-dividing cells can be
analysed on the basis of the percentage of the total unit complement of the cell that is found
in each individual row. If the assumption is made that ciliary rows are uniformly spaced over
a dorsal field whose width is independent of the number of rows, then it can be shown that
units are distributed among rows according to a relatively invariant spatial pattern. The
form of this pattern remains the same in the face of variation in the absolute number of rows
and of units.
3. Prior to cell division new units develop anterior and posterior to old units situated
within the equatorial zone of each row. About one-half of the original units are included
within this zone. The cell fission line develops within this zone such that the total number of
units passed to the anterior and posterior division products are about equal.
4. The pattern according to which units of different zones (proliferating and non-proliferating) are distributed among different rows has been mapped in cells that have completed
the process of proliferation of units but have not yet completed cell division. The results of
this mapping show that the pattern of distribution of units in the equatorial zone at the
conclusion of proliferation is not the same as the overall pattern in non-dividing cells. Further
analysis indicates that the geometry of proliferation can be most simply represented as a
result of two superimposed processes, one of which is the recruitment of old units into the
zone of proliferation, while the other is the intensity of proliferation, i.e. the number of new
units formed adjacent to each old unit. Both recruitment and intensity have constant values
in the central region of the dorsal field, while recruitment is higher and intensity lower near
both margins. The recruitment and intensity distributions are mutually nearly reciprocal,
with slight asymmetries that formally account for the more dramatic asymmetry of the pattern
of non-dividing cells.
5. A dualistic hypothesis is formulated for the control of the formation of new ciliary units
within ciliary rows. The position of each new unit is largely controlled locally in relation to
pre-existing units, while the decision of whether or not new units will develop at all, and how
many will be formed, depends on superimposed positional systems operating within the
context of the entire dorsal surface.
1
Author's address: Department of Zoology, University of Iowa, Iowa City, Iowa 52242,
U.S.A.
554
J. FRANKEL
INTRODUCTION
Ciliated protozoa provide excellent examples of formation of complex
patterns in the absence of internal partitions, and are therefore useful for asking
questions concerning whether the rules governing pattern formation in multicellular systems (Wolpert, 1969, 1971) can apply to unicellular organisms also.
Results of microsurgical analyses of the positioning of compound feeding and
locomotory organelle systems in large ciliates (Uhlig, 1960; Tartar, 1962; JerkaDziadosz, 1974) and of morphometric analyses of the placement of contractile
vacuole pores in certain smaller ciliates (Nanney, 1966c, 1972; Kaczanowska,
1974) suggest a positive answer. These results are consistent with Wolpert's
positional information hypothesis (Frankel, 1974), although, as pointed out by
Sonneborn (1974), critical evidence for a two-step process of establishment of a
positional coordinate system followed by its interpretation is lacking in ciliates.
At a minimum, however, positioning of these organelle systems involves relational assessments in which relatively long distances are measured, generally
between two or more reference points (Frankel, 1975). These rules apply in
particular to organelle systems that are formed anew during developmental
processes, generally at sites spatially removed from pre-existing organelles or
organelle systems of the same kind.
Most ciliates, in addition to fabricating certain specialized systems once in
every cell cycle, also maintain other structures, the longitudinal ciliary rows,
which considered as ensembles are spatially continuous and maintain their
integrity from one cell generation to the next. In these, new units develop within
a differentiated structural framework that is capable of indefinite persistence
and propagation within a clone. In this system, unlike those referred to above,
positional controls appear to involve local determination within small parts of
the cell surface, in which the position and orientation of newly arising structures
(ciliary units or parts thereof) is tightly correlated with that of specific microscopically visible pre-existing structures (ciliary basal bodies and their accessory
fibers) (Dippell, 1968; Allen, 1969). A consequence of this inductive mode of
positional control is the indefinite propagation of geometrical inversions of
ciliary rows in Paramecium aurelia (Beisson & Sonneborn, 1965); another consequence is the inheritance of the pre-existing number of ciliary rows, as has
been observed in Tetrahymena pyriformis (Nanney, 19666) and Euplotes minuta
(Heckmann & Frankel, 1968; Frankel, 1973a, b).
Even in the ciliary rows, however, large-scale developmental control systems
are superimposed upon the localized processes of orientation of new ciliary
units. Mechanisms exist for bringing about changes in number of ciliary rows
(Chen-Shan, 1969, 1970; Nanney, 1972; Frankel, 19736); these changes are not
fully random, but rather are constrained within (or directed toward) a range of
stable row numbers (Nanney, 19666, 1968; Frankel, 1972, 1973c) that is under
genie control (Heckmann & Frankel, 1968; Frankel, 1973c). The total number
Distribution of ciliary units in Euplotes
555
of ciliary units is probably also under genie control (Heckmann & Frankel, 1968).
In Tetrahymena this number is homeostatically regulated with the number of
units per row decreasing as the total number of rows increase, so that the number
of units per cell tends to remain constant (Nanney, 1971a; Nanney & Chow,
1974). Most pertinent in the present context, there appear to be spatial patterns
of distribution of units among rows that reflect overriding large-scale positional
systems. The clearest published example of this is the predictable distribution of
two kinds of ciliary units, with one and two basal bodies per unit respectively,
over the surface of Paramecium aurelia (Gillies & Hanson, 1968; Sonneborn,
1970). This distribution is the same in normal and inverted rows, indicating that
the distribution is not determined by the ciliary row itself, but is rather fixed in
relation to more distant reference structure(s), probably in the region of the
feeding organelles (Sonneborn, 1970). Another means of describing distribution
patterns is to consider how the ciliary units are distributed among ciliary rows.
An analysis of this by Nanney & Chow (1974) indicated that such distribution
patterns may be rather constant within most syngens (sibling species) of T.
pyriformis; however, their study included an assessment of only a portion of the
total ciliary row pattern (7 out of 14-24 rows) at a single developmental stage
(pre-di vision).
The present investigation is an attempt to pursue the issue of spatial distribution of ciliary units further by a detailed quantitative analysis including all
ciliary rows in both non-dividing and dividing cells. Such an analysis can be
carried out in hypotrich ciliates of the genus Euplotes, in which the total number
of rows and units is reasonably small, most rows are on a single (dorsal) surface
of the cell so that total counts can be made in suitably prepared material, and
new units develop within a restricted portion of each row during a delimited
time interval. Further, in the course of several years' study of ciliary patterns in
Euplotes minuta a wide variety of material has become available in which there
is considerable variation in number of both rows and units, thus fulfilling a
prerequisite to a meaningful analysis of patterns of distribution. The analysis
to be presented demonstrates a relatively invariant pattern of spatial allocation
of units among rows. This pattern, like the arrangement of 'singlet' (one basal
body) and 'doublet' (two basal body) units in P. aurelia, appears to be causally
independent of the specific rows themselves. Study of dividing cells further
suggests an important role for the lateral boundaries of the dorsal region in
regulating this pattern. The suggestion is therefore made that the area occupied
by the ciliary rows can, like many 'field' systems in developing multicellular
organisms, be described within the conceptual framework of the positional
information model.
MATERIALS AND METHODS
The organisms used in this study were strains A23, K7 and VF17 of Euplotes
minuta, plus progenies of various crosses amongst these strains. The present
35
EMB
33
556
J. FRANKEL
Dorsal
Ventral
Cirri
Fig. 1. Diagrams of the arrangement of ciliature on the vential and dorsal surfaces
of Euplotes minuta. The dark masses on the ventral surface represent groupings of
cilia (cirri and membranelles), whereas the dots represent sites of single short cilia.
The cell here depicted has nine ciliary rows. Cells with a smaller or greater number
of rows show the same pattern of arrangement of rows, with wider or narrower
spacing of rows.
analysis is based on preparations made in 1966-70, utilizing the Chatton-Lwoff
silver nitrate procedure (Frankel & Heckmann, 1968) and the protargol method
(Frankel, 1973 b). Most of this material has been considered earlier (Heckmann
& Frankel, 1968; Frankel, 1973 a, b) in other contexts. Details concerning the
stocks, culture procedures, methods of crossing, and circumstances under which
the cytological preparations were made can be found in the above papers.
