/ . Embryol. exp. Morph. Vol. 42, pp. 261-274, 1977 2 6 1Printed in Great Britain © Company of Biologists Limited 1977

Proportional control oforganelle position by a mechanism which similarly

monitors cell size of wild type and conicalform-mutant Tetrahymena

BY DENIS H. LYNN1

From the Department of Zoology, The University,St Andrews, Scotland

SUMMARYDistance between mouthparts of dividing cells of wild type and conical form-mutant

Tetrahymena thermophila (formerly T. pyriformis syngen 1) is directly proportional to cellsize. This distance is related to cell length in both wild type and conical cells although theproportionality is different in each cell type. However, for both wild type and conical cellsthe distance between mouthparts is directly and similarly proportional to the product ofcell length and cell width which is an estimate of cell size. Evidence has been obtainedwhich suggests that the new mouthparts are positioned with reference to the anteriormouthparts rather than to either pole of the cell. Determination of the site of the newmouthparts 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 dorso-ventrally flattened ciliates such as the hypotrichs Euplotes and Urostyla andthe cyrtophorine Chilodonella. In these ciliates, the field boundaries are con-sidered coincident with the border between dorsal and ventral surfaces. Thedevelopment of cirral primordia in Urostyla altered by microsurgery (Jerka-Dziadosz & Frankel, 1969; Jerka-Dziadosz, 1974, 1977), the spatial distri-bution of ciliary units on the dorsal surface of Euplotes (Frankel, \915a), andpositioning of contractile vacuole pores in Chilodonella (Kaczanowska, 1974,1975) have been explained with reference to these boundaries. However, it isdifficult to define field boundaries in 'radially' symmetrical ciliates such asTetrahymena 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 specifythe 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 oralprimordium or mouthparts in a regulative manner, proportional to the bodyor cell length (Lynn & Tucker, 1976). This relationship was demonstrated withlog dividers and first and second post-starvation dividers of Tetrahymenacorlissi. Since these three types of dividers are similarly shaped, it is not possibleto test whether body length or overall body size (e.g. surface area, volume,biomass) is the determining parameter nor what the reference points for oralprimordium positioning are. Genetically related ciliates of different shapewould be necessary for this purpose. Doerder, Frankel, Jenkins & De Bault(1975) have isolated a form-mutant of Tetrahymena thermophila. This form-mutant, named conical, results from the action of a single recessive gene whichchanges the overall cell shape from ovoid to conical and places the anteriormouthparts on average further from the anterior pole. The gene apparentlydoes not affect cortical characteristics such as number of ciliary rows, numberof 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 themechanism responsible for positioning the oral primordium, then it is possibleto test the hypothesis that primordium position is determined with referenceto overall body size rather than to some single linear parameter such as celllength.

This report will demonstrate that similar proportional positioning of theoral primordium of both wild-type (co+) and conical (co) cells occurs both asa function of cell length and, more importantly, as a function of cell sizeestimated by the product of cell length and cell width. The reference pointfor the measurement is apparently the anterior mouthparts rather than theanterior pole of the cell.

MATERIALS AND METHODSCulture techniques

Tetrahymena thermophila (formerly Tetrahymena pyriformis syngen 1, seeNanney & McCoy, 1976) strain D was obtained from Dr D. L. Nanney andcultured axenically in 2-0 % proteose-peptone with 0-5 % yeast extract (PP) orin tryptone-dextrin-vitamin-salts (TS) medium (Frankel, 1965) at 28 °C. Cellsin TS medium were sampled only once in mid-log phase, while cells in PPmedium 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 logdividers and first and second post-starvation dividers of T. corlissi have beendescribed (Lynn & Tucker, 1976).

Pattern regulation in Tetrahymena 263

Staining and measurement

Both T. thermophila and T. corlissi were stained by the Chatton-Lwoff wet-silver procedure, following the directions of Frankel & Heckmann (1968) forT. thermophila and those of Corliss (1953) for T. corlissi.

Measurement of silver-stained organisms was performed in two ways. DrJ. Frankel generously supplied the original data obtained during the descriptionof the co mutant (Doerder et al. 1975). Measurements of cell proportions ofone sample of each genotype, cultured in TS medium, were made with anocular micrometer as described in the original paper.

