/. Embryol exp. Morph. Vol. 49, pp. 167-202, 1979
Printed in Great Britain © Company of Biologists Limited 1979
A mutant of Tetrahymena thermophila
with a partial mirror-image duplication of cell
surface pattern
I. Analysis of the phenotype
By MARIA JERKA-DZIADOSZ 1 AND JOSEPH FRANKEL 2
From the Department of Zoology, University of Iowa
SUMMARY
Cells of a mutant clone, CU-127, of Tetrahymena thermophila (formerly T. pyriformis,
syngen 1) manifest three anatomical abnormalities. First, the stable number of ciliary
meridians is 21-25, above the usual number (17-21) in this species. Second, up to 30 % of the
cells have two oral apparatuses (OAs), one normal and the other abnormal. Third, more
than one-half of the cells possess two distinct sets of contractile vacuole pores (CVPs). In
some living cells two contractile vacuoles are seen. These abnormalities have persisted unchanged during more than 500 generations of vegetative propagation, and are similarly
expressed in subclones. The normal and abnormal OAs are topographically segregated, with
normal OAs developing along the 'primary oral axis' and abnormal OAs developing along
a 'secondary oral axis' that is situated 170° of the cell circumference to the cell's right of the
primary oral axis. CVPs always appear within this 170° arc and never within the complementary 190° arc to the left of the primary oral axis.
A unique feature of the CU-127 clone is the commonly expressed mirror image reversal of
the structural pattern of OAs that develop along the secondary oral axis. The primordia of
such OAs initially appear (as usual) to the cell's left of a ciliary meridian, but as membranelles
develop they frequently come to be oriented in a mirror image of the normal pattern, and an
undulating membrane sometimes develops on the wrong (left) side of the oral primordium.
When two sets of CVPs are formed, their average positions are roughly equidistant with
respect to the two oral axes, with the two sets located 50-60° to the right and left respectively
of the primary and secondary oral axes. Such cells are thus bilaterally symmetrical about
a plane defined by the central longitudinal axis and the halfway point between the two
CVP sets (see Fig. 25). This plane bisects the cell into a normal and a 'reversed' half-cell.
However, only oral asymmetry and large-scale CVP positioning are subject to such reversal;
all ciliary meridians remain of normal asymmetry and all CVPs are situated on the left side
of CVP meridians. The fact that major aspects of large-scale cellular organization can be
reversed while the 'fine-positioning' associated with the ciliary meridians remains normal
indicates that the two aspects of cell organization are distinct.
1
Author's address: Department of Cell Biology, M. Nencki Institute of Experimental
Biology, Pasteura 3, Warsaw 02-093, Poland.
2
Author's address (for reprints): Department of Zoology, University of Iowa, Iowa City,
Iowa 52242, U.S.A.
168
M. JERKA-DZIADOSZ AND J. FRANKEL
INTRODUCTION
Positioning of cell surface organelle systems in ciliated protozoa involves
interactions taking place over very small as well as relatively large intracellular
distances. There has been considerable discussion in the literature over the
degree to which the mechanisms governing long-range positioning differ from
those controlling short-range positioning (Frankel, 1974, 1975; Jerka-Dziadosz,
1974; Lynn & Tucker, 1976; Lynn, 1977; Sonneborn, 1975). The paradigm for
short-range positional interactions is the propagation of the ciliary meridian.
Within this ensemble new structures such as basal bodies and accessory fibrillar
and microtubular systems are positioned in definite spatial relations to nearby
pre-existing structures. The proof of the determinative role of the pre-existing
topographic arrangement within ciliary meridians was the demonstration that
an 180° inversion of that arrangement is faithfully propagated. This was shown
first in Paramecium (Beisson & Sonneborn, 1965) and later in Tetrahymena
(Ng & Frankel, 1977; Ng & R. Williams, 1977). In Tetrahymena it was further
ascertained that the geometry of the ciliary meridian also controls the finepositioning of the contractile vacuole pore (Ng, 1977, 1978) as well as aspects of
the positioning of cortically situated mitochondria (Aufderheide, 1978).
Investigation of positioning of structures such as new oral apparatuses has been
carried out mainly by microsurgical experiments on large ciliates, such as
Stentor (Tartar, 1962; Uhlig, 1960) and urostylids (Jerka-Dziadosz, 1974, 1977).
The results of these experiments suggest that positioning over long intracellular
distances may involve 'gradient-fields' that may be analogous to those
operating in multicellular development (Frankel, 1974, 1975; Jerka-Dziadosz,
1974). In Tetrahymena, morphometric analyses of the cell latitudes at which
new oral apparatuses are formed (Lynn & Tucker, 1976; Lynn, 1977) and of
the longitudes at which new contractile vacuole pores appear (Nanney, 1966 a)
suggest that both structures are determined relationally as joint functions of
reference points (or axes) and aspects of overall cell size.
A decisive judgement of the relationship of long-range positioning systems to
the better-understood short-range systems could be made if it were possible to
rotate or reverse either system within the spatial domain of another. One way
of doing this would be to obtain a propagated geometrical reversal of some
aspect of large-scale cellular organization. A propagated reversal of asymmetry
of feeding structures has already been obtained in mirror-image doublets of
hypotrich ciliates (Faure-Fremiet, 1945; Tchang, Shi & Pang, 1964; Tchang &
Pang, 1965; Dryl & Totwen-Nowakowska, 1972), but these cases have not
been subjected to detailed cytological analysis. Recently, however, we have
encountered a clone of Tetrahymena thermophila that indefinitely perpetuates
the capacity to manifest a mirror-image reversal of oral structures at a welldefined cellular position and also maintains a corresponding reversal of contractile vacuole pore positions. Since in Tetrahymena (unlike the hypotrichs
Mirror-image duplication in Tetrahymena
169
referred to above) both oral structures and contractile vacuole pores develop
in the close neighborhood of ciliary meridians (reviewed in Frankel & Williams,
1973), this reversal achieves the desired superimposition of two positional
systems. The cytological analysis to be presented here shows that these two
systems are indeed separate and dissociable. The accompanying paper (Frankel
& Jenkins, 1979) demonstrates that the unique reversal of asymmetry in this
clone is under genie control, and provides further evidence for the separation
of short-range and long-range positional controls.
MATERIALS AND METHODS
The CU-127 clone of Tetrahymena thermophila (Nanney & McCoy, 1976)
that is the subject of this investigation was obtained in December 1976 from the
laboratory of Dr P. Bruns. It had previously been subjected to mutagenesis in
10 /Ag/ml of iV-methyl-A^'-nitrosoguanidine, followed by short-circuit genomic
exclusion, a protocol designed to select homozygous cells rapidly (Bruns,
Brussard & Kavka, 1976). CU-127 was one of a subset of morphologically
abnormal clones among a larger number of clones that had been screened for
temperature sensitivity following the above-mentioned mutagenesis and genomic
exclusion protocol (Bruns & Sanford, 1978). All but one of the experiments were
performed on a sample received directly from Dr Bruns' laboratory in December
1976. One experiment (cf. Table 6; Table 7, II) was carried out on another sample
that had first been sent by Dr Bruns to Dr D. L. Nanney's laboratory for cryopreservation and was received from there in February 1977; this is designated
CU-127 (111.).
The media used in experiments were all axenic, and consist of four different
recipes: TGVS: 0-3% bacto-tryptone (Difco), 0-5% glucose, vitamins, and
salts (Frankel, 1965); 1 % PPY: 1 % proteose peptone (Difco) plus 0 1 % yeast
extract (Difco); 2 % PPY: 2 % proteose peptone plus 0-5 % yeast extract; Dryl's:
Dryl's inorganic medium made up as described by Nelsen & DeBault (1978).
Stocks were maintained at 28 °C in axenic tube cultures containing 5 ml of
1 % PPY or TGVS medium, with transfer daily or every second day [weekly for
CU-127 (111.)]. Fernbach flasks (500 ml, Jena Glaswerk) containing 100 ml of
medium were inoculated with one to five drops of 1-day-old tube culture. Flasks
containing TGVS medium were inoculated with cells from TGVS tubes,
while flasks with 1 % PPY or 2 % PPY medium were inoculated from 1 % PPY
tubes. The flask cultures were incubated for 20-24 h at 28° ± 1 °C to yield
mid-log phase cultures (cell density 15000-50000 per ml). In some cases such
cultures were shifted to a bath set at 40° ±0-1 °C (39-5 °C inside flask) and
maintained at that temperature for various durations (usually 4-5-5 h), following
which, in certain experiments, the flask was returned to 28 °C for 1-5 h. Samples
were fixed at various intervals.
In a few experiments cultures were fixed after being allowed to enter stationary
170
M. JERKA-DZIADOSZ AND J. FRANKEL
phase by 48 h continuous maintenance at 28 °C, or by 24 h following a shift of
a mid-log phase culture from 28 to 40 °C. In still other experiments, cells grown
in 1 % PPY at 28 °C were washed by centrifugation and transferred to Dryl's
inorganic medium and maintained under various temperature regimens.
In one experiment, the cells were maintained in 1 % PPY and TGVS tubes,
with daily transfer, for 10 successive days at 39-5 °C. After the tenth such serial
tube-culture had reached late-log phase, the contents of the entire tube were
poured into a Fernbach flask containing 100 ml of medium, kept at 39-5 °C for
3 h, and fixed. These cultures had thus spent about 100 cell generations at 39-5 °C
prior to fixation.
