Analysis of the temperature-sensitive period of the short-1 mutation

affecting stomatogenesis in Paramecium tetraurelia

LAI-WA TAM and STEPHEN F. NG*

Zoology Department, University of Hong Kong, Hong f\ong

•Author for correspondence

Summary

Reduction in the length of the oral apparatusproduced by the temperature-sensitive mutationshort-1 (shl) involved suppressed growth of theoral primordium in all stages of development.Temperature shift-up and heat-shock exper-iments revealed that the temperature-sensitiveperiod of this mutation coincided with nearly theentire stomatogenic phase (stages 1-6) in sexualreproduction. Low- and high-sensitivity phaseswere noted, corresponding to the periods of slow

(stages 1 and 2) and rapid (stage 3 to stage 6)elongation of the oral primordium, respectively.The action of shl is thus concentrated afterstage 2. The mutation hypothetically results indefective membrane growth and extension in theoral primordium, leading to restriction in incor-poration of basal bodies into the developingmembranelles.

Key words: Paramecium, genetics, development, mutant,oral apparatus.

Introduction

The oral apparatus is the most prominent organelle ofciliated protozoa, and is of taxonomic, phylogeneticand developmental significance (see Corliss, 1967;Hanson, 1974). It has been extensively exploited inexperimentation and conceptualization in a variety ofcontexts in the developmental biology of ciliates (e.g.see Bakowska et al. 1982a,b; Buhse, 1966; Buhse &Zeuthen, 1974; Frankel, 1961, 1967, 1984; Frankelel al. 1984; Golinska, 1978, 1984; Grimes, 1982;Hanson, 1955, 1962; Jerka-Dziadosz, 1974, 1983;Sonneborn, 1963, 1970a; Tartar, 1960; Tchang &Pang, 1977, 1981).

The development of the oral apparatus (stomato-genesis) of Paramecium tetraurelia has been the focusof our studies for the following reasons. First, stomato-genesis is under the influence of the micronucleus.This is in contrast to the commonly held idea that themicronucleus in ciliates participates in nuclear reorgan-ization only during sexual reproduction (Ng & Mikami,1981; Ng & Newman, 1984a,6; Tarn & Ng, 1986).Second, the development and perpetuation of the oralapparatus during cell division is known to be affectedby the pre-existing oral apparatus (Hanson, 1955,1962; Hanson & Ungerleider, 1973; Sonneborn, 1963,

Journal of Cell Science 88, 241-250 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

1970a). Stomatogenesis thus offers a system for study-ing the interaction between factors of genie (micro- andmacronuclear) and non-genic nature (the pre-existingpattern). Third, in common with other ciliates, Para-mecium develops a new oral apparatus during bothasexual and sexual reproduction. The development ofthe same organelle in two different phases of the life-cycle raises the fundamental question of how stomato-genic controls are integrated into two different (asexualand sexual) developmental programs.

The stomatogenic process in P. tetraurelia in celldivision has been described in some detail by Kaneda &Hanson (1974), Jones (1976) and Shi (1980), and insexual reproduction by Ng & Newman (1984a). Tofurther our understanding of the control of stomato-genesis we have undertaken the approach of geneticdissection and a number of temperature-sensitive sto-matogenic mutants have been isolated (Tarn & Ng,1987). The stomatogenic properties of one such mutantcollected, short-1 (genotype shl/shl), which showsreduction in the length of the oral apparatus, membra-nellar irregularities, pleiotropic effects on cell size, celldivision and macronuclear division (for details, seeTarn & Ng, 1987), are the subjects of the presentinvestigation. The advantage of studying the shl geneis its high penetrance in both sexual and asexual cycles.

241

Mutations expressed during the sexual cycle are par-ticularly suitable for the characterization of stomato-genic events and temperature-sensitive period, assynchronization of conjugation can easily be achieved,and the timing of morphogenetic events in conjugationhas been well-established (Ng & Newman, 1984fl).Demarcation of the temperature-sensitive period, theinterval within which a shift to the restrictive tempera-ture can result in mutant expression, may throw lighton the timing and mode of gene action, and in turn onthe control of development.