RESULTS
1. Non-dividing cells
(a) Variation in number of ciliary rows and number of ciliary units
The ciliary rows are situated on the dorsal and lateral surfaces of this dorsoventrally flattened ciliate (Fig. 1). Each row consists of a number of longitudinally
aligned ciliary units. The units each consist of a stubby cilium or 'bristle'
(Hammond, 1937) plus at least one basal body (electron microscopical investigation of the related E. eurystomus has revealed that each unit is made up of a
complex of two adjacent basal bodies plus accessory fibrils (Ruffolo, 1972)).
The units are clearly visualized with both the silver nitrate and protargol procedures. When parallel samples from the same culture are prepared with each of
these two very different methods, the number of units counted is very nearly
the same in both (cf. Frankel, 19736).
The amount of variation in ciliary pattern observed within a particular sample
of a clonal culture of E. minuta is generally rather limited. Usually no more than
two corticotypes (number of rows) are found, and for non-dividing cells of a
Distribution of ciliary units in Euplotes
557
given corticotype the coefficient of variation in number of units is generally in
the vicinity of 5%. This uniformity is partly due to the fact that increase in
number of units takes place only in a clearly recognizable interval of development prior to cell division (Wise, 1965; Hufnagel & Torch, 1967; Heckmann &
Frankel, 1968; see also below), so that as long as only non-dividing cells are
considered, variation ascribable to progression through the cell cycle does not
occur. Neither does there appear to be substantial variation associated with
phases of culture growth; cells from starving cultures have as many ciliary units
as do cells from well-fed cultures, presumably because the units acquired prior
to the last cell division before exhaustion of food are simply retained, with no
resorption and no additions (or, less likely, with resorption and addition
precisely balancing one another).
Despite this limited short-term variation in a clone, the total variation observed
in the course of long-term study of a number of stocks and genotypes is considerable. Most of the major causes of this variation have been dealt with in
detail in earlier publications (Heckmann & Frankel, 1968; Frankel 1913 a, b)
but will be briefly recapitulated below.
The number of rows has been observed to vary for four reasons. First, every
non-aged strain examined has the capacity for stable propagation of more than
one corticotype; for example, strain K7 may display either eight or nine rows.
Within this 'stability range' the pre-existing number of rows tends to be nongenically inherited, yielding diversity between as well as within sub-clones
(Frankel, 1973a). Secondly, non-autogamous strains (i.e. K7 and VF17) undergo
ageing if allowed to divide for several hundred fissions without being supplied
with suitable mates, and during clonal senility the number of rows becomes less
well regulated and many cells with lower corticotypes (seven and even six rows)
appear (Frankel, 1973 a). Thirdly, there is a genically determined difference
between the non-autogamous strains and the autogamous strain A23 in the
stable number of rows; the former have a usual row number of eight to nine,
the latter nine to ten, rarely even eleven (Heckmann & Frankel, 1968; Frankel,
1973a). Finally, the strain K7 was found to harbor a recessive gene (bbd) which
when made homozygous resulted in a reduction in number of rows from the
standard eight to nine to a broader spectrum of six to nine (Frankel, 1913 b).
Thus, the overall variation of numbers of rows observed in E. minuta is six to
eleven, with the corticotypes seven, eight, nine and ten sufficiently abundant for
detailed analysis.
There are four known causes of variation in number of ciliary units. The
first is directly related to the number of ciliary rows. Cells within a clone that
differ in number of rows also tend to differ in number of units, with the larger
number of rows associated with a greater number of units; E. minuta, unlike
Tetrahymenapyriformis (Nanney, 1971 a; Nanney & Chow, 1974), does not fully
compensate for an increased number of rows by an equivalent reduction in
number of units. There is, however, a partial compensation, since the difference
35-2
558
J. FRANKEL
in average number of units between two corticotypes is generally less than the
number of units found in an average row. The second source of variation is the
relatively small random fluctuation in number of units observed among cells
of a given corticotype within a given culture. Thirdly, systematic temporal
variations have occasionally been observed during the course of long-term
culture of clones, especially in two situations. One was a progressive increase
with increasing clonal age in the number of units observed in homozygotes for
the bbd allele (Frankel, 19736, p. 346), and the other was a significant reduction
in number of units that took place in all non-autogamous stocks during a onemonth interval in late 1969. In the latter case the change had no relation to the
clonal life cycle, since a parallel decrease of 10 % or more was observed both in
old K7 lines and in a newly established Fx between K7 and VF 17 ; it is more
likely that a change in growth conditions which occurred at that time (a switch
from weekly to every-other-day feeding of cells) was somehow responsible for
the change in number of units. These two examples are both exceptions to a
generally observed stability over time in number of units observed within a
particular line of descent. Fourthly and finally, the cell's genotype can strikingly
influence the number of units. This influence, too, was uncovered in two very
different contexts: one was a great diversity of number of units per row among
different backcross progeny ((A23 x VF17) x A23, Heckmann & Frankel (1968),
table 9), a diversity that is probably caused by a segregation of genes affecting
number of ciliary units independently of ciliary rows (Heckmann & Frankel,
1968; Frankel, 1973tf); the other resulted from homozygosity for the bbd gene,
which reduces the number of units even more strikingly than the number of
rows while also causing displacements and irregularities in the positioning of
units (Frankel, 19736). The outcome of all of these types of variation is that
counts of ciliary units vary over a three-fold range, from a low of 42 to a high
of 129. If units are tallied separately for different corticotypes, and the resultant
counts then averaged within each sample or related group of samples (thus
removing the first two sources of variation from the analysis), the variation is
still considerable (Table 1).
It should finally be added that cells vary considerably in size, with newly fed
cells being very much larger than those which are exhausting their food supply.
This size variation has little or no effect on any aspect of ciliary pattern.
(b) Distribution of ciliary units among ciliary rows
The considerable observed variation in number of rows and number of units
permits an analysis directed at discerning a possible uniformity of spatial distribution that underlies the diversity outlined above. The first step of such an
analysis is a tabulation of spatial patterns within reasonably uniform subsets of
the overall assemblage of 1813 non-dividing cells in which numbers of both
units and rows have been counted. In making up this tabulation three rules are
employed: (a) each corticotype is tabulated separately, so that in effect a separate
Distribution of ciliary units in Euplotes
559
analysis is carried out for 7-, 8-, 9-, and 10-rowed cells, (b) Within each corticotype, the percentage of the total number of units found in each row is tabulated.
For example, in the first set of cells in Table 1 (VF17, November 1969), an average
of 76-4 units are distributed among seven rows as follows: row 1 8-58, 2 10-16,
3 11-00, 4 10-50, 5 11-25, 6 13-75, and 7 11-17. Reduced to a percentage basis,
this becomes 1 11-2, 2 13-3, etc. (see Table 1). This mode of tabulation allows
direct comparison of the distribution of units among groups of cells with very
different total numbers of units, (c) Groups are set up to include sets of related
cells with similar numbers of units. Each group consists, at a maximum, of one
of the three original strains (K7, VF17 or A23) or the progeny of a cross. Further
subdivision is made when samples have been taken several years apart. When
sampling has been more frequent, data of different samples are lumped except
in those few cases when there were large differences between subsets of samples
(for example, for strain K7 the homogeneous counts of the samples taken in
January, March and April 1970 are lumped, while the results of the significantly
higher November 1969 sample are tabulated separately). F x progeny are handled
similarly, with the progeny of a given cross between strains (always homogeneous) entered together. The two backcross progeny are subdivided according
to the nature of their heterogeneity (cf. section 1 a): the continuously varying
(A23 x VF17) x A23 ('BC-1' in Table 1) progeny clones are arbitrarily subdivided
into 'high', 'medium', and 'low' lines, while the discontinuously varying
(K7 x VF17) x K7 ('BC-2' in Table 1) progeny clones are more objectively subdivided into normal and bbd (basal body deficient) lines. The outcome of this
selective lumping procedure is a somewhat arbitrary parcelling out of cells into
groups of unequal membership. A possibly more objective procedure with less
lumping would have created a more unwieldy tabulation without changing the
outcome in any meaningful way.
