J. Embryol. exp. Morph. 90, 287-309 (1985)
287
Printed in Great Britain © The Company of Biologists Limited 1985
Changes in the distribution of membranous organelles
during mouse early development
BERNARD MARO*, MARTIN H. JOHNSON,
SUSAN J. PICKERING
Department of Anatomy, University of Cambridge, Downing Street,
Cambridge CB2 3DY, U.K.
AND DANIEL LOUVARD*
Institut Pasteur, Paris, France
SUMMARY
The unfertilized oocyte, fertilized egg and early embryo (2-cell to 16-cell) of the mouse have
been examined immunocytochemically for the distribution of antigens associated with the
endoplasmic reticulum, the lysosomal and acidic vesicle fraction (lOOkD antigen), Golgi
apparatus (135kD antigen) and coated vesicles (clathrin). The distribution of these antigens has
also been examined in isolated 8-cell and 16-cell-stage blastomeres of various ages and
phenotypes. Endoplasmic reticulum is detected only weakly in the oocyte and egg, but is seen
abundantly at later stages both in association with the nuclear membrane and evenly distributed
throughout the cytoplasm, except in regions of cell: cell apposition from which it is excluded.
Intracellular clathrin is associated with the spindle in mitotic and meiotic cells. During
interphase, clathrin is distributed throughout the cell until the mid-8-cell stage when it is
concentrated into the apical region of the cell under the region of membrane at which a surface
pole of microvilli will form subsequently. Thus, the cytoplasmic polarization of clathrin precedes
overt polarization at the surface. At mitosis, the clathrin relocates to the spindle and is
distributed to both daughter cells. It resumes an apical location beneath the surface pole of
microvilli in polar daughter 1/16 cells, but remains dispersed in apolar daughter 1/16 cells. Both
the lysosomal and Golgi antigens are distributed throughout the cytoplasm until the early 16-cell
stage. In pairs of 16-cell blastomeres both antigens aggregate in a single cluster and do so
whether the surface phenotype of the blastomeres is polar or apolar. The position of this cluster
is not consistently related to the point of contact with the other cell in the pair but there is a
suggestion that in cells with a polar surface phenotype the polar foci of Golgi/lysosomal antigens
are located between the nucleus and the surface pole at earlier time points, but shift to a position
between the basolateral membrane and the nucleus at the later time point. In intact 16-cell
embryos also, the aggregated Golgi/lysosomal antigens of polar cells appear to localize to the
basal region. The distributions of these various organelles in embryonic cells reported here show
a number of differences from those reported previously for mature, differentiated cells.
INTRODUCTION
Many differentiated cells manifest a highly asymmetric organization that is
dependent partly upon continuing cell contact, v/ithin for example an epithelial
*C.N.R.S., Paris, France.
Key words: mouse, blastomere, clathrin, Golgi, lysosome, endoplasmic reticulum.
288
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
layer, and is partly intrinsic to the structure of the cell (e.g. Ziomek, Schulman &
Edidin, 1980). Various approaches to the study of how this cell asymmetry is
developed and maintained are available including the use of cell lines, such as the
MDCK cell line, which can modulate some epithelial properties reversibly in vitro
(Van Meer & Simons, 1982), and the examination of polarized, epithelial cells
after their isolation and manipulation (Ziomek et al. 1980). However in these
model systems polarity is not generated de novo from a truly symmetric precursor
cell. De novo polar organization of cells develops first early in embryogenesis with
the formation of the primary epithelial germ layers and the delamination of
extraembryonic epithelia. The earliest evidence of this process in the mouse
embryo has been detected at the 8-cell stage, during which elements of the cell
surface (Handyside, 1980), cytoskeleton (Johnson & Maro, 1984), and endocytotic
processing pathway (Reeve, 1981; Fleming & Pickering, 1985) undergo a radical
reorganization to convert a non-polar cell to a highly polarized cell over a period of
8-10 h (Ziomek & Johnson, 1980). Elements of this polarity are conserved at
division (Johnson & Ziomek, 1981), and the polarity is elaborated and stabilized at
the 16- and 32-cell stages to generate the definitive trophectodermal epithelium
(Fleming, Warren, Chisholm & Johnson, 1984; Fleming & Pickering, 1985). In this
paper, we report on the immunocytochemical localization of various antigens
specific to membranous organelles (endoplasmic reticulum, the acid vesicle/
lysosome compartment, coated vesicles, the Golgi apparatus) concerned with
endocytotic and biosynthetic activity and on the changes that occur during the
early stages of cell polarization.
MATERIALS AND METHODS.
1. Recovery of embryos
MF1 female mice (3-5 weeks; Olac) were superovulated by injections of 5i.u. of pregnant
mare's serum gonadotrophin (PMSG; Intervet) and human chorionic gonadotrophin (hCG;
Intervet) 48 h apart. The females were paired overnight with HC-CFLP males (Hacking &
Churchill) and inspected for vaginal plugs the next day. Unfertilized and fertilized eggs were
recovered from females at 14-16 h post hCG; 2-cell and 4-cell embryos were recovered at
46-50h post hCG; 8-cell embryos were derived by overnight culture of 2- to 4-cell embryos;
early 16-cell embryos were recovered at 65-70 h post hCG.
