/. Embryo/, exp. Morph. Vol. 49, pp. 89-102, 1979
Printed in Great Britain © Company of Biologists Limited 1979
gO,
Sorting out behaviour of
disaggregated cells in the absence of morphogenesis
in Dictyostelium discoideum
By M. TASAKA 1 AND I. TAKEUCHP
From Department of Botany, Faculty of Science, Kyoto University
SUMMARY
Cells disaggregated from slugs of Dictyostelium discoideum were cultured in Bonner's salt
solution in roller tubes. Cells rapidly stuck together to form an amorphous loose agglutinate
which was later transformed into a spheroidal tight agglutinate surrounded by slime sheath
material. Prespore cells in the loose agglutinate underwent partial dedifferentiation by starting
to decompose their specific antigen until formation of the tight agglutinate, in which the
antigen was resynthesized. During the process, there was some decrease in the proportion of
prespore cells.
Changes in the distribution of prespore and prestalk cells in the agglutinates were examined
by using immunocytochemical staining. They were randomly distributed in the early agglutinates, but became well separated in 4 h agglutinates in such a way that prestalk cells were completely enveloped by prespore cells. Prestalk cells later came outside to be partially enveloped
and finally occupied a hemisphere side by side with prespore cells. During the process, cells in
one or two outer layers differentiated into prestalk or stalk cells. Similar changes in the distribution pattern were observed, when labelled prestalk cells were cultured with unlabelled
prespore cells and their distribution in co-agglutinates was followed by autoradiography. It
was concluded from these results that the majorities of prestalk and prespore cells isolated
from slugs are sorted out in agglutinates without changing their original cell types, and that
the sorting-out occurs in the complete absence of polar structures and movement of the cell
mass. The distribution patterns in agglutinates of prestalk and prespore cells were discussed
with reference to intercellular adhesion among/between them.
INTRODUCTION
Upon depletion of the food supply, amoebae of the slime mould Dictyostelium discoideum aggregate chemotactically to form a slug-shaped cell mass.
The slug finally constructs a fruiting body consisting of a terminal spore mass
on a supporting cellular stalk. During formation of the fruiting body, the
anterior cells of the slug differentiate into stalk cells, while the posterior cells
differentiate into spores. It has been revealed by various means that the anterior
prestalk cells of a slug have characteristics different from the posterior prespore
cells. When sections of D. discoideum slugs are stained with fluorescein1
Authors'' address: Department of Botany, Faculty of Science, Kyoto University, Kyoto
606, Japan.
90
M. TASAKA AND I. TAKEUCHI
conjugated antiserum produced against spores of D. mucoroides, the prestalk
cells have no staining while the prespore cells are strongly stained in cytoplasmic granules (Takeuchi, 1963, 1972).
It has been shown that when prestalk and prespore cells in the slug are displaced or mixed they are sorted out to occupy their original positions. This was
first shown by Bonner (1952) who grafted vitally stained cells from the anterior
end onto the posterior end of a colourless migrating slug and found that the
coloured cells move up to the anterior positions as the slug migrates. The finding
was later refuted by Farnsworth & Wolpert (1971), but recently confirmed by
Yamamoto (1977). On the other hand, Takeuchi (1969) who mixed cells disaggregated from the anterior and the posterior portions of slugs and allowed
them to reaggregate on agar found that they are sorted out during slug formation to occupy their original positions in a reformed slug.
The present work was attempted to examine whether or not such sorting out
of cells is brought about by morphogenetic movements of a cell mass, i.e.
formation and migration of a polarized slug. For this purpose, prestalk and
prespore cells disaggregated from slugs were cultured in roller tubes to produce
round spherical co-agglutinates, in which their distributions as well as states of
differentiation were followed immunocytochemically and autoradiographically.
As a result, it was found that the two cell types are sorted out in an agglutinate
completely devoid of polar structure and movement with most cells maintaining
the original cell types.
