DEVELOPMENTAL

BIOLOGY

81,336-341(1981)

Positional

Control of Nuclear

Differentiation

in Paramecium

SIMONE GRANDCHAMPANDJANINEBEISSON Centre de Gk&ique

Molbulaire,

Centre National

de la Rechew&

Scientifique,

91190 G&w-Yvette,

France

Received January 2, 1980; accepted in revised form May 27, 1980

Nuclear reorganization, which results in the differentiation between macronuclear anlagen and micronuclei during autogamy or conjugation in Paramecium tetraurelia, was compared in wild-type cells and in two mutants, mic.& and kin241, which form abnormal numbers of macronuclear anlagen and micronuclei. Our observations show that all macronuclear anlagen derive from the nuclei positioned at the posterior pole of the cell at the second postzygotic division. This posterior localization is transient and correlated with a marked change in cell shape and decrease of cell length. These results suggest that cytoplasmic or cortical factors precisely located in the posterior pole are essential to trigger macronuclear differentiation and that the control of nuclear positioning is dependent upon precise modifications of cell shape.

INTRODUCTION

Most ciliated Protozoa contain two types of nuclei which differ markedly in ploidy, structure, and function. The diploid micronucleus has condensed, practically inactive chromatin and represents the “germ line”; the polyploid macronucleus is the site of transcription but its genetic continuity is limited to the intervals of vegetative multiplication between sexual processes. It is then broken down, digested, and replaced by a new macronucleus which develops from the micronuclear line. In Paramecium tetraurelia, the highly polyploid (ca. 800 n) macronucleus develops at each sexual process (whether conjugation or autogamy) in the following manner. The diploid zygotic nucleus divides twice, and within minutes after the second mitosis, two daughter nuclei are committed toward the macronuclear state while the other two retain their micronuclear status. In the macronucleus, polyploidization proceeds by rapid rounds of DNA synthesis, and transcription soon begins (Berger, 1973). The micronucleus, however remains diploid and probably completely inactive (Pasternak, 1967) in the course of subsequent cellular divisions. ’ What triggers and controls the divergent fates of genetically identical nuclei within the same cytoplasm is still unknown. However, one factor seems to be the position of the nuclei at the end of the second postzygotic division. In Tetrahymena py@iformis (Nanney, 1953) and in Colpidium and Leucophrys (Maupas, 1889), the two nuclei located at the anterior pole of the cell have been shown to become the macronuclei. In Paramecium, Hertwig (X389), Chao (unpublished data cited by Sonneborn, 1955), and Jones (1956) noted that in some cells, the two macronuclear anlagen were located 0012-1606/81/020336-06$02.00/O Copyright All rights

Q 1981 by Academic Press, Inc. of reproduction in any form resewed.

in the posterior cell pole, but the generality of the phenomenon was not ascertained. We demonstrate here, by cytological observation of wild-type cells of P. tetraurelia and of two mutants forming abnormal numbers of micro and macronuclei at nuclear reorganization, that all macronuclei originate from the posteriorly located nuclei. Our observations suggest that the commitment toward differentiation into macronuclear anlagen depends on a very short stay of the nuclei at the posterior pole of the cell. MATERIALS

AND METHODS

Strains. The strains used in these experiments were the following: a wild-type strain, stock d4-2, of P. tetraurelia according to Sonneborn’s (1975) nomenclature, and two mutants, mic4.4 and kin.241, derived from this wild-type strain. The mic& was obtained after a uv mutagenesis and first screened as a slow-growing mutant (Ruiz, unpublished). Its major phenotypic properties, as listed in Sonneborn (1974) are an abnormal nuclear reorganization regularly yielding 4 micronuclei:4 macronuclear anlagen instead of the normal 22, as well as disorders in the cortical pattern. The kin241 was also obtained after dv mutagenesis and screened as a thermolethal, slow-growing mutant. This mutation is highly pleiotropic, affecting most aspects of cellular morphogenesis (Beisson et al., 1976) and in particular nuclear reorganization. The kin.2-41may have variable numbers of macronuclei and micronuclei, ranging from the normal 22 up to 12 macronuclear anlagen with a modal value of 4:4. Both mutations are monogenic recessive nuclear mutations and they belong to two independent loci. A cytological study of meiosis and nu-