Measurements of the PP cultured T. thermophila and T. corlissi were madeusing 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 distancesmeasured on ventrally oriented specimens whose long axis was approximatelyon 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 1-2have been included.

RESULTS

Positioning of oral primordium in TS cultured T. thermophila

The oral primordium begins to develop several /im posterior to the anteriormouthparts and adjacent to a ciliary row (kinety 1) which runs between thetwo sets of mouthparts in organisms at stages 1-2 of fission (Fig. 1). By theend of stage 2 the three membranelles are not yet apparent and kineties in thepresumptive furrow region have not yet been interrupted.

The distance between mouthparts (d, Fig. 1) in dividing co+ and co organismsat fission stages 1-2 is proportionately related to body length (/, Fig. 1). TheTS cultured co+ cells varied between 37-46/^m in length by 12-24 ^m inwidth (»v, Fig. 1), and d ranged from 6 to 11 jam; the co cells varied from27-39 /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 ofco+ 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 areassessing cell size by the same mechanism (see Introduction), then someestimate of cell size should prove similar for both types. The product of celllength 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 suggeststhat the positional mechanism determines the location of the oral primordiumas a function of cell size, rather than cell length, since the former character issimilar and the latter character is different for the two genotypes while thedistance between mouthparts remains the same.

Regression analysis has been used to demonstrate the relationship between

264 D. H. LYNN

20 M

Anterior mouthparts

Developing posteriormouthparts

CO CO

Fig. 1. Schematic drawing of wild type (co+) and conical form-mutant (co) dividersof Tetrahymena thermophila based on average measurements of 24 silver-stainedspecimens 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 thebody width.

cell length and distance between mouthparts in dividing T. corlissi (Lynn &Tucker, 1976). The TS sample was not optimal for such analysis becauseof the small number of cells measured and the restricted range of variation inthese crucial parameters. A significant regression of d on / is not obtained forco+ but is for co. For the latter, however, the intercept on the y-axis is sig-nificantly 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 byco+ cells alone. Most importantly, co+ and co measurements considered jointlyyield 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 estimateof cell size.

The anterior mouthparts in co+ and co are not positioned at similar distancesfrom the anterior end of the cell; in co+ cells the average preoral distance is3-8 /im while in co cells it is 6-2 jim (Table 1). The oral primordium is positionedat a similar, though statistically significantly different, distance from the anteriorend in co+ and co cells (Table 1), while it is an average of 14-4 //m and 5-4 /imrespectively 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 thermophilawild type (co+) and conical form-mutant (co) cells grown in tryptone-saltsmedium*

Character

Body length, /(/mi)Body width, w (/mi)Distance between mouthparts,d (/mi)

Preoral distance (j<>m)Preprimordium distance (/mi)Postprimordium distance (/mi)Ratio, d\lBody length x body width,

/ x w (/mi2)

(Meant42-220-2

9-2J

3-821-214-40-218

850-6J

S.E.

0-4610-4500-253

00980-2990-3800006

19-2

Range

37-4612-246-11

3-518-2312-18

0-133-0-262552-1056

f

Meant

35-5240

9-3$

6-223-85-40-260

853-8$

CO

S.E.

0-5140-3480-347

01970-3670-3900008

19-9

Range

27-3920-274-12

4-719-272-10

0148-0-308567-1053

* Original data were kindly supplied by Dr J. Frankel, Department of Zoology, Universityof 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 nextsection 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 demon-strable in co+ and co individually as well as jointly. Several different samplesof ciliates were fixed and stained 3 years after the original isolation of theco mutant and 2 years after the TS experiment described above. In this secondsample totalling 50 individuals of each genotype, co+ and co cells have divergedin cortical characteristics from the cells in the first isolation. For example, themodal 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) and18(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 thesewere considered quite similar (Tables 3, 5 in Doerder et ah 1975). This degreeof variation in cortical features has been observed over an extended timewithin co+ strains of T. pyriformis (Frankel, 1972, personal communication).Furthermore, co+ and co cells which were cultured in the very rich PP mediumare substantially larger than in the previous experiment in the less nutrient-richTS medium. The co+ cells have become differentially wider so that their widthis no longer significantly less than that of co cells (Table 2). However, thecharacteristic difference between the ovoid shape of co+ cells and the conicalshape of co cells (Fig. 1) remains undiminished.