MATERIALS AND METHODS
Organism and culture
D. discoideum strain NC 4 was used in this study. Spores of the strain were
inoculated with Escherichia coli B/r on a standard nutrient agar medium
(Bonner, 1947). Amoebae were harvested at the stationary growth phase and
washed by repeated centrifugations (1500 rev./min, 1-5 min). To obtain slugs,
washed amoebae were streaked on non-nutrient agar (2%) and incubated at
21 °C. To obtain preculminating cell masses, amoebae were grown, at 21 °C,
with E. coli in a nutrient liquid medium on a rotary shaker (150 rev./min).
After being washed, 107 amoebae were resuspended in 0-5 ml KCl-containing
phosphate buffer (K-P buffer: 20mM-KCl, 10 mM phosphate buffer, pH 6-5)
and deposited on a 5-5 cm Whatman No. 50 filter paper supported by three
pieces of 5-5 cm Toyo No. 2 filter paper saturated with K-P buffer. Development
was allowed to proceed at 21 °C, and preculminating cell masses were collected
after 17 h of incubation.
Roller tube culture of slug cells
Migrating slugs were washed by two centrifugations (500 rev./min, 30 sec)
in K-P buffer to be freed of unaggregated amoebae and disaggregated at 4 °C
by forced pipetting in EDTA-P buffer (1 mM EDTA, 20 mM-NaCl, 40 mM
Sorting out behaviour of disaggregated cells in Dictyostelium
91
phosphate buffer, pH 7-0). The buffer was then filtered through nylon mesh
(23 /«n openings) to remove cell clumps. Disaggregated cells were washed, with
Bonner's salt solution (BSS: lOmM-NaCl, 10mM-KCl, 3 mM-CaCl2) and
resuspended in BSS at 1-7 x 106 cells/ml. Roller tube culture of disaggregated
cells was made as described previously (Takeuchi, Hayashi & Tasaka, 1977).
Each tube containing 3 ml of the cell suspension was rolled at 21 °C, at 28 rev./
min. Agglutinates formed were collected at intervals by centrifugations at
500 rev./min for 30 sec.
Fractionation of presumptive cells
The method employed was a modification of that previously used for D.
mucoroides cells (Oohata & Takeuchi, 1977). Stepwise density gradients consisted of Urografin (Schering) solutions of specific gravities, p 1-21 (1 mD,
p 1-17 (2-5 ml), p 1-61 (2 ml), and p 1-15 (2 ml), which were adjusted to fractionate D. discoideum cells disaggregated at the preculmination stage. Disaggregated
cells were suspended in 1-5 ml EDTA-P buffer at 3xlO 8 cells/ml and were
centrifuged as described by Oohata & Takeuchi (1977). Cells were collected
from bands by syringes and washed with BSS by three centrifugations at
2000 rev./min for 5, 3, and 2 min.
Immunocytochemistry and microfluorometry
Immunocytochemical staining of prespore cells was made with fluorescein
isothiocyanate (FITC)-conjugated antiserum produced against spores of D.
mucoroides, as described by Takeuchi (1963). Agglutinates were disaggregated
in a 50 mM Tris-bufFered (pH 7-2) solution of 0-1 % pronase containing 25 mM
dimercaptopropanol (Takeuchi & Yabuno, 1970). After being washed, disaggregated cells were placed on a coverglass, fixed in methanol and stained. The
preparations were observed in a dark field with a Reichert fluorescence microscope (Zetopan). Fluorescence intensities of stained cells were determined using
a Reichert microspectrophotometer attached to the microscope, as described by
Hayashi & Takeuchi (1976). For histological staining, agglutinates were fixed,
embedded in Paraplast (Sherwood) and sectioned 3-5 /mi thick. The sections
were stained with the antiserum and observed.