336

BRIEF NOTES

clear reorganization in these mutants is described elsewhere (Grandchamp, in preparation). Culture conditions. Cells were grown according to the usual procedures (Sonneborn, 1970) in a Scotch grass infusion bacterized by Klebsiella pneumoniae, supplemented with 0.4 pg/ml p sitosterol (Merck). Culture temperature was 27 or 15°C. The populations studied were derived from single autogamous cells, and examination of nuclear reorganization was made on either autogamous cells or on conjugating animals. Autogamy occurs in all cells of a clone when the food is exhausted, but the onset of autogamy is not quite synchronous and therefore an “autogamous population” comprises all stages of nuclear reorganization processes. In contrast, a good synchrony can be obtained in pairs of conjugants isolated 1 hr 30 min after mixing of two sexually reactive populations of complementary mating types (Sonneborn, 1970). Cytological technics. Whole cells were put on albuminized slides and allowed to dry. After fixation in an ethanol-acetic acid mixture (3:l) for 20 min and hydrolysis in 1 N HCl(6O”C) for 11 min, the preparations were stained in a mixture of 10 ml of 0.5% Azure A with 0.15 g sodium metabisulfite and 1.5 ml 1 N HCl for 30 min (Delamater, 1951). Measurements. Three types of measurements were carried out on Azure A-stained cells at Xl000 on a phase-contrast microscope: (a) cell length, (b) the diameters of macronuclear anlagen corresponding to the maximum diameter of the stained chromatin mass, and (c) the distances of macronuclear anlagen from the posterior pole of the cells. RESULTS The main steps of nuclear reorganization in Paramecium tetraurelia are represented in Fig. 1: the time course of the events is indicated on the basis of observations of wild-type conjugants maintained at 27°C as described under Materials and, Methods. The nuclear processes are accompanied by changes in cell length which reaches a minimum during stages 8-10, as shown in Fig. 2. This extreme reduction in cell length is correlated with a marked change in the shape of the posterior part of the cell which appears “truncated.” This feature is transient: by stage 11, the posterior part of the cell regains a more or less conical shape, and by stage 12 the cell length has returned to its initial value. The new information presented here concerns the behavior of the nuclei just after completion of the second mitosis of the zygotic nucleus at stages 8-10 of Fig. 1. The existence of stage 10, in which the spindles have disappeared, the nuclei have not yet moved away from their polar location and the young macronuclear an-

337

lagen are already differentiated, was first established by observation of the mutant mic4.4. The occurrence of stage 10 in wild-type cells was then ascertained, and its functional significance corroborated by observation of the mutant kin.241 which forms abnormal numbers of macro- and micronuclei. The observations on wildtype cells and on the two mutants mic44 and kin241 (illustrated in Figs. 3-5) will be presented successively.

Wild Type Figures 3a-e show the succession of nuclear events corresponding to the stages 8-12 of Fig. 1. The situation of Fig. 3c was observed only once among over 200 autogamous cells grown at 27°C. Assuming that stage 10 is a very transient one, we examined populations undergoing autogamy at 15°C with the hope that the duration of this stage might be increased. At this temperature many “short truncated” cells were indeed found and selectively examined. Among this subpopulation, 30% of the cells were at stage 10. Then, in order to ascertain whether in all cells the macronuclear anlagen had indeed started to differentiate in the posterior pole, before moving away, the diameters of the nuclei were measured and plotted against the distance from the posterior pole. Figure 6 shows that (1) a positive correlation exists between the diameters of macronuclear anlagen and their distance from the posterior pole and (2) most significantly none of the smallest 2pm macronuclear anlagen was found beyond 15-20 pm away from the posterior pole. These facts support the conclusion that macronuclear anlagen always originate from the posterior pole and that their differentiation is triggered before they move away toward more central locations within the cell. mic 44 The mutant mic4.4 regularly forms, in more than 90% of cases, four micronuclei and four macronuclear anlagen at nuclear reorganization. This abnormality results from the formation of two zygotic nuclei instead of one, as described elsewhere (Grandchamp, in preparation). Except for this initial difference from the wild-type situation, nuclear reorganization proceeds normally. The other main difference between wild-type cells and mic44 ones is that ca. 10% of unselected autogamous cells are in stage 10. Figures 4a-c show representative images of the stages 9, 10, and 12 and unambiguously indicate that in this mutant, all macronuclear anlagen derive from the posterior products of the second postzygotic mitotic division.