Examination of Table 1 reveals that within each corticotypic class there is a
general uniformity in the percentage of units allocated to each ciliary row.
This uniformity is striking in row no. 3 and all of the rows to its right, somewhat
less so in rows 1 and 2. In addition, the bbd lines of BC-2 (italicized in the table)
differ somewhat from the rest. Apart from these lines, it is clear that even a
substantial difference in total number of units does not influence the pattern of
distribution of these units among different rows (compare, for example, the
'high' and 'low' lines of BC-1). It is further clear that the number of units in
different rows is not the same. Regardless of corticotype, the two rows on the
left (rows 1 and 2) have the smallest number of units, while the second row from
the right (row n - 1 ) has the largest number.
All of the data of Table 1, except those for the bbd lines, are plotted on a
common ordinate and abscissa in Fig. 2. Construction of the abscissa is predicated on two assumptions: that the side-to-side spacing of rows over the
dorsal surface is uniform and that the width of the surface is not correlated with
the number of rows. The latter assumption is supported by measurements
85-7
961
102-2
1101
96-6
1040
106-3
111-2
961
95-5
99-3
101-5
1161
101-5
92-9
1021
vii, ix. 67
xi. 69
vii. 66
xi. 69
i, iii, vii. 70
?. 66
xi. 69
xi. 69
i, ii, iii, iv, vii. 70
vii. 66
viii. 66
ii, iv, vii. 70
xi. 67
xi. 67
xi. 67
viii. 66
VF 1 7
VF 17
K7
K7
K7
9
BC-2 w.t.
BC-1 high
BC-1 med.
BC-1 low
(A 23 x K 7 ) F 2
JN.7 X / \ 2 3
VF17xK7
VF17xK7
VF 17 x A 23
A23
A23
81-5
83-1
98-4
90-8
105-6
90-8
96-4
58-4
700
vii, ix. 67
xi. 69
xi. 69
i, iii, vii. 70
xi. 69
i, ii, iii, iv, vii. 70
i, ii, iv, vii. 70
i, ii, iii. 70
iv, vii. 70
VF 1 7
VF 17
K7
K7
VF17xK7
VF 17 x K 7
BC-2 w.t.
BC-2 bbd
BC-2 bbd
8
76-4
910
85-7
54-7
62-5
iv, vii. 70
i, 11,111. 70
xi. 69
xi. 69
iv. 70
Average total
no. of
units
VF 1 7
K7
K7
BC-2 bbd
BC-2 bbd
Strain
Date(s)
examined
7
Corticotype
(no. of
rows)
11-2
130
12-2
11-4
110
1
9-9
10-5
10-4
10-3
110
10-7
10-8
8-9
9-3
1
8-5
8-6
8-6
9-5
8-9
8-2
8-2
9-7
8-9
8-5
9-2
90
9-6
9-3
9-4
9-8
1
14-4
13-9
14-5
16-5
15-4
3
131
12-6
12-5
12-6
12-3
12-4
12-3
146
132
3
115
11-4
110
11-4
10-9
10-8
10-7
110
10-7
110
110
10-8
10-6
10-7
10-8
10-7
2
90
8-3
9-2
8-8
9-3
9-2
8-9
8-2
9-4
9.4
9-6
9.4
9-3
9-3
9-2
9.4
3
13-3
13-7
14-3
12-9
12-5
2
118
10-4
11-6
11-5
115
10-8
10-9
95
10-5
2
4
121
11-7
11-7
11-3
11-5
11-5
11-2
11-5
11-3
115
11-5
11-4
10-9
11-3
11-3
11-1
13-7
13-7
13-9
14-3
14-4
4
12-7
12-3
12-3
12-4
12-4
12-5
12-5
13-6
130
4
5
116
11-2
11-4
10 9
11-4
11-3
110
111
11-3
11-5
11-1
114
111
112
111
110
14-7
14-4
13 9
14-7
14-7
5
121
12-5
12-4
12-3
121
12-3
12-4
140
13-3
5
6
11-2
10-8
10-8
11-2
11-1
11-2
10-9
10-9
110
111
10-9
112
10-9
111
10-8
10-9
180
17-6
171
16-9
17-3
6
13-4
12-7
12-8
13-2
12-7
131
13-2
119
13-4
6
7
121
121
12-3
11-6
12-2
121
12-8
120
120
121
11-8
120
12-3
12-3
12-2
11-8
8
140
14-9
14-3
13-9
13-8
14-7
150
14-8
14-3
14-4
140
140
14-5
14-2
14-5
14-2
14-6
13-7
141
13-3
14-8
8
1
15 6 11-3
16 9 12-2
15-8 12-1
15-4 12-3
160 120
161 12-2
15-7 12-3
14-7 12-9
15-6 11-7
7
Percentage of units in row
9
9-9
10-9
10-6
11-4
10-9
111
11-2
108
111
10-5
111
10-8
10-8
10-7
10-8
10-9
Table 1. Distribution of ciliary units among ciliary rows in different populations o/Euplotes minuta
20
15
50
20
39
34
70
26
113
84
10
143
50
52
40
53
50
20
40
81
60
97
166
26
64
12
10
16
40
52
tallied
nf roll"
Number
r
>
O
10
Corticotype
(no. of
rows)
BC-1 high
BC-1 med.
BC-1 low
(A23 x K.7) r2
A23
A 23
Strain
1
71
7-7
8-3
8-2
7-5
8-8
Average total[
no. of
units
113-1
112-2
1190
113-4
1031
110-4
3
9-7
9-3
9-3
9-6
9-2
9-2
2
6-8
6-5
7-6
8-2
7-4
81
10-2
10-2
10-2
10-2
101
100
4
10-3
10-3
9-8
101
10-4
101
5
10-2
100
101
9.9
10-2
100
6
BC-2 = F x (K7 x VF17) x K 7 :
w.t. = normal progeny clones (Frankel, 19736).
bbd = basal body deficient progeny clones (Frankel, 19736).
10-3
10-2
9-8
100
10-7
10-2
7
Percentage of units in row
Key to backcrosses
BC-1 = F 1 (A 2 3 xVF 1 7 )xA 2 3 :
high = nos. 22, 28 (Heckmann & Frankel, 1968, table 9).
med. = nos. 2, 4, 9, 69, 99 (Heckmann & Frankel, 1968, table 9).
low = nos. 11, 37, 83 (Heckmann & Frankel, 1968, table 9).
?. 66
xi. 69
xi. 67
xi. 67
xi. 67
viii. 66
Date(s)
examined
Table 1 (cont.)
115
11-8
11-4
11-2
121
111
8
13-7
13-7
13-4
12-8
12-9
130
9
10-2
10-4
100
9-7
9-5
9-5
10
64
50
17
47
30
50
Number
of cells
tallied
ON
W
562
J. FRANKEL
18
16
I 14
10
• fi
8-rowed cells
9-rowed cells
10-rowed cells
1
1
1
2
3
2
2
4
3
3
4
5
5
4
6
5
5
6
7
6
6
7
7
r
7-rowed cells
7
8
9•
•
10
Row position
Fig. 2. Distribution of ciliary units among ciliary rows in normal cells of Euplotes
minuta. Separate distribution curves are shown for 7-, 8-, 9- and 10-rowed cells.
The points plotted are the means for the cell groups of normal (non-bbd) cells
given in Table 1. The three groups of 7-rowed cells and sixteen groups of 9-rowed
cells are represented by D, while the seven groups of 8-rowed cells and six groups
of 10-rowed cells are represented by • . The abscissae are constructed by dividing
four identical line segments that represent the width of the cell into equal subdivisions, the number of these subdivisions being determined by the number of
rows; each row is then placed in the center of its subdivision (or 'row territory').
