/ . Embryol. exp. Morph. Vol. 42, pp. 261-274, 1977
Printed in Great Britain © Company of Biologists Limited 1977
261
Proportional control of
organelle position by a mechanism which similarly
monitors cell size of wild type and conical
form-mutant Tetrahymena
BY DENIS H. LYNN 1
From the Department of Zoology, The University,
St Andrews, Scotland
SUMMARY
Distance between mouthparts of dividing cells of wild type and conical form-mutant
Tetrahymena thermophila (formerly T. pyriformis syngen 1) is directly proportional to cell
size. This distance is related to cell length in both wild type and conical cells although the
proportionality is different in each cell type. However, for both wild type and conical cells
the distance between mouthparts is directly and similarly proportional to the product of
cell length and cell width which is an estimate of cell size. Evidence has been obtained
which suggests that the new mouthparts are positioned with reference to the anterior
mouthparts rather than to either pole of the cell. Determination of the site of the new
mouthparts is not related to the number of basal bodies between the two sets of mouthparts.
INTRODUCTION
Analysis of pattern formation in ciliates has been largely confined to dorsoventrally flattened ciliates such as the hypotrichs Euplotes and Urostyla and
the cyrtophorine Chilodonella. In these ciliates, the field boundaries are considered coincident with the border between dorsal and ventral surfaces. The
development of cirral primordia in Urostyla altered by microsurgery (JerkaDziadosz & Frankel, 1969; Jerka-Dziadosz, 1974, 1977), the spatial distribution of ciliary units on the dorsal surface of Euplotes (Frankel, \915a), and
positioning of contractile vacuole pores in Chilodonella (Kaczanowska, 1974,
1975) have been explained with reference to these boundaries. However, it is
difficult to define field boundaries in 'radially' symmetrical ciliates such as
Tetrahymena and Paramecium (see Nanney, 1968; Sonneborn, 1974; Frankel,
1974). The interaction of positional signals (e.g. diffusing morphogens)
emanating from one or several reference points could be sufficient to specify
the pattern of cortical structures in these ciliates.
1
Author's address: Department of Zoology, University of Guelph, Guelph, Ontario,
Canada, NIG2W1
262
D. H. LYNN
It has been established that dividing Tetrahymena position the new oral
primordium or mouthparts in a regulative manner, proportional to the body
or cell length (Lynn & Tucker, 1976). This relationship was demonstrated with
log dividers and first and second post-starvation dividers of Tetrahymena
corlissi. Since these three types of dividers are similarly shaped, it is not possible
to test whether body length or overall body size (e.g. surface area, volume,
biomass) is the determining parameter nor what the reference points for oral
primordium positioning are. Genetically related ciliates of different shape
would be necessary for this purpose. Doerder, Frankel, Jenkins & De Bault
(1975) have isolated a form-mutant of Tetrahymena thermophila. This formmutant, named conical, results from the action of a single recessive gene which
changes the overall cell shape from ovoid to conical and places the anterior
mouthparts on average further from the anterior pole. The gene apparently
does not affect cortical characteristics such as number of ciliary rows, number
of ciliary units within these rows, and positions of contractile vacuole pores
(Doerder et al. 1975). If it is assumed that this gene also does not affect the
mechanism responsible for positioning the oral primordium, then it is possible
to test the hypothesis that primordium position is determined with reference
to overall body size rather than to some single linear parameter such as cell
length.
This report will demonstrate that similar proportional positioning of the
oral primordium of both wild-type (co+) and conical (co) cells occurs both as
a function of cell length and, more importantly, as a function of cell size
estimated by the product of cell length and cell width. The reference point
for the measurement is apparently the anterior mouthparts rather than the
anterior pole of the cell.
MATERIALS AND METHODS
Culture techniques
Tetrahymena thermophila (formerly Tetrahymena pyriformis syngen 1, see
Nanney & McCoy, 1976) strain D was obtained from Dr D. L. Nanney and
cultured axenically in 2-0 % proteose-peptone with 0-5 % yeast extract (PP) or
in tryptone-dextrin-vitamin-salts (TS) medium (Frankel, 1965) at 28 °C. Cells
in TS medium were sampled only once in mid-log phase, while cells in PP
medium were sampled repeatedly from mid-log to early stationary phase.