2. Preparation and handling of single cells
2-cell embryos were recovered at 48 h post hCG and cultured in Medium 16 containing
4 m g m r 1 BSA (M16+BSA) (Whittingham & Wales, 1969) under oil for 13 h at 37°C in 5 %
CO2 in air. All 4-cell embryos were then exposed briefly to acid Tyrode's solution (Nicolson,
Yanagimachi & Yanagimachi, 1975) to remove the zona pellucida, rinsed in Medium 2+BSA
(Fulton & Whittingham, 1978), and placed in Ca2+-free M2+6mgml~1 BSA for 5-45 min,
during which time they were disaggregated to single 4-cell blastomeres (1/4 cells) using a flamepolished micropipette. Cells were cultured on Sterilin tissue culture dishes in drops of
M16+BSA under oil at 37°C in 5 % CO2 in air. The cultures were inspected hourly for evidence
of division to 2/8 pairs, and couplets were removed, designated Oh old, and cultured in
M16+BSA as natural 2/8 pairs.
Membranous organelle distribution in mouse embryos
289
Late 8-cell embryos were recovered at 64 h post-hCG and were disaggregated to single 8-cell
blastomeres (1/8 cell) as described above. Couplets of 16-cell blastomeres (2/16 natural pairs)
were selected as above. In some experiments, 2/16 pairs were cultured in the presence of a
monoclonal antibody to cadherin (Yoshida-Noro, Suzuki & Takeichi, 1984; also called
uvomorulin, L-CAM) in order to avoid the envelopment of the apolar cell by the polar cell that
would otherwise occur (Ziomek & Johnson, 1981).
In one series of experiments, whole 8-cell embryos were freed from their zonae, disaggregated
to single or paired blastomeres in Ca2+-free medium and the blastomeres analysed immediately.
3. Immunocytochemistry
Surface polarity was assessed by incubation of cells or embryos in SOjugml"1
tetramethylrhodamine-labelled succinyl Concanavalin A (SOfigml'1 M2+BSA: TMRTC-SConA, Polysciences) for 5min at room temperature, followed by two to three washes in
M2+BSA. Labelled cells were then placed in specially designed chambers exactly as described
previously (Maro, Johnson, Pickering & Flach, 1984) for fixation with 3-7% formaldehyde
followed by extraction with 0-25 % Triton X-100. After washing, cells were incubated with
affinity-purified polyclonal, rabbit antibody to clathrin, the major coat protein of coated vesicles
(Louvard etal. 1983), to a 135kD antigen associated with the Golgi apparatus (Louvard, Reggio
& Warren, 1982), to an endoplasmic reticulum antigen (Louvard et al. 1982) or to a lOOkD
protein associated with the acid vesicle/lysosomal fraction (Reggio et al. 1984). A second layer
of fiuorescein-labelled anti-rabbit immunoglobulin was used to visualize the bound antibody.
The detailed characteristics of the procedures have been reported previously (Maro et al. 1984).
Samples were mounted in Citifluor (City University, London) in order to reduce fading of
fluorescent labels and viewed on a Leitz Ortholux II microscope with filter set L2 for FITClabelled reagents and N2 for TMRTC-labelled reagents. Photographs were taken on Kodak Tri
X film using a Leitz Vario-orthomat photographic system.
RESULTS
1. Organelle distribution in whole embryos
We first studied the distribution of organelle antigens in permeabilized whole
embryos from the unfertilized egg to the 16-cell stage. Each antigen examined
showed a pattern of distribution that varied characteristically with time.
(a) Intracellular clathrin
Clathrin was distributed in a diffuse granular pattern throughout early development (Fig. lb,c,d,e) but with two exceptions. First, when meiotic (Fig. la) or
mitotic (Fig. lh,i) cells were examined, clathrin distribution corresponded closely
with that of the spindle, and remaining areas of the cytoplasm were relatively free
of clathrin. Second, during the late 8-cell stage clathrin appeared to be more
concentrated in the apical region of the blastomeres (Fig. If). However, this
localization was difficult to resolve clearly in whole mounts.
(b) Golgi apparatus
The Golgi antigen was also distributed diffusely throughout the cytoplasm of
early embryonic cells (Fig. 2a-e). However, at all stages the granular foci of Golgi
antigen tended to be larger than those observed with the anti-clathrin antibody,
Fig. 1. Clathrin distribution in whole embryos as revealed by use of anti-clathrin antibody and FITC-labelled anti-IgG for embryonic
stages (a) unfertilized egg, note staining of the second metaphase spindle, (b) 1-cell embryo, note the strong staining of the second
polar body and otherwise diffuse cytoplasmic stain, (c) 2-cell embryo, note relative lack of staining adjacent to zone of cell contact,
(d) 4-cell embryo, (e) early 8-cell embryo, (f) late compacting 8-ceU embryo, note concentration of clathrin at outward facing apical
end of the cell, (g) 16-cell embryo, (h) 4-cell embryo, note clathrin concentration in the spindle area of the mitotic cell on the right, (i)
16-cell embryo, note staining of the spindle. (Mags a-h x470; i X90.)
1
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Membranous organdie distribution in mouse embryos
291
and this was particularly marked at the 8-cell and 16-cell stages. Moreover at 16cell stages there appeared to be a greater concentration of Golgi-antigen at the
centre of the embryo (Fig. 2e), a location corresponding to the totally enclosed
inside cells and/or to the basal regions of outer cells.
(c) Endoplasmic reticulum
The antiserum to endoplasmic reticulum antigen stained the nuclear membrane
region intensely, together with a diffuse granular staining throughout the cytoplasm that was somewhat weaker at the 1-cell stage than subsequently (Fig. 2f-h).
Otherwise, no convincing evidence of temporal change or spatial asymmetry in
staining pattern was observed with use of this antiserum, although in many cases
the most peripheral areas of the blastomeres can appear to stain more weakly.
However, it was difficult to be certain that this appearance was not simply due to
the greater density of positive cytoplasm at the centre of the embryo.