(Heckmann & Frankel, 1968, p. 33; Frankel, 19736, p. 350), while the former
assumption has not been confirmed rigorously but seems justified by general
examination of the cells (e.g. see photographs in Heckmann & Frankel, 1968;
Frankel, 19736). The resulting plots show that all corticotypes share a common
pattern of distribution of units among rows, characterized by a dip on the left,
a plateau in the center, and a sharp peak near the right margin of the cortical
field. The curves for the different corticotypes differ only in that they are offset
on the vertical axis, due to the simple arithmetic fact that the fewer the number
of rows the greater the average percentage of units in each row. This offset can
be eliminated by creating a separate ordinate scale for each corticotype, and then
adjusting the scales relative to one another so as to exactly compensate for the
difference, for example, by moving the ordinate for the 9-rowed cells to a value
10/9 that of the ordinate for the 10-rowed cells. Once this is done, the four
curves of Fig. 2 collapse into one (Fig. 3). The fit of the points to a common
curve is excellent over all portions of the field except perhaps for the portion
occupied by row 2, where there is some irregularity, with 7-rowed cells having
relatively more and 10-rowed cells relatively fewer units than might be expected.
Distribution of ciliary units in Euplotes
14
563
20
"14 .16 "18
2 12 s
"l4 •16
•12
'12 .14
•10
5
~\2 " 8 - "10
. 8
"10 3>
D
10 9 8 7
1 io
•>
7-rowed cells
2
1
8-rowed cells
9-rowed cells
1
10-rowed cells
\
3
5
4
6
7
1
1
1
2
3
2
4
3
4
5
5
6
6
7
7
8
9
8
1
4
10
5
6
Row position
Fig. 3. Replotting of the data of Fig. 2 with two modifications: (a) each point
represents the overall average of the means of separate groups shown in Fig. 2:
© = 7-rowed, • = 8-rowed, O = 9-rowed, C = 10-rowed; (b) a separate
ordinate is provided for each number of rows, with all four ordinates mutually
adjusted so as to compensate for the inverse correlation between number of rows
and percentage of units in a row (see text).
16
1,4
•E
>2
S10
8
7-rowed cells
8-rowed cells i
2
3
4
5
Row position
6
7
8
Fig. 4. Distribution of ciliary units among ciliary rows in basal body deficient
(bbd) cells of E. mimita. The method of plotting is the same as in Fig. 3. Each group
of bbd cells (italicized data in Table 1) is plotted separately. Open symbols represent
groups of 7-rowed cells, filled symbols represent groups of 8-rowed cells. Circles
represent BC-2 bbd, January, February and March 1970, squares represent BC-2
bbd, April and July 1970 (cf. Table 1).
The generally good fit strongly suggests that there is a common pattern that all
cells obey, regardless of the specific number of units and rows that actually
happen to be present in a given cell. This is particularly clear on the right side.
For example, if one considers the third row from the right (n - 2), the points in
the original plot (Fig. 2) are at different positions in relation to the 'right peak',
being halfway up the slope in the 10-rowed cells, at progressively lower positions
564
J. FRANKEL
Normal cells
la
lb
No. of
rows
lc
2a
3a
sA
7
513
25-6
20-5
8
52-7
32-1
9
65 1
\
Other
Total
no.
0
2-6
0
91
31
0-4
2-5
514
20-9
7-1
5-5
0-9
0-6
820
39
10
73-3
13 6
31
6-2
2-3
1-5
258
Av.
62-2
23-4
7-4
4-7
10
1-4
1631 =
V
93 0
bbd cells
j 1
No. of
rows
' i = '!' 2
''3 ' 4
5
* ^ , »• •
7
59-8
8-7
4-3
8
63 3
3-3
7-8
2-2
4.4
6
»-•
7
/ .
8
9
Total
no.
4-3
3-3
0
11
16 3
92
0
5-6
2-2
0
13-3
90
182 = 2
Fig. 5. Census of patterns of unit number in the ciliary rows on the right side of the
cell. The patterns for the foui right rows (n— 3 to n) are given for normal cells (top),
and patterns for the three right rows (n —2 to n) are given for basal body deficient
(bbd) cells (bottom). The patterns that are encountered are indicated in diagrammatic
form at the top of each column. Where only the three right rows are considered
(bbd, bottom), the nine possible patterns are: 1: n — 2 < n — 1 > n; 2: n —2 =
n - 1 > n; 3: n - 2 > n - 1 > n; 4: n - 2 > n - 1 = n; 5: n - 2 = n - 1 = n;
6: n - 2 > n - 1 = n; 7: n - 2 < n - 1 < n; 8: n - 2 = n - 1 < n; 9: n - 2 >
n — 1 < n. In normal cells, where the four right rows are considered, the patterns
shown are: la: n — 3 < n — 2 < n — l > n ; l b : n — 3 = n — 2 < n — l > n ; lc:
n - 3 > n - 2 < n - 1 > n; 2a: n - 3 < n - 2 = n - 1 > n; 3a: n - 3 < n - 2 >
n - 1 > n. The 22 other possible permutations involving four rows are not shown,
since they were manifested in few or no cells. All data in the body of the table are
given in percentages, the actual number of cells tallied is given in the right column.
in 9- and 8-rowed cells, and near the base in 7-rowed cells. In the second collapsed
plot (Fig. 3) these points fit into a common smooth curve, with the point for
the n - 2 (5th) row of 7-rowed cells perfectly juxtaposed on the point for the
n - 3 (7th) row of 10-rowed cells. This suggests that when the cell decides how
many units to allocate to row n - 2 (or to any other row) it does not merely
count rows from the nearest edge of the dorsolateral field, but instead measures
the relative position of that row on the dorsolateral surface.
The distribution of units in the basal body deficient (bbd) lines does not
precisely fit the same pattern set by the other lines. A collapsed plot of the bbd
data, constructed in the same way as Fig. 3, is given in Fig. 4. The general pattern
is not radically different from that found in normal cells, but the right peak is
Distribution of ciliary units in Euplotes
565
lower, and there is a hump near the left side as well. This difference in pattern
does not appear to reflect any alteration in the shape of the dorsal surface, as
cells of normal and bbd lines appear indistinguishable in form.
Thus far, we have only considered averaged distributions of units based on
groups of at least ten cells each. This leaves unanswered the question of the
extent to which each individual cell follows the general spatial design depicted
in Fig. 3. A partial answer to this question was achieved by censusing the frequency of occurrence of the most striking element of the pattern, the 'right
peak'. This was accomplished by a 'nearest neighbor' analysis: in each cell the
pattern was assayed semi-quantitatively by recording the relation of the number
of units in a given row to that in the immediately neighboring row(s). The
results of this analysis are given in Fig. 5 (see figure legend for the specific
criteria employed). The normal (non-bbd) cells are strikingly uniform in pattern,
with nearly 99 % of the cells manifesting only 5 out of the 27 patterns that are
logically possible in this form of analysis. These five patterns are each distinguished by a 'right peak'. The n - 1 row has the highest number of units (pattern
1) in 93 % of the normal cells. Among these, the n — 2 row has a number of units
intermediate between those of its neighbors (pattern la) in 62% of the cells.
It is noteworthy that pattern 1 a becomes more prevalent and the alternative
patterns 1 b and 1 c become less so as the total number of rows increases from
seven to ten, which is what one would expect by virtue of the fact that the
relative position of the n — 2 row shifts further to the right (and hence higher
up on the slope of the 'right peak') as the total number of rows increases. This
also explains why the only other reasonably common pattern (2a in Fig. 5), in
which the 'right peak' is broadened to include both the n — 1 and n —2 rows, is
more often encountered in 9- and 10-rowed cells than in 7- and 8-rowed cells
(^2 _ 6-i95 p < 0-02). The pattern-census thus reinforces the conclusion that a
relatively constant underlying distribution of unit-forming potential is present
in virtually every cell, a distribution which is obeyed rather than controlled by
the developmental processes occurring in individual ciliary rows.