Materials and methods

Strains and culture methods

The object of the present investigation is a temperature-sensitive mutant of Parameciuni tetraurelia, designated asshort-1 (genotype shl/shl), which develops oral apparatusesof reduced length (Fig. 1). This mutant was derived from thestandard stock 51, by ultraviolet mutagenesis (Tarn & Ng,1987). Two opposite mating types (VII and VIII) were usedthroughout the study.

Stock 51, mating types VII and VIII, were used forreference.

Bacterized Cerophyl medium (2-5 gl~' , phosphate-buffered at about pH7), supplemented with 5mgl~'

stigmasterol was used. Culture methods followed that ofSonneborn (1950, 19706).

Conjugation and heat treatment of conjugantsThe study of stomatogemc events and delineation of thetemperature-sensitive period were done with conjugatingcells. Post-autogamous cultures were fed in tubes and mildlystarved on the third day to induce mating reactivity. Mating-reactive cells of both mating types were mixed in Petri dishesat 27CC and the clumping reaction started immediately. Atabout 1 -25 h after mixing, fresh medium was added to themating cultures to eliminate loose pairing and further aggluti-nation, so that synchronized conjugation was attained. Tightpairs were picked up beginning at about 1*5 h. Samples of 50pairs each were collected in 0-2 ml medium in glass de-pression slides, or in capillary tubes sealed at one end. Atvarious times after the second hour of mixing, these cellsamples were heat-treated in a 34-5°C or 33-5°C water-bath,for defined periods (see Results). Depression slides werecontained in a sealed box submerged under water. Capillarytubes were dropped into a beaker immersed in the water-bathto permit quick equilibration of temperature. It should benoted that temperatures above 33 °C have the effect ofeliminating mating reactivity, so that temperature shift-upscould not be started before tight pairs had formed. Nuclearreorganization and stomatogenesis in conjugating cells arecomplete about 6-75-7 h after mixing at 27°C. Pair-separ-ation occurs at 5-5-5-75 h, but is usually speeded up by10—20 min at the restrictive temperatures.

Fig. 1. Oral apparatuses of a wild type (A,B represent two focal levels) and two short-1 mutant cells (C,D). Each oralapparatus consists of three oral membranelles, quadrulus (q), dorsal and ventral pemculi (dp, vp). The horizontal linesrepresent the antero-posterior extent of the quadrulus, which is taken as a measurement of the length of the oral apparatus.The variable food-vacuole-forming region (fvf) posterior to the posterior limit of the quadrulus is excluded frommeasurement. The posterior S-spiral of the quadrulus is absent in the two short-1 cells (C,D); D illustrates thedisorganized oral membranelles in some of the short-1 mutant cells. The inclined lines define the level where a break occursin the membranelles, leading to a lateral shifting of the fragments so that the anterior fragment of the dorsal peniculus hassome of its basal body rows lined up with those of the posterior fragment of the quadrulus (arrow in D). Silverimpregnation. X1800.

242 L.-W. Tarn and S. F. Ng

Cytology

The samples were fixed for silver impregnation (Chatton &Lwoff, 1936; Corliss, 1953) at defined times in each exper-iment. The oral apparatus was clearly revealed by thismethod. The lengths of the oral apparatus and oral primor-dium were measured with an ocular micrometer under X 1000phase-contrast optics. The length of the oral apparatus wasassessed by measuring the antero—posterior span of thequadrulus (Fig. 1). The length of the oral primordiumrepresented the antero—posterior extent, but not the absolutelength, of the developing membranelles, which are curved(see Fig. 2).

Statistics

The mean lengths of the oral apparatuses for differentsamples were compared by one-tailed Student's /-tests (Sokal& Rohlf, 1969). A SPSS-X multiple regression program wasemployed in the computation of linear and curvilinear

regressions, and also in tests of curvihnearity by /•'-tests ofsignificant improvements of fit over a linear model with theintroduction of second- and third-order polynomials. TheF-tests for the null hypothesis that the kth order termcontributes no substantial improvement of fit is given by:

F_ (R2 with &th order term) — (R2 without 6th order term)(1 - R2 with *th order term)/(A' - k - 1)

with 1 and (A'—k — 1) degrees of freedom; R, regressioncoefficient (see also Zar, 1974).