Autoradiography
To label amoebae, they were grown with a thymidine-requiring mutant
strain (B3) of E. coll in a nutrient liquid medium containing [3H]thymidine
(5 /tCi/ml, Radiochemical Centre, Amersham) plus 5 /*g/ml thymidine. After
being washed, cells were allowed to develop on filter papers. Labelled prestalk
and unlabelled prespore cells were fractionated from preculminating cell masses
as described, mixed at 1:3 ratio and incubated in roller tubes. Agglutinates
formed were fixed in Famer's fixative and sectioned 5 /on thick. The preparations were dipped in liquid emulsion, Sakura NR-M2, in a dark room. They
92
M. TASAKA AND I. TAKEUCHI
were stored in a black box at room temperature for 2 weeks and developed in
Sakura Conidol-X for 6 min at 21 °C, fixed in Coni fixer and washed with
distilled water.
RESULTS
Formation of agglutinates from disaggregated slug cells
When disaggregated slug cells were cultured in BSS in roller tubes, cells
quickly stuck together to form amorphous agglutinates such as shown in Fig.
1 A. When placed on a slide glass, cells of these agglutinates adhered to the
glass and tended to disperse. They were thus similar to the 'loose' agglutinates
which form in an early roller tube culture of washed vegetative amoebae
(Takeuchi et al. 1977). When the culture was continued, cells in the loose
agglutinate secreted on its surface viscous material like the slime sheath surrounding the slug. The agglutinate now assumed a smooth spheroidal shape and
no longer showed a tendency to be dispersed on glass (Fig. 1B). Such changes in
the agglutinate appear comparable to those from the 'loose' to the 'tight'
agglutinates which have previously been described in a roller tube culture of
washed vegetative amoebae (Takeuchi et al. 1977). Formation of tight agglutinates was completed after 3-4 h of culture. Although no apparent changes of the
agglutinates were discernible during further culture, an increasing number of
cells adjacent to the outer sheath were observed to form heavy cellulose walls
and large vacuoles in a fashion characteristic of stalk cells (Fig. 1C). On rare
occasions, a few spore-like cells appeared in the agglutinates after 12 h of
culture.
Changes of prespore cells in agglutinates
Taking advantage of the fact that the prespore cells of the migrating slug were
specifically stained in cytoplasmic granules with heteroplastic antispore serum
(Takeuchi, 1963), changes in the proportion of prespore cells and their antigenic
contents in agglutinates were examined during the roller tube culture. Agglutinates were collected at various times of culture. Cells disaggregated therefrom
were stained with the FITC conjugated anti-Z). mucoroides spore serum, and the
numbers of cells with and without stained granules were counted as described
above. The ratio of prespore to total cells was initially about 75 % (in slugs),
but gradually decreased in agglutinates to about 63 % within 5-6 h of culture
(Fig. 2 A). Fluorescence intensities of individual cells were microspectrophotometrically determined, and their frequency distributions at indicated times were
shown in Fig. 3. The frequency distribution pattern of 0 h cells indicates that
cells comprising slugs were grouped into two classes: those of high fluorescence
intensities corresponded to prespore cells and those of low to prestalk cells
(Hayashi & Takeuchi, 1976). When the culture was continued, the former
gradually decreased both in number and fluorescence intensity, with the concomitant increases of the latter. Thus, the distribution became too spread out to
Sorting out behaviour of disaggregated cells in Dictyostelium
Fig. 1. Agglutinates formed in roller tube culture of disaggregated slug cells. The
culture was conducted according to the Method section. (A) 2h agglutinate:
amorphous loose agglutinates without slime sheath material. (B) 6h agglutinate:
a spheroidal tight agglutinate with surrounding sheath. (C) Crushed 12 h agglutinate:
mature stalk cells are differentiated in the outer layer.