(d) lOOkD membrane antigen
The antiserum to the lOOkD protein associated with the acid
organelle/lysosome fraction gave a diffuse granular pattern of reaction (Fig. 2i-l),
but, as for Golgi-antigen, the granular foci were larger at 8- and 16-cell stages
(Fig. 2k, 1). Moreover, in some late 8-cell embryos and most 16-cell embryos, there
was a concentration of lOOkD antigen in clumps at the centre of the embryo
(Fig. 2k,l).
(e) Summary
The use of the whole embryo mounts allows a general assessment of changing
temporal and spatial patterns of antigen distribution, and suggests that for
clathrin, Golgi and lOOkD antigens changes occur at the 8-cell stage and later. In
order to visualize these changes more clearly, we used pairs of 8-cell or 16-cell
blastomeres. This approach reduces background interference from fluorescent
emission outside the plane of focus and also permits a more accurate temporal
staging of blastomeres within the fourth and fifth developmental cell cycles.
2. Organelle distribution in pairs of blastomeres
Preparations of isolated 4-cell and 8-cell blastomeres were made, cultured and
examined at hourly intervals for evidence of division to yield two 0 h 8-cell or 16cell blastomeres (a 2/8 or 2/16 pair). Pairs were then cultured for up to 11 h before
being examined for their surface phenotype (assessed by binding of TMRTCsuccinyl-Con A) and the distribution of organelles. Surface phenotype in 2/8 pairs
was categorized as being apolar if Con A was uniformly bound and polar if Con A
binding was restricted to the apical region of the cell (compare Fig. 4b apolar with
Fig. 4k, polar). For 2/16 pairs, three surface phenotypes were defined, namely
polar in which Con A binding was restricted to a limited area of membrane (e.g.
j
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g
E
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Fig. 2. Distribution of Golgi antigen (G: a-e), endoplasmic reticulum antigen (E: f-h) and lOOkD antigen (L: i-1) in whole embryos
revealed by use of specific antibodies and FTTC-labelled anti-IgG for embryonic stages, (a) unfertilized egg; (b) 2-cell embryo;
(c) 4-cell embryo; (d) 8-cell embryo; (e) 16-cell embryo, note aggregates of Golgi staining; (f) 1-cell embryo, note pronuclear
membrane staining with anti-ER; (g) 8-cell embryo; (h) 16-cell embryo; (i) unfertilized egg; (j) 2-cell embryo; (k) 8-cell embryo, note
aggregates of staining for lOOkD protein; (1) 16-cell embryo. (Mag. x470.)
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Membranous organelle distribution in mouse embryos
293
\ and
Homogeneous
Polar £
Zonal
Rinc
Aggregates
Coincident
Opposite
Random
Polar
Fig. 3. Schematic summary of distribution patterns of organelles in pairs of 8- and 16cell blastomeres. The surface pole in 1/16 blastomeres is indicated by the black
hatching.
upper cell Fig. 7d), bright apolar, in which the surface is brightly labelled over all
or most of its surface (e.g. upper cell Fig. 7b) and dull apolar, in which a uniform
weak labelling was observed (e.g. lower cell Fig. 7b). We have shown previously
(Johnson & Ziomek, 1981) that the phenotype of a 2/16 couplet depends upon the
way in which the polarized 1/8 cell divides. Thus if the cleavage plane is oriented
perpendicular to the axis of polarity one bright and one dim apolar cell result, and
the bright area 'shrinks' over a l h period to form a discrete pole (e.g. Fig. 7b
converts to Fig. 7d). If the division plane is oriented along the axis of polarity of
the 1/8 blastomere, two polar cells result (e.g. Fig. 7f,h). Five patterns of
organelle distribution were observed, and these are indicated schematically in
Fig. 3.
(a) Clathrin in 2/8 pairs
The changing distribution of intracellular clathrin in relation to surface polarity
at the 8-cellstage is summarized in Table 1. Three points emerge from these data.
First, the incidence of surface polarity increased with time (Table 1, column 9);
this result confirms previous observations (Ziomek & Johnson, 1980). Second, the
distribution of intracellular clathrin was mainly zonal soon after division (Table 1,
line 1, columns 3 and 4), but thereafter the proportion of cells homogeneous or
zonal for clathrin declined whilst the proportion polar for clathrin increased
(summarized in Table 1, column 10). Third, cells were detectably polarized for
intracellular clathrin before showing evidence of surface polarity (Table 1, compare columns 9 and 10). Fig. 4 shows examples of cells that were apolar at their
294
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
surface but homogeneous (Fig. 4c,d,e), zonal (Fig. 4a,b) or polar (lower cell
Fig. 4f,g and Fig. 4h,i lower blastomere) for clathrin distribution. In addition,
when the surface pole was present, it invariably overlay the pole of clathrin
(Fig. 4j,k and upper cell Fig. 4h,i) and in almost all cases examined this dual pole
was opposite to the point of contact with the partner cell (e.g. Fig. 4j,k).
Moreover, clathrin concentration in the polar region was not confined to the
cytoplasm, the membrane overlying the pole of clathrin-positive vesicles also
staining clearly (e.g. Fig. 4h,j). Enhanced membrane staining for clathrin was also
observed in regions of cell apposition.
The redistribution of clathrin from homogeneous to polar pattern, and its
relationship to the surface pole of Con A binding, are not artefacts of the in vitro
culture of 2/8 pairs, as was shown by the staining patterns of blastomeres isolated
from precompact 8-cell and from compacted late 8-cell embryos (Fig. 41,m), in
which a homogeneous clathrin distribution predominated in the former and a
polar pattern in the latter.