Results

Temperature-sensitivity of sh 1

The short-1 mutant possessed slightly shorter oralapparatuses and cell length compared to the wild typeat 27°C (Table 1). However, pronounced expression of

1

• : « : • ' •

<<•":•

< '• V?< . ; • • ;

«/

pf

Fig. 2. Stomatogenic stages 1-7 in conjugation in P. tetraurelia (adapted from Ng & Newman, 1984a). Stage 1 (0-5 h ofconjugation). Gradual proliferation of basal bodies in an oral anlage field (af) on the right vestibular wall between theinnermost right vestibular kinetics (rv) and the endoral kinety (ek). Stage 2 (5-5-5 h). Alignment of the basal bodies intoparallel rows in the anlage field, pc, paroral cone of the mate. Stage 3 (5-5-5-75 h). Development into a 6-row hook-shapepromembranelle. Stage 4 (5-75-6-15 h). Addition of basal body rows to the promembranelles and transformation into aC-shaped tripartite promembranelle with 12 rows. Stages 5-7 (6-15-6-5 h): Stage 5. Longitudinal stretching of thepromembranelle; appearance of the buccal cavity; the quadrular rows becoming spaced apart at the anterior and theposterior part developing into a spiral shape. Stage 6. Further expansion of the buccal cavity; differentiation of the ribbedwall (ra>) on its right side (contiguous to the right vestibule, rv); development of the oral membranelles into the maturepattern. Stage 7. Appearance of the postoral fibres (pf); the oral apparatus reaches its full length, <?, quadrulus; dp, dorsalpeniculus; vp, ventral peniculus. Stages 1, 6, left view; stages 2, 3, left-ventral view; stage 4, right-ventral view; stages 5,7, ventral view.

Paramecium stomatogenic mutant 243

Table 1. Comparison of the lengths of the oral apparatus in asexual and sexual reproduction, and cell length,between the short-1 mutant and wild type, at 27°C and 35°C

Temperature(°C)

27

35

Parameter

Oral lengthAsexualSexual

Cell length

Oral lengthAsexual|Sexual

Cell length

Mean ± S.D.

28-9 ±0-6526-8 ± 1-65

lll-3±5-l

28-3 ±0-9826-1 ±201

119-014-2

Wild typef

(»)

(21)(18)(18)

(20)(22)(18)

[range] (jim)

[28-30][24-30]

[102-120]

[27-31-5][23-29]

[104-120]

Mean ± S.D.

24-9 ±1-6825-3 ± 1-35

100-6 ±4-7

14-1 ±3-8620-4 ±1-3570-1 ± 13-6

Short-1

(")

(22)(22)(18)

(26)(19)(21)

[range] (/im)

[23 -28 ] "[23-28]*[89-105]"

[8 -5 -22]"[18-5-22]"

[ 4 4 - 9 6 ] "

Data adapted from Tarn & Ng (1987; Tables 2 and 3).| Wild type exhibited a very slight reduction in oral length during asexual reproduction (after three fissions) at 35°C, compared to 27°C

(sexual 0 -02>P>0-01 , sexual 0 - 2 > P > 0 - 1 ) .JThc oral length of short-1 was reduced to a larger extent in asexual reproduction at 35 °C.•0-005>P>0-001 , "0-001 >P (one-tailed Mests), short-1 and wild-type compared.

this mutant was brought about by elevating the tem-perature to 35 °C, which produced a much greaterextent of reduction of these parameters. It is thus clearthat the mutant is temperature-sensitive. Hence, ex-pression of the phenotype can be analysed by applyinghigh temperature at different stages of development ofthe oral apparatus.

Stotnatogenesis during conjugation in sh 1Stomatogenesis during conjugation of wild-type para-mecia has been reported in detail (Ng & Newman,1984a; see Fig. 2 and legend). How does the stomato-genic process of short-1 deviate from the normalprogramme?