kin 241 In the mutant kin241 nuclear reorganization yields a wide range of situations. At late stages, one might

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VOLUME 81, 1981

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FIG. 1. The main steps of nuclear reorganization in wild-type cells at 27°C. Stage 1: represents a premeiotic cell with one macronucleus (M) and two micronuclei (m). Stages Z-5: the two micronuclei are in metaphase (2), yielding eight haploid nuclei (3); from stage 3, the macronucleus begins to fragment. One micronucleus forms a zygotic nucleus (zn) in the paroral cone (PC) and the other seven disintegrate. In different cells at stage 4, the spindle may be seen with either a longitudinal, transverse or oblique orientation with respect to the cell long axis. At stage 5, the zygotic nucleus may still be seen at the paroral cone or further away. Stages 6-8: first and second postzygotic divisions; at stage 8, the two mitotic spindles (ms) extend from pole to pole; the macronucleus is now fragmented (Mf). Stage 9: the spindles are no longer visible. Stage 10: the two macronuclear anlagen (Ma), already differentiated, are located at the posterior pole of the cell and the two micronuclei are located at the anterior pole. Stages 11-12 the two micronuclei and two macronuclear anlagen migrate toward the center of the animal.

observe some cells containing two micronuclei and two macronuclear anlagen (2:2), a majority of 4:4 cells, and about lo-30% cells with any number of nuclei from 0 up to 20; among the latter category, there are cells containing unequal numbers of macronuclear anlagen and

801,

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012345678 Hours

FIG. 2. Variation of cell length during nuclear reorganization of wild-type cells. The measurements were carried out on populations of autogamous cells fixed and stained with Azure A. Each point represents the mean value (with its standard error) calculated for a 30cell sample. The cell mean length is plotted as a function of time after conjugation; the t = 0 point corresponds to the mixture of sexually reactive populations of complementary mating types. The later points correspond, respectively, to stages 2,3,4, + 5,6,7,8 + 9 + 10, 11, and 12 as defined in Fig. 1.

micronuclei, e.g., 5:3, 6:2, 4:0, 0:4, etc. At early stages, i.e., just after the second postzygotic division, two classes of cells are present: (1) cells displaying two or four long parallel spindles stretching from pole to pole, similar to those shown in Fig. 3a; (2) cells in which the spindles are no longer present and which contain more than four nuclei, randomly located in the cytoplasm and all retaining the aspect and diameter of micronuclei. It is not yet known whether this latter class corresponds to an absence of polar localization at the end of second postzygotic division (defective elongation or positioning of the spindles?) or to a delay in macronuclear differentiation (nuclei moving away from their polar sites before differentiation was triggered?). Whatever the case may be, it was observed that within an autogamous population of kin2.41 cells, the proportion of stage 12 cells showing impaired nuclear differentiation (i.e., unequal numbers of macronuclei and macronuclear anlagen) was the same as that of stage 8 cells displaying a nonpolar localization of still undifferentiated nuclei. For example, in one preparation we observed, out of 60 late stages, 41 (70%) which contained equal numbers of macronuclear anlagen and micronuclei but 19 which contained unequal numbers of macronuclear anlagen and micronuclei, and, out of 43 early stages, 28 (66%) which contained nuclei normally positioned at the poles and 15 which contained 8 or more dispersed micronuclei not yet engaged in macronuclear differentiation. The simplest interpretation of this correlation is to assume that when the nuclei do not reach the polar sites or