(b) Clathrin in 2/16 pairs
The analysis of intracellular clathrin distribution in 2/16 pairs is complicated by
the cell heterogeneity at the 16-cell stage. Thus, two populations of cells exist that
differ in surface phenotype (bright or polar; dull and apolar) as well as in
properties, developmental fate and lability (reviewed Johnson, 1985). One such
property is the tendency after 4 to 8 h in culture of the polar cells to envelope the
apolar cells (e.g. see Fig. 8e-h), this being a reflection of the role of polar cells
Table 1. Clathrin distribution in natural pairs of 8-cell blastomeres in relation to
surface polarity
* Patterns of intracellular clathrin distribution (%)
in cells with a surface phenotype that is
Scored cells
(1)
Age
Idumber
(l)O-lh
(2)2-3h
(3)3-4h
(4)5-6h
(5)7-8h
(6) 9-10 h
(7)9-10ht
(8) 11-12 h
12
40
56
36
58
41
91
24
(3)
H
Apolar
(4)
Z
(5)
P
16
10
5
3
1
15
24
0
84
55
20
28
12
10
0
8
0
35
66
33
39
24
11
17
(6)
H
Polar
(?)
Z
(8)
P
0
0
0
0
5
0
4
0
0
0
0
3
2
0
0
13
0
0
9
33
41
51
61
62
summary oi
% of cells showing
(10)
(9)
Surface Clathrin
poles
poles
0
0
9
36
48
51
65
75
0
35
75
66
80
75
72
79
*H: homogeneous; Z: zonal; P: poles; No cells scored as ring or aggregate - see Fig. 3 for
illustration of this classification.
t Group 7 provided a control for the experiment summarized in Table 2 (see text); cells were
incubated in the presence of antibody to cadherin.
Membranous organelle distribution in mouse embryos
295
4
V V
m
Fig. 4. Pairs of natural 2/8 blastomeres derived by division of a 1/4 blastomere (a-k),
and 3x1/8 blastomeres obtained by disaggregation of a late 8-cell embryo (l,m),
double labelled to reveal patterns of clathrin distribution (a,c,d,f,h,j,l) and Con A
binding (b,e,g,i,k,m). (a,b) 0-1 h old pair of cells both zonal for clathrin and apolar at
their surface; (c,d,e) 0-1 h old pairs of cells homogeneous for clathrin and apolar at
surface; (f,g) 3-4 h old pair of cells both apolar at surface whilst lower is polar and
upper intermediate between zonal and polar for clathrin; (h,i) 5-6 h old pair of cells
upper polar for clathrin and at surface, lower polar for clathrin only; (j,k) 7-8h old
pair of cells both polar for clathrin and at surface; (l,m) all three cells polar for clathrin
and at surface. (Mag. X700.)
(3)
68
23
23
9
30
13
0
0
0
0
0
0
0
0
0
0
8
95
100
100
Polar
(4)
12
35
13
44
8
0
0
0
H
(5)
47
65
70
44
69
12
13
0
R
(6)
41
0
0
0
20
0
0
0
A
(7)
0
0
17
12
3
88
87
100
P
(8)
-
100
97
92
92
Co.
—
-
0
0
0
0
Op.
-
0
3
8
8
Lat.
75
0
0
2
23
8
Adj.
—
0
0
100
74
15
23
Op.
25
100
0
24
62
69
Lat.
* H: homogeneous; R: ring; A: aggregates; P: poles; Co: coincident; Op: opposite; Lat: lateral; Adj: adjacent (no cells were scored as zonal);
for explanation see Fig. 3.
t Pairs were cultured in presence of an antibody to cadherin in order to avoid the envelopment of the apolar cell by the polar cell - see text.
(7)5-6ht
(8)8-9ht
Apolar Cells
(5) 0-1 h
(6)5-6h
(4)8-9hf
(3)5-6ht
Bright
Age
1dumber apolar
Polar Cells
74
92
(1) 0-1 h
5
40
(2)5-6h
(2)
Scored cells
(1)
Patterns of
surface Con A
distribution (%)
Clathrin poles subcategorized
in relation to
in relation to
contact point
ivith other cellI
surface pole
(10)
(9)
* Patterns of intracellular clathrin distribution (%)
Table 2. Clathrin distribution in natural pairs of 2/16 blastomeres in relation to surface polarity
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Membranous organdie distribution in mouse embryos
297
-..*
f
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Fig. 5. Pairs of 8-cell blastomeres derived by division of a 1/4 blastomere double
labelled to reveal patterns of Concanavalin A binding (d,f,h) and Golgi antigen
distribution (a-c,e,g) at (a,b) 0-1 h-dispersed Golgi antigen snowing some localization
at poles of spindle in B; (c,d) 6-8 h, Golgi antigen aggregated in clumps in a couplet in
which one cell is polarized and the other non-polarized at the surface (e-h), 9—10 h, all
cells polar at surface but having aggregate clumps of Golgi antigen internally
(Mag.x700.)
in situ as precursors of the trophectoderm of the 32-cell blastocyst stage (Ziomek &
Johnson, 1982). Envelopment makes scoring of surface and intracellular phenotype more difficult. In most cases therefore we incubated the 2/16 couplets in the
presence of a monoclonal antibody to cadherin (see Materials & Methods), a
surface homotypic, Ca 2+ -dependent, adhesion molecule; this antibody prevented
cells from flattening on each other and blocked the process of envelopment
(c.f. Fig. 8e-h with Fig. 8i-l). It did not interfere, under the conditions used here,
with polarization of surface or intracellular organelles at the 8-cell stage (compare
lines 6 and 7 in Table 1; also Johnson, 1985).