This conclusion does not appear to apply with the same force to the bbd
segregant. In these cells virtually all of the 27 possible patterns were manifested
by the four right rows, so to prevent a too unwieldy presentation of data only
the three rows on the extreme right (rows n to n - 2 ) are included in Fig. 5
(bottom), reducing the number of possible pattern permutations to nine, all of
which are found. A ' right peak' is observed only in about 2/3 of the cells (pattern
1, plus some of the cells with patterns 2 and 3). Nearly 15% of the bbd cells
instead display the exact opposite, a 'right trough' (pattern 9), a feature that
was not observed in even one normal cell. This substantial variation in pattern
may be related to the difference between the average patterns shown in Figs. 3
and 4. In bbd cells there is sufficient 'noise' in the distribution of units among
rows that it is difficult to know with certainty what the true pattern really is.
For example, the 'right peak' is lower in Fig. 4 (bbd) than in Fig. 3 (normal),
566
J. FRANKEL
Proter
Eo
Opisthe
Nondeveloping
EarlyLate
development
Fig. 6. A schematic diagram of mode of proliferation of ciliary units in a ciliary row.
Each dot represents a unit. Eo represents the number of ciliary units in the equatorial
zone at the beginning and Ed the number at the end of the process of unit proliferation within that zone. A and P indicate the number of units in the anterior and
posterior zones within which no new units are formed. For further explanation,
see the text.
but the lowering of the bbd mean values results from the presence of a substantial
minority of cells with no right peak at all, while a majority have a peak that is
typically of near-'normal' height. The 'noisiness' of the pattern in bbd cells is
probably due to the marked irregularity and unreliability of the process of new
unit formation, which is one of the primary phenotypic attributes of the bbd
lines (Frankel, 19736).
2. Dividing cells
(a) The developmental sequence
During the process of cell-surface development the old ciliary units of each
row are retained and an approximately equal number of new ones are added.
The addition occurs exclusively in the equatorial zone of the cell immediately
prior to the onset of division furrowing. Previous studies have shown that new
ciliary units are added at short distances anterior and posterior to existing cilia
(Hufnagel & Torch, 1967; Ruffolo, 1972). Basal bodies develop first, then cilia
Distribution of ciliary units in Euplotes
567
Proter
Opisthe
Fig. 7. Camera-lucida drawing of the dorsal surface of a protargol-stained normal
cell during a late pre-division stage (stage V). This cell has eight ciliary rows, and
all except no. 1 (which is tucked under the left margin) are shown. The borders
between the equatorial zone of proliferation (Ed) and the anterior (A) and posterior
(P) non-proliferating regions are indicated by short-dashed lines, while the presumptive fission line, separating future proter and opisthe, is indicated by the longdashed line. The portions of the equatorial proliferating zone destined for anterior
division product (proter) and posterior division product (opisthe) are labelled E(la
and Edp, respectively.
grow out (Ruffolo, 1972). Unit proliferation begins on the right side (cf. Wise,
1965), but quickly spreads leftward to involve about half of the pre-existing
units, situated in the equatorial region of every ciliary row. The pattern of unit
proliferation in each row is schematically illustrated in Fig. 6. Initially one new
unit appears next to each old one in the equatorial region, giving rise t o ' doublets'
of units. One can also discern 'triplets' and occasional 'quadruplets' in somewhat more advanced cells, but the discrete 'unit clusters' tend to lose their
separate identity as proliferation continues and all units, new and old, instead
become uniformly and closely spaced within the zone of proliferation (Ed in
Fig. 6). The only discontinuity within this zone is the gap that marks the fission
line, which is the site of the future division furrow. While the unit proliferation
is proceeding, complex developmental events are taking place on the ventral
surface, and a DNA replication band is progressing through the macronucleus.
This constellation of events allows for easy monitoring of developmental stages
in appropriately prepared material (cf. Frankel, 1973 b, fig. 1). Tallies of ciliary
units at specific stages allow determination of the time at which proliferation of
ciliary units within ciliary rows ceases (late stage IV). The overall dorsal configuration of a cell in stage V, after the end of unit proliferation but before the
beginning of division, is shown in Fig. 7. Tallies were made of the number of
units in the equatorial zone in which proliferation has recently been completed
(Ed), further subdivided into a region destined for the anterior division product
(Eda) and a region destined for the posterior division product (E dp ), and of the
J. FRANKEL
Table 2. Distribution of ciliary units among regions in developing cells
A23*
i
Regionf
BC-2*
A
^
BC-1*
K
i
\
Average
9-row
10-row
9-row
8-row
9-row
%
A
A
A
r
*
> <
^ (
\ i
*
\ i
\ in each
Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. region
31-8 ±2-5
(15-6%)
71-0 ±4-9
(34-9 %)
Proter total 102-8 ±5-6
(50-5 %)
J
78-6 ±4-8
dp
(38-6%)
22-1 ±2-3
(10-8 %)
Opisthe
100-6 ±4-8
total
(49-5 %)
203-5 ±9-5
Grand
total
32
n
Non106-3 ±7-4
dividers:!:
70
n
97-2
No. units
added§
181-9 ±4-9
30-2 ±3-1
(14-9 %)
69-8 ±3-9
(34-6%)
100-0 ±5-0
(49-5 %)
81-3 ± 5 0
(40-3 %)
20-6 ±2-9
00-2%)
101-9±4-l
(50-5 %)
201-9 ±8-4
217-9 ±10-3
19
U2-2±6-9
16
92-9 ±5-4
28
98-7 ±5-9
12
105-7 ±6-3
50
1081
30
891
40
103-3
30
112-3
360 ±2-9
06-3%)
76-3 ±4-8
(34-6%)
112-3±5-2
(510%)
82-3 ±4-2
(37-3 %)
25-7 ±2-4
(M-7%)
1080 ±5-2
(490 %)
220-3 ±9-6
29-4 ±2-2
06-2%)
63-4 ±3-5
(34-8 %)
92-8 + 3-1
(51-0%)
66-4 ±3-7
(36-5 %)
22-7 ±2-2
(12-5%)
89-1 ±3-3
(490%)
30-3 ± 2 1
03-9%)
76-8 ±5-3
(35-2%)
107-1 ±5-5
(49-1 %)
88-0±5-5
(40-4 %)
22-8 ±2-2
(10-5 %)
110-8±5-6
(50-9 %)
15-4
34-8
50-2
38-6
111
49-8
* Key to strains employed:
A23 = Cells from eleven parallel A23 sublines, fixed November 1969 (Frankel, 1973 a,
table 2).
BC-1 = Fj (A23 x VF17) x A23, no. 11, fixed November 1967 (Heckmann & Frankel,
1968, table 9).
BC-2 = F1 (K7 x VF17) x K7, five normal progeny lines, fixed April 1970.
f See Fig. 7 for explanation of abbreviations.
t Non-dividing cells separately tallied from the same preparations as those used in the
tally of dividing cells.
§ 'Grand total' of dividing cells minus 'non-dividers'. This is equal to Ed — Eo (Fig. 9)
summed over all rows.
number of units in the anterior (A) and posterior (P) zones in which no unit
proliferation has occurred. There was only occasional uncertainty in the assignment of units at the margins between these zones and the E d region. These
tallies allow assessment of the number of units destined for the anterior division
product or proter (Eda + A) and for the posterior division product or opisthe
(E dp + P), and make possible additional computations which will be considered
subsequently. Only normal cells are considered here, since some difficulty is
encountered in demarcating the various zones in the very irregular bbd cells.
(b) Regional distribution of ciliary units
Before considering the distribution of ciliary units in dividing cells on a row-
Distribution of ciliary units in Euplotes
569
by-row basis, the overall allocation of units to the different zones will first be
described. The relevant data are presented in Table 2. These data represent
results of tallies of cells at stages between the end of unit proliferation (late stage
IV) to the latest stage at which the E d region can be clearly distinguished from
the A and P zones (early division, stage VI), utilizing those groups of wet silver
impregnated (A23, BC-1) and protargol stained (BC-2) samples in which the
appropriate stages were found in sufficient numbers. The tabulated results allow
three main conclusions to be drawn. The first is a confirmation of the conclusion
arrived at earlier (Wise, 1965; Heckmann & Frankel, 1968) that new units are
added only during the interval of development just prior to division. This is
seen by comparing the 'proter total' and 'opisthe total', which represent the
numbers of units in the incipient division products, to the average number of
units found in randomly selected non-dividers tallied on the same slides. These
totals are similar, indicating that the full complement of units is produced prior
to cell division and few if any are added later. As a corollary of this, the number
of units added during unit development (bottom line of Table 2) is similar to
the complement of units in a non-dividing cell. This fact will be important in
the subsequent analysis of the distribution of new units among different rows
(section 2c, below).
