changes in the distribution of membranous organelles during

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

SUMMARYThe 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 theendoplasmic reticulum, the lysosomal and acidic vesicle fraction (lOOkD antigen), Golgiapparatus (135kD antigen) and coated vesicles (clathrin). The distribution of these antigens hasalso been examined in isolated 8-cell and 16-cell-stage blastomeres of various ages andphenotypes. Endoplasmic reticulum is detected only weakly in the oocyte and egg, but is seenabundantly at later stages both in association with the nuclear membrane and evenly distributedthroughout 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. Duringinterphase, clathrin is distributed throughout the cell until the mid-8-cell stage when it isconcentrated into the apical region of the cell under the region of membrane at which a surfacepole of microvilli will form subsequently. Thus, the cytoplasmic polarization of clathrin precedesovert polarization at the surface. At mitosis, the clathrin relocates to the spindle and isdistributed to both daughter cells. It resumes an apical location beneath the surface pole ofmicrovilli in polar daughter 1/16 cells, but remains dispersed in apolar daughter 1/16 cells. Boththe lysosomal and Golgi antigens are distributed throughout the cytoplasm until the early 16-cellstage. In pairs of 16-cell blastomeres both antigens aggregate in a single cluster and do sowhether the surface phenotype of the blastomeres is polar or apolar. The position of this clusteris not consistently related to the point of contact with the other cell in the pair but there is asuggestion that in cells with a polar surface phenotype the polar foci of Golgi/lysosomal antigensare located between the nucleus and the surface pole at earlier time points, but shift to a positionbetween the basolateral membrane and the nucleus at the later time point. In intact 16-cellembryos also, the aggregated Golgi/lysosomal antigens of polar cells appear to localize to thebasal region. The distributions of these various organelles in embryonic cells reported here showa number of differences from those reported previously for mature, differentiated cells.

INTRODUCTION

Many differentiated cells manifest a highly asymmetric organization that isdependent 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 isdeveloped and maintained are available including the use of cell lines, such as theMDCK cell line, which can modulate some epithelial properties reversibly in vitro(Van Meer & Simons, 1982), and the examination of polarized, epithelial cellsafter their isolation and manipulation (Ziomek et al. 1980). However in thesemodel systems polarity is not generated de novo from a truly symmetric precursorcell. De novo polar organization of cells develops first early in embryogenesis withthe formation of the primary epithelial germ layers and the delamination ofextraembryonic epithelia. The earliest evidence of this process in the mouseembryo has been detected at the 8-cell stage, during which elements of the cellsurface (Handyside, 1980), cytoskeleton (Johnson & Maro, 1984), and endocytoticprocessing pathway (Reeve, 1981; Fleming & Pickering, 1985) undergo a radicalreorganization to convert a non-polar cell to a highly polarized cell over a period of8-10 h (Ziomek & Johnson, 1980). Elements of this polarity are conserved atdivision (Johnson & Ziomek, 1981), and the polarity is elaborated and stabilized atthe 16- and 32-cell stages to generate the definitive trophectodermal epithelium(Fleming, Warren, Chisholm & Johnson, 1984; Fleming & Pickering, 1985). In thispaper, we report on the immunocytochemical localization of various antigensspecific to membranous organelles (endoplasmic reticulum, the acid vesicle/lysosome compartment, coated vesicles, the Golgi apparatus) concerned withendocytotic and biosynthetic activity and on the changes that occur during theearly stages of cell polarization.

MATERIALS AND METHODS.

1. Recovery of embryosMF1 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 wererecovered from females at 14-16 h post hCG; 2-cell and 4-cell embryos were recovered at46-50h 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 cells2-cell embryos were recovered at 48 h post hCG and cultured in Medium 16 containing

4mgmr 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 flame-polished micropipette. Cells were cultured on Sterilin tissue culture dishes in drops ofM16+BSA under oil at 37°C in 5 % CO2 in air. The cultures were inspected hourly for evidenceof division to 2/8 pairs, and couplets were removed, designated Oh old, and cultured inM16+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-cellblastomeres (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 amonoclonal antibody to cadherin (Yoshida-Noro, Suzuki & Takeichi, 1984; also calleduvomorulin, L-CAM) in order to avoid the envelopment of the apolar cell by the polar cell thatwould otherwise occur (Ziomek & Johnson, 1981).