Tetrahymena corlissi strain WT, clone TC-2, was cultured axenically in 2-0 %
proteose-peptone and 0-1 % yeast extract. The methods for obtaining log
dividers and first and second post-starvation dividers of T. corlissi have been
described (Lynn & Tucker, 1976).
Pattern regulation in Tetrahymena
263
Staining and measurement
Both T. thermophila and T. corlissi were stained by the Chatton-Lwoff wetsilver procedure, following the directions of Frankel & Heckmann (1968) for
T. thermophila and those of Corliss (1953) for T. corlissi.
Measurement of silver-stained organisms was performed in two ways. Dr
J. Frankel generously supplied the original data obtained during the description
of the co mutant (Doerder et al. 1975). Measurements of cell proportions of
one sample of each genotype, cultured in TS medium, were made with an
ocular micrometer as described in the original paper.
Measurements of the PP cultured T. thermophila and T. corlissi were made
using a Leitz filar ocular micrometer mounted on a Leitz Ortholux microscope.
For T. thermophila, cell proportions were measured from three (co+) or four
(co) slides representing cells fixed in middle to late log phase. The distances
measured on ventrally oriented specimens whose long axis was approximately
on the horizontal plane are as shown in Doerder et al. (1975; fig. 14, p. 247)
or in Lynn & Tucker (1976; fig. 1, p. 37). Only specimens in division stages 1-2
have been included.
RESULTS
Positioning of oral primordium in TS cultured T. thermophila
The oral primordium begins to develop several /im posterior to the anterior
mouthparts and adjacent to a ciliary row (kinety 1) which runs between the
two sets of mouthparts in organisms at stages 1-2 of fission (Fig. 1). By the
end of stage 2 the three membranelles are not yet apparent and kineties in the
presumptive furrow region have not yet been interrupted.
The distance between mouthparts (d, Fig. 1) in dividing co+ and co organisms
at fission stages 1-2 is proportionately related to body length (/, Fig. 1). The
TS cultured co+ cells varied between 37-46/^m in length by 12-24 ^m in
width (»v, Fig. 1), and d ranged from 6 to 11 jam; the co cells varied from
27-39 /un in length by 20-27/tm in width, and d ranged from 4 to 12/tm
(Table I). Although there is a significant difference between cell lengths of
co+ and co cells, d is not significantly different for the two cell types. Hence,
the ratio of dto /differs in co+ and co cells (Table 1). If co+ and co cells are
assessing cell size by the same mechanism (see Introduction), then some
estimate of cell size should prove similar for both types. The product of cell
length and cell width, / x w, for co+ and co cells is not significantly different
(Table 1), although these cells are shaped very differently (Fig. 1). This suggests
that the positional mechanism determines the location of the oral primordium
as a function of cell size, rather than cell length, since the former character is
similar and the latter character is different for the two genotypes while the
distance between mouthparts remains the same.
Regression analysis has been used to demonstrate the relationship between
264
D. H. LYNN
20 M
Anterior mouthparts
Developing posterior
mouthparts
CO
CO
Fig. 1. Schematic drawing of wild type (co+) and conical form-mutant (co) dividers
of Tetrahymena thermophila based on average measurements of 24 silver-stained
specimens of each type (data from Doerder et al. 1976 kindly supplied by Dr J.
Frankel). d is the distance between mouthparts; / is the body length; w is the
body width.
cell length and distance between mouthparts in dividing T. corlissi (Lynn &
Tucker, 1976). The TS sample was not optimal for such analysis because
of the small number of cells measured and the restricted range of variation in
these crucial parameters. A significant regression of d on /is not obtained for
co+ but is for co. For the latter, however, the intercept on the y-axis is significantly different from zero. When d is compared to an estimate of cell size,
/ x w, significant proportionality is again exhibited by co cells alone but not by
co+ cells alone. Most importantly, co+ and co measurements considered jointly
yield a significant common relationship represented by the equation
d = 0-00746 (/)(w) +2-837.