This question was answered by following the devel-opment of the oral apparatus in conjugating short-1cells. Heat treatment was started at the second hourafter the initiation of conjugation, i.e. in stomatogenicstage 1, about 3 h before the start of stage 2 (see Fig. 2).Sampling for cytological examination began at thefourth hour, at 15-min intervals, until the exconjugantsresumed feeding (at about the sixth hour). Thesesamples showed that the morphology of the developingprimordium at all stages of stomatogenesis was notdifferent from normal, except for size reduction. Thelength of the oral primordium at various stages ofdevelopment was measured and the results are shownin Fig. 3. It is obvious that the developing oralprimordium of short-1 was significantly shorter thanthat of wild type at all stages of stomatogenesis(P< 0-001). This suggests that the reduction in thefinal length of the oral apparatus (membranelles andbuccal cavity) was a result of either one of twoalternative modes: (1) suppression of the expansion ofthe oral primordium at all stages of its development, or(2) a severe restriction of the growth of the primordiumat the early stages (e.g. stages 1 and 2), producing a

long-lasting effect on its lengthening in subsequentstages. The second alternative necessitates that themost temperature-sensitive period coincides with, orprecedes, the early stages. However, temperature shift-up and heat-shock experiments detailed below indicatethe contrary.

Temperature shift-up experimentsThe temperature-sensitive period (TSP) of shl wasdelineated by shift-up experiments. At different timesof conjugation at 27°C, cells were transferred to therestrictive temperature and maintained until the timeof fixation, usually at 8-11 h after mixing the twomating-types, when development of the oral apparatusshould have been completed. Parallel samples ofmating short-1 cells maintained at 27°C throughoutserved as controls. The length of the oral apparatus indifferent samples was assessed and the means wereplotted against the time at which the shift-ups began(Fig. 4). Initially, experiments (nos 1-4, Fig. 4A)were carried out in depression slides and at therestrictive temperature of 34-5°C. This, however,produced some anomalous results (nos 2, 4, Fig. 4A,the 4th h sample). Also, wild-type cells in 35°C tend toproduce slightly shorter oral apparatuses (see Table 1).Therefore, subsequent experiments (nos 5, 6, Fig. 4B)were performed in capillary tubes for better control oftemperature, and at 33-5°C, at which wild-type cellswere little affected.

The end-point of the TSP was defined by the earliesttime at which a shift to the restrictive temperature doesnot result in any reduction in the length of the oralapparatus. Of the six experiments, four indicated thatthe end-point of the TSP was located within 6-6-5 hafter the initiation of conjugation (nos 1, 2, 3, 5,Fig. 4). This corresponds to stomatogenic stages 6 and7 (Fig. 2), when the oral apparatus is approaching its

244 L.-W. Tarn and S. F. Ng

35-1

30-

E3E 25-

o

o•S 15 '

10-

Wild type

Short-1

15

40

2 3 4a 4x 4b 4 y 4c 5a 5b 6 7 8Stages in stomatogenesis

Fig. 3. The mean length and standard error of the oral primordium at different stages of development of short-1 (at34-5°C) and wild-type cells (at 27°C). For stages in stomatogenesis, refer to Fig. 2. Values for wild-type are adapted fromNg & Newman (1984a). 4x, conjugants connected by thin bridge; 4y, exconjugants. Within a stage, a, b, c denote earlierand later substages. Samples sizes are indicated in each bar.

final length (see Fig. 3). In experiments 4 and 6, thelast heat-treatment samples (at the sixth hour) did notreveal the end-point of the TSP. This could be due to alonger stomatogenic cycle in these cases, so that thecells in the last samples had not yet reached' the finalphase of elongation of the oral apparatus in stages 6 and7, when the heat treatment was applied.