In another experiment, a CU-127 culture growing in 1 % PPY was subjected
to clonal expansion. Thirty single cells were removed by a micropipette from
a log-phase flask culture growing in 1 % PPY at 28 °C, and were individually
inoculated into depressions of three-spot depression slides, each containing
0-5 ml pen-strep 1 % PPY (Frankel, Jenkins, Doerder & Nelsen, 1976). They
were kept in these depressions at 25° for 2 days. These 30 subcultures were
transferred from the depressions to culture tubes each containing 5 ml of 1 %
PPY medium, and maintained at 25° for 2 further days. The 30 cultures were
then fixed for silver impregnation as they were entering stationary phase,
25-30 generations after the original single-cell isolation. All but two of the
30 subclones were terminated at the time of fixation. One subclone (CU-127-1)
was retained and used in further experiments.
Cells were prepared for counting as described earlier (Frankel, 1965) and
counted in a model A Coulter Counter (Coulter Electronics). Food vacuole
formation was assayed by uptake of carmine particles. Silver impregnation was
performed according to Frankel & Heckmann (1968) with slight modifications
(Nelsen & DeBault, 1978). Measurements of silver impregnated specimens were
made with a filar micrometer eyepiece (American Optical). Protargol staining
was done according to the procedure of Ng & Nelsen (1977). Cells were fixed
for scanning electron microscopy in 1 % osmic acid for 3 min, and submitted
to critical point drying following the procedure of Ruffolo (1974) with the
omission of amyl acetate. Cells were observed in a Cambridge stereoscan S4
microscope.
RESULTS
1. General comparison of the phenotype of CU-127 and wild-type cells
Cells of the CU-127 clone were 'originally scored as flat cells' (J. W. McCoy,
personal communication). This flattening is generally not very pronounced, and
CU-127 cells otherwise differ only slightly from wild-type cells in size and form.
Like wild-type (WT) cells, they have a single macronucleus and generally
a single micronucleus (Fig. 9 a). The rate of population growth and the density
attained at stationary phase are similar for CU127 and WT. CU-127 grows well
at high temperature (39-5 °C), and differs from WT only in tending to die after
Mirror-image duplication in Tetrahymena
171
F I G U R E S 1 AND 2
Scanning electron micrographs of CU-127 cells. The bars in both micrographs
represent 10 /.cm.
Fig. 1. A polar view of a cell with two oral apparatuses (OAs), indicated by arrows.
The OA on the viewer's right is the structurally normal primary OA, while the OA on
the viewer's left is the secondary OA. The arrows point to the first membranelle of
each of these two OAs. Note that the secondary OA has three membranelles, but no
undulating membrane.
Fig. 2. A lateral view of a dividing cell with one OA and two oral primordia. The
normal oral axis is at the left (viewer's right) edge of the cell, where both OA and OP
are visible. The secondary oral axis is at the right (viewer's left) edge of the cell,
where only an OP is present. Note the single set of contractile vacuole pores (CVPs)
neai the posterior end of the cell (arrow).
prolonged maintenance at 39-5 °C without transfer, which is the probable basis
on which CU-127 was originally selected as a 'temperature sensitive' putative
mutant (Bruns & Sanford, 1978).
CU-127 differs dramatically from WT in three major aspects of cortical
pattern. First, the number of ciliary meridians is higher in CU-127 cells than in
WT cells, with typically 21-25 in the former and 17-21 in the latter. The high
meridian number in CU-127 cells is stable even when cells are propagated by
frequent transfer, conditions under which WT populations that include cells
with unusually high ciliary meridian numbers tend to return to a 'stability
center' near 19 ciliary meridians (Nanney, 19666, 1968; Frankel, unpublished
observations). Ciliary meridians of CU-127 are of normal polarity and
asymmetry, and also of normal ultrastructural organization.
A second striking cortical anomaly manifested by CU-127 cells is the presence
of secondary abnormal oral areas at a well defined location nearly 180° opposite
172
M. JERKA-DZIADOSZ AND J. FRANKEL
OA-1
** / •• &* * M* *
jiff
/
' 4
/
• • / «•
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/ /
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Mirror-image duplication in Tetrahymena
173
from the normal OA (Figs. 1, 4). A certain proportion of dividing CU-127 cells
produce a similarly abnormal secondary oral primordium (OP) nearly opposite
from the normal OP (Fig. 2). On the side of the cell with the normal OA and
OP, both the anatomy and mode of development of OA are indistinguishable
from those of WT cells. The longitudinal strip of cell surface along which the
normal OA is located anteriorly and the OP is developing subequatorially prior
to cell division is here called the 'primary oral axis'. Correspondingly, on the
opposite side of the cell the longitudinal strip of the cell where the abnormal OA
is located and along which the abnormal OP develops is termed the 'secondary
oral axis'. This is unambiguously distinguished by the abnormality of oral
structures and their irregular occurrence (see section 2b). The secondary oral
axis also lacks a cytoproct such as is normally observed at the posterior end of
the primary axis.
Finally, the majority of CU-127 cells possess two sets of contractile vacuole
pores (CVPs). A cell is said to possess one CVP set if all CVPs are situated near
adjacent meridians. All WT cells have one CVP set situated immediately to the
left1 of the posterior ends of one or two adjacent meridians that are located
approximately 22 % of the cell circumference to the right of meridian no. 1
(Nanney, 1966a; Nanney, Chow & Wozencraft, 1975). Some CU-127 cells
possess a single CVP set situated at the position usual in normal cells (Figs. 2, 4,
and the posterior cell in Fig. 5), but the majority have two CVP sets separated
by one, two, three or four ciliary meridians (Fig. 3 and anterior cell in Fig. 5).
The mean number of CVPs within each set in cells with two sets (1-57 and 1-63
for the left and right site respectively) approaches the mean number of CVPs in
cells that have only one set (1-84). Each of the two CVP sets is at least transiently
underlain by a separate contractile vacuole, as two such vacuoles are frequently
1
Throughout this report, right and left refer to the observer's right and left, assuming
that he stands inside the animal so that his anterior-posterior axis coincides with that of the
animal, and he keeps turning around his own longitudinal axis to face the surface of the
animal.
Figs. 3-5. Silver impregnated CU-127 cells. The bar in each micrograph represents
10 /tm.
Fig. 3. A cell with two CVP sets (arrows) separated by four ciliary meridians
lacking CVPs.
Fig. 4. A cell with two OAs and one CVP set (arrow). The apparent reversal of the
orientation of oral structures in the primary OA (OA-1) is a focusing artifact.
Fig. 5. A dividing cell with two CVP sets just anterior to the division furrow (small
arrows), and one CVP set near the posterior end (large arrow). Note that the two
CVP sets of the anterior fission product are positioned to the right and left of the
single CVP set of the posterior fission product.
Fig. 6. A living CU-127 cell photographed under bright field optics. Two contractile
vacuoles, indicated by arrows, are evident just anterior to the division furrow, while
one is present at the posterior end of the cell. The bar represents 10 /on.
12
KMB 49
174
M. JERKA-DZIADOSZ AND J. FRANKEL
Table 1. Number of OAs and CVP sets in CU-127 cells under various
growth conditions
Phenotype
Tpm.
\J VCI 11 °/
perature in oral 1 OA 1 OA 2 OA 2 O A (
2 CVP
Growth. regimen* devel- 1 CVP 2 CVP 1 CVP 2 CVP
(°Q
phase
opment set
sets
set
sets 2 OAs sets
I till
Medium
TGVS
TGVS
1 %PPY
1 %PPY
2%PPY
2%PPY
2%PPY
Log
Stat
Log
Stat
Log
Stat
Log
28
28
28
28
28
28
39-5
0 /
/o
30
0
24
0
27
5
27
Il\/Al
'
41
44
41
40
17
24
36
46
54
44
60
55
69
59
6
1
6
0
7
1
1
7
0
9
0
21
6
4
13
1
15
0
28
7
5
53
54
53
60
76
75
63
* The 28° cultures were fixed after growth of flask cultures to mid-log phase 1:20-24 h)
or to early stationary phase (48 h). The 39-5° culture was sampled after continuous growth
at that temperature for about 100 cell generations (see Methods).
observed at the same cell latitude in CU-127 cells (Fig. 6); this situation has
not previously been reported for Tetrahymena. The two neighboring contractile
vacuoles empty their contents asynchronously.
The 'fine-positioning' (Ng, 1977) of CVPs is always normal in CU-127 cells:
CVPs are observed close to the left side of ciliary meridians or sometimes
directly on the axis of meridians, but not on the right side of the meridians
(Figs. 2-4).
Although they superficially resemble homopolar doublets, induced by division
blockage (Faure-Fremiet, 1948) or by failure of separation of conjugating cells
(Nanney et al. 1975) that are in the process of reverting to singlets (Nanney,
1966b; Nanney et al. 1975), the CU-127 cells are profoundly different from
such doublets in two respects. First, the expression of the CU-127 phenotype
has been stable for over 500 generations of propagation in our laboratory, with
no substantial augmentation nor diminution of the degree of doubleness of
CVP sets or OAs. Second, instead of possessing two similar normal oral axes,
CU-127 cells possess a primary oral axis that is completely normal plus a secondary oral axis that is strikingly abnormal (see Results, sections 3 and 4).