The ^-intercept is not significantly different from zero (t — 1-65; D.F. = 46).
Thus, the distance between mouthparts is exactly proportional to this estimate
of cell size.
The anterior mouthparts in co+ and co are not positioned at similar distances
from the anterior end of the cell; in co+ cells the average preoral distance is
3-8 /im while in co cells it is 6-2 jim (Table 1). The oral primordium is positioned
at a similar, though statistically significantly different, distance from the anterior
end in co+ and co cells (Table 1), while it is an average of 14-4 //m and 5-4 /im
respectively from the posterior pole of these two cell types. Although this
Pattern regulation in Tetrahymena
265
Table 1. Summary of measurements made on dividing Tetrahymena thermophila
wild type (co+) and conical form-mutant (co) cells grown in tryptone-salts
medium*
CO
Character
(
Meant
f
S.E.
Range
Meant
S.E.
Range
27-39
Body length, /(/mi)
42-2
35-5
37-46
0-461
0-514
20-27
Body width, w (/mi)
0-450
0-348
20-2
240
12-24
4-12
Distance between mouthparts,
0-253
6-11
0-347
9-3$
9-2J
d (/mi)
0098
3-5
6-2
0197
4-7
Preoral distance (j<>m)
3-8
18-23
19-27
Preprimordium distance (/mi)
0-299
23-8
0-367
21-2
Postprimordium distance (/mi) 14-4
0-380
12-18
5-4
0-390
2-10
0-260 0008 0148-0-308
Ratio, d\l
0-218 0006 0-133-0-262
Body length x body width,
552-1056 853-8$ 19-9
567-1053
850-6J 19-2
/ x w (/mi2)
* Original data were kindly supplied by Dr J. Frankel, Department of Zoology, University
of Iowa, Iowa City, Iowa, U.S.A.
t Sample size for co+ and co cells is 24.
X /-test of sample means reveals no significant difference at P = 005 (Simpson, Roe &
Lewontin, 1960).
suggests that the anterior pole could be an important reference point, the next
section will demonstrate this to be unlikely.
Positioning of oral primordium in PP cultured T. thermophila
A second experiment was made to determine if proportionality was demonstrable in co+ and co individually as well as jointly. Several different samples
of ciliates were fixed and stained 3 years after the original isolation of the
co mutant and 2 years after the TS experiment described above. In this second
sample totalling 50 individuals of each genotype, co+ and co cells have diverged
in cortical characteristics from the cells in the first isolation. For example, the
modal number of kineties is now 20(17-22; n = 50) for co+ and 16(13-19;
n = 50) for co cells where previously these were 19(16-21; n = 196) and
18(14-21; n = 253) respectively, while the number of postoral rows is 1-4
(1-2; n = 30) for co+ and 1-1 (1-2; n = 30) for co cells where previously these
were considered quite similar (Tables 3, 5 in Doerder et ah 1975). This degree
of variation in cortical features has been observed over an extended time
within co+ strains of T. pyriformis (Frankel, 1972, personal communication).
Furthermore, co+ and co cells which were cultured in the very rich PP medium
are substantially larger than in the previous experiment in the less nutrient-rich
TS medium. The co+ cells have become differentially wider so that their width
is no longer significantly less than that of co cells (Table 2). However, the
characteristic difference between the ovoid shape of co+ cells and the conical
shape of co cells (Fig. 1) remains undiminished.
266
D. H. LYNN
Table 2. Summary of measurements made on dividing Tetrahymena thermophila wildtype (co+) and conical form-mutant (co) cells grown in proteose-peptone medium*
co+
Character
Body length, / (/*m)
Body width, w (/tm)
Distance between mouthparts,
Preoral distance (/*m)
Preprimordium distance (/*m)
Postprimordium distance (/.tm)
Ratio, d\l
Body length x body width,
Ixw 0«m2)
Meant
CO
Range
S.E.
Meant
49-9
29-2J
12-9
0-669
0-276
0-335
39-9-58-2
240-33-3
9-4-17-7
41
0101
2-8-6-0
41-8
29-8J
118
S.E.