EMB 49
93
94
M. TASAKA AND I. TAKEUCHI
prespore to total
o
1 70
0
T
—o
1
rat
o
o
M 50
1
1
— '
1
1
1
Time culture (h)
Fig. 2. (A) Changes in the ratio of prespore to total cells in agglutinates during
roller tube culture of disaggregated slug cells. The culture was conducted as described in the Method section. Agglutinates formed were collected at intervals, and
cells disaggregated from them were stained with the fluorescein-conjugated antiD. mucoroides spore serum. Prespore cells were identified as those having stained
granules. The value at 0 h represents the ratio in slugs. The horizontal bar indicates
the time of appearance of tight agglutinates, and the vertical ones standard deviations. (B) Changes in the fluorescence intensities (arbitary unit) of prespore cells
in agglutinates during the culture. The average fluorescence intensities were calculated from each histogram shown in Fig. 3, regarding cells exceeding 15 in intensity
as the prespore cells.
Sorting out behaviour of disaggregated cells in Dictyostelium
95
20
10
20
10
••s
20
3 10
20
10
8h
10
0
30
60
90
Fluorescence intensity of a cell
Fig. 3. Frequency distributions of the fluorescence intensities of cells comprising
agglutinates at various times of roller tube culture. Agglutinates were collected at
times indicated, and cells disaggregated from them were stained with the fluorescein
conjugated antiserum. The fluorescence intensity of a cell was measured with a
microphotometer and is expressed as a relative value to a standard.
be clearly bimodal, as observed with cells of 4 and 6 h agglutinates (Fig. 3).
Formation of tight agglutinates, however, was soon followed by increases in
number and intensity of prespore cells, resulting in restoration of a bimodal
distribution. On the basis of these histograms, the average fluorescence intensity
of prespore cells, indicative of their antigen content, was calculated at each
culture time, regarding cells exceeding 15 (arbitrary unit) in intensity as the
prespore cells. As shown in Fig. 2B, the average fluorescence intensity began to
7-2
96
M. TASAKA AND I. TAKEUCHI
decrease after 2 h of culture, but showed a considerable increase after formation
of tight agglutinates to the level equal to that of prespore cells contained in the
slug.
Distribution of presumptive cells as revealed by immunocytochemistry
Changes in the distribution of prespore and prestalk cells in agglutinates were
examined by means of immunocytochemical staining of sections of agglutinates
fixed at various times of culture. In 2 h (loose) agglutinates, prespore cells were
either clustered or intermingled with unstained, probably prestalk cells (Fig.
4A). In 4 h (tight) agglutinates covered by stained sheath, prespore cells were
well separated from and completely enveloped unstained (prestalk) cells (Fig.
4B). The latter was confirmed by serial sections not to come in contact with any
part of the surface of an agglutinate. Later, however, the unstained region came
outside to the surface to be partially enveloped by prespore cells after about 6 h
of culture (Fig. 4C). Simultaneously, many cells in one or two outer layers
became unstained and some of them differentiated into stalk cells. Final distribution of both types of cells was attained in 8 h agglutinates, in which prespore
and prestalk cells occupied each hemisphere (Fig. 4D). All the cells in outer
layers were now either prestalk or stalk cells.
Distribution of presumptive cells as revealed by autoradiography
To examine whether the above-described changes in distribution of the two
cell types are mainly attributable to rearrangement of cells whose original differentiated states are maintained or to dedifferentiation of cells followed by their
redifferentiation after formation of agglutinates, labelled prestalk cells were
mixed with unlabelled prespore cells and their locations in co-agglutinates were
traced by autoradiography. Amoebae were labelled with [3H]thymidine as
described in the Method section. Autoradiography of these cells revealed grains
concentrated on the nucleus, except for some scattered in the cytoplasm,
probably on mitocondria.
Separation of prestalk from prespore cells. To fractionate the two cell types,
cells disaggregated from preculminating cell masses were centrifuged through a
discontinuous Urografin gradient. Preculminating cell masses were used,
because they gave better separation than slugs. After centrifugation, three bands
formed at interfaces, as shown in Fig. 5. Cells in each band were stained with
the fluorescent antispore serum to identify prespore cells. As shown in Table 1,
most cells in the band I were unstained, while those in the band III were stained.