The data summarizing the distribution of intracellular clathrin in 2/16 pairs is
summarized for each cell subpopulation in Table 2. Four points emerge from these
data. First, immediately after division, most cells regardless of surface phenotype
showed a non-polar distribution of clathrin (Table 2, lines 1 and 5; Fig. 7a,b).
Second, at later stages most cells with a polar surface phenotype also manifested a
298
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
polar distribution of clathrin (Table 2, columns 4 and 8, lines 2-4). Third, in these
cells the clathrin and surface poles were almost always scored as coincident
(Fig. 7c-f); in cells cultured in the presence of the antibody to cadherin the
clathrin tended to cluster between the nucleus and the surface pole, but when
flattening and envelopment occurred the clathrin-positive staining was displaced
laterally as the nucleus became located closer to the surface membrane. The
surface and clathrin poles showed no consistent relationship to the contact point
with the other cell (Table 2, columns 9 and 10, lines 2-4; this latter result confirms
and extends a previous report; Johnson & Ziomek, 1981). Fourth, at later stages,
most cells with an apolar surface phenotype did not have a polar distribution of
clathrin (Table 2, columns 5-8, lines 6-8).
L
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•
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Fig. 6. Pairs of 8-cell blastomeres derived by division of 1/4 blastomeres double
labelled to reveal patterns of surface Con A binding (b,d,f,h) and either endoplasmic
reticulum (E: a,c) or lOOkD antigen distribution (L: e,g). (a,b) 2-3 h; both cells
homogeneous for ER and apolar at the surface. (c,d) 9-10 h; both cells zonal for ER,
upper cell clearly polar at the surface, lower cell less clearly so. (e,f) 9-10 h; both cells
homogeneous for lOOkD protein and polar at the surface. (g,h) 9-10 h; both cells polar
at the surface, upper cell zonal for lOOkD protein and the lower cell provides a rare
example of a blastomere polar for lOOkD protein. (Mag. x700.)
Membranous organelle distribution in mouse embryos
c
• •
299
c
C
Fig. 7. Couplets of 2/16 blastomeres derived from division of a polarized 1/8
blastomere and cultured for a varying number of hours. For each consecutive pair of
figures the first is stained with antiserum to an organelle, and the second is the Con A
binding pattern. Throughout cells that are polar for Con A are indicated with a solid
arrowhead. (a,b) 0-1 h, anti-clathrin (C) - note bright larger polar cell and pale smaller
cell. Clathrin is dispersed around the nuclei or diffusely in the cytoplasm. (c,d) 5-6 h,
anti-clathrin - in the polar cell the clathrin is apical and lateral to the nucleus whereas it
tends to distribute all round the nucleus in the apolar cell. (e,f) 5-6h, anti-clathrin,
both cells polar, note the sharp surface membrane staining for clathrin at the poles.
(g,h) 8-9 h, anti-Golgi (G) after incubation in antibody to cadherin - the aggregate of
Golgi antigen (open arrowhead) in each of the polar cells in this pair are located in
different positions. In the upper cell, the Golgi antigen and the surface pole are
coincident whereas in the lower they are separated by the nucleus (Mag. xllOO.)
(c) Golgi in 2/8 pairs
The data on the distribution of the Golgi antigen with time and in relation to
surface polarity are summarized in Table 3 and illustrated in Fig. 5. No clear trend
towards a polar organization of the Golgi antigen is evident. Immediately after
division the Golgi antigen appeared in many cells to concentrate in a single polar
aggregate in association with the spindle pole (Table 3, line 1; Fig. 5a,b).
However, thereafter the Golgi antigen was dispersed throughout the cell in
multiple aggregates of varying size and distribution (Fig. 5c-h).
300
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
8
^
E
E
/\
j
k
I
Fig. 8. Couplets of 2/16 blastomeres, derived by division of a polarized 8-cell
blastomere, and cultured for a varying number of hours. For each pair of consecutive
figures the first shows the staining with antiserum to endoplasmic reticulum (E: a-d) or
lOOkD lysosomal antigen (L: e-1) and the second the staining pattern observed with
Concanavalin A. (a,b) 5-6h old, zonal distribution of endoplasmic reticulum, upper
cell polar at surface; (c,d) 5-6h in antibody to cadherin, homogeneous distribution of
endoplasmic reticulum, upper cell polar at surface; (e,f) 5-6h old, polar cell has
enveloped the apolar cell, note intense lOOkD (lysosomal) antigen concentrated
adjacent to nucleus of inner cell, and aggregates of antigen in cytoplasmic processes of
outer cells (arrowheads) distant from outer cell nucleus (arrow); (g,h) 5-6h old pair in
which outer polar cell (arrowhead) is in process of enveloping the nonpolar inner cell.
In both cells lysosomal antigen is concentrated in a para-nuclear focus; (i,j) 8-9h old
pair incubated in antibody to cadherin, each cell with a single, polar focus of lysosomal
antigen, opposite to the surface pole in the upper cell (open arrowhead); (k,l) - similar
to previous pair except that lower cell shows a nonpolar surface and a homogeneous
distribution of lysosomal antigen. (Mag. xllOO.)
Membranous organdie distribution in mouse embryos
301
(d) Golgi in 2/16 pairs
Data for the distribution of Golgi antigen in the two cell subpopulations
identifiable at the 16-cell stage are summarized in Table 4, from which four points
emerge. First, with time Golgi antigen changed from a dispersed into an increasingly aggregated organization and ultimately into a single aggregate (designated polar in Table 4; columns 8 and 9, Fig. 7g,h). Second, this concentration of
the Golgi antigen occurred regardless of whether cells had a polar or an apolar
surface phenotype. Third, the polar aggregate was not obviously or consistently
related to the contact point with the other cells (Table 4; column 11). Fourth, in
cells that had surface poles, the Golgi antigen was more often on the axis of
polarity than off it (Table 4, column 10). Moreover at 5-6 h, and especially in pairs
in which flattening and envelopment occurred, the Golgi was mainly located
coincident with the surface pole (e.g. Fig. 7g, upper cell) whereas at the later time
point a more basal location opposite to the pole was more frequent, (e.g. Fig. 7g,
lower cell).