0-574
0-309
0-313
Range
34-1-50-4
240-351
8-0-17-4
5-5
25-7J
0-367
21-0-30-5
16-8
0-435
9-2-23-2
0-257 0004
0-196-0-313
1461-9 29-5
1104-6-1933-2
0151
40-100
0-356 21-0-32-3
2-9-11-7
7-9
0-345
0-279 0005 0-200-0-349
25-9J
1247-9
26-3
830-6-1638-2
8
10
Number of basal bodies in Kl
0-246
4-11
0-378
5-18
* Silver-stained specimens kindly provided by Dr J. Frankel, Department of Zoology, University of
Iowa, Iowa City, Iowa, U.S.A.
t Sample size for co+ and co is 50.
% /-test of sample means reveals no significant difference at P = 005 (Simpson et al. 1960).
20-0
100
500
40-0
60-0
Body length
Fig. 2. The relationship between the distance between the mouthparts (d) and the
body length (/) for 50 dividing organisms of co+ and 50 co dividers. The lines
fitted by linear regression analysis have the equations d = 0-447 (/) —6-96 for co+
and d = 0-447 (/) —9-38 for co. ; co+; A, co.
Pattern regulation in Tetrahymena
267
200 -
100
8000
10000
12000
14000
16000
Body length X body width (jum2)
18000
20000
Fig. 3. The relationship between the distance between the mouthparts (d) and the
body length-body width product (/ x w), an estimate of cell size or surface area for
100 dividing organisms (50 each of co+ and co). The best line fitted by linear
regression analysis has the equation d = 000907 (/) (w). • , co+; A., co.
Since relative cell size has changed since the TS experiment, the comparison
of Ixw reveals a significant difference between co+ and co cells (Table 2).
However, these changes in cortical features and shape have not greatly affected
the spacing of the mouthparts in the two cell types. The average distance is
quite similar though significantly different in the two cell types (Table 2).
Regression analysis of the PP experiment demonstrates that here also there
is a proportional relationship between d and / in these genetically different
strains of T. thermophila (Fig. 2). Comparison of the ratio d\l shows a distinct
difference in proportionality of these two parameters for co+ and co cells
(Table 2). Yet, the slopes of the regression lines (Fig. 2) are not significantly
different. If d is regressed upon the estimate of cell size Ixw, co+ and co cells
fall along the same line which goes through the origin (Fig. 3). The two lines
fitted to co+ and co separately (given as d = 0-00973 (/) (w)—1-31 for co+, and
d = 0-00851 (/)(w) + l-09 for co) do not fit the scatter of points significantly
better than the single line d = 000907 (/) (w) for co+ and co (where F = 2-43 <
2-68 a t P = 0-05 for D.F. = 3/99).
Since / x vv is a rather crude approximation of cell size, a better estimate
was derived using calculus. Measurements of the profiles of 10 co+ and 20 co
cells were used to derive equations for the cell shape. By integration, volumes
and surface areas were calculated for each cell and d was then regressed upon
268
D. H. LYNN
300 h
t
200
100
i 500-0
2000-0
25000
30000
35000
Body length X body width (//m3)
40000
45000
Fig. 4. The relationship between the distance between the mouthparts (d) and the
body length-width product (Ixw) for 106 dividing organisms of Tetrahymena
corlissi. The line fitted by linear regression analysis has the equation d = 000469
(/) (M>) + 5-15. A, first dividers; • , log dividers; • , second dividers.
these estimates. These further estimates of cell size were not able to explain
any more of the variation than the / x w estimate.
As in the previous experiment, preoral and postprimordium distances in
+
co and co are very different. However, the preprimordium distances are
not significantly different (Table 2). Again, this suggests that the anterior pole
could be an important reference point. If the preprimordium distance, p, is
regressed upon / or Ixw, each genotype lies on a different line. In neither
case does a single line suffice. For example, p = 0-0111 (/)(w) + 9-40 for co+
and p = 0-00941 (/)(w) +14-17 for co are significantly better at accounting
for the variation at P = 0-05 than the single line p = 0-00775 (/)(w) +15-29
for co+ and co jointly. In addition, none of these lines demonstrates exact
proportionality as the j-intercepts are very significantly different from zero.