339

FIG. 9. Nuclear reorganization in wild-type cells. The old macronucleus has broken down into a number of fragments (Mf). (a-e) Corresponds respectively to the stages 8-12 of Fig. 1. Thick arrows indicate macronuclear anlagen and thin arrows micronuclei. (a-b) Show two nuclei at the anterior pole of the cell and two at the posterior pole; the mitotic spindles (ms) are visible in (a). (c) Shows two young macronuclear anlagen still in the posterior pole of the cell. (d) The two macronuclear anlagen and the two micronuclei have migrated away from the poles. In (e), the macronuclear anlagen are already well developed. FIG. 4. Nuclear reorganization in mutant mic 44. (a-b) Correspond, respectively, to stages 9 and 10 of Fig. 1. Thick arrows indicate macronuclear anlagen and thin arrows micronuclei. (a) Shows four nuclei at the anterior pole of the cell and four at the posterior pole; (b) shows four young macronuclear anlagen at the posterior pole of the cell. (c) Corresponds to stage 12 of Fig. 1; four micronuclei and four welldifferentiated macronuclear anlagen are present. FIG. 5. Nuclear reorganization in the mutant kin 241.Thick arrows indicate macronuclear anlagen and thin arrows micronuclei. (a and b) Represent two cells at early stages i.e., just after the second postzygotic division: 8 nuclei in (a) and 16 in (b) are dispersed randomly in the cytoplasm. (c) Is an example of a cell containing an unequal number of macronuclear anlagen and micronuclei: 53.

move away before they have started the differentiation is impaired.

to differentiate,

DISCUSSION

In order to analyze the factors responsible for the differentiation between macro- and micronuclei which

occurs during sexual processes (autogamy and conjugation) in P. tetraurelia, we have compared nuclear reorganization in wild-type cells and in two mutants forming abnormal numbers of macro- and micronuclei. The long spindles of the second postzygotic mitosis push the sister nuclei to the poles of the cell. The end of this mitosis (Fig. 1, stage 8) is correlated with a

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DEVELOPMENTALBIOLOGY

VOLUME81,198l

I

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Diometer

sf

onlogen

.

b

In pm

FIG. 6. Correlation between macronuclear anlagen diameters and their distance from the posterior pole. The diameters are measured to the nearest half pm. The line was fit by a linear squares method, without transformaiton of the data which should have homogenized the variances.

truncated shape of the posterior part of the cell and a maximum reduction in cell length (Fig. 2) that lasts about 20 min. Then by stage 11, the posterior end of the cell returns to its conical shape, cell length begins to return to its initial value, and already differentiated macronuclear anlagen are located anywhere within the cell. In wild-type cells maintained at 27”C, already differentiated macronuclear anlagen are rarely found at their polar site, but their polar origin is demonstrated by the observed positive correlation between their size and their distance from the posterior pole (Fig. 6). In wild-type cells maintained at 15”C, the much higher probability of observing stage 10 indicates that low temperature slows down the migration of macronuclear anlagen from the pole and the recovery of the initial cell length and shape, without delaying macronuclear

differentiation. Similarly, the high frequency of observation of stage 10 found among autogamous mic.44 cells suggests that this mutation does not affect macronuclear differentiation but delays cell elongation and migration of the macronuclear anlagen from the pole. These observations on wild-type and mic.44 cells demonstrate that the macronuclei regularly develop from the posterior nuclei and that during their short stay (which is certainly less than 20 min) in the posterior cell pole, macronuclear differentiation (i.e., DNA synthesis, transcription, and changes in chromatin organization) is triggered. That this polar localization is indeed essential for macronuclear differentiation is strongly suggested by the observation of the mutant kin241 in which unequal numbers of macro- and micronuclei are correlated with a nonpolar localization of