In contrast, the band II was a mixture of stained and unstained cells. It was thus
concluded that about 95 % of cells in the bands I and III were prestalk and
prespore cells respectively. It was noticed during the experiments that the
specific gravities of disaggregated cells are considerably affected by many
factors, such as the conditions of slug formation, the method of disaggregation,
the number of cells layered on the gradient and so on. If different conditions are
Sorting out behaviour of disaggregated cells in Dictyostelium
Fig. 4. Sections of agglutinates stained with the fluorescein-conjugated antiserum.
Agglutinates were fixed and sectioned after various times of roller tube culture. (A)
2 h agglutinate: prespore cells (stained) and prestalk cells (unstained) are distributed
randomly. (B) 4 h agglutinate: preslalk cells are completely enveloped by prespore
cells, and the agglutinate is covered by slime sheath (stained). (C) 6 h agglutinate:
the prestalk cell mass is partially enveloped by prespore cells. (D) 8 h agglutinate:
the prestalk and prespore cell masses occupy each hemisphere. Cells in outer one
or two layers of the agglutinate are prestalk or stalk cells.
97
98
M. TASAKA AND I. TAKEUCH1
Cell-suspending
medium
115
bandl
116
band II
1-17
band III
121
Fig. 5. A centrifugal pattern on a stepwise Urografin gradient of cells disaggregated
at the predomination stage. Three bands (I, IT and HT) formed at interfaces.
Table 1. The numbers of stained and unstained cells in the bands I and III produced
by fractionation of disaggregated cells on a Urografin gradient
Band I
Expt. 1
Expt. 2
Expt. 3
Expt. 4
Band III
The numbers of
stained cell
The numbers of
unstained cell
The numbers of
stained cell
The numbers of
unstained cell
16
(5-9)
16
(5-2)
9
(2-6)
61
(66)
257
(94-1)
291
(94-8)
342
(97-4)
850
(93-4)
418
(931)
361
(91-9)
306
(93-9)
1224
(971)
31
(69)
32
(81)
20
(61)
37
(2-9)
Cells were collected from the band I and TIT (cf. Fig. 5) and stained with the fluoresceinconjugated antiserum. The results of four independent experiments are shown. Figures
in parentheses indicate percentages of cells to total number of cells.
used, the densities of Urografin solutions should be adjusted to obtain clear
separation of the two cell types.
Distribution of labelled cells in agglutinates. Labelled prestalk cells and unlabelled prespore cells were mixed and cultured in roller tubes as described
above. Agglutinates formed were fixed and sectioned at subsequent times.
Distributions of the two types of cells in agglutinates were examined using the
Sorting out behaviour of disaggregated cells in Dictyostelium 99
•••*•••
*
v
••«•**
/
^ '
•*
•
Fig. 6. Autoradiographs of sections of agglutinates. Labelled prestalk cells and unlabelled prespore cells were mixed and cultured in roller tubes. Co-agglutinates
formed were fixed and sectioned at intervals, and their autoradiographs were
prepared. (A) 2 h agglutinate: original prestalk (labelled) and prespore (unlabelled)
cells are randomly dispersed in the agglutinate. (B) 4h agglutinate: original prestalk cells are accumulated in the inside of the agglutinate. (C) 8 h agglutinate:
most original prestalk cells are partially enveloped by original prespore cells.
100
M. TASAKA AND I. TAKEUCHI
method of autoradiography. Labelled prestalk cells were first scattered randomly
in 2 h (loose) agglutinates (Fig. 6A), indicating that both prestalk and prespore
cells adhered without any selection. In 4 h (tight) agglutinates, however, the
majority of labelled prestalk cells were concentrated in the centre (Fig. 6B).