2. Analysis of expression of CU-127
(a) Expression in non-dividing cells under different conditions
The expression of secondary OAs and double sets of CVPs by CU-127 cells
was analyzed in combinations of three media, two growth phases (log and
stationary), and several temperature regimens. Typical results are shown in
Table 1. Three basic findings emerge. First, expression is not temperature
sensitive: cultivation at high temperature, either continuously (Table 1) or for
Mirror-image duplication in Tetrahymena
1-OA/1-OP
1-OA/2-OP
2-OA/l-OP
175
2-OA/2-OP
To t;il
0
14
13
0
1 0
1
0
0
1
11
66
{7-8)
83
0
0
0
76
1
22
U4-5)
(.8-5)
Fig. 7. Tabulation of configurations of OAs, oral primordia, and CVPs in dividing
CU-127 cells. Each of the 16 sketches schematically represents one of the 16 possible
configurations of OAs and CVP sets in dividing cells. Each black dot indicates a CVP
set. The boldface numbers indicate actual sums; the italicized numbers in parentheses give values expected on the basis of random associations of numbers of OAs
and oral primordia assuming that the two are independently determined. For
example, in series A the proportion of 1-OA is 52/66 or 0-787, while the proportion
of 1-OP is 37/66 or 0-561; the proportion of joint occurrence of 1-OA and 1-OP
assuming independence is 0-787x0-561, or 0-442. This leads us to expect that
0-442 of the total of 66 cells, or 29-2 cells, should have both 1-OA and 1-OP. (A) A
culture grown in 1 % PPY to mid-log phase at 28 °C, then fixed. (B) A culture grown
in 2 % PPY to mid-log phase at 28 °C followed by exposure to 39-5 °C for 5-5 h
followed by return to 28 °C for 1-5 h, then fixed. (C) The aggregated results from
tallies of various other samples, all in log phase at the time of fixation.
short periods (not shown), does not in itself raise or lower the expression of
doubleness of CVP sets. Second, expression of secondary OAs (but not of
double CVP sets) is invariably very low in stationary phase. Third, expression
of secondary OAs and of double CVP sets are mutually independent: the
proportion of the four possible joint phenotypic classes (1OA/1 CVP, 1 OA/2
CVP, 2 OA/1 CVP, 2 OA/2 CVP) within every sample is close to what would
be expected on the basis of random combinations of the frequency of expression
of each separate trait. In general, except for the scarcity of secondary OAs in
stationary phase, the similarity of expression of OA and CVP anomalies under
the investigated conditions is more striking than any differences (a tendency for
higher expression in 2 % PPY does not attain statistical significance).
176
M. JERKA-DZIADOSZ AND J. FRANKEL
Table 2. Percentage of CU-127 cells with two sets of organelles
Oral apparatus
,
Contractile vacuole pore (CVP)
Dividing cells
Sample*
A. (1 % PPY)
B. (2 % PPY)
C. Various
Anterior
(OA)
21
42
30
Posterior
(OP)
44
53
37
Nondividing
Cellsf
15
31
18
Dividing cells
Non,
*
> dividing
Anterior Posterior
cellst
80
86
66
55
70
53
53
74
61
* See legend of Fig. 7.
t Percentages apply to randomly chosen non-dividing cells from the same samples (A, B)
or the same set of samples (C) used in the analysis of dividing cells.
(b) Expression in dividing cells
Dividing CU-127 cells were analyzed in order to gain insight into the
dynamics of propagation of the secondary oral structures and double CVP sets.
The results of an analysis of two samples, plus the remaining collection of
relevant data from numerous other samples, are tabulated in Fig. 7 according
to membership in each of 16 possible combinations of OAs and CVP sets (it
should be noted that a dot in the diagrams indicates a set of CVPs, not a single
CVP). Fifteen of the 16 possible combinations have in fact been observed. OA
and CVP patterns are largely independent of each other, and there are no
invariant associations between the two division products for either oral or CVP
patterns (cf. Figs. 2 and 5). Ascertainment of whether numbers of OAs and CVP
sets are randomly associated in anterior and posterior division products
demands a more searching analysis. First, the aggregate proportion of secondary
OAs and double CVP sets in anterior and posterior division products separately
was computed by summing across the appropriate columns in Fig. 7 and adjusting the totals to percentages; these percentages are shown in Table 2, where they
are also compared to the corresponding percentages observed in non-dividing
cells. It is evident that dividing cells manifest a higher expression of secondary
oral primordia and generally also of double anterior CVP sets than do nondividing cells. The relative excess of doubleness of newly formed structures (oral
primordia, anterior CVP sets) suggest that newly formed structures may not
always persist. The next step is to use the aggregate percentages of individual
traits to compute the expected frequencies of random combinations, and then
compare the expected to the observed proportions. This comparison is shown
for the oral structures in Fig. 7 (observed values bold, expected numbers
italicized and in parentheses). The association is clearly not random, as there
is a highly significant excess of homogeneous combinations (1 OA/1 OP,
2 OA/2 OP) and a deficiency of heterogeneous combinations (1 OA/2 OP,
Mirror-image duplication in Tetrahymena
177
2 OA/1 OP). The same is true, though in a less dramatic fashion, for CVP sets
(not shown). This suggests some tendency to propagate the pre-existing state of
expression. Finally, the striking asymmetry between the two heterogeneous
combinations, with the 1 OA/2 OP class much more common than the
2 OA/1 OP class, is a reflexion of the fact that dividing CU-127 cells have a
higher frequency of secondary oral primordia than of secondary OAs (Table 2).
A similar excess of the 2 CVP(ant)/l CVP(post) class relative to the 1 CVP(ant)/
2 CVP(post) class was observed. In a sample of 50 dividing cells of CU-127 (111.)
fixed 16 months later all of the above findings, excepting only the excess of
2 CVP(ant)/! CVP(post) over 1 CVP(ant)/2 CVP(post), were confirmed. The
observations summarized in Fig. 7 may thus be presumed to reflect constant
characteristics of the CU-127 clone.
In a different approach to the problem of continuity of states of expression,
a single CU-127 culture was expanded into 30 subclones by selection of single
celJs, with fixation and silver impregnation of the subclones 25 to 30 generations
later. The expression of the OA and CVP abnormality was indistinguishable in
these subclones.
(c) Expression during oral replacement
Oral replacement is an alternative mode of oral development (Frankel &
Williams, 1973), typically observed in T. thermophila during stationary phase
(Kaczanowski, 1976) and during transformation to a 'rapid swimmer' phenotype (Nelsen, 1978). CU-127 cells also undergo oral replacement under both of
these circumstances. The normal primary OA undergoes typical oral replacement with involvement of the undulating membrane (Frankel, 1969). In CU-127
cells undergoing the 'rapid swimmer' transformation, the abnormal secondary
OA, which generally lacks an undulating membrane (see section 4) is replaced
by an oral primordium that is formed adjacent to its right postoral meridian,
generally some distance back from its anterior end. In some cases, transforming
CU-127 cells may form an abortive division furrow on the side of the cell with
the secondary oral axis while simultaneously undergoing typical oral replacement on the side with the primary oral axis. In contrast, CU-127 cells entering
stationary phase generally undergo replacement only of the primary OA, while
the secondary OA is simultaneously resorbed without being replaced. This is
probably one reason for the low proportion of secondary OAs in stationary
phase cells.
(d) Relation of secondary OAs and double CVP sets to cell size and number o
ciliary meridians.
CU-127 cells in predivision stages of oral development [stages 1-5, see Frankel
& Williams (1973) and section 4 below] were scored for OA and OP number
and cell length and width. The results, presented in Table 3, show that the
expression of secondary oral structures is differently related to cell size and to
178
M. JERKA-DZIADOSZ AND J. FRANKEL
Table 3. Association between ciliary meridian number, cell size, and number
of oral structures in CU-127 cells cultured in 2%PPY
Ciliary imeridian no.
Cell width
Cell length
configuration
Mean
±S.D.
n
Mean
±S.D.
n
1 OA-1 OP
1 OA-2 OP
1 OA Total
22-56*
23-20*
±0-71
±0-77
32
15
47-25
48-90
47-88t
±2-51
±3-83
±3-16
32
20
52
r
±S.D.
n
±2-77
±3-28
±2-96
32
20
52
±3-07 22 35-50
±3-52
53-74
53-05
±3-86 22 34-56
±4-59
±3-57 44 35-07$ ±4-07
53-39|
* Ciliary meridian no. , 1 OPv. 2 OP; t, = 2-44, 0-025 > P > 001.
t Cell length, 1 OA v. 2OA; / = 800, P < 0001
$ Cell width, 1 OA v. 2OA; / = 3-90, P < 0001
22
22
44
2 OA-1 OP
2 OA-2 OP
2 OA Total
22-80*
2305*
±0-98
±0-97
21
19
Mean
3200
32-67
32-26$
number of ciliary meridians. Cells with two OAs are significantly longer and
wider than cells with one OA. Within each OA class there is no association
between number of OPs and cell size. Increased size is therefore at most
a consequence, and not a cause, of stomatogenic activity along the secondary
oral axis.