The beginning of TSP is usually revealed by tem-perature shift-down experiments, which involve incu-bation at the restrictive temperature at an early stageand subsequent shifts to the permissive temperature.However, early heat treatment of conjugants up to2h after the initiation of conjugation is impossiblebecause it results in the separation of the conjugantsand termination of conjugation (see Materials andmethods). Nevertheless, given this limitation, the earlyTSP may be identified by defining the latest time atwhich a shift-up produces the maximum effect onreduction of oral length. In fact, shift-up experiments1, 3, 5, 6 and also 7(1) (see below) indicated that themean length of the oral apparatuses continued todecrease with earlier shifts (Figs 4, 5A). Furthermore,the earliest heat treatment did produce the mostmarked effect in reduction of oral length (beginning atthe second hour of conjugation in expts 4, 5, 6, and also7(1), see below; third hour, expt 3; 2-5—3 h, expt 1).

While these observations do not allow us to pinpointthe start of the TSP, they suggest that the TSPincluded the third hour, and probably extended intothe second hour, corresponding to stomatogenic stage1, which lasts for 4-5-5 h (Ng & Newman, 1984a).Furthermore, as concluded previously, the TSP lastedfor about 6— 6-5 h after the initiation of conjugation.Hence the entire TSP should comprise stomatogenicstages 1-6 or 7.

To delineate the TSP in exact stomatogenic terms,two additional temperature shift-up experiments, 7(1)and 8(1) (Fig. 5), were performed at 33-5°C, in whichcell samples at the beginning, as well as at the end, oftemperature shifts were also fixed for silver impreg-nation to define the stomatogenic stages. The resultsagreed with the conclusion from the previous exper-iments: the TSP covered stomatogenic stage 1 tostage 6.

A common feature of the eight experiments was atransition in the slopes of the curves around 5-5-5 hafter conjugation. The relation between final orallength and the time at which temperature shift-upoccurred in each experiment was best described byfitting a curvilinear regression (see Materials andmethods). Significant improvements of fit over a linear

Paramecium stomatogenic mutant 245

25

24

~ 23E

I 22D.D.

21

- 20o

19

18

17

16

27°C Controls

B

-

-

-

i

5 ? ' ' ' '

1 1

ff

1 1

4 5 6 7 0 1 2 3Time at which heat treatment begins (h after initiation of conjugation)

Fig. 4. The final mean length of the oral apparatuses attained in short-1 after heat treatment of conjugants beginning atvarious times since initiation of conjugation. A. Experiments 1-4, 34-5°C; B, experiments 5, 6, 33-5°C. Each plotrepresents an experiment and is numbered; s, separation of conjugants. Vertical bar indicates ±standard error of mean.Parallel samples kept at 27°C throughout serve as controls. Except where marked by f, all points are significantly lowerthan the 27°C control in each experiment (P<0-05, one-tailed /-test). Anomalous results in expts 2, 4 are marked by ?.

model were achieved with either a second-order poly-nomial (expts 1, 2, 5, 7(1), 8(1), and also 4 excludingthe fourth hour subset of data: sample sizes, 161, 226,124, 275, 427, 99 cells, respectively; F-tests, no. 2,P < 0 - 0 1 ; no. 1, P<0-005; others, P<O001) , orthird-order (expts 3, 6: sample sizes, 46, 97 cells,respectively; F-tests: P<O025 and P < 0 0 5 , respect-ively). Furthermore, experiments 7(1) and 8(1)showed that the transition in the slopes of the curvesoccurred around stomatogenic stages 2 and 3 (Fig. 5).The transition suggests that the TSP consists of twophases: an initial low-sensitivity phase, comprisingstomatogenic stages 1 and 2, subsequently transform-ing into a high-sensitivity phase in stages 3-6. The bestway to test the sensitivity of each interval within theTSP is, however, by heat-shock experiments.

Heat-shock experiments

Heat-shock experiments 7(11) and 8(11) were per-formed, in parallel with temperature shift-up exper-iments 7(1) and 8(1). Cell samples were exposed to heatat 33-5°C for 30min at different times of conjugation,and then fixed for silver impregnation at 10-11 h afterthe initiation of conjugation. Parallel samples were

fixed to ascertain the stomatogenic stages at the begin-ning and end of each heat-shock treatment. Short-1mating pairs maintained at 27 °C throughout served ascontrols.