(e) Endoplasmic reticulum in 2/8 and 2/16 pairs
In general the endoplasmic reticulum antigen showed an apolar distribution at
all time points and in all cell types examined (Table 5). Only two deviations from
this pattern were observed. First, as cells flattened on each other at the 8-cell or 16cell stage, a zone free of endoplasmic reticulum developed adjacent to the zone of
contact in some polar cells (Table 5, columns 5 and 10; Fig. 6a-d; Fig. 8a,b).
When flattening was reduced in the presence of the monoclonal antibody to
cadherin, zonal clearance of antigen was reduced (Table 5, column 10 compare
lines 3 and 4, also compare Fig. 8a and 8c). Second, in some polar cells the apical
cytoplasmic zone also appeared to be relatively deficient in ER antigen (e.g.
Fig. 6c, upper cell).
Table 3. Golgi distribution in 2/8 natural pairs of blastomeres in relation to surface
polarity
* Patterns of intracellular Golgi distribution (%) in cells with a surface
phenotype that is
Scored cells
Apolar
Polar
(10)
(6)
(7)
(2)
(3)
(4)
(5)
(8)
(9)
(1)
Age
Number
H
Z
A
P
H
A
P
Z
(l)O-lh
(2)2-4h
(3)4-6h
(4)6-8h
(5) 9-10 h
20
16
18
34
34
45
50
44
35
21
0
38
56
24
0
35
12
0
12
12
20
0
0
0
3
0
0
0
9
32
0
0
0
3
0
0
0
0
9
18
0
0
0
9
15
* H: homogeneous; Z: zonal; A: aggregates; P: poles; no cells were scored as ring stained (for
illustration of classification see Fig. 3).
31
14
23
0
1
5
14
21
4
15
37
19
A
(8)
0
36
57
66
0
37
51
77
P
(9)
-
80
58
39
Co.
-
10
9
44
Op.
-
10
33
17
Lat.
25
58
20
0
42
15
Adj.
25
9
10
80
16
20
Op.
50
33
70
20
42
65
Lat.
*H: homogeneous; Z: zonal; R: ring; A: aggregates; P: poles; Co: coincident; Op: opposite; Lat: lateral; Adj: adjacent (see Fig. 3 for
illustration of categories).
t Pairs were cultured in presence of an antibody to cadherin in order to inhibit the envelopment of the apolar cell by the polar cell - see text.
$ In two-thirds of these cases, cells were enveloping an apolar cell, during which process bright Con A binding spreads over the whole surface see Ziomek & Johnson, 1982 and Fig. 8e,f.
0
0
0
0
66
45
6
13
0
0
0
0
Apolar Cells
(5) 0-1 h
(6)5-6h
(7)5-6ht
(8)8-9hf
0
0
0
0
52
37
5
4
0
7
0
0
44
4
7
0
60
22
21
15
R
(7)
Z
(6)
H
(5)
Patterns of
surface Con A
distribution (%)
Scored cells
(3)
(2)
(4)
(1)
Bright
Age
Number apolar Polar
Polar Cells
96
52
4
(1) 0-1 h
27
81
19*
(2)5-6h
27
8
92
(3)5-6hf
26
8
92
(4)8-9hf
rS
W
r
o
c
>
5w
O
25
O
o
HH
VJ
•«—1
C/J
o25
V)
X
E
o
<—t
O
>
B
Golgi poles subcategorized
in relation to
contact point
in relation to
with other cell
surface pole
(10)
(11)
Table 4. Golgi distributioni in natural pairs of 2/16 blastomeres in relation to surface polarity
* Patterns of intracellular Golgi distribution (%)
302
Membranous organelle distribution in mouse embryos
303
(f) lOOkD membrane antigen in 2/8 pairs
The distribution of the lOOkD antigen is recorded in Table 6. As for the Golgi
antigen, little evidence of redistribution to a focal, polar state was evident during
the 8-cell stage, most cells showing a homogeneous granular pattern (Fig. 6e,f).
Only rarely was a polar localization of antigen observed (Fig. 6g,h).
Table 5. Endoplasmic reticulum distribution in natural pairs of blastomeres in relation
to surface polarity
* Patterns of endoplasmic reticulum distribution (%) in cells with a
surface phenotype that is
(1)
Stage
Scored cells
(2)
(3)
Age Number
(4)
Apolar
Polar
(5) (6) (7) (8) (9) (10) (11) (12)
H
Z
R
A
P
H
Z
R
A
(13)
P
2/8
(1)
(2)
2-3 h
9-10 h
28
34
11
35
75
6
0
0
7
0
7
0
0
44
0
15
0
0
0
0
0
0
2/16
(3)
(4)
5-6 h
5-6 hf
54
24
44
46
0
8
0
0
0
0
0
0
13
42
43
4
0
0
0
0
0
0
*H: homogeneous; Z: zonal; R: ring; A: aggregates; P: poles (see Fig. 3 for illustration of
patterns).
t Pairs were cultured in presence of antibody to cadherin in order to avoid the envelopment of
the apolar cell by the polar cell - see text.