266 D. H. LYNN

Table 2. Summary of measurements made on dividing Tetrahymena thermophila wild-type (co+) and conical form-mutant (co) cells grown in proteose-peptone medium*

Character

Body length, / (/*m)Body width, w (/tm)Distance between mouthparts,

Preoral distance (/*m)Preprimordium distance (/*m)Postprimordium distance (/.tm)Ratio, d\lBody length x body width,

Ixw 0«m2)Number of basal bodies in Kl

Meant

49-929-2J12-9

4125-7J16-80-257

1461-9

8

co+

S.E.

0-6690-2760-335

01010-3670-4350004

29-5

0-246

Range

39-9-58-2240-33-39-4-17-7

2-8-6-021-0-30-59-2-23-2

0-196-0-3131104-6-1933-2

4-11

Meant

41-829-8J118

5-525-9J

7-90-279

1247-9

10

CO

S.E.

0-5740-3090-313

01510-3560-3450005

26-3

0-378

Range

34-1-50-4240-3518-0-17-4

40-10021-0-32-32-9-11-7

0-200-0-349830-6-1638-2

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

40-0 500 60-0

Body length

Fig. 2. The relationship between the distance between the mouthparts (d) and thebody length (/) for 50 dividing organisms of co+ and 50 co dividers. The linesfitted 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 18000 20000Body length X body width (jum2)

Fig. 3. The relationship between the distance between the mouthparts (d) and thebody length-body width product (/ x w), an estimate of cell size or surface area for100 dividing organisms (50 each of co+ and co). The best line fitted by linearregression analysis has the equation d = 000907 (/) (w). • , co+; A., co.

Since relative cell size has changed since the TS experiment, the comparisonof Ixw reveals a significant difference between co+ and co cells (Table 2).However, these changes in cortical features and shape have not greatly affectedthe spacing of the mouthparts in the two cell types. The average distance isquite similar though significantly different in the two cell types (Table 2).

Regression analysis of the PP experiment demonstrates that here also thereis a proportional relationship between d and / in these genetically differentstrains of T. thermophila (Fig. 2). Comparison of the ratio d\l shows a distinctdifference in proportionality of these two parameters for co+ and co cells(Table 2). Yet, the slopes of the regression lines (Fig. 2) are not significantlydifferent. If d is regressed upon the estimate of cell size Ixw, co+ and co cellsfall along the same line which goes through the origin (Fig. 3). The two linesfitted to co+ and co separately (given as d = 0-00973 (/) (w)—1-31 for co+, andd = 0-00851 (/)(w) + l-09 for co) do not fit the scatter of points significantlybetter 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 estimatewas derived using calculus. Measurements of the profiles of 10 co+ and 20 cocells were used to derive equations for the cell shape. By integration, volumesand 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 40000 45000

Body length X body width (//m3)

Fig. 4. The relationship between the distance between the mouthparts (d) and thebody length-width product (Ixw) for 106 dividing organisms of Tetrahymenacorlissi. 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 explainany more of the variation than the / x w estimate.

As in the previous experiment, preoral and postprimordium distances inco+ and co are very different. However, the preprimordium distances arenot significantly different (Table 2). Again, this suggests that the anterior polecould be an important reference point. If the preprimordium distance, p, isregressed upon / or Ixw, each genotype lies on a different line. In neithercase 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 accountingfor the variation at P = 0-05 than the single line p = 0-00775 (/)(w) +15-29for co+ and co jointly. In addition, none of these lines demonstrates exactproportionality as the j-intercepts are very significantly different from zero.

The number of basal bodies in kinety 1 between the mouthparts has beencounted in silver-stained co+ and co cells (Table 2). Silver-stained basal bodycounts 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 269significant difference in this number in co+ and co cells although a similardistance separates the mouthparts (Table 2). As observed in T. pyriformisstrain W (Lynn & Tucker, 1976), there is a great deal of variation in thenumber 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 anotherspecies of Tetrahymena, individuals of T. corlissi were measured. There is asignificant 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 pro-portional manner when all three cell types are included, the best fit line foreach cell type does not coincide exactly with this regression line.

DISCUSSION

Proportional distance assessment

A mechanism which proportionately assesses cell length has been suggestedto position the oral primordium in dividing T. corlissi (Lynn & Tucker, 1976).The results of the present study demonstrate that a similar mechanism whichmonitors at least cell length also determines the position of the oral primordiumin dividing T. thermophila co+ and co cells.