341

BRIEF NOTES

still undifferentiated nuclei after the second postzygotic division. A positional control of macronuclear differentiation has been demonstrated in Tetrahymena ~r@rrnis (Nanney, 1953), Co&i&urn, and Leucophrys (Maupas, X389),and quite recently in l? caudatum (Mikami, 1980). This could therefore be a common feature in ciliates, resembling the positional control of nuclear differentiation in metazoa and in particular of the polar cells in drosophila (Illmensee and Mahowald, 1974; Mahowald, 1977; Waring et al., 1978). The interest of the results reported here is twofold. (1) They suggest that nuclear differentiation is controlled by strictly localized cytoplasmic factors whose nature remains to be determined and could be established by the same techniques as those used to demonstrate the role of polar granules in the differentiation of polar cells in Drosophila. Preliminary experiments show that amputation of the posterior part of the cells at stages 4-6 (Fig. 1) results in absence of macronuclear anlagen. (2) Our observations also open up a way to analyze the factors which control the precise positioning required for nuclear differentiation to be triggered. The correlation between the polar positioning of the nuclei and the shortened and modified shape of the cell suggests that cytoskeletal components controlling cell shape and connecting the nuclei and the cell surface might be essential in bringing the nuclei to the poles and then releasing them. It is of particular interest to note that in the mutant kin2.41, the cells in which the nuclei do not reach a polar localization at the end of the second division do not show the typical shortened shape. It is also of interest to point out that the two mutations (mic.44 and kin241), which display abnormal nuclear reorganization, are also characterized by defects in their surface organization (irregular localization of cilia and trichocyst attachment sites). Further studies of these and similar mutants should permit us

to analyze the relationships between the organization of the cell cortex and the control of nuclear positioning. REFERENCES BEISSON,J., ROSSIGNOL, M., RUIZ, F., ADOU~E, A., and GRANDCHAMP, S. (1976). Genetic analysis of morphogenetic processes in Paramecium: A mutation affecting cortical pattern an nuclear reorganization. J. Protozool. 23,3A. BERGER,J. D. (1973). Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia. Chromosoma (Berlin)

42,247-268.

DELAMATER,E. D. (1951). A staining and dehydrating procedure for the handling of microorganisms. Stain Technol. 26,199-204. HERTWIG,R. (1889). Uber die Konjugation der Infusorien. Abh. Bayer. Akad. Wiss. 17, 151-233. ILLMENSEE,K., and MAHOWALD,A. P. (1974). Transplantation of pasterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Nat. Acad Sci. USA 71.1016-1020. JONES,K. W. (1956). Nuclear differentiation in Paramecium. Ph.D. Thesis, Department of Biology, Aberystwyth University, Wales. MAHOWALD,A. P. (1977). The germ plasm of Drosophila: An experimental system for the analysis of determination. Amer. 2001. 17, 551-563. MAUPAS, E. (1889). Le rajeunissement karyogamique chez les cilibs. Arch. 2001. Exp. Gen. Ser. 2-7,149-517.

MIKAMI, K. (1980). Differentiation of somatic and germinal nucleus correlated to intracellular localization in Paramecium candatum exconjugants. Develop. Biol. 80, 46-55. NANNEY, D. L. (1953). Nucleo-cytoplasmic interactions during conjugation in Tetrahymena Biol. Bull. 105, 133-148. PASTERNAK,J. (1967). Differential genie activity in Paramecium aurelia. J. Exp. Zool. 165, 395-417. SONNEBORN, T. M. (1954). Patterns of nucleo-cytoplasmic integration in Paramecium. Caryologia 1,307-325. SONNEBORN,T. M. (1955). Heredity, development and evolution in Paramecium. Nature (London) 175, 1100-1106. SONNEBORN, T. M. (1970). Methods in Paramecium research. Methods Cell Physiol. 4, 241-339. aurelia. In “Handbook of SONNEBORN,T. M. (1974). Paramecium Genetics” (R. King, ed.), Vol. 2, pp. 469-594. Plenum, New York. SONNEBORN, T. M. (1975). The Parmeeium aurelia complex of 14 sibling species. Trans. Amer. Micros. Sot. 94,155-178. WARING, G. L., ALLIS, C. D., and MAHOWALD,A. P. (1978). Isolation of polar granules and the identification of polar granule-specific protein. Develop. Biol. 66, 197-206.