A comparison of unit totals in different regions of cells which have just completed development leads to two further conclusions. One is that the number of
units destined for proter and opisthe are equal. The second is that the dividing
cell is not symmetrical with respect to the allocation of units between proliferating
and non-proliferating zones respectively. The anterior non-proliferating region
(A) always has a greater number of units than the posterior non-proliferating
region (P), and, exactly compensating for this inequality, a larger proportion
of the units in the proliferation zone (Ed) is allocated to the posterior division
product (Edp) than to the anterior product (Eda). This suggests a precise regulation of the site of the fission line such that the total number of units is divided
equally between the division products, even though the equatorial zone is
divided unequally as a consequence. It is not, however, clear why the cell bothers
to create these compensated asymmetries in the allocation of ciliary units. One
possibility is that the E d zone is centrally positioned with respect to the axial
measurements of the cell, and that as a consequence of this the A region in
which the units are somewhat closer together (particularly in the two rows on
the extreme right) winds up with more units than the more thinly populated P
region. If correct, this would imply that the spatial territory of the presumptive
proter is initially somewhat smaller than that of the presumptive opisthe; such
a small difference could easily be compensated by differential growth during the
division process.
It is interesting to note that in Paramecium trichium, in which unit proliferation
within ciliary rows occurs in an equatorial zone as in Euplotes (Gillies & Hanson,
1968), there is initially a larger number of units in the presumptive opisthe than
570
J. FRANKEL
12
14 .16
14
12
10
—£—=— A —• -ft
12
•10
10
e
. 8
• 8
14
16 18
14,16
•E 12
12
10
10
14
12
10
. 8
8
6
. 6
' 4
A+P
4
10 9 8
C A
D
O
•
8-rowed cells
9-rowed cells
10-rowed cells
3
4
5
6
7
10
Row position
Fig. 8. Distribution of ciliary units in different regions of dividing cells among
ciliary rows. The mode of plotting the data is the same as in Fig. 3. Ed indicates the
number of units within the equatorial zone of proliferation, A + P the sum of the
units in the anterior and posterior non-proliferating regions (cf. Fig. 6). The data
plotted are based on tallies of cells in late pre-division (late IV and V) and early
division (VI) stages, in which unit proliferation has been completed. Filled symbols
represent 8-rowed cells, open symbols 9-rowed cells, and half-filled symbols, 10rowed cells. Circles represent A23 cells, triangles cells of BC-1, and squares cells of
BC-2 (see Table 2 for further information on the material used and sample sizes).
in the proter, which is compensated by a more intense unit proliferation in the
proter, so that ultimately proters and opisthes obtain an equal number of units
(Suhama, 1971).
(c) Distribution of ciliary units among ciliary rows
The dividing cells considered above will now be analysed from the perspective
of the distribution of ciliary units among different ciliary rows. A table comparable to Table 1 (not shown) was constructed for the five groups of cells
Distribution of ciliary units in Euplotes
571
indicated in Table 2, and the results are plotted in Fig. 8, employing the same
conventions used in the construction of Fig. 3. The distribution of units among
rows in the non-proliferating regions (A + P) and in the equatorial zone of
proliferation (Ed) displays a regular spatial pattern despite variation in numbers
of rows and units. However, the patterns in the Ed and in the (A + P) regions
are mutually dissimilar and also differ from the distribution observed in nondividing cells (Fig. 3). This difference is manifested primarily at the 'edges' of
the distribution; the number of units in rows 1 and n is unexpectedly high in
the E d region and correspondingly low in the (A + P) regions. This suggests that
the mechanics of proliferation of units are not identical in all rows, but rather
that whatever process controls proliferation is distributed unequally over
different longitudinal sectors of the dorsal surface.
To analyse these inequalities, we will obtain measures of the recruitment of
pre-existing units into the zone of proliferation and of the intensity of proliferation within this zone. The measure of recruitment, termed Eo, cannot be assayed
directly, but can be calculated by subtracting the number of units outside of the
equatorial zone of proliferation (A + P) from the total number of units present
in non-dividing cells that were tallied separately on the same slides (cf. Table 2).
This procedure gives a valid measure of the number of units in the presumptive
equatorial zone provided that one assumes (a) that the non-dividing cells tallied
are all destined to enter division, i.e. there is no subset of non-cycling cells with
an atypical number of units, and (b) that the units outside of the proliferating
equatorial zone remain constant in number, with no net addition or loss of
units. The first assumption is justified by results of earlier single cell cloning
studies which showed that virtually all cells from non-senile clones can divide
(Frankel, 1973 a, p. 87), while the second is likely in view of the results considered
in the previous section as well as the general absence of pairs of closely spaced
ciliary units in the A and P regions. Given a valid estimate of number of preexisting units recruited into the zone of proliferation (Eo), the number of units
added within each row during the process of development is then simply equal
to E d - Eo, i.e. the number of units in the equatorial zone at the end of proliferation minus the number present at the beginning (this computation is nontautologous because Eo was arrived at by comparison of developing cells with
an independent set of non-developing cells). The final step is to compute the
intensity of proliferation, that is, the average number of new units formed
adjacent to each pre-existing one. This is done by dividing the number of units
added during development by the number present at the beginning, i.e. (Ed — Eo)/
Eo. The three parameters are interrelated, since recruitment times intensity
equals total number of new units formed:
/Ed-E0\
This relationship does not entail a parallel distribution of the three measures,
36
EMB 33
572
J. FRANKEL
18
14 16
14 ,16
12
14
12
10
Eo (recruitment)
12
10
10
12
14 k16
12 14
10
fia
12
10
10
•
AO
E d —Eo (no. added)
©
10 9
8
2-5
20
1-5
Eo
(intensity)
10 L
8-rowed cells
9-rowed cells
10-rowed cells
5
6
Row position
10
Fig. 9. Distribution of calculated parameters of unit proliferation in the same groups
of cells as were considered in Fig. 8. The mode of plotting the data is the same as in
Fig. 8, except for the Ed —Eo/Eo parameter the ratios are plotted directly. For
explanation of the symbols, see legend for Fig. 8 and text.
since one may obtain a given arithmetic product in many different ways (e.g.
| x 2 and I x 4 both equal 1).
Examination of Fig. 9 shows that the spatial distributions of the three parameters are indeed quite different. First, it is not surprising that the pattern of
distribution of the number of units added during development (Ed - Eo, middle
curve) is basically identical to the pattern of distribution of units in non-dividing
cells shown in Fig. 3 (the causes for the scatter in the left portion of the graph
will be considered in section 3). This identity is required to maintain the constancy of the distribution of ciliary units from one cell generation to the next.
However, the distributions of recruitment (Eo) and intensity ((Ed - Eo)/Eo) are
very different both from one another and from the distribution of the product
Distribution of ciliary units in Euplotes
573
of the two, Ed — Eo. The recruitment function (Eo) has high values at the two
margins (rows 1 and n). This means that in these rows the proportion of units
involved in the proliferation process is greater than in the other rows; this
accounts for the surprisingly high values of E d in these rows (Fig. 8). The shape
of the intensity function is nearly the inverse of that of recruitment. It is to be
noted that the intensity function, being a ratio, is expressed differently from the
others; the ordinate gives an average of the number of new units formed per
pre-existing unit in the equatorial zone. In most rows, this ratio is slightly
greater than two, meaning that an average of somewhat more than two new
units develop near each old one. This approximate triplication of units compensates for the fact that only about half of the units of a row are recruited into
the proliferating zone. In the rows at the margins, where the higher recruitment
causes more than half of the old units to become involved in proliferation, the
intensity function is correspondingly lower, so that fewer new units are induced
to form near each old one.