The number of basal bodies in kinety 1 between the mouthparts has been
counted in silver-stained co+ and co cells (Table 2). Silver-stained basal body
counts are a good estimate of the true basal body number between mouthparts
(Lynn & Tucker, 1976; Lynn, unpublished observations). There is a statistically
Pattern regulation in Tetrahymena
269
+
significant difference in this number in co and co cells although a similar
distance separates the mouthparts (Table 2). As observed in T. pyriformis
strain W (Lynn & Tucker, 1976), there is a great deal of variation in the
number of basal bodies even within strains (Table 2).
Positioning of oral primordium in T. corlissi
To test that a similar relationship between d and cell size exists in another
species of Tetrahymena, individuals of T. corlissi were measured. There is a
significant regression when d\s regressed upon / x w for these 3 types of dividers
(Fig. 4). Although, as is apparent from Fig. 4, d is related to / x w in a proportional manner when all three cell types are included, the best fit line for
each cell type does not coincide exactly with this regression line.
DISCUSSION
Proportional distance assessment
A mechanism which proportionately assesses cell length has been suggested
to position the oral primordium in dividing T. corlissi (Lynn & Tucker, 1976).
The results of the present study demonstrate that a similar mechanism which
monitors at least cell length also determines the position of the oral primordium
in dividing T. thermophila co+ and co cells.
The ratio d\l is different for each strain (Tables 1, 2). As co+ and co cells
have been demonstrated to be quite similar for a number of cortical characteristics (Doerder et at. 1975), the differences in djl ratios and in assessment
of of as a function of / do not in themselves refute the assumption that the
same mechanism positions the oral primordium in both cell types. Therefore,
it is assumed that the co gene has not altered the fundamental positioning
mechanism. In fact, the regression of d on / reveals that both co+ and co cells
have the same proportion of cell length contributing to the estimation of d
(Fig. 2) and thus, perhaps, share a similar underlying positional mechanism.
Cell length may not be the crucial parameter which is the reference for the
distance assessment, since djl ratios are different and the same proportion of
/ contributes to the estimation of d (Fig. 2). A simple estimate of cell size is
the product of / x w which is likely to be an estimate of cell surface area rather
than cell volume or biomass. Undoubtedly, it would be highly correlated with
all three. In the TS experiment, co+ and co are on average not significantly
different when d and / x w are compared (Table 1). In the PP experiment,
although d and / x w are now different since the two genotypes have diverged
morphologically, regression analysis clearly shows that the cells could be
making an identical proportional assessment of / x w (Fig. 3). A relationship
to cell size is exhibited by different sized dividers of T. corlissi (Fig. 4).
For several other reasons, cortical surface area is likely to be the component
of a ciliate's size which is used to determine the distance which the oral
l8
EMB 42
270
D. H. LYNN
primordium is from the anterior mouthparts. Many ciliates are able to change
shape rapidly and consequently volume varies considerably. The ciliate cortex
and cell surface are much more stable, being resistant to deformation since
they are composed of a complex array of basal bodies, microtubules, and
microfilaments. If the mechanism of size assessment requires a stable cell
parameter, surface area (or / x w?) is more likely than volume. Already the cell
surface has been implicated in the control of cellular events. De Terra (1974,
1975) demonstrated that the cortex of Stentor can control nuclear division.
Transplanted cortical components, especially the oral region of the cortex,
have an inhibitory or inductive effect on the cell to which they are transplanted
(Uhlig, 1960; Tartar, 1961). It is possible that changes in surface area (perhaps
correlated with changes in number of ciliary rows?) might also be a factor in
the distribution of basal bodies among ciliary rows in Tetrahymena. The
researches of Nanney and co-workers (Nanney, 1971; Nanney & Chow, 1974)
have demonstrated that as the number of ciliary rows increases, the number
of basal bodies within a row decreases. However, the exact relationships
between number of ciliary rows and number of basal bodies per row to cell
length, cell width, and distance between mouthparts have yet to be analysed.
Reference points for oral primordium position
The old or anterior mouthparts of ciliates have been considered an important,
and by some, an essential reference point for positional determination of
developing cortical structures. Kaczanowska (1974, 1975) has concluded
this from her studies of contractile vacuole pore positioning in Chilodonella.