The results are shown in Fig. 5A(II), B(II). Heatshocks given within stage 1 to stage 2 of stomatogenesisdid not have any effect, whereas those given withinstage 2 to stage 6 produced a significant reduction in thelength of the oral apparatus, in agreement with thetemperature shift-up experiments. On the other hand,the shift-up experiments have established that the TSPextended into stage 1; the ineffectiveness of heat-shocktreatment in stage 1 could be due to reversibility of theeffect of exposure to heat for 30min. Examples of suchphenomena were found in Drosophila (Foster, 1973).This explanation also agrees with the low temperature-sensitivity of stages 1 and 2 (as revealed by the shift-upexperiments), so that prolonged heat treatment instages 1 and 2 is required to bring about a significantreduction in oral length.

Although the heat-shock experiments indicated thatthe transition between the low- and high-sensitivityphases occurred around stages 2 and 3, the boundarybetween the two phases cannot be defined in exactstomatogenic terms in our experiments. This is because

246 L.-W. Tarn and S. F. A'g

25-1

§_24-

a.g-23-

"o 22-JSSoc

20'

A. Experiment 7

(II) Heat-shock ( { > « _ _ _

(I) Shift-up

(6)22%

3

(1)-

4 5Hours after initiation of conjugation

Stomatogenic stages

(6)83%"(5b) 17%

25"I B. Experiments

E 24'3

oJZ

S 2 1 -U.

20-

J27-C(*>) Control

3

(1)-

4 5Hours after initiation of conjugation

"(1) (1) (2)Stomatogenic stages

(4a) 92 % (4b.c)_(5) 64 % (7) 73 % ( | | ,(4b) 8 * (6) 36% (8) 27%

Fig. S. The final mean length of the oral apparatuses attained in short-1 when heat treatments (33-5°C; shift-ups (I), heat-shocks (II)) were applied at defined stomatogenic stages during conjugation. A. Expt 7; B, expt 8. Vertical bars indicatethe standard error of mean. For both shift-up experiments, 7(1) and 8(1), the sixth-hour sample is the earliest one withlength not significantly smaller than the 27°C control. For the heat-shock experiments 7(11) and 8(11), horizontal barsindicate each 30-min period of exposure to heat. Values in parentheses underneath the abscissa represent the stomatogenicstages at 27CC at the beginning of heat treatments (shift-ups or heat-shocks). Values in parentheses next to the bars indicatethe stomatogenic stages at the end of heat-shock, which were usually a little bit more advanced than those in cells at 27°C,as stomatogenesis was speeded up at higher temperature (see Materials and methods). In some samples (sample sizes,10—30 cells), variation in the stomatogenic stages among the cells was found and the percentage of cells in each stage in thesame sample is specified. Values significantly lower than those of the 27°C control are marked by asterisks (one-tailed/-test): • P < 0 - 0 5 ; • • />< 0-005; • • • />< 0-0001.

Paramecium stomatogenic mutant 247

stage 2 usually spans less than 0-5 h (Ng & Newman,1984a), and conjugants heat-shocked for 0-5 h begin-ning at stage 2 have proceeded to stages 3 and 4 at theend of the heat-shock period (Fig. 5A(II), B(II)).Thus, although these samples indicated that heat-shock was effective in reducing oral length, it is notclear if the stage 2 oral primordium was affected to alarge extent by the treatment. It is nevertheless clearthat stage 3 falls within the high-sensitivity period,since heat-shocks ending or beginning at stage 3produced significant reductions of oral length(Fig.SA(II)).

Discussion

The action of the sh 1 gene

The shl gene produces a reduction in the length of thebuccal cavity and oral membranelles, the latter obvi-ously consisting of fewer basal bodies than normal (cf.Fig. 1A,C). The reduction in oral length is manifestedin all stomatogenic stages, the oral primordium beingreduced in length throughout (Fig. 3). Furthermore,the temperature-sensitive period of shl consists of twophases: a low-sensitivity phase, including stomatogenicstages 1 and 2, during which basal bodies accumulate inthe oral anlage field, and the primordium increases onlyslowly in size; and a high-sensitivity phase, fromstomatogenic stage 3 to stage 6, covering the period ofrapid expansion of the oral primordium (from stage 4to stage 6), when more basal bodies are added and theybecome aligned and progressively close-packed to formthe membranellar rows. The low-sensitivity phase hasnot been defined to our satisfaction, partly because ofvarying responses of the cells in some of the tempera-ture shift-up experiments (nos 2 and 4, Fig. 4; no. 8(1),Fig. 5), and partly because of technical limitations inour experimentation (see Materials and methods),prohibiting a precise definition of the beginning of thetemperature-sensitive period. Nevertheless, it is clearthat the temperature-sensitive period includes the thirdhour (and maybe the second) from the beginning ofconjugation, and hence stomatogenic stage 1.