The ratio d\l is different for each strain (Tables 1, 2). As co+ and co cellshave been demonstrated to be quite similar for a number of cortical charac-teristics (Doerder et at. 1975), the differences in djl ratios and in assessmentof of as a function of / do not in themselves refute the assumption that thesame mechanism positions the oral primordium in both cell types. Therefore,it is assumed that the co gene has not altered the fundamental positioningmechanism. In fact, the regression of d on / reveals that both co+ and co cellshave 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 thedistance 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 isthe product of / x w which is likely to be an estimate of cell surface area ratherthan cell volume or biomass. Undoubtedly, it would be highly correlated withall three. In the TS experiment, co+ and co are on average not significantlydifferent 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 divergedmorphologically, regression analysis clearly shows that the cells could bemaking an identical proportional assessment of / x w (Fig. 3). A relationshipto 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 componentof 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 changeshape rapidly and consequently volume varies considerably. The ciliate cortexand cell surface are much more stable, being resistant to deformation sincethey are composed of a complex array of basal bodies, microtubules, andmicrofilaments. If the mechanism of size assessment requires a stable cellparameter, surface area (or / x w?) is more likely than volume. Already the cellsurface 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 (perhapscorrelated with changes in number of ciliary rows?) might also be a factor inthe distribution of basal bodies among ciliary rows in Tetrahymena. Theresearches of Nanney and co-workers (Nanney, 1971; Nanney & Chow, 1974)have demonstrated that as the number of ciliary rows increases, the numberof basal bodies within a row decreases. However, the exact relationshipsbetween number of ciliary rows and number of basal bodies per row to celllength, 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 ofdeveloping cortical structures. Kaczanowska (1974, 1975) has concludedthis from her studies of contractile vacuole pore positioning in Chilodonella.Sonneborn (1974) arrived at the same conclusion in his review of ciliatemorphogenesis.

In co+ and co cells, the preprimordium and postprimordium distances aredifferent 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 notdemonstrate a similar relationship for both genotypes. If the anterior pole isthe reference point, these two results suggest that a different assessment ofcell size operates in each genotype. On the other hand, the interoral distanced is the same when / x w is the same (Table 1) and there is a similar relationshipwhen d is regressed on / or / x w for both genotypes. Thus, if the anteriormouthparts are the reference point, these results suggest that the same exactassessment of cell size is operating in each genotype. Since both genotypesare similar in a number of other cortical characters (Doerder et al. 1975), thissecond alternative is preferred. The mo3 mutant of T. thermophila is arrestedduring division so that chains of cells are formed (Frankel, Jenkins & De Bault,1976). The cells within the chain which do not have an independent anteriorpole 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 mo3mutant will provide a crucial test between the above alternative hypotheses.

Pattern regulation in Tetrahymena 271However, 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 cellsare on average and predictably at different distances from the anterior poleand 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 aftercytokinesis to yield the co+ and co phenotypes and another mechanism deter-mines 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 theabsolute number of cortical units or basal bodies from the anterior mouth-parts. The variability in number of basal bodies between mouthparts of thesestrains of T. thermophila and in T. corlissi (Lynn & Tucker, 1976) seems topreclude this possibility. Frankel (personal communication) has isolated amutant disl which has a highly disorganized kinety pattern, including kinety 1,and yet this strain manifests normal positioning of the oral primordium. Thisclearly 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 cellswhich have been morphologically altered by microsurgery. The pattern isexplained in terms of gradients re-establishing themselves in an altered field.Frankel (1974, 1975b) has discriminated explicitly between the concept of agraded distribution of a property and gradients of diffusing morphogens. Atthis time, Frankel prefers to discuss the phenomenon of pattern formationemploying the abstract concept of a graded property.

There are two major obstacles to the establishment of a morphogeneticdiffusion gradient in ciliates, assuming that an appropriate source can bechosen. First, is there sufficient time in the cell cycle to establish a gradient bydiffusion? Secondly, is the cytoplasm ever free from cyclotic movementswhich would prevent the establishment or reduce the equilibrium stability ofsuch a gradient?