Sonneborn (1974) arrived at the same conclusion in his review of ciliate
morphogenesis.
In co+ and co cells, the preprimordium and postprimordium distances are
different for cells of the same average size (i.e. when / x w is the same, Table 1).
Moreover, the regression of preprimordium distance on / or / x w does not
demonstrate a similar relationship for both genotypes. If the anterior pole is
the reference point, these two results suggest that a different assessment of
cell size operates in each genotype. On the other hand, the interoral distance
d is the same when / x w is the same (Table 1) and there is a similar relationship
when d is regressed on / or / x w for both genotypes. Thus, if the anterior
mouthparts are the reference point, these results suggest that the same exact
assessment of cell size is operating in each genotype. Since both genotypes
are similar in a number of other cortical characters (Doerder et al. 1975), this
second alternative is preferred. The mo3 mutant of T. thermophila is arrested
during division so that chains of cells are formed (Frankel, Jenkins & De Bault,
1976). The cells within the chain which do not have an independent anterior
pole still place an oral primordium at some distance from the old mouthparts.
What this distance is related to is presently uncertain. It is likely that the mo3
mutant will provide a crucial test between the above alternative hypotheses.
Pattern regulation in Tetrahymena
271
However, this is not to say that the anterior pole is irrelevant in shape and
pattern formation. The facts that the anterior mouthparts in co+ and co cells
are on average and predictably at different distances from the anterior pole
and that these cells have different cell shapes suggest that some mechanism,
perhaps under control of the co gene, alters the overall cortical patterning.
The above discussion has assumed that one mechanism shapes the cell after
cytokinesis to yield the co+ and co phenotypes and another mechanism determines the position of the oral primordium.
The measurement for oral primordium position could proceed along kinety 1.
However, the spacing of the mouthparts is unlikely to involve a count of the
absolute number of cortical units or basal bodies from the anterior mouthparts. The variability in number of basal bodies between mouthparts of these
strains of T. thermophila and in T. corlissi (Lynn & Tucker, 1976) seems to
preclude this possibility. Frankel (personal communication) has isolated a
mutant disl which has a highly disorganized kinety pattern, including kinety 1,
and yet this strain manifests normal positioning of the oral primordium. This
clearly indicates that kinety 1 is not essential for positioning the oral primordium.
Case for a diffusing morphogen
Jerka-Dziadosz (1974) has presented an analysis of modified 'sand-hill'
models to account for the pattern of cortical development in Urosty/a cells
which have been morphologically altered by microsurgery. The pattern is
explained in terms of gradients re-establishing themselves in an altered field.
Frankel (1974, 1975b) has discriminated explicitly between the concept of a
graded distribution of a property and gradients of diffusing morphogens. At
this time, Frankel prefers to discuss the phenomenon of pattern formation
employing the abstract concept of a graded property.
There are two major obstacles to the establishment of a morphogenetic
diffusion gradient in ciliates, assuming that an appropriate source can be
chosen. First, is there sufficient time in the cell cycle to establish a gradient by
diffusion? Secondly, is the cytoplasm ever free from cyclotic movements
which would prevent the establishment or reduce the equilibrium stability of
such a gradient?
There is an obvious choice for a source in the differentiated ciliate. It is the
anterior oral apparatus. There is strong evidence from the researches of Uhlig
(1960) and Tartar (1961) that the oral apparatus has the properties of a
morphogenetic 'source'. Indirect evidence is provided by analysis of pattern
formation in Chilodonella (Kaczanowska, 1974), T. corlissi (Lynn & Tucker,
1976), and T. thermophila (see Results) that the anterior oral apparatus is a
primary reference point for the determination of the position of the new oral
apparatus.
Crick (1971) has suggested that the time t in hours required to establish
a gradient by diffusion is given by the following relationship
«Jt ^ x
and thus
t ^ x 2,
18-2
272
D. H. LYNN
where x is the distance in millimetres over which the gradient occurs. In the
larger species T. corlissi, individuals rarely reach 100 /im or 10"1 mm in length;
thus, the minimal time required is 10~2 h or less than 40 sec. Sequential divisions
without intervening growth can occur in this species within 2 h of each other
(Lynn, 1975). Even a small fraction of this time is ample to establish a gradient
by diffusion.