The main emphasis of our results is that the effect ofthe shl gene is concentrated after stomatogenic stage 2.This means that the shl gene acts mainly during therapid expansion of the oral primordium and incorpor-ation of basal bodies into the developing membranelles.These processes necessarily involve the growth andextension of the pellicular membrane of the oralprimordium, and the initiation and maturation of basalbodies and their subsequent incorporation into thedeveloping membranelles. The details of these pro-cesses in Paramecium are not fully understood. Never-theless, we present the following speculation as aworking hypothesis.

The initiation and maturation of basal bodies involvea series of morphogenetic steps documented at theultrastructural level (Dippell, 1968). We do not thinkthat shl primarily creates defects in these steps per se,as evident from another detail of the stomatogenicprocess at stage 4. In addition to extending in anantero-posterior direction, the stage 4 primordiumalso develops from six basal body rows to one possess-ing 12 rows, eventually divided into three groups offour rows each (Fig. 2). In stage 4, spaces are seenbetween adjacent rows in the six-row oral primordiumand there are indications that the increase in thenumber of basal body rows involves addition of basalbodies between adjacent rows (Ehret & de Haller,1963; Gillies & Hanson, 1968; Ehret & McArdle, 1974;Ng & Newman, 1984a). While the precise site of originof these basal bodies is unknown, we can be sure thattheir formation and incorporation into the oral mem-branelles are unaffected by shl, because as a rule themutant develops three oral membranelles, each carry-ing four rows of basal bodies (Fig. 1C). This arguesagainst the notion that the shl mutation introduces aprimary defect in the initiation and maturation of oralbasal bodies perse. Instead, we favour the view that theprimary defect resides in the restriction of membranegrowth and extension of the oral primordium, primar-ily in the antero-posterior direction. This limitationimposed by oral membrane topography can explainwhy basal body incorporation is restricted in theantero-posterior direction, but is possible laterallybetween basal body rows in the oral primordium.

Evidently, a more detailed study of the dynamics ofbasal body addition in the oral primordium throughoutstomatogenesis, especially at the ultrastructural level,would be imperative to further our understanding ofthe mode of action of shl. A parallel approach would beto exploit the pleiotropism of the shl mutation (Tarn &Ng, 1987). In addition to reduction in the length of theoral apparatus, the length of the cell is also markedlyreduced (Table 1). It should be noted that the oralapparatus is a specialized cortical region. As such, itsstructural components have much in common withthose in the cortex, including membranes, basalbodies, and other cytoskeletal elements, though thedeployment of these in the two are different. Thereduction in the lengths of the oral apparatus andthe cell suggests that the two are affected basically bythe same lesion. If so, the mode of action of shl may beassessed through the study of cellular growth anddivision, which also involve basal body proliferationand membrane growth on the cell cortex. Duringdivision, the formation of new cortical units involveselongation of existing units (membrane growth) withinbasal body rows near the fission zone, and simul-taneously sub-surface formation of new basal bodiesand their emergence anterior to pre-existing basal

248 L.-W. Tarn and S. F. Ng

bodies (Dippell, 1968; Kaneda & Hanson, 1974).Further extension of the cortical units and partitioningof basal bodies within the rows subsequently divideeach original unit into two or more new ones. Thesomatic basal body rows on the cell surface are moreamenable to analysis in connection with the shl mu-tation.

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(Received 27 April 1987 -Accepted 4 June 1987)

250 L.-W. Tarn and S. F. Ng