Later in 8 h agglutinates, labelled cells came outside to be partially enveloped
by unlabelled cells (Fig. 6C). Some labelled cells were also observed scattered
on the surface of the agglutinates, but most of them were those adsorbed on the
slime sheath after its formation. It is thus concluded that the majority of labelled
cells which had previously been prestalk cells in the slug followed the same
pattern of distribution as that of cells unstained by immunocytochemical staining (cf. Fig. 4). This indicates that most prestalk cells are sorted out in the newly
formed agglutinates without changing their differentiated state. When labelled
prespore and unlabelled prestalk cells were mixed in a roller tube culture, their
distribution pattern underwent the same type of changes as described above.
DISCUSSION
When cells disaggregated from slugs were cultured in roller tubes, they first
formed a loose agglutinate which later became tight by secreting slime sheath
material on its surface. During the period of loose agglutinates, both the proportion of prespore cells and their antigenic contents decreased, indicating that
cells in such an agglutinate underwent the same process of dedifferentiation as
completely isolated cells (Takeuchi & Sakai, 1971). After formation of a tight
agglutinate, however, prespore cells resynthesized the antigen, but their proportion remained about 60 %. That this ratio is characteristic of such a submerged
agglutinate was shown by the following fact. When slug cells disaggregated by
the use of pronase-dimercaptopropanol were cultured, the transition from a
loose to a tight agglutinate was delayed about 2 h. As a result, the proportion of
prespore cells decreased to about 25 % in loose agglutinates, but the final proportion in tight agglutinates was about 60% (unpublished).
Recently, roller tube or rotary shake cultures of washed vegetative amoebae
were conducted by Forman & Garrod (1977), Sternfeld & Bonner (1977) and
Takeuchi et al. (1977), who observed differentiation of prespore cells, spores and
stalk cells in agglutinates kept under submerged conditions. In their roller tube
culture of washed vegetative amoebae, Takeuchi et al. (1977) found the same
type of transition from a loose to a tight agglutinate as observed in the present
study and proposed that formation of tight agglutinates is a prerequisite for
differentiation of prespore cells. The proportion of prespore cells in such an
agglutinate attained to about 50 %.
The distribution of prestalk and prespore cells in agglutinates was investigated
by two different methods, immunocytochemical staining for prespore cells and
autoradiographical tracing of labelled prestalk or prespore cells. The fact that
the distributions as revealed by both the methods were essentially the same
Sorting out behaviour of disaggregated cells in Dictyostelium 101
indicates that the majority of presumptive cells are rearranged in an agglutinate
without changing their original cell types. However, this does not completely
exclude occurrence of the conversion of cell types in agglutinates. In fact, the
conversion, though on a small scale, is suggested to have occurred by a certain
decrease during the culture in the proportion of prespore cells. The decrease was,
at least in part, attributable to ^differentiation of prespore cells located in the
outer layers of agglutinates into prestalk or stalk cells. The reason why they
underwent such differentiation is unknown, but the phenomenon parallels with
the fact that the prespore cells next to the agar surface in a migrating slug lose
the prespore antigen during migration (Takeuchi et ah 1977).
The present study showed that prestalk cells are first sorted out inside to be
completely enveloped by prespore cells, but that they later move outside to be
only partially enveloped. The fact that such sorting out proceeded in a cell mass
which, unlike the slug, underwent no morphogenetic movement suggests that
it may have been brought about by differential intercellular adhesion.
Steinberg (1963") described in his differential adhesion hypothesis that sorting
out behaviour of embryonic cells is attributed to differences in the strength of
their intercellular adhesion. According to his hypothesis, the fact that prespore
cells completely enveloped prestalk cells in the 4 h agglutinate indicates that the
work of adhesion among prestalk cells (Wpst-pst) was greater than that among
prespore cells (Wpsp-psp) and that the work between prespore and prestalk
cells (Wpsp-pst) was in between: Wpst-pst > Wpsp-pst > Wpsp-psp. Furthermore, the changes from complete to partial envelopment of prestalk cells as
observed in later agglutinates are interpreted to indicate that the work of
adhesion among the same types of cells was relatively increased during the
culture than that between the different types to become Wpst-pst > Wpsp-psp
> Wpsp-pst. That prestalk cells are more adhesive than prespore cells agrees
well with the previous observations made by Maeda & Takeuchi (1969),
Takeuchi & Yabuno (1970) and Yabuno (1971).