Cells with two OAs did not, in this experiment, have a significantly greater
average number of ciliary meridians than cells with one OA. Instead, cells with
two oral primordia had a number of ciliary meridians that was slightly but
significantly greater than cells with one OP. Hence, development of a secondary
OP may sometimes be associated with a gain of one or occasionally more ciliary
meridians. Other data (Table 5) suggest that cells double for both OAs and CVP
sets have (on the average) about one more ciliary meridian than cells that are
single for both characteristics.
3. Cytogeometry of CU-127
(a) Location of the secondary OA
The secondary OA, when present, appears to a first approximation to be
located 180° opposite to the primary OA (Fig. 1). A more careful examination
indicates that this is not strictly true. To quantitate the relative positions of the
OAs, we will follow a convention whereby the primary oral axis is operationally
defined by the right postoral ciliary meridian of the primary OA, and the
secondary oral axis is similarly defined by the right postoral meridian of the
secondary OA.
If one assumes equal spacing of ciliary meridians, then cells in which the two
OAs are located precisely opposite each other on the cell circumference should
be separated by an equal number of ciliary meridians on both sides. For
Mirror-image duplication in Tetrahymena
179
Table 4. Relative location of primary and secondary OAs in CU-127
cells with 2 OAs
Ciliary meridian number
A
Number
of
Total
Right*
Left*
cells
20
21
22
10
10
10
11
10
11
11
12
11
12
13
10
11
12
11
13
12
13
12
14
13
13
1
2
6
7
2
38
35
24
2
12
1
23
24
25
26
The data in this table are based on those of the cells from experiments 1 and 2 (cf. Table 5)
in which two OAs are present and the position of both can be ascertained.
* 'Right' and 'left' refer to distances between the primary and secondary oral axes
measured in intermeridional intervals to the right and left respectively of the primary oral
axis. For conventions of measurement, see the text.
example, in a cell with 24 ciliary meridians, the secondary oral axis should be
12 intermeridional spaces distant from the primary axis in both directions. Such
equality is observed in slightly fewer than 50 % of the cells with an even total
number of ciliary meridians (Table 4). In the remainder, the distance in ciliary
meridians between the primary and secondary oral axis is less when measured
to the right of the primary axis than when measured to the left; for example,
most cells with 24 ciliary meridians have 11 intermeridional intervals between
the two oral axes measured to the right of the primary axis, and 13 intermeridional intervals between the two oral axes measured to the left of the
primary oral axis (Table 4). In cells with an odd total number of ciliary meridians,
equality of distance between oral axes on the two sides is inherently impossible,
and in these cases all cells manifest a slightly shorter distance between oral axes
to the right of the primary axis than to the left of the primary axis (Table 4).
An opposite inequality (shorter distance to the left of the primary axis than to
the right) was never observed. In the experimental series summarized in Table 4
most cells with 2 OAs had 11 or 12 intermeridional intervals on the side of the
cell to the right of the primary oral axis (the side bearing the CVPs). In the much
later examination of CU-127 (111.), when virtually all cells possessed 22 ciliary
meridians (cf. Table 7), 90 % of the cells with 2 OAs manifested 10 intermeridional intervals on the side of the cell to the right of the primary oral axis,
12 on the left. It thus appears that some rather precise geometric instructions
180
M. JERKA-DZIADOSZ AND J. FRANKEL
Table 5. CVP cytogeometry of CU-127
Number of
Position of CVP sets*
Ciliary merid.
Line
Expt.
no.
Culture
conditions
CU-127
CU-127
CU-127-1
CU-127-1
CU-127
CU-127
CU-127-1
CU-127-1
CU-127
CU-127
CU-127-1
CU-127-1
CU-127
CU-127
CU-127-1
CU-127-1
la
lb
2a
2b
la
lb
2a
2b
la
lb
2a
2b
la
lb
2a
2b
% PPY-log-28c
% PPY-log-t
% PPY-log-28c
-log-t
%PPY- log-28°
%PPY- •log-t
% PPY •Iog-28C
%PPY- log-t
% PPY-log-28°
% PPY-•log-t
% PPY-•log-28°
% PPY- log-t
% PPY- log-28°
%PPY- log-t
%PPY- log-28°
X PPY- log-t
CVP
OAs sets Mean +s.D. CVP1*
2
22-66
22-50
23-23
22-56
23-52
22-40
24-14
23-75
22-96
2
2
2
2
2
2
2
22-82
23-76
23-56
23-90
23-17
24-09
23-84
±1-20
±0-62
±0-65
±1-22
±1-13
±1-13
±0-37
±0-44
±1-13
±0-86
±0-60
±0-58
±0-75
±0-86
±0-94
+ 0-37
CVP CVP2*
Sample
size
79-8"
84-5°
76-7°
861°
84-8°
76-1°
85-8°
83-8a
62-9°
52-7°
60-9°
54-5°
56-3°
52-2°
61-4°
55-3°
24
18
26
25
17
15
7
16
(89-7")§ 116-5°
(821°)
(85-63)
(83-3°)
(79-93)
(790°)
(86-6°)
(82-3°)
111-5°
110-4°
112-1°
31
28
25
25
103-5°
105-8°
111-8°
109-3°
22
28
11
19
* For conventions of measurement, see Fig. 8 and text.
t Grown to mid-log phase at 28 °C, followed by exposure to 39-5 °C for 5-5 h, followed by return to
28°Cfor 1-5 h, then fixed.
t Grown to mid-log phase at 28 °C, followed by exposure to 39-5 °C for 4 h, then fixed.
§ Midpoint between two CVP sets indicated in parentheses. See Fig. 8.
must govern the positioning of the secondary OA relative to the primary OA
such that the secondary OA is seldom placed much more or less than 170° to
the right of the primary OA.
(b) Arrangement of CVP sets
One of the unique features of the CU-127 clone is the location of its two CVP
sets on the cell circumference. Positions of CVP sets analyzed in a set of four
experiments are tabulated according to numbers of OAs and of CVP sets present
in the cell (Table 5). The measurements are expressed in terms of the central
angle convention of Nanney (1966a). Computation of this angle is made with
reference to a plane extending from the central longitudinal axis of the cell to
the right postoral meridian of the primary oral axis. The 'central angle' is
measured at the intersection of this plane and a horizontal line extending from
the central axis to the midpoint within the CVP set (for conventions used in ascertaining this midpoint, see Nanney, 1966a). The central angle is thus a measure
of the position of each CVP set on the cell circumference relative to an oral
axis. Three further remarks must be made about this measure, (a) Measurement
Mirror-image duplication in Tetrahymena
OA-2
181
OA-1
OA-1
CVP-?*-—1—•" CVP-1
OA-1
<CVP>
CVP
CVP-2
(CVP)
CVP-1
Fig. 8. Diagrammatic representation of various geometric possibilities of positions
of OAs and CVPs in CU-127 cells. The dashed vertical line in the diagram of the
cell represents the central longitudinal axis of rotation. A horizontal circle embracing
the cell circumference is shown in the posterior region of the cell, and is projected
outwards to diagrams of four configurations of OAs and CVPs. These four configurations, from left to right, correspond to the four classes from top to bottom
(1 OA-1 CVP, 2 OA-1 CVP, 1 OA-2 CVP, 2 OA-2 CVP) in Table 5. The labelling
of OAs and CVPs follows the same conventions as in Tables 5 and 7, and the angles
a and /? are those tabulated in Table 7 (Group 1).
is to the right of the primary oral axis, (b) Measurement of surface position of
CVP sets is made by counting ciliary meridians, not by measuring actual surface
distances, so the unit of distance is thus intermeridional intervals, not micrometers. As ciliary meridians appear to be uniformly spaced around the cell
circumference, the former is proportional to the latter, (c) The 'central angle'
convention is purely descriptive, and carries no mechanistic implications other
than indicating that there is some general proportionality of positioning (see
Nanney, 1966 a).
All CVPs in CU-127 cells are situated in the space between the two oral axes
that is to the right of the primary axis. As indicated earlier, either one or two
sets of CVPs are present within this space. In CU-127 cells with only one set of
CVP's, the average position of the midpoint of that set with respect to the plane
defined by the primary oral axis is 82°, or 23 % of the entire cell circumference
(Table 5, Fig. 8). This is very close to the 75-80° (20-22 %) characteristic of
normal T. thermophila (Nanney, 1967b\ Nanney et al. 1975; Frankel, 1972). In
CU-127 cells with two sets of CVPs, the average midpoint of the set closer to
182
M. JERKA-DZIADOSZ AND J. FRANKEL
Table 6. Position of newly established CVP sets
Position of CVP sets
Type of cell
Number
of OAs
Anterior component of divider
1
Non-dividing cell (control)
2
1
2
CVP1
51-4°
(51-4°)
45-0°
50-6°
51-3°
CVP 2
Sample
size
109-8°
(109-3°)
100-9°
100-6°
98-4°
28
(23)*
12
28
13
* The subset in which the posterior as well as the anterior component of the dividing cell
has only one OA.