In one series of experiments, whole 8-cell embryos were freed from their zonae, disaggregatedto single or paired blastomeres in Ca2+-free medium and the blastomeres analysed immediately.

3. ImmunocytochemistrySurface polarity was assessed by incubation of cells or embryos in SOjugml"1

tetramethylrhodamine-labelled succinyl Concanavalin A (SOfigml'1 M2+BSA: TMRTC-S-ConA, Polysciences) for 5min at room temperature, followed by two to three washes inM2+BSA. Labelled cells were then placed in specially designed chambers exactly as describedpreviously (Maro, Johnson, Pickering & Flach, 1984) for fixation with 3-7% formaldehydefollowed by extraction with 0-25 % Triton X-100. After washing, cells were incubated withaffinity-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 lOOkDprotein associated with the acid vesicle/lysosomal fraction (Reggio et al. 1984). A second layerof 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 offluorescent labels and viewed on a Leitz Ortholux II microscope with filter set L2 for FITC-labelled reagents and N2 for TMRTC-labelled reagents. Photographs were taken on Kodak TriX 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 wholeembryos from the unfertilized egg to the 16-cell stage. Each antigen examinedshowed a pattern of distribution that varied characteristically with time.

(a) Intracellular clathrin

Clathrin was distributed in a diffuse granular pattern throughout early devel-opment (Fig. lb,c,d,e) but with two exceptions. First, when meiotic (Fig. la) ormitotic (Fig. lh,i) cells were examined, clathrin distribution corresponded closelywith that of the spindle, and remaining areas of the cytoplasm were relatively freeof clathrin. Second, during the late 8-cell stage clathrin appeared to be moreconcentrated in the apical region of the blastomeres (Fig. If). However, thislocalization was difficult to resolve clearly in whole mounts.

(b) Golgi apparatus

The Golgi antigen was also distributed diffusely throughout the cytoplasm ofearly embryonic cells (Fig. 2a-e). However, at all stages the granular foci of Golgiantigen tended to be larger than those observed with the anti-clathrin antibody,

to O o 33 en O Z on o

1 I

Fig.

1.

Cla

thri

n di

stri

buti

on i

n w

hole

em

bryo

s as

reve

aled

by

use

of a

nti-

clat

hrin

ant

ibod

y an

d FI

TC

-lab

elle

d an

ti-Ig

G f

or e

mbr

yoni

cst

ages

(a)

unf

ertil

ized

egg

, no

te s

tain

ing

of t

he s

econ

d m

etap

hase

spi

ndle

, (b

) 1-

cell

embr

yo, n

ote

the

stro

ng s

tain

ing

of t

he s

econ

d Q

pola

r bo

dy a

nd o

ther

wis

e di

ffus

e cy

topl

asm

ic s

tain

, (c

) 2-

cell

embr

yo, n

ote

rela

tive

lack

of

stai

ning

adj

acen

t to

zon

e of

cel

l con

tact

, C

(d)

4-ce

ll em

bryo

, (e

) ea

rly

8-ce

ll em

bryo

, (f)

lat

e co

mpa

ctin

g 8-

ceU

em

bryo

, not

e co

ncen

trat

ion

of c

lath

rin

at o

utw

ard

faci

ng a

pica

l >

end

of th

e ce

ll, (

g) 1

6-ce

ll em

bryo

, (h)

4-c

ell e

mbr

yo, n

ote

clat

hrin

con

cent

ratio

n in

the

spin

dle

area

of

the

mito

tic c

ell o

n th

e ri

ght,

(i)

g16

-cel

l em

bryo

, no

te s

tain

ing

of t

he s

pind

le.

(Mag

s a-

h x4

70; i

X90

.)

Membranous organdie distribution in mouse embryos 291

and this was particularly marked at the 8-cell and 16-cell stages. Moreover at 16-cell stages there appeared to be a greater concentration of Golgi-antigen at thecentre of the embryo (Fig. 2e), a location corresponding to the totally enclosedinside cells and/or to the basal regions of outer cells.

(c) Endoplasmic reticulum

The antiserum to endoplasmic reticulum antigen stained the nuclear membraneregion intensely, together with a diffuse granular staining throughout the cyto-plasm that was somewhat weaker at the 1-cell stage than subsequently (Fig. 2f-h).Otherwise, no convincing evidence of temporal change or spatial asymmetry instaining pattern was observed with use of this antiserum, although in many casesthe 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 tothe greater density of positive cytoplasm at the centre of the embryo.