Table 6. lOOkD protein distribution in 2/8 natural pairs of blastomeres in relation to
surface polarity
protein distribution
* Patterns of intracellular lOOkD
:
(95?) in cells with a surface phenotype that is
Polar
Apolar
Scored cells
(4)
(5)
(6)
(7)
(8)
(2)
(3)
(1)
P
Number
H
Z
H
Z
P
Age
(l)O-lh
(2)3-4h
(3)6-7h
(4) 9-10 h
(5)9-10ht
22
20
58
47
46
86
70
34
17
28
14
20
16
23
2
0
0
0
0
0
0
0
38
28
52
0
10
3
23
11
0
0
9
9
7
*H: homogeneous; Z: zonal; P: poles; no cells scored as ring or aggregate (see Fig. 3 for
illustration of classification).
t Control for 2/16 experiment: pairs were cultured in presence of antibody to cadherin - see
text.
304
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
(g) lOOkD membrane antigen in 2/16 pairs
The distributions of lOOkD antigen in the two cell subpopulations identifiable at
the 16-cell stage are summarized in Table 7. Four points emerge from these
data. First, with time the lOOkD antigen concentrated into a single aggregate
(designated polar in Table 7, column 9; Fig. 8g-l). Second, this concentration
occurred regardless of the cell surface phenotype. Third, the polar aggregate was
not obviously or consistently related to the point of contact with the other cell
(Table 7; column 11). Fourth, in cells that had surface poles the lOOkD antigen
tended to lie along the axis of polarity (Table 7; column 10). In the 5-6 h group
there was a particularly high incidence of flattening and envelopment and the
lysosomes tended to concentrate in the 'arms' of the outer cell processes that
extend round the apolar cell (see Fig. 8e). In pairs cultured in the presence of the
antiserum to cadherin the lysosomal antigen was concentrated initially between
the nucleus and the pole, but at the later time point had shifted to the opposite or
basal side of the nucleus in many polar cells scored (Table 7, lines 3 and 4, column
10; Fig. 8i,e,k).
DISCUSSION
The process of de novo polarization of blastomeres in the mouse early embryo is
of central importance to the generation of cell diversity in the blastocyst (Johnson,
1985) and of considerable interest as a cell biological phenomenon. The polarization process is oriented by contact signals from other cells (Ziomek & Johnson,
1980), and is initiated at a characteristic stage of development. The acquisition of
polarized features by the cell occurs progressively, new polar features being
acquired, and established polar features being elaborated, at successive 8-, 16- and
32-cell stages (see Johnson, 1985; Fleming etal 1984; Fleming & Pickering, 1985).
In this paper we have examined the changing distribution with time of four
membranous organelles, as inferred from antigenic distribution, and have
detected three distinctive patterns of change.
The levels of the endoplasmic reticulum antigen detected appear to increase
after the 1-cell stage but otherwise the antigen was distributed uniformly throughout the cytoplasm at all stages examined except adjacent to contact zones with
other cells. A similar distribution is observed in differentiated cells (Louvard et al.
1982). Exclusion of actin (Johnson & Maro, 1984) and myosin (Sobel, 1983) from
contact zones has also been described previously, and in this study intracellular
clathrin was likewise excluded from contact zones.
Cytoplasmic clathrin also showed a dispersed distribution (other than in contact
zones) except in two situations. First,' in all cells that were polarized (or polarizing)
at their surface, clathrin also accumulated in a focal aggregate or pole that was
located immediately underneath the surface pole. Moreover, this polarity of
clathrin preceded by several hours the occurrence of detectable polarity at the
0
0
14
12
14
13
16
5
49
4
11
12
49
34
23
32
A
(8)
R
(7)
U
48
63
43
0
74
72
77
P
(9)
_
_
_
_
_
17
84
29
Co.
_
_
_
_
_
70
8
49
Op.
_
_
_
_
_
13
8
22
Lat.
50
50
25
53
27
18
Adj.
28
8
19
20
8
24
Op.
22
42
56
27
65
58
Lat.
*H: homogeneous; Z: zonal; R: ring; A: aggregates; P: poles; Co: coincident; Op: opposite; Lat: lateral; Adj: adjacent (see Fig. 3 for
illustration of classification).
t Pairs were cultured in presence of an antibody to cadherin in order to avoid the envelopment of the apolar cell by the polar cell - see text.
$ In 85 % of cases polar cells had enveloped apolar cells (see Fig. 8e,f), and so have an overall bright phenotype.
0
0
0
0
51
18
0
13
0
0
0
0
Anolar Cells
(5) 0-1 h
(6)5-6h
(7)5-6ht
(8)8-9ht
0
0
0
0
0
6
0
0
36
3
0
6
35
29
22
37
Z
(6)
H
(5)
Patterns of
surface Con A
Scored cells
distribution (%)
(3)
(4)
(2)
(1)
Bright
Age
Number apolar Polar
Polar Cells
(1) 0-1 h
49
92
8
(2)5-6h
31
42*
58
36
5
95
(3)5-6hf
(4)8-9hf
65
2
98
lOOkD poles subcategorized
in relation to
in relation to
contact point
surface pole
with other cell1
(10)
(11)
* Patterns of intracellular lOOkD protein distribution (%)
Table 7. lOOkD protein distribution in natural pairs of 2/16 blastomeres in relation to surface polarity
o
1
9
OS
to
©
S'
3
©"
§
©
Is
306
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
surface. The redistribution of intracellular clathrin coincided with that reported
for endosomes (Fleming & Pickering, 1985) and for filamentous, cytoplasmic actin
(Johnson & Maro, 1984), both of which also polarized in advance of the cell
surface and which also colocalized with clathrin. It seems probable that these three
events are linked either causally to each other or via some underlying mechanism
that affects each. The reorganization of clathrin, endosomes and actin thus
provides an early indication of cell polarization at the 8-cell stage. However,
clathrin polarity is not as stable as the later developing surface pole. Thus, at
mitotic (or meiotic) division, the clathrin redistributed from its polar location to
the spindle; an association between microtubules or tubulin and coated vesicles or
clathrin has also been observed in other mitotic and interphase cell types (Imhof
et al. 1983; Kelly et al. 1983; Louvard & Reggio, 1981; Louvard et al. 1983;
Pfeffer, Drubin & Kelly, 1983). The association between the spindle and clathrin
presumably ensures that the latter is distributed to each daughter cell in a 2/16
couplet, in which it is then relocated in a polar distribution only in the progeny cells
that had surface poles. Thus, at the 16-cell stage the surface pole appears to act as,
or be associated with, an organizing focus for cytoplasmic polarity. In most nonpolar cells of 2/16 couplets, clathrin remained distributed throughout the cell with
no obvious polar cluster. In a few cells, a polar cluster did form after 5-9 h at the
16-cell stage but only in cells treated with antibody to cadherin. Under such
conditions, envelopment of the apolar cell is prevented, and it is known from
previous work that non-enveloped apolar 1/16 cells will start to develop elements
of polarity at about this time (Ziomek & Johnson, 1982). The polar clustering of
clathrin could therefore represent the earliest manifestations of this regulative
polarization.