This work was in part supported by grants-in-aid to I. T. from the Ministry of Education
of Japan (No. 248010) and Takeda Science Foundation.
REFERENCES
J. T. (1947). Evidence for the formation of cell aggregates by chemotaxis in the
development of the slime mold Dictyostelium discoideum J. exp. Zool. 106, 1-26.
BONNER, J. T. (1952). The pattern of differentiation in amoeboid slime molds. Am. Nat. 86,
79-89.
FARNSWORTH, P. A. & WOLPERT, L. (1971). Absence of cell sorting out in the grex of the
slime mould Dictyostelium discoideum. Nature, Lond. 231, 329-330.
FORMAN, D. & GARROD, D. R. (1977). Pattern formation in Dictyostelium discoideum II.
Differentiation and pattern formation in non-polar aggregates. /. Embryol. exp. Morph.
40, 229-243.
HAYASHI, M. & TAKEUCHI, I. (1976). Quantitative studies on cell differentiation during
morphogenesis of the cellular slime mold Dictyostelium discoideum. Devi Biol. 50, 302-309.
BONNER,
102
M. TASAKA AND I. TAKEUCHI
Y. & TAKEUCHI, I. (1969). Cell differentiation and fine structures in the development
of the cellular slime molds. Develop. Growth & Differentiation 11, 232-245.
OOHATA, A. & TAKEUCHI, I. (1977). Separation and biochemical characterization of the two
cell types present in the pseudoplasmodium of Dictyostelium miicoroides. J. Cell Sci. 24,
1-9.
STEINBERG, M. S. (1963). Reconstruction of tissues by dissociated cells. Science, N.Y. 141,
401-408.
STERNFELD, J. & BONNER, J. T. (1977). Cell differentiation in Dictyostelium under submerged
conditions. Proc. natn. Acad. Sci. U.S.A. 74, 268-271.
TAKEUCHI, I. (1963). Immunochemical and immunohistochemical studies on the development
of the cellular slime mold Dictyostelium mucoroides. Devi Biol. 8, 1-26.
TAKEUCHI, I. (1969). Establishment of polar organization during slime mold development.
Nucleic Acid Metabolism, Cell Differentiation and Cancer Growth (ed. E. V. Cowdry &
S. Seno), pp. 297-304. Oxford and New York: Pergamon Press.
TAKEUCHI, I. & YABLNO, K. (1970). Disaggregation of slime mold pseudoplasmodia using
EDTA and various proteolytic enzymes. Expl Cell Res. 61, 183-190.
TAKEUCHI, I. & SAKAI, Y. (1971). Dedifferentiation of the disaggregated slug cell of the
cellular slime mold Dictyostelium discoideum. Develop. Growth & Differentiation 13, 201210.
TAKEUCHI, I. (1972). Differentiation and dedifferentiation in cellular slime molds. Aspects of
Cellular and Molecular Physiology (ed. K. Hamaguchi), pp. 217-236. Tokyo: Univ. Tokyo
Press.
TAKEUCHI, I., HAYASHI, M. & TASAKA, M. (1977). Cell differentiation and pattern formation
in Dictyostelium. Development and Differentiation in the Cellular Slime Moulds (ed. P.
Cappuccinelli & J. M. Ashworth), pp. 1-16. Amsterdam: Elsevier/North-Holland.
YABUNO, K. (1971). Changes in cellular adhesiveness during the development of the slime
mold Dictyostelium discoideum. Develop. Growth & Differentiation 13, 181-190.
YAMAMOTO, M. (1977). Some aspects of behavior of the migrating slug of the cellular slime
mold Dictyostelium discoideum. Develop. Growth & Differentiation 19, 93-102.
MAEDA,
(Received 28 June 1978, revised 8 September 1978)