The data in this table are based on a single sample of CU-127 (111.) that was grown at
28 °C in a flask culture of 1 % PPY and fixed in mid-log phase. Only cells with 2 CVP sets
are included.
the primary oral axis (CVP set 1) is situated at a position described by a central
angle of about 55°, and that of the other (CVP set 2) at an angle of about 110°,
both measured to the right of the primary oral axis (Table 5, Fig. 8). The mean
halfway point between these two CVP sets (Table 5, values in parentheses) is
about the same as the average position of the CVP midpoint in those cells with
only one CVP set.
It should be stressed that the average CVP positions tabulated in Table 5 and
depicted in Fig. 8 do not faithfully represent the positions encountered in each
individual CU-127 cell. The distribution of locations of CVP sets is quite broad.
When there are two CVP sets they may be close together or far apart. When there
is only one CVP set, it may be positioned about halfway between the two OAs
or it may be positioned off-center, occasionally even at a location typical for
either CVP set in those cells that have two sets. However, the distribution of
positions of CVP sets in cells with one CVP set is broadly monomodal, with 76 %
of the CVP sets situated between 65° and 95° to the right of the primary oral
axis. In cells with two CVP sets, 74 % of these sets are located outside of these
limits. Hence the geometrical construct of Fig. 8, though idealized, is not
artifactual (see also Fig. 5).
Both the means and distributions of CVP locations indicate that the positions
of CVP sets are unrelated to whether or not a secondary OA is present (Table 5).
However, the measurements described thus far were made on cells with established CVP sets, and it is possible that sets of CVPs initially formed in a cell
with two OAs might subsequently come to reside in a cell with only one OA,
either by resorption of the secondary OA or by original formation of CVPs in
an anterior division product with two OAs followed by subsequent segregation
of these CVPs at the next fission into a posterior division product of a cell that
had formed only one oral primordium. For this reason, when CU-127 (111.) was
examined, the CVP cytogeometry of dividing as well as of non-dividing cells was
Mirror-image duplication in Tetrahymena
183
Table 7. Relational geometry of CVPs in CU-127 cells with 2 OAs
and 2 CVP sets
CVP distances
A
Group*
1
11
Ciliary
OA 1 to CVP 1
m priH —
111CI l i i
A
ian
no.
Sample Mean
size distancef
OA 2 to CVP 2
\
1
A
S.D.
fit
—
]
—
—
±0-95
±0-76
+ 0-96
57-2°
65-3°
64-5°
69-8°
64-7°
2
15
19
7
44
1
2
5
2
10
1
4
10
1
16
—
63-1°
—
62-8°
1
28
—
29
—
1
—
1
—
3
1
4
S.D.
at
Mean
distancef
21
22
23
24
25
23-69
1
4
21
34
10
70
2-75
3 04
3-41
3-63
3-80
3-54
±0-58
±0-55
±0-50
±0-92
49.70
53-4°
54-5°
54-7°
53-8°
4-25
3-50
417
4-30
4-65
4-26
20
22
24
22-00
1
32
1
34
2-75
2-81
4-75
2-87
±0-63
—
46-0°
—
46-9°
3-25
3-86
3-75
3-84
±0-96
±0-50
—
a < p a = fi a > fi
* Group I includes those of the 2 OA-2 CVP set cells from expts. 1 and 2 (cf. Table 5) for which
complete data on positions of both OAs and CVP sets are available. Group II includes the 2 OA-2 CVP
set cells from the CU-127 (III.) sample (cf. Table 6). In Group 11 the anterior and posterior components
of dividing cells are included together with the non-dividing cells. Group II was fixed about 16 months
later than Group I.
t CVP distance as measured in intermeridonal intervals (to obtain CVP positions in the sense of
Nanney (1966a, 19676), add 1 to the CVP distances).
t 'Central angles' defined with reference to the right postoral meridians of OA 1 (a) and OA 2 (fi).
For clarification, see Fig. 8 and text.
assessed. The major question is whether, in those anterior moieties of dividing
cells which possess two CVP sets, the position assumed by these sets is affected,
by the presence or absence of oral structures along the secondary axis. The
results, presented in Table 6, show that (a) the average position of the two CVP
sets is similar in prospective anterior division products with newly formed CVPs
to what it is in non-dividing cells with established CVPs and (b) the location of
the two newly developed CVP sets is similar in cells with one and two sets of
oral structures. Secondary oral structures thus exert a minimal direct influence,
if any, on the positioning of the CVP sets to the left of the secondary oral axis.
If one switches one's frame of reference from the primary to the secondary
oral axis, one can construct a reference plane extending from the central cell
axis to the right postoral ciliary meridian of the secondary OA, and compute
the position of CVP sets to the left of that plane. When one examines the position
of the single CVP set of cells with 2 OAs and 1 CVP set in this manner, the
average distance of the CVP midpoint to the left of the secondary oral axis is
only slightly greater than the distance measured to the right of the primary
oral axis (data not shown). When a similar calculation is carried out for cells
184
M. JERKA-DZIADOSZ AND J. FRANKEL
9a
9b
Fig. 9 (a) A protargol-stained cell showing the primary OA and OP. M-l, M-2, and
M-3 indicate membranelles 1, 2, and 3 respectively. Note the posterior tapering of
M-l. UM = undulating membrane. The single macronucleus is seen out of focus in
the center of the cell, the dividing micronucleus is on the viewer's right. The bar
represents 10 /tm. (b) The arrangement of basal bodies in a normal, completed oral
apparatus. Redrawn from McCoy (1974). The bar represents 10 fim.
with 2 OAs and 2 CVP sets, it becomes apparent that CVP 2 is situated at
a somewhat larger distance to the left of the secondary oral axis (angle /?, Fig. 8)
than is CVP 1 to the right of the primary oral axis (angle a, Fig. 8) (Table 7).
The data in Table 7 are consistent with the assumption that positioning of
CVPs in CU-127 as in wild-type cells takes place through measurement of
relative rather than absolute distances, so that the 'central angles' remain the
same as number of ciliary meridians vary. This assumption received further
support from a limited analysis of progeny clones homozygous for the 'Janus'
allele that determines the unique geometrical confirmation described here but
allows considerable variation in number of ciliary meridians (Frankel &
Jenkins, 1979); janus cells with as few as 18 or as many as 31 ciliary meridians
differ little if at all from each other or from CU-127 in the relative placement
of OAs and CVPs.
Mirror-image duplication in Tetrahymena
185
4. Secondary oral structures
(a) Structure and function of secondary OAs
As mentioned earlier (Results, section \b), while primary OAs of CU-127
cells are indistinguishable from OAs of WT cells (Fig. 9), secondary OAs are
structurally abnormal. The abnormalities consist of varying combinations of
three basic defects: (a) absence of oral components, typically the undulating
membrane (see Fig. 9 b for normal components and their arrangement),
(b) reduced size or apparent fragmentation of components, and (c) reversed
asymmetry of components. A characteristic, though by no means universal,
combination of defects is present in the secondary OA shown in Fig. 1, in which
the undulating membrane is absent and the three membranelles manifest the
reverse of the usual right-left asymmetry, i.e. they slant from the anterior-left
to the posterior-right rather than the typical anterior-right to posterior-left
(cf. Fig. 9). A few abnormal OAs manifest an undulating membrane at the left
margin (Fig. 10) rather than the right margin as is normal. However, some
secondary OAs are nearly normal, whereas others are very tiny, with only one
or two membranellar fragments, suggestive of ongoing resorption.
The generally reduced size and commonly encountered abnormal configuration
of secondary OAs makes it relatively easy to distinguish primary and secondary
OAs in living cells, even when observed under bright field optics. When fed
with carmine powder in 1 % PPY medium, CU-127 cells with two OAs formed
food vacuoles only with their primary OAs. The membranelles of secondary
OAs appeared stiffly outstretched and did not beat properly. Secondary OAs
sometimes do possess a buccal cavity (Fig. 1) but lack the deep fibers described
by Nilsson & Williams (1966). It is not known whether they possess a food
vacuole forming region.
(b) Development of secondary oral primordia
A secondary OP initially appears as a field of basal bodies to the left of a ciliary
meridian (Fig. 10) or midway between two ciliary meridians (Fig. 11). If an
anterior secondary OA is also present, the ciliary meridian(s) in question can be
identified as the right postoral meridian (Fig. 10) or the right and left postoral
meridians for the above two situations respectively (the primary OP, like the
OP of WT cells, appears to the left of the right postoral meridian). In no case
did a secondary OP arise adjacent to the right side of a ciliary meridian, and in
all CU-127 cells the ciliary meridians were of the usual polarity and asymmetry,
with longitudinal microtubule bands to their right (Figs. 10, 11) and new basal
bodies forming anterior to old ones (Figs. 10-12). When a secondary OP was
present it appeared at the same cell latitude as did the primary OP. Except for
their common tendency to appear midway between two ciliary meridians, there
was nothing atypical revealed by light microscopy about the position or
186
M. JERKA-DZIADOSZ AND J. FRANKEL
Mirror-image duplication in Tetrahymena
187
geometry of secondary oral primordia at early stages of their development. The
secondary oral primordia were, however, invariably smaller than their primary
counterparts.