There is also an appropriate time in the cell cycle in some ciliates when
cyclosis, the rapid movement of endoplasm, ceases. In Paramecium, cyclosis
ceases during division at the time when the oral primordium is migrating
and the macronucleus is dividing (Sikora & Kuznicki, 1976). This stability
lasts for 5-15 min. Although this phenomenon has not been observed in
Tetrahymena and would have to occur in the middle of the cell cycle when
the oral primordium develops, a cessation of equal time would be more than
adequate to establish a gradient by diffusion. For ciliates, this condition may
not be necessary as there is no cyclosis in the epiplasm and cortical ectoplasm
which are themselves very stable cytoplasmic regions (Sibley & Hanson, 1974).
Diffusion of a morphogen might occur through this ectoplasmic region of the
cytoplasm and thus be unaffected by endoplasmic cyclosis. Even if the properties
of the ectoplasm are somewhat different from the general properties of cytoplasm assumed in Crick's analyses (1970, 1971), there are at least two orders
of magnitude more time available between divisions than the minimal time
required by the model.
The anterior oral apparatus of Tetrahymena could be the source of a
morphogen which diffuses through the ectoplasmic regions of the cell. There
is sufficient time in the cell cycle for a diffusion gradient to reach an equilibrium
stability. If a 'circular gradient' exists in Tetrahymena (Nanney, 1966, 1968;
Nanney, Chow & Wozencraft, 1975) as it seemingly does in Stentor (Uhlig,
1960) and if it extends from the ' stomatogenic kinety' around the cell, the
position of the oral primordium could be exactly specified by the end-boundary
of the circular gradient and by the diffusing morphogen.
I would like to thank Dr Joseph Frankel for his enthusiastic encouragement, for his
careful criticisms, and especially for his generous provision of original data and silverstained specimens. I am also indebted to Dr John B. Tucker and Mr C. D. Sinclair for their
advice and criticism.
This research has been supported bygrantsB/SR/88418 and B/SR/5894.5 from the Science
Research Council (U.K.) and by grant No. 08485 from National Institutes of Health (U.S.)
awarded to J. Frankel. The author was supported as a NATO Postdoctoral Fellow by the
National Research Council of Canada.
REFERENCES
J. O. (1953). Silver impregnation of ciliated protozoa by the Chatton-Lwoff
technic. Stain Technol. 28, 97-100.
CRICK, F. (1970). Diffusion in embryogenesis. Nature, Lond. 225, 420^22.
CRICK, F. (1971). The scale of pattern formation. In Control Mechanisms of Growth and
Differentiation (ed. D. D. Davies & M. Ball). Symp. Soc. expl. Biol. 25, 429-438.
Cambridge: Cambridge University Press.
CORLISS,
Pattern regulation in Tetrahymena
273
N. (1974). Cortical control of cell division. Science, N.Y. 184, 530-537.
N. (1975). Evidence for cell surface control of macronuclear DNA synthesis in
Stentor. Nature, Lond. 258, 300-303.
DOERDER, F. P., FRANKEL, J., JENKINS, L. M. & DE BAULT, L. E. (1975). Form and pattern
in ciliated protozoa. Analysis of a genie mutant with altered cell shape in Tetrahymena
pyriformis, syngen 1. /. exp. Zool. 192, 237-258.
FRANKEL, J. (1965). The effect of nucleic acid antagonists on cell division and oral organelle
development in Tetrahymena pyriformis. J. exp. Zool. 159, 113-148.
FRANKEL, J. (1972). The stability of cortical phenotypes in continuously growing cultures
of Tetrahymena pyriformis. J. Protozool. 19, 648-652.
FRANKEL, J. (1974). Positional information in unicellular organisms. /. theor. Biol. 47,
439-481.
FRANKEL, J. (1975o). An analysis of the spatial distribution of ciliary units in a ciliate,
Euplotes minuta. J. Embryol. exp. Morph. 33, 553-580.