There is an obvious choice for a source in the differentiated ciliate. It is theanterior oral apparatus. There is strong evidence from the researches of Uhlig(1960) and Tartar (1961) that the oral apparatus has the properties of amorphogenetic 'source'. Indirect evidence is provided by analysis of patternformation in Chilodonella (Kaczanowska, 1974), T. corlissi (Lynn & Tucker,1976), and T. thermophila (see Results) that the anterior oral apparatus is aprimary reference point for the determination of the position of the new oralapparatus.

Crick (1971) has suggested that the time t in hours required to establisha gradient by diffusion is given by the following relationship

«Jt ^ x and thus t ^ x2,18-2

272 D. H. LYNN

where x is the distance in millimetres over which the gradient occurs. In thelarger 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 divisionswithout 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 gradientby diffusion.

There is also an appropriate time in the cell cycle in some ciliates whencyclosis, the rapid movement of endoplasm, ceases. In Paramecium, cyclosisceases during division at the time when the oral primordium is migratingand the macronucleus is dividing (Sikora & Kuznicki, 1976). This stabilitylasts for 5-15 min. Although this phenomenon has not been observed inTetrahymena and would have to occur in the middle of the cell cycle whenthe oral primordium develops, a cessation of equal time would be more thanadequate to establish a gradient by diffusion. For ciliates, this condition maynot be necessary as there is no cyclosis in the epiplasm and cortical ectoplasmwhich are themselves very stable cytoplasmic regions (Sibley & Hanson, 1974).Diffusion of a morphogen might occur through this ectoplasmic region of thecytoplasm and thus be unaffected by endoplasmic cyclosis. Even if the propertiesof the ectoplasm are somewhat different from the general properties of cyto-plasm assumed in Crick's analyses (1970, 1971), there are at least two ordersof magnitude more time available between divisions than the minimal timerequired by the model.

The anterior oral apparatus of Tetrahymena could be the source of amorphogen which diffuses through the ectoplasmic regions of the cell. Thereis sufficient time in the cell cycle for a diffusion gradient to reach an equilibriumstability. 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, theposition of the oral primordium could be exactly specified by the end-boundaryof the circular gradient and by the diffusing morphogen.

I would like to thank Dr Joseph Frankel for his enthusiastic encouragement, for hiscareful criticisms, and especially for his generous provision of original data and silver-stained specimens. I am also indebted to Dr John B. Tucker and Mr C. D. Sinclair for theiradvice and criticism.

This research has been supported bygrantsB/SR/88418 and B/SR/5894.5 from the ScienceResearch 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 theNational Research Council of Canada.

REFERENCES

CORLISS, J. O. (1953). Silver impregnation of ciliated protozoa by the Chatton-Lwofftechnic. 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.

Pattern regulation in Tetrahymena 273DE TERRA, N. (1974). Cortical control of cell division. Science, N.Y. 184, 530-537.DE TERRA, 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 Tetrahymenapyriformis, syngen 1. /. exp. Zool. 192, 237-258.

FRANKEL, J. (1965). The effect of nucleic acid antagonists on cell division and oral organelledevelopment in Tetrahymena pyriformis. J. exp. Zool. 159, 113-148.

FRANKEL, J. (1972). The stability of cortical phenotypes in continuously growing culturesof 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 impregnationprocedure 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 cycleprocesses 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 positionalinformation and preformed structures in formation of cortical pattern. Ada Protozool. 12,239-274.

JERKA-DZIADOSZ, M. (1977). Temporal coordination and spatial autonomy in regulationof 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 primordiain 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 Chilodonellacucullulus (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 duringdetermination 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 numbersof ciliary rows. / . exp. Zool. 178, 177-182.

NANNEY, D. L. & CHOW, M. (1974). Basal body homeostasis in Tetrahymena. Am. Naturalist108, 125-139.

NANNEY, D. L. & MCCOY, J. W. (1976). Characterization of the species of the Tetrahymenapyriformis complex. Trans. Am. microsc. Soc. 95, 664-682.

NANNEY, D. L., CHOW, M. & WOZENCRAFT, B. S. (1975). Considerations of symmetry inthe cortical integration of Tetrahymena doublets. /. exp. Zool. 193, 1-14.

SIBLEY, J. T. & HANSON, E. D. (1974). Identity and function of a subcortical cytoskeletonin 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.

274 D. H. LYNN

SIMPSON, 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 morpho-genesis. 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.

{Received 25 April 1977, revised 16 June 1977)