Oral primordia in late stages of differentiation often manifested reversed
right-left asymmetry. This can be seen by comparing protargol stained stage-5
secondary oral primordia (Figs. 12-14) with a similarly stained primary OP
fixed at an equivalent stage (Fig. 9 a). Reversed membranelles are seen in Fig. 12,
and reversed undulating membranes in Figs. 13 and 14. The reversed undulating
membrane in Fig. 14 bears a single row of cilia as does a normal undulating
membrane (Nilsson & Williams, 1966), and the cilia of that row are short, as is
characteristic of normal stage-5 oral primordia (Buhse, Stamler & Corliss,
1973; Ruffolo & Frankel, 1972). The subtler details of the reversed oral
primordia, less clearly evident on the photographs, suggest that the secondary
oral primordia, though defective, are true mirror-images of the complete primary
oral primordia. The cilia of the undulating membrane shown in Fig. 14 appear
on close examination to be attached to the outermost of the two staggered rows
F I G U R E S 10-14
Protargol-stained preparations of CU-127 cells, focused on the secondary oral
primordia. All photographs are printed in the same orientation, with the cell
anterior at the top of the page and viewer's right corresponding to cell's left. In each
micrograph the bar represents 10 fim.
Fig. 10. A cell with a secondary OA and an incipient (stage 1) oral primordium (OP).
Only the undulating membrane (UM) of the anterior OA is in focus. The OP is
situated to the cell's left of the right postoral ciliary meridian. Note that new
unciliated basal bodies within ciliary meridians near tbe OP are situated immediately
anterior to old ciliated ones (arrows). Longitudinal microtubule bands are faintly
visible to the right (viewer's left) of the ciliary meridians.
Fig. 11. A cell lacking a secondary OA, with a stage-1 secondary OP (thick arrow)
situated midway between two ciliary meridians. Thin arrows point out longitudinal
microtubule bands situated to the right of the two ciliary meridians on either side of
the OP.
Fig. 12. A cell with a differentiated (stage late-4) secondary OP. The two upper arrows
show the tapering of the first membranelle, with three rows of basal bodies at
the left-anterior end, two rows at the right-posterior end. The ciliary meridian to the
cell's right of the OP is interrupted by the membranelles, while the meridian to the
left of the OP remains continuous. The posterior arrow indicates a new unciliated
basal body anterior to an old ciliated one.
Fig. 13. A portion of a cell with a secondary OP. Although only tiny fragments of
membranelles remain, the well differentiated state of the undulating membrane
(TJM) and the equatorial discontinuities in the ciliary meridians indicate that this
is an advanced (stage 5) oral primordium, possibly undergoing resorption. Note
the diagonal couplets of basal bodies within the UM, and the posterior continuity of
the UM with the ciliary meridian situated to the cell's left of the remainder of the OP.
Fig. 14. A portion of a dividing cell, with a secondary OP. The undulating membrane
(UM) bears a single row of short cilia. Ciliated membranelles (out of focus) are
visible to the right of the UM.
188
M. JERKA-DZIADOSZ AND J. FRANKEL
a
e/
• f-
^
*•*•<&
y •••••• •••
15
Mirror-image duplication in Tetrahymena
189
of basal bodies, mirroring the analogous configuration of undulating membranes
of normal oral primordia. The third, outermost row of basal bodies within the
first membranelle terminates at a somewhat more anterior position than do the
other two rows (Fig. 9 b). This feature is readily observed in favorable protargol
preparations of stage-5 primary oral primordia (it is responsible for the 'taper'
of the first membranelle of the OP in Fig. 9 a) and is present in mirror-image in
those stage-5 secondary OPs that have well-developed first membranelles
(Fig. 12).
Examination of the geometrical configurations of basal bodies and cilia
during intermediate stages of development gives further indications of mirror
asymmetry of development of secondary oral primordia. During normal oral
development basal bodies within the OP become associated into couplets
(McCoy, 1974) that later organize into membranelles (initially double rows of
basal bodies) beginning at the anterior-left margin of the OP (Fig. 15). In
a secondary OP the couplets are formed but their association into membranelles
tends to occur in a less regular fashion than in a normal OP; however, there is
a definite tendency for differentiation of membranelles to begin at the anteriorright margin of the OP, with progression posteriorly and later to the left - the
mirror image of the normal geometry of membranelle assembly (Fig. 16).
Examination of ciliary outgrowth with scanning electron microscopy adds
further evidence for a geometrical pattern of development of secondary oral
primordia that often approximates a mirror image of the normal pattern. We
have confirmed in primary oral primordia the observations of Buhse et al. (1973)
that the three constituent basal body rows of membranelles sprout cilia in an
ordered sequence. Cilia grow out first on the innermost row, then the middle
row, and finally the outermost row of each membranelle. Figure 17(6) shows an
early stage of this process (at a time when there are still only two rows of basal
bodies within each membranelle) in a primary OP. The innermost, right row of
cilia within each membranelle is partly grown out, while the outer, second row
to its left consists only of short stubs. Figure 18(6) illustrates a secondary OP
in a similar stage of development: again, one sees nearly longitudinal rows of
partly grown cilia adjacent to rows of ciliary stubs, but now each row of stubs
Figs. 15 and 16. Photographs and drawings of protargol preparations of CU-127
cells focused on oral primordia in an early stage of formation of oral membranelles
(stage 3). Photographs are shown above camera lucida drawings of the same preparations. Orientation is the same as in Figs. 10-14. In each illustration the bar
represents 10 /tm.
Fig. 15 (a) A photograph of a stage-3 primary OP. The arrow indicates the region of
initiation of membranelle differentiation, (b) A camera lucida drawing of the same
preparation.
Fig. 16 (a) A photograph of a stage-3 secondary OP. The arrow indicates the region of
probable initiation of membranelle differentiation, (b) A camera lucida drawing of
the same preparation.
13
EMB
49
M. JERKA-DZIADOSZ AND J. FRANKEL
Mirror-image duplication in Tetrahymena
191
is to the right of the adjacent row of cilia. This same mirror-image pattern of
ciliary outgrowth in primary and secondary oral primordia is evident at a later
stage, after the third row of basal bodies has formed within each membranelle
(cf. Williams & Frankel, 1973) and the third, outermost row of cilia is growing
out. In a primary OP this outermost row of stubby cilia is seen on the leftanterior margin of each developing membranelle (Fig. 19); in a secondary OP
this short outermost row is on the right-anterior margin (Fig. 20). The pattern
of ciliary outgrowth in secondary oral primordia is somewhat less clear than in
primary primordia mainly because the membranelles are smaller and closer to
one another in the secondary primordia, with the third membranelle often
missing.
One further aspect of this geometric reversal of oral differentiation in secondary oral primordia becomes evident when one examines the relationship of the
developing oral primordia to the surrounding ciliary meridians. As mentioned
earlier, secondary as well as primary oral primordia typically (though not
invariably) begin their development to the left of a 'stomatogenic' ciliary
meridian. As differentiation of membranelles proceeds, normal oral primordia
tend to remain closely associated with that ciliary meridian (Fig. lid). At later
stages the portion of that meridian which is adjacent to the oral primordium
loses its cilia, merges with the primordium, and presumably participates in the
differentiation of the undulating membrane (Figs. 19, 21 a). Simultaneously, the
differentiating membranelles interrupt the continuity of the ciliary meridian
immediately to the left of the OP (Fig. 21 a). The evolving relationship between
secondary OPs and. the adjacent ciliary meridians is quite different. The differentiating secondary OP becomes dissociated from the 'stomatogenic' meridian
to its right (Fig. 18 a). Later, if substantial membranelles are formed, these
membranelles interrupt the continuity of the meridian to its right (Figs. 12, 21 b)
F I G U R E S 17 AND 18
Scanning electron micrographs of CU-127 cells, showing oral primordia in an early
stage of membranelle formation (stage early-4). Whole cells are shown above,
enlargements of their oral primordia below. Orientation of cells is as in the preceding
figures. The bars in the upper micrographs represent 10/tm, those in the lower
micrographs represent 1 /tm.
Fig. 17 (a) A cell with a primary OA and a stage early-4 primary OP. Note the close
association of the OP with the adjacent right postoral ciliary meridian, (b) Enlargement of OP. Three membranelles are developing in the anterior-left portion of the OP.
Each membranelle has one row of partly-grown cilia and one row of ciliary stubs
(arrows) to its anterior-left.
Fig. 18 (a) A cell with a stage early-4 secondary OP, and lacking a secondary OA.
Note the absence of close contact of the OP with the ciliary meridian to its right. An
associated CVP set is seen near the posterior end of the cell (arrow), (b) Enlargement
of OP. Membranelles are developing on the right side of the OP. Each membranelle
has one row of partly-grown cilia plus one row of ciliary stubs (arrows) to its right.
Developing membranelles are more closely packed together than in a normal OP.
13-2
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M. JERKA-DZIADOSZ AND J. FRANKEL
Mirror-image duplication in Tetrahymena
193
in much the same way that the membranelles of the primary OP interrupt the
continuity of the meridian to its left. The comparison of Figs. 21 (a) and (b),
representing primary and secondary oral axes of the same cell, indicates this
particularly clearly. The configuration on the other (left) side of the secondary
OP is more variable: in a few cases the meridian to the left of a secondary OP
abuts on a reversed undulating membrane (Fig. 13) much as the meridian to the
right of the primary OP does on the normal undulating membrane; more
typically, no well organized undulating membrane is produced by the secondary
OP, and the ciliary meridian to the left of the OP tends to remain uninterrupted
(Figs. 12,216).