FRANKEL, J. (19756). Pattern formation in ciliary organelle systems of ciliated protozoa.
In Cell Patterning (ed. R. Porter & J. Rivers). Ciba Fdn. Symp., pp. 25-49. London:
Elsevier.
FRANKEL, J. & HECKMANN, K. (1968). A simplified Chatton-Lwoff silver impregnation
procedure for use in experimental studies with ciliates. Trans. Am. microsc. Soc. 87,
317-321.
FRANKEL, J., JENKINS, L. M. & DE BAULT, L. E. (1976). Causal relations among cell cycle
processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants.
J. Cell Biol. 71, 242-260.
JERKA-DZIADOSZ, M. (1974). Cortical development in Urostyla. II. The role of positional
information and preformed structures in formation of cortical pattern. Ada Protozool. 12,
239-274.
JERKA-DZIADOSZ, M. (1977). Temporal coordination and spatial autonomy in regulation
of ciliary pattern in double forms of a hypotrich ciliate Paraurostyla weissei. J. exp. Zool.
200, 23-32.
JERKA-DZIADOSZ, M. & FRANKEL, J. (1969). An analysis of the formation of ciliary primordia
in the hypotrich ciliate Urostyla weissei. J. Protozool. 16, 612-637.
KACZANOWSKA, J. (1974). The pattern of morphogenetic control in Chilodonella cucullulus.
J. exp. Zool. 187, 47-62.
KACZANOWSKA, J. (1975). Shape and pattern regulation in regenerants of Chilodonella
cucullulus (O.F.M.). Acta Protozool. 13, 343-360.
LYNN, D. H. (1975). The life cycle of the histophagous ciliate, Tetrahymena corlissi Thompson,
1955. J. Protozool. 22, 188-195.
LYNN, D. H. & TUCKER, J. B. (1976). Cell size and proportional distance assessment during
determination of organelle position in the cortex of the ciliate Tetrahymena. J. Cell Sci.
21, 35-46.
NANNEY, D. L. (1966). Cortical integration in Tetrahymena: an exercise in cytogeometry.
/. exp. Zool. 161, 307-317.
NANNEY, D. L. (1968). Cortical patterns in cellular morphogenesis. Science, N.Y. 160,
496-502.
NANNEY, D. L. (1971). The constancy of cortical units in Tetrahymena with varying numbers
of ciliary rows. /. exp. Zool. 178, 177-182.
NANNEY, D. L. & CHOW, M. (1974). Basal body homeostasis in Tetrahymena. Am. Naturalist
108, 125-139.
NANNEY, D. L. & MCCOY, J. W. (1976). Characterization of the species of the Tetrahymena
pyriformis complex. Trans. Am. microsc. Soc. 95, 664-682.
DE TERRA,
DE TERRA,
NANNEY, D. L., CHOW, M. & WOZENCRAFT, B. S. (1975). Considerations of symmetry in
the cortical integration of Tetrahymena doublets. /. exp. Zool. 193, 1-14.
J. T. & HANSON, E. D. (1974). Identity and function of a subcortical cytoskeleton
in Paramecium. Arch. Protistenk. 116, 221-235.
SIKORA, J. & KUZNICKI, L. (1976). Cytoplasmic streaming within Paramecium aurelia. IV.
Cyclosis during binary fission and conjugation. Acta Protozool. 15, 173-178.
SIBLEY,
274
D. H. LYNN
G. G., ROE, A. & LEWONTIN, R. C. (1960). Quantitative Zoology. New York:
Harcourt, Brace.
SONNEBORN, T. M. (1974). Ciliate morphogenesis and its bearing on general cellular morphogenesis. In Actualites Protozoologiques, vol. i (ed. P. de Puytorac & J. Grain), pp. 327-355.
France: University of Clermont-Ferrand.
TARTAR, V. (1961). The Biology o/Stentor. London: Pergamon.
UHLIG, G. (1960). Entwicklungsphysiologische Untersuchungen zur Morphogenese von
Stentor coeruleus Ehrbg. Arch. Protistenk. 105, 1-109.
SIMPSON,
{Received 25 April 1977, revised 16 June 1977)