The distributions of the lOOkD (acidic organelle/lysosome) and the Golgi
antigens are similar to each other but differ from that of clathrin, an observation in
striking contrast to the situation in fully differentiated cells in which clathrin and
Golgi antigen tend to colocalize (Louvard & Reggio, 1981; Louvard et al. 1983),
and in which the acid vesicle antigen can be detected in endosomes and coated
vesicles (Reggio et al. 1984). These differences presumably relate to the relative
immaturity of processing pathways in blastomeres (Fleming & Pickering, 1985).
Both the lOOkD and Golgi antigens were dispersed up until the 16-cell stage,
although during the 8-cell and early 16-cell stage the antigens were increasingly
aggregated into fewer, larger clumps. During mitosis, the antigens distributed to
both poles of the spindle, thereby presumably ensuring transmission of each
organelle to both progeny. During the 16-cell stage, each antigen became
organized into a single (polar) clump, but did so regardless of the surface
phenotype of the cell. Thus, unlike the focal and polar distribution of clathrin, that
of the Golgi/lysosomal antigens did not appear to be related to the development of
polarity but more to the maturation of endocytic and secretory function in the
cells. Analysis of the maturing endocytic pathway at this time supports the view
that major changes in the organization of and capacity for lysosomal processing
Membranous organdie distribution in mouse embryos
307
occur during the 16-cell stage (Fleming & Pickering, 1985), with the first appearance of secondary lysosomes. However, although the concentration of Golgi/
lysosomal antigens into a single aggregated focus represents a maturational change
in both polar and apolar cell types at the 16-cell stage, the location at which the foci
of antigen developed in cells with polar surface phenotypes may be related to the
axis of polarity of each cell in this subpopulation. Thus, in antibody-treated polar
cells there was the suggestion of a shift from an initially mainly apical to a later
mainly basal location. In non-antibody-treated pairs there is a suggestion that,
unlike clathrin, the lysosomal and Golgi antigens localize in the enveloping arms
of the polar cells away from the nuclei. In intact embryos, the aggregates of
antigen appear to locate basally. Independent cytochemical evidence also suggests
that at the late 16- and early 32-cell stages lysosomal-like bodies and the Golgi
apparatus locate basally (Fleming & Pickering, 1985) or in the enveloping arms of
polar, trophectoderm cells (Fleming etal. 1984).
Alignment of the Golgi apparatus along the axis of polarization through the cell
has been observed in differentiated cells, in which an association with the
microtubule organizing centre is also reported (Carpen, Virtanen & Saksela, 1982;
Kupfer, Dennert & Singer, 1983). However, in late 8-cell mouse blastomeres the
MTOC is located apically (unpublished observations by B. Maro and S. J.
Pickering), again stressing that early embryonic cells may not have established the
range of interorganelle associations characteristic of more mature cells. It is also
noteworthy, that in embryonic chick corneal epithelium the Golgi apparatus shifts
from an apical to basal position during two periods each correlating in time with
the appearance of an acellular collagenous matrix beneath the epithelium
(Trelstad, 1970). Significantly, in this regard, in mouse embryos, all three polypeptide subunits of laminin are first synthesized and secreted basolaterally from
the 16-cell stage (Leivo, Vahari, Timpl & Wartiovaara, 1980; Cooper &
MacQueen, 1983).
Thus, a temporal sequence for the development of polarity in mouse early
blastomeres may be emerging. Actin, endosomal and clathrin redistribution are
evident early in the 8-cell stage. The surface polarization becomes evident later in
the 8-cell stage. Golgi and lysosomal bodies align on the polar axis during the 16cell stage at the same time as endocytic processing pathways mature. In the
accompanying paper (Johnson & Maro, 1985) we describe experiments to disrupt
selectively elements of this sequence of maturation, and as a result we propose a
model for the process of polarization.
We wish to thank our research colleagues and Professor S. J. Singer for their critical advice
and reading of the manuscript, Gin Flach, Ian Edgar and Sheena Glenister for their technical
assistance, and Dr M. Takeichi for supplying the antibody to cadherin. The work was supported
by grants to M. H. Johnson and P. R. Braude from the Medical Research Council and the
Cancer Research Campaign. B. Maro is a visiting EMBO Research Fellow.
308
B. MARO, M. H. JOHNSON, S. J. PICKERING AND D. LOUVARD
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{Accepted 19 June 1985)