It should be emphasized that the above account has deliberately emphasized
the cases in which development of the secondary OP is most complete and its
reversed character best manifested. Not all secondary oral primordia are of this
type. A tally of 36 silver impregnated cells with advanced (stage 5) primary oral
primordia that also possessed secondary oral primordia revealed that 14 of the
secondary oral primordia were right-left reversed much as described above, 4
were nearly normal (not reversed), 1 was ambiguous (with an undulating membrane on both sides), and 17 were highly incomplete or defective, suggesting either
retardation in development or ongoing resorption. An example of a secondary
oral primordium that would be scored as nearly normal is shown in Fig. 22,
while Fig. 23 indicates an 'ambiguous' primordium with nearly transverse
Figs. 19-20. Enlarged scanning electron micrographs of CU-127 oral primordia at a
late stage of membranelle formation (stage early-5). Orientation is as in preceding
figures. Bars represent 1 /*m.
Fig. 19. A primary OP in stage early-5. The three membranelles (M-l, M-2, and
M-3) possess two rows of partly grown out cilia apiece, plus one row of ciliary stubs
(open arrow) on the outermost (anterior-left) margin of each membranelle. The
undulating membrane (UM) is undergoing alignment of its single ciliary row.
Fig. 20. A secondary OP in stage late-4 or early-5. Only the first membranelle (M-l)
is fully developed, and the second membranelle (M-2) is short. Ciliary stubs (open
arrow) are situated on the anterior-right margins of these membranelles. The cilia
on the left side of the OP (right of micrograph) are not organized in the configuration typical of a UM, and may instead represent an abnormal membranelle.
Fig. 21. Two sides of the same CU-127 cell oriented as in preceding figures, (a) The
side with the primary oral axis. A normal OA is present. The OP is in stage late-4.
Membranelles are well differentiated and the UM is undergoing alignment of its
cilia. The two arrows indicate the anterior and posterior ends of the portion of the
left postoral ciliary meridian that is interrupted by the membranelles of the OP.
(b) The same cell rotated to reveal the secondary oral axis. An OA is lacking. The
OP is highly abnormal, with one irregular reversed membranelle situated between
the arrows that delineate the portion of the ciliary meridian to the right of the OP
that is interrupted. Note that the ciliary meridian to the left of the OP (extreme
right of the photograph) bends around the OP without any interruption. Bars
represent 10 /*m.
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M. JERKA-DZIADOSZ AND J. FRANKEL
23
F I G U R E S 22 AND 23
Enlarged scanning electron micrographs of secondary CU-127 oral primordia at late
stages of membranelle formation. Orientation is as in preceding figures. The bars
represent 1 /im.
Fig. 22. An OP with three small membranelles (M-l, M-2, and M-3) that are approximately transverse in orientation. The clump of short cilia on the right side of the OP
might represent a partly organized undulating membrane (UM).
Fig. 23. An OP with two membranelles (M-l, M 2), oriented somewhat more transversely than is normal. Remaining cilia are scattered on both sides of the posterior
portion of the primordium, suggesting ambivalence in positioning of the undulating
membrane.
membranelles and indications of undulating membrane development on both
sides.
In many cases in which secondary oral primordia were small or development
was apparently arrested, ciliary meridians to the right and left of the oral
primordia both remained uninterrupted.
Whereas only a small proportion of differentiating secondary oral primordia
are geometrically nearly normal, at least one-half of the fully developed
secondary OAs have membranelles with normal or near-normal orientation.
This difference is probably due mainly to selective resorption of the most
abnormal oral structures; however, some reorientation of membranelles may
Mirror-image duplication in Tetrahymena
195
also take place during late stages of oral development. Advanced oral primordia
with 'ambiguous' orientation, as in Fig. 23, may be undergoing such
reorientation.
DISCUSSION
(A) Demonstration of a reversed cortical field
The CU-127 clone is characterized by unique abnormalities in the structure
of oral apparatuses (OAs) and in the positioning of contractile vacuole pores
(CVPs) juxtaposed on structurally normal ciliary meridians. We will argue that
the characteristic abnormalities of this clone are manifestions of a propagated
morphogenetic field of reversed asymmetry. Abnormalities of the pattern of
oral development in Tetrahymena that have previously been observed (Frankel,
1964; Frankel, Nelsen & Jenkins, 1977 and unpublished; Kaczanowski, 1976;
Nelsen, 1970) did not manifest any change of the fundamental asymmetry of
the OA. In CU-127 cells, however, oral primordia (OP) frequently develop with
a clear reversal of left-right asymmetry. Although such oral structures were not
analyzed at the ultrastructural level, examination of features such as the relative
lengths of oral membranelles and of ciliary rows within membranelles suggests
that the reversal is a true situs inversus; i.e. the abnormal oral primordia cannot
be rotated so as to superimpose them on the normal ones. Although the basis of
this situs inversus is itself unknown, it is independent of the structural asymmetry of the ciliary meridians. The ciliary meridians adjacent to the reversed
oral primordia are characterized by normal configurations of accessory microtubule bundles, and by normal polarity of basal body proliferation. A further
symptom of the normal morphogenetic influence of these ciliary meridians is
that oral primordia destined to become abnormal initially develop adjacent to
the normal (left) side of a 'stomatogenic meridian', and only become reversed
during the later development of membranelles. The reversed oral primordia
are never complete, and there are intermediate cases that are suggestive of
a conflict between two incompatible systems of pattern control. The resulting
abnormal OAs are then readily resorbed. The resorption is probably a nonspecific response to abnormality of oral structure (cf. Frankel, 1964; Nelsen,
1970).
CU-127 cells produce both abnormal OAs and also normal ones that are in
no known way different from OAs of wild type cells. The two types of OAs are
spatially segregated from one another. The fully normal OAs are generated
along one longitudinal axis, here called the 'primary axis'. Stomatogenesis
never fails along this axis, and the OAs produced there are always normal.
Abnormal OAs are generated only along a 'secondary axis' that is almost but not quite - opposite to the primary axis. The secondary axis is visibly
abnormal in two ways, first in the nature of the OAs produced there (never
completely normal, frequently reversed), and second in the sporadic manifestation of stomatogenic activity. Oral structures may be completely absent along
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M. JERKA-DZIADOSZ AND J. FRANKEL
the secondary axis of a dividing cell, or there may be an OA but no OP, or an
OP without an OA, or both an OA and an OP. The production of oral structures
along the secondary axis appears to reflect a dynamic state, with a probability
of stomatogenesis during division that is always less than one, and a probability
of resorption of previously formed OAs that probably depends on the degree of
abnormality, and perhaps also the age, of the OA.
Although the expression of oral development along the secondary axis is
variable, the location of that axis as indicated by the circumferential position
of OAs that do develop is remarkably constant. This suggests the existence of
a continuously propagated morphogenetic field which underlies the secondary
structures. The mode of expression of double CVP sets provides further
evidence for the constant existence of such a field, and the geometry of this
expression as well as that of the secondary OAs indicates that this field is
reversed in CU-127 cells. A clear understanding of this conclusion requires
a comparison of CVP cytogeometry in CU-127 and wild-type cells. In wild-type
T. thermophila (= T. pyriformis, syngen 1) the position of the CVP midpoint is
at an angle of close to 80° to the right of the oral axis as defined by the right
postoral ciliary meridian (Nanney, 19676; Frankel, 1972). When the position
of the oral axis undergoes ' slippage' the location of the CVP set undergoes
a corresponding shift (Nanney, 1967a). This geometrical association, as well as
the fact that CVP positioning involves assessment of & proportionate rather than
an absolute distance around the cell circumference, led to the notion that oral
and CVP positioning are coordinately controlled by a morphogenetic field of
some kind (Nanney, 1966a, 1967a). Homopolar doublets of genotypically wild
type cells behave as two separate (Nanney, 1966a) though not entirely independent (Nanney et al. 1975) domains with respect to CVP positioning, with
each component 'semicell' of the duplex positioning its CVP set as if it were an
entire singlet cell. The angle between each of the two similar oral axes of a
typical Tetrahymena doublet and the corresponding CVP midpoint is thus close
to 40° of the approximately 180° width of the semicell, rather than 80° of the
360° circumference of the duplex (Fig. 24, top). The CVP cytogeometry of
CU-127 cells with one CVP set is remarkably similar to that of wild-type cells,
provided that one ignores the secondary OAs (Fig. 24, bottom left). However,
CU-127 'doublets' (cells with two OAs and two CVP sets) manifest a remarkable difference from wild-type doublets: in CU-127 cells there is one CVP set
associated with the primary OA as in a typical doublet, and a second CVP set
that is associated with the secondary OA, but invariably to its left rather than its
right (Fig. 24, bottom right). The reversed direction of CVP determination in
CU-127 doublets suggests that these cells might have a morphogenetic field
underlying the secondary oral axis that is of reversed, handedness.
We are now in a position to demonstrate why the postulated morphogenetic
field underlying the secondary oral axis must be continuously propagated in
a manner that is independent of its expression. First, the expression of double
Mirror-image duplication in Tetrahymena
Singlet.
