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
338
DEVELOPMENTAL BIOLOGY
5h40
VOLUME 81, 1981
gh
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,
1
1
1
1
I
1
1
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
340
DEVELOPMENTALBIOLOGY
VOLUME81,198l
I
1
I
I
1
2
3
4
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.