If the recruitment and intensity functions were precise mirror images of one
another, then all rows would acquire an equal number of new units, and the
pattern of distribution of units among rows would quickly become horizontal.
However, the two measures are not perfect mirror images. This asymmetry is
particularly clear at the positions just internal to the two margins. For example,
the recruitment function rises gradually on the right while the intensity function
falls off more sharply but only at the extreme right edge. This particular asymmetry allows row n — 1 to be unlike all of the others and have a high value of
recruitment without a sacrifice of intensity. It is this that is formally responsible
for the striking peak in E d — Eo and consequently in the overall number of units
(Fig. 3) at that position. In this case, then, a strikingly asymmetrical patternsingularity can be accounted for by a relatively modest difference in two complementary gradients extending from the boundary of the dorsal field. What this
analysis has achieved, therefore, is to show that the complex spatial distribution
of units in a mature cell can be analytically subdivided into a product of two
simpler distributions that represent extensive and intensive qualities of the
developing system.
3. Effects of gains and losses of ciliary rows on the pattern of distribution
of ciliary units
Gains and losses of ciliary rows take place within clonal lines of descent.
Such changes are frequent in bbd lines, less so in normal clones (Frankel, 19736).
However, changes do occur in normal lines, and in some cases are systematically
biased in a single direction, as for example the decrease from ten to nine rows
in A23 clones prevented from undergoing autogamy (Heckmann & Frankel,
1968), the decline from nine to eight and seven rows in ageing K7 clones (Frankel,
1973 a), and the increase from nine to ten rows in certain progeny of BC-1
((A23 x YF17) x A23, cf. Heckmann & Frankel (1968)). In each of these cases,
36-2
574
J. FRANKEL
although a trend of change in row number can unmistakeably be deduced by
comparison of counts of successive samples, the rate of change per fission is
sufficiently low that individual dividing cells in the process of losing or gaining
rows are not commonly found. What limited observations have been made
suggest that rows may be added or lost at any position. However, in 10-rowed
A23 cells regulating downward to nine rows there are clear indications that the
second row from the left (no. 2) is preferentially lost. Thus, incomplete second
rows with few units have commonly been observed in non-dividing A23 cells
(cf. figs. 5 and 6 in Heckmann & Frankel, 1968), and several dividing cells have
been observed in which new units of row no. 2 are only present in one of the
two presumptive daughter cells.
We can now consider how the pattern of distribution of units among rows
adjusts when the number of rows changes. In general, adjustment must be fairly
rapid in order to yield the high uniformity of pattern indicated by Fig. 5; with
one exception to be noted below, this uniformity was just as great in groups of
cells that had undergone recent shifts in number of rows as in other groups
in which there had been no systematic shifts. An example will illustrate the
implications of this uniformity. In an earlier study, one dividing cell had been
found that was in the process of adding a tenth row at the extreme right (Heckmann & Frankel, 1968, fig. 7A). In this cell, the row with the greatest number
of units was still no. 8. However, we can be fairly confident that if this cell had
been allowed to survive, its 10-rowed progeny would have adjusted so that
row no. 9, now shifted from the former position of row ' n ' (extreme right) to
the new position of row ' n - 1 ' (second from right), would have acquired the
higher number of units appropriate to that new position, while row no. 8,
shifted from the 'n - 1 ' to the ' n - 2 ' position, would have regulated to a lower
number of units. Had such an adjustment not taken place, then even a modest
frequency of row-additions near the right margin would have yielded a greater
variability of pattern than was in fact observed.
It now remains to consider one exception to this regularity that was encountered in normal (non-bbd) cells. It will be recalled that the only position at which
the data points did not provide an excellent fit to the 'collapsed' curve of unit
distribution (Fig. 3) was in the region of row no. 2; it can be further seen that
the most divergent point is the one representing 10-rowed cells (half-filled
circles). This divergence is due to a 'dip' in the curve at the position of row 2
for 10-rowed cells (Fig. 2), a dip attributable in part to low values in 10-rowed
A23 cells (Table 1), which also show anomalously low unit numbers in row 2
of developing cells (Figs. 8 and 9). These low average values are a consequence
of the relatively frequent occurrence of incomplete second rows with low
numbers of units, which in turn reflects the probable tendency to lose a row at
that site. It should be noted that once this row is lost the former row no. 3
becomes row no. 2, and the proportion of units in this row correspondingly
regulates downward (Table 1, Fig. 2). Hence the most conspicuous irregularity
Distribution of ciliary units in Euplotes
575
in the pattern of distribution of units among rows is at least partly explicable
on the basis of a tendency toward preferential loss of a row at that site, and
serves to illustrate rather than contradict the general principle of adjustment
of pattern to position.
(There is only one other major discrepancy, and that is the anomalously high
number of units found in row 1 in the E d zone of BC-2 clones (represented by
the squares in Fig. 9). In this case, more units are added during development
than would be necessary to make up the unit complement observed in nondividing cells. The only plausible explanation is that in this special case a few
units may be resorbed after division.)
DISCUSSION
A major conclusion of this study is that there is a dual control of formation
of ciliary units within ciliary rows. On the one hand, new units appear in definite
spatial relations to old ones, and these spatial relations are the basis of the
observed continuity of ciliary rows (Frankel, 1973 b). On the other hand, whether
or not new units develop at all, and how many develop, is dependent on position
along the longitudinal and transverse axis of the cell. Along the longitudinal
axis proliferation occurs only in an equatorial zone that embraces about one-half
of the original units. This equatorial zone is not situated exactly midway in the
cell in terms of number of units, as more non-proliferating units are invariably
found at the anterior than the posterior end, but it may well be midway in terms
of distance on the surface, as the anterior units are more closely spaced than
the posterior ones. Along the transverse axis, the number of units formed within
a given row depends on the position of that row on the cell surface. This positiondependence is demonstrated by the commonality of the pattern irrespective of
number of units and number of rows (Results, section 1 b), and is reinforced by
considerations of pattern adjustment following gain or loss of a row (Results,
section 3). The proportion of the total potential for formation of ciliary units
that is allocated to a particular row depends on where that row is in relation
to the boundaries of the dorsal cell territory. Of course, once the equatorial
portion of each row obtains its allotment of unit-precursors, it positions the
new units according to its local symmetry and polarity.
Inequalities in number of units in different ciliary rows are not unique to
Euplotes, and may be expected in any but the most radially symmetrical ciliate
species. Such inequalities have been documented in Paramecium trichium (Gillies
& Hanson, 1968; Suhama, 1971) and in Tetrahymena pyriformis (Nanney &
Chow, 1974). In both cases, the conclusion was drawn that the number of units
is a direct reflexion of the length of the row: Gillies & Hanson (1968, p. 16)
assert' The number of cortical units per kinety varies according to the length of
the kinety', while for Nanney & Chow (1974, p. 129) 'The assignment of basal
bodies among the index rows may be viewed as a function - either a causal or
576
J. FRANKEL
consequential correlate - of the shape of the cell.' I regard this view as an oversimplification, at least when applied to Euplotes. Although measurement over
the somewhat curved surface of the cells is difficult, it is still apparent that the
n - 1 row, with by far the largest number of cortical units, is not longer than the
row to its immediate left ( n - 2 ) ; row n, on the extreme right, is considerably
shorter than the central rows yet has very nearly as many units. The reason why
the number of units in the two right rows is greater than would be expected on
the basis of the length of these rows was already appreciated by Borror (1962)
in his taxonomic re-description of E. minuta. He stated ' the anterior three to
five bristles in rows no. 8 and 9 are more closely set than elsewhere' (Borror
was considering 9-rowed cells). This characteristic crowding of units at the
anterior end of the two right rows was obvious in virtually every normal (i.e.
non-bbd) cell that I have observed (cf. Fig. 1). On the left side of the cell there
is a closer correspondence between number of units and length of the row,
with the rows progressively shorter toward the left due to the curvature of the
membranelle band (Fig. 1), and the number of units correspondingly smaller.