197
Doublet
CVP-2
WT
OA
OA-2
OA-1
CVP-1
CVP
OA-1
OA-1
(OA-2)
CVP-2
CVP-1
CVP
Fig. 24. Comparison of CVP cytogeometry of wild type (WT) and CU-127 cells with
one and two sets of CVPs. Illustrative conventions are as in Fig. 8. Configurations
of WT cells are shown in the top half of the diagram, those of CU-127 cells in the
bottom half; cells with single CVP sets on the left, those with double sets on the right.
The angles indicated are averages for cells with a total of 24 ciliary meridians. Data
for W.T. are those of Nanney et al. (1975) for 13 +13 row doublets extrapolated to
12 + 12 row doublets; data for CU-127 are from Table 7, with the angle between
OA 2 and CVP 2 modified as indicated in Fig. 25 and accompanying text.
sets of CVPs and of secondary OAs are largely independent of each other
(Results, section 2a). Second, the positions of CVP sets, whether one or two,
are the same irrespective of whether or not secondary OAs are present (Results,
section 3 b and Tables 5 and 6). These two observations rule out any direct
influence of secondary OAs themselves on positioning of CVPs and strengthen
the conclusion that the secondary OAs and the associated CVP positions are
both separate manifestations of a reversed underlying morphogenetic field.
A corollary of this conclusion is that the reference point(s) for the cell longitudes
at which CVPs and probably also new oral structures are positioned are not the
OAs themselves, though, spatially correlated with them when they are present.
Is the mutual arrangement of these two morphogenetic fields completely
symmetrical? The results summarized in Table 7 suggest a negative answer, as
the average distance from the secondary oral axis to the midpoint of the second
CVP set is greater than the average distance from the primary oral axis to the
midpoint of the first CVP set. However, in the Results (section 3 b) the oral axes
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M. JERKA-DZIADOSZ AND J. FRANKEL
14
13
—/OA-1
Fig. 25. The bilateral symmetry of CU-127 cells with two OAs and two CVP sets.
(a) A schematized polar projection of a CU-127 cell with 23 ciliary meridians, 2 OAs,
and 2 CVP sets viewed from above so that the anterior end is in the center. The
primary oral axis is on the viewer's right side of the diagram, the secondary oral
axis on the viewer's left. Oral primordia are shown undergoing membranelle
development. Ciliary meridians are represented as rows of dots (basal bodies)
radiating from the center (cell apex). The average positions of the midpoints of the
two CVP sets on the cell circumference are indicated by blackened circles. The two
oral reference axes are shown as dashed lines, the primary axis along meridian no. 1,
the secondary axis between meridians 11 and 12 (see text), (b) A more highly
schematized representation of the bilateral symmetry of the cell shown in (a). The
two oral axes and the positions of the two CVP sets are shown. The plane of bilateral
symmetry is indicated by the heavy vertical line.
were arbitrarily defined by the positions of the right postoral meridians. If we
instead consider the actual course of development in the two OAs, the right
postoral ciliary meridian of the secondary oral axis is not equivalent to the
corresponding ciliary meridian of the primary oral axis. Due to the right-left
reversal of membranelle development, the membranelles of the secondary OP
come to abut upon the right postoral meridian of the secondary axis much as
the membranelles of the primary OP abut on the left postoral meridian of the
primary axis (Figs. 21, 25 a). The longitudinal axis that has the same geometrical
relation to the secondary oral structures as the right postoral meridian does to
the primary oral structures is most likely situated approximately midway
between the ciliary meridians to the right and left of the secondary OP. The
positions of these two equivalent axes are shown by the heavy dashed lines in
Fig. 25(a). Using this construct, the secondary oral axis is now approximately
one-half of an intermeridional interval to the left of where it had earlier been
Mirror-image duplication in Tetrahymena
199
placed. This, then, provides a justification for subtraction of 0-5 from the
positions of CVP sets located to the left of the secondary axis, lowering the
average distances from the 4-26 and 3-84 recorded in Table 7 to 3-76 and 3-34,
both not much greater than the respective mean distances of 3-54 and 2-87
from the primary oral axis to its associated CVP set (this adjustment was
already incorporated into Fig. 24). This correction allows us to schematize
a CU-127 cell that has two OAs as being bisected by a mirror-plane separating
two bilaterally symmetrical halves (Fig. 25b). These halves may be considered
as two separate and mutually symmetrical domains under the control of the
morphogenetic fields underlying the respective oral axes, one normally directed
and the other reversed. The two halves are also capable of manifesting some
physiological independence, as when one half attempts to divide while the other
undergoes oral replacement following a shift to starvation conditions (Results,
section 2 c).
The geometrical idealization shown in Fig. 25 requires one important qualification with regard to position of CVP sets. Many CU-127 cells, even those
with two OAs, have only one CVP set, at a position that is the same as in normal
singlets. In those CU-127 cells that have two CVP sets, the average positions of
these sets are further away from the oral axes than they are in typical WT
doublets (Fig. 24, bottom right). However, these averages conceal a broad range
of variation in CVP positions, with reference angles ranging from 35 to 75°.
CU-127 cells with two CVP sets can therefore be viewed as expressing a continuum of cytogeometric states with respect to positioning of CVPs, ranging
between the limits of a 'doublet' condition with the reference angles typical of
WT doublet cells (but with one of the angles reversed in direction) to a condition
closely approaching a 'singlet' state (e.g. with only one ciliary meridian separating the two CVP sets). Then, the apparent 'singlet' state of CU-127 cells
could be viewed as an actual doublet state in which the two oppositely directed
reference angles reach a limit at which the two sites of CVP determination
become superimposed, so that only one CVP set can be scored. In fact, such sets
often display clear indications of actual doubleness, such as the appearance of
CVPs along four adjacent ciliary meridians, a condition never found in wild-type
cells yet one which has to be scored as a 'single' set according to our objective
scoring rules. On this view, the true percentage of double CVP sets in CU-127 cells
may really be 100 %; what varies is the type of expression of these two sets, depending on a varying degree of interaction between the two underlying morphogenetic fields. When there is no interaction, each system operates as if the other
did not exist, and thus positions CVPs at an angle of about 85°, as in singlet
cells (but with one angle reversed, hence the resulting superimposition). When
there is maximal interaction, the cell is 'divided up' between two non-interpenetrating morphogenetic fields as in Fig. 25(b), and the cells's CVP cytogeometry is the same as that of a wild-type homopolar doublet cell except for
the reversed handedness of one of the fields. Somewhat surprisingly, the
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M. JERKA-DZIADOSZ AND J. FRANKEL
strength of the interaction between the two morphogenetic fields as manifested
by the varying CVP reference angles is largely independent of the expression of
oral development along the secondary axis.
(B) Duality of control of cortical asymmetry
The reversal that generates (or possibly reflects) bilaterality of cellular
organization in CU-127 cells affects asymmetry of OAs and the large-scale
aspect of the positioning of the CVP sets which was earlier shown to be relationally determined (Nanney, 1966a). It is essential to note that the bilaterality is
not all-encompassing, as it does not extend to the structure of the ciliary
meridians, to the side of the meridian on which the OP first appears, and to the
'fine positioning' of CVPs relative to adjacent ciliary meridians (cf. Ng, 1977).
Thus, one aspect of cell organization is reversed in one cell-half while another
aspect remains normal. This dissociation demonstrates that the two aspects of
cell organization are to a considerable degree independent of each other. Another
consequence of this same independence is presented in the accompanying paper
(Frankel & Jenkins, 1979), in which the kinetics of expression of cortical reversal
and of change in number of ciliary meridians are shown to be mutually
independent following acquisition or loss of homozygosity for the controlling
janus gene. We therefore suggest that what remains normal in the CU-127 clone
is a short-range positional system directly associated with ciliary meridians,
whereas the aspect subject to reversal is a long-range system comparable to
the 'gradient-fields' operating in multicellular development.
We should finally note that the existence of a dual system of positional
controls in ciliates is supported by substantial earlier evidence derived mainly
from microsurgical studies of larger ciliates (e.g. see Jerka-Dziadosz, 1974;
Frankel, 1974). The experimental result that most closely approximates the
geometrical situation in thd CU-127 clone is an operation performed by Uhlig
(1960) in which the development of reversed oral structures was brought about
within a context of normal ciliary meridians (see interpretation in Frankel,
1974, pp. 466-467). In Stentor the reversal of oral asymmetry is demonstrably
related to a right-left reversal of the 'zone of stripe contrast' (Tartar, 1956,
1960) that is thought to be a manifestation of an underlying circular gradient
(Uhlig, 1960). Two greatly different methods of experimentation applied to
Tetrahymena and Stentor respectively have thus supported remarkably similar
conclusions.
The authors would like to thank Mr Richard J. Hollis for suggesting one of the experiments, and Drs J. Wynne McCoy and Stephen F. Ng for advice concerning analysis and
interpretation of the data. We also wish to express our appreciation for helpful criticisms of
various drafts of the manuscript by Drs Karl Aufderheide, Giinter Cleffmann, Anne W. K.
Frankel, Krystina Golinska, Leslie M. Jenkins, Andrzej Kaczanowski, Denis Lynn,
J. Wynne McCoy, David L. Nanney, E. Mario Nelsen, Stephen F. Ng, and Norman E.
Williams. This research was supported by grant no. HD 08485 from the U.S. National
Institutes of Health.
Mirror-image duplication in Tetrahymena
201
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{Received 18 July 1978, revised 26 September 1978)