Even here it is doubtful whether the correspondence is exact, since length of
rows appears to be reduced more drastically than number of units. It thus seems
that the mechanism that governs the spatial distribution of unit-forming potential
in the equatorial zone of dividing cells does not respond directly to the total
length of each longitudinal sector. Rather, the units produced are fitted into the
space available, with differential degrees of crowding of units ensuing wherever
the contours of the distribution of unit-forming capacity do not correspond
precisely to the geometric contours of the cell. This in turn indicates that in
cases of more precise correspondence, in which units end up evenly spaced, the
match of cell shape and unit pattern is a result of a natural selection for a specific
end result (even spacing), rather than a mechanistically inevitable outcome of
the process by which units are formed.
It remains to consider how the distribution of unit formation is actually
brought about. It is obviously based on regional differences in what is happening
in the equatorial zone of unit proliferation. The total number of new units formed
in a row must logically depend on the number of old units that participate in
the proliferation process ('recruitment'), and on the number of new units
produced adjacent to every old unit ('intensity'). These functions could conceivably be identical for all rows, but in fact they are not: in general, the recruitment function is highest and the intensity function lowest near the margins of
the dorsal ciliary field (Fig. 9). The rather considerable differences in number of
units produced in specific rows is a result of mutual asymmetries in the spatial
distribution of the recruitment and intensity functions; for example, the gentler
slope of the decline in recruitment compared to the increase in intensity near
the right margin provides a formal explanation for the striking peak in number
of units in the n - 1 row.
One obvious feature of the distribution of the recruitment and intensity
Distribution of ciliary units in Euplotes
577
functions (Fig. 9) is that the values of these functions grade more or less steeply
from the margins to a central region in which the values are uniform. This
permits the suggestion that events at the margin might be important in controlling the characteristics of unit proliferation. It is unlikely that these events
are results of inductive influences emanating from specific cytologically visible
structures. Although the anterior portion of the left margin is marked by the
prominent membranelle band, the posterior portion of the left margin as well
as the entire right margin are characterized by no specific bounding structure,
but only an abrupt discontinuity in the pattern of silver staining lines (the
'argyrome' of Tuffrau (1960), cf. also Wise (1965)) that in fact correspond to
membrane ridges rather than fibrillar structures (Ruffolo, 1972). This patterndiscontinuity demarcates the dorsal region covered by ciliary rows from the
ventral territory characterized by the prominent cirri (cf. Fig. 1). The ciliature
occupying these two territories is sharply different in structure, in pattern of
development (Frankel, 1973 c), and even in the conditions under which development may be triggered; for example, during conjugation (Hammond, 1937) and
autogamy (Frankel, unpublished) the ventral surface of Euplotes undergoes two
rounds of replacement of cirri, while the dorsal surface remains developmentally
quiescent. The dorsal and ventral fields may be considered as analogous to
two segments, with the discontinuities between them constituting segmentboundaries. These discontinuities might serve as 'boundary zones' in the sense
of Wolpert (1969), which are responsible for specifying positional information
of adjacent regions. This positional information may in turn be interpreted to
bring about the previously noted gradients in the recruitment and intensity
functions. However, the existence of positional information in this system has
not been positively established, and it could be that the observed gradients are
results of direct interactions of parts of the dorsal field. The latter interpretation, however, makes it somewhat more difficult to account for the observed
regularity (size-compensation) of the pattern in cells differing considerably in
size (cf. Wolpert, 1969, p. 6).
Comparable systems might also exist in other ciliates. In Paramecium trichium,
for example, the number of units in rows of the left and right sides is similar,
but at least in prospective anterior fission products the proportion of units
recruited into the zone of proliferation is considerably smaller on the left than
on the right side, while the number of new units produced per old unit is correspondingly greater on the left side than on the right (Suhama, 1971, text-fig. 1).
The possible significance of such regional differences in controlling the total
number of units is unclear in Paramecium, since the number of rows and units
is so large as to make a row-by-row analysis almost impossibly tedious. In
Tetrahymena pyriformis, in which there are considerable data on the distribution
of units in pre-dividing cells (Nanney & Chow, 1974), unit proliferation takes
place at all times in the cell cycle (Nanney, 19716) and in all regions of the
ciliary row (Perlman, 1973). The observed inequalities in number of units in
578
J. FRANKEL
different rows of this organism would be maintained if in every cell cycle one
new unit were allowed to form near each old one. It is not known whether
regulation of unit formation in Tetrahymena is really this simple. There are
some indications that it is not, since two adjacent nascent ciliary units] are
sometimes seen (Nanney, personal communication).
A final comment should be made concerning the evolutionary stability of
the distribution patterns being considered here. There appears to be a striking
constancy in all normal Euplotes minuta, embracing both the non-autogamous
and autogamous strains which almost certainly constitute separate subspecies
or incipient species (Luporini & Nobili, 1967). Further, E. minuta collected by
Borror on the New Hampshire coast, distant from the Mediterranean sites at
which the progenitors of the stocks considered here were collected, also display
a maximum number of units at the second row from the right (Borror, 1962).
Euplotes vannus is also characterized by a pattern of distribution of ciliary units
similar to that of E. minuta, with a prominent 'right peak' in the n—1 row
(unpublished observations by the author). E. vannus and E. minuta resemble
one another in general ciliary pattern (Borror, 1962), in systems of mating type
determination (Heckmann, 1963; Nobili, 1966), and further can conjugate with
one another, although the pairs are inviable (Nobili, 1964). Hence they are
almost certainly very closely related species. On the other hand, limited data
from other Euplotes species, all of which are different in numerous respects from
E. vannus and E. minuta, reveal diverse patterns of distribution: E. raikovi
(Washburn & Borror, 1973), E. daidelos (Diller & Kuonaris, 1966), and E.
aediculatus (unpublished observations by the author) all lack a 'right peak',
and have distributions of units that differ from those of E. minuta and E. vannus,
and from each other, in other respects as well.
A parallel situation appears in Tetrahymena pyriformis. Similar patterns of
distributions of ciliary units among seven ventral ciliary rows were found by
Nanney & Chow (1974) in all twelve syngens (sibling species) of T. pyriformis.
Patterns of different strains within syngens were indistinguishable for all but
two syngens (one of the two syngens was undergoing a change in number of
rows when fixed, making clear judgment impossible in that case). Some differences were observed between syngens, but these were rather subtle. Extensive
molecular differences among syngens ofT. pyriformis make plausible the suggestion that they are of more ancient evolutionary divergence than is typically the
case with sibling species swarms (see Nanney & Chow).
Taking the Euplotes and Tetrahymena data together, it appears that the
spatial rules which govern the manner in which available units are distributed
among different rows are highly conservative, certainly far more stable than the
aggregate number of units and rows. They are not, however, immutable. It
would be interesting to see whether evolutionary transitions in pattern among
reasonably closely related species involve alterations in the intensity or the
recruitment functions separately, or in both simultaneously.
Distribution of ciliary units in Euplotes
579
I would like to thank Professor Klaus Heckmann for introducing me to the organism and
providing valuable collaboration as well as the laboratory facilities in which the experimental
work was done. Mrs Elizabeth Merrick and Miss Ingeborg Sonntag provided indispensable
technical assistance. Drawings were executed by Dr Anne W. K. Frankel and Ms Diane
Knight. Drs Anne Frankel, Klaus Heckmann and David L. Nanney read the manuscript
and provided helpful suggestions.
Research supported by the Alexander von Humboldt Foundation (Bonn-Bad Godesberg,
German Federal Republic) and by the U.S. National Science Foundation (research grants
GB-6242 to Dr K. Heckmann and GB-32408 to J. Frankel). The material analysed in this
report was prepared in part by Dr Heckmann at the Southwest Center for Advanced Studies
(now University of Texas at Dallas) and in part by the author at the Division of Cell Research
of the Zoological Institute of the Tubingen University.
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{Received 24 June 1974)