(d) lOOkD membrane antigen

The antiserum to the lOOkD protein associated with the acidorganelle/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, therewas 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 changingtemporal and spatial patterns of antigen distribution, and suggests that forclathrin, Golgi and lOOkD antigens changes occur at the 8-cell stage and later. Inorder to visualize these changes more clearly, we used pairs of 8-cell or 16-cellblastomeres. This approach reduces background interference from fluorescentemission outside the plane of focus and also permits a more accurate temporalstaging 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 andexamined at hourly intervals for evidence of division to yield two 0 h 8-cell or 16-cell blastomeres (a 2/8 or 2/16 pair). Pairs were then cultured for up to 11 h beforebeing examined for their surface phenotype (assessed by binding of TMRTC-succinyl-Con A) and the distribution of organelles. Surface phenotype in 2/8 pairswas categorized as being apolar if Con A was uniformly bound and polar if Con Abinding was restricted to the apical region of the cell (compare Fig. 4b apolar withFig. 4k, polar). For 2/16 pairs, three surface phenotypes were defined, namelypolar in which Con A binding was restricted to a limited area of membrane (e.g.

G

b

8 to W

GE

EE

g

o as o as on o O r o

jI

Fig.

2.

Dis

trib

utio

n of

Gol

gi a

ntig

en (

G: a

-e),

end

opla

smic

ret

icul

um a

ntig

en (

E: f

-h)

and

lOO

kD a

ntig

en (

L: i

-1)

in w

hole

em

bryo

sre

veal

ed b

y us

e of

spe

cifi

c an

tibo

dies

and

FT

TC

-lab

elle

d an

ti-Ig

G f

or e

mbr

yoni

c st

ages

, (a

) un

fert

ilize

d eg

g; (

b) 2

-cel

l em

bryo

;(c

) 4-

cell

embr

yo;

(d)

8-ce

ll em

bryo

; (e

) 16

-cel

l em

bryo

, no

te a

ggre

gate

s of

Gol

gi s

tain

ing;

(f)

1-c

ell

embr

yo,

note

pro

nucl

ear

mem

bran

e st

aini

ng w

ith a

nti-

ER

; (g

) 8-c

ell e

mbr

yo; (

h) 1

6-ce

ll em

bryo

; (i)

unf

ertil

ized

egg

; (j)

2-c

ell e

mbr

yo; (

k) 8

-cel

l em

bryo

, not

eag

greg

ates

of

stai

ning

for

lO

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tein

; (1

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-cel

l em

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. (M

ag.

x470

.)


\ and

Homogeneous Zonal Rinc

Coincident Opposite

Polar £ Polar

Aggregates

Random

Fig. 3. Schematic summary of distribution patterns of organelles in pairs of 8- and 16-cell blastomeres. The surface pole in 1/16 blastomeres is indicated by the blackhatching.

upper cell Fig. 7d), bright apolar, in which the surface is brightly labelled over allor most of its surface (e.g. upper cell Fig. 7b) and dull apolar, in which a uniformweak 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 theway in which the polarized 1/8 cell divides. Thus if the cleavage plane is orientedperpendicular to the axis of polarity one bright and one dim apolar cell result, andthe bright area 'shrinks' over a l h period to form a discrete pole (e.g. Fig. 7bconverts to Fig. 7d). If the division plane is oriented along the axis of polarity ofthe 1/8 blastomere, two polar cells result (e.g. Fig. 7f,h). Five patterns oforganelle distribution were observed, and these are indicated schematically inFig. 3.

(a) Clathrin in 2/8 pairs

The changing distribution of intracellular clathrin in relation to surface polarityat 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, thedistribution of intracellular clathrin was mainly zonal soon after division (Table 1,line 1, columns 3 and 4), but thereafter the proportion of cells homogeneous orzonal for clathrin declined whilst the proportion polar for clathrin increased(summarized in Table 1, column 10). Third, cells were detectably polarized forintracellular clathrin before showing evidence of surface polarity (Table 1, com-pare columns 9 and 10). Fig. 4 shows examples of cells that were apolar at their


surface but homogeneous (Fig. 4c,d,e), zonal (Fig. 4a,b) or polar (lower cellFig. 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 polewas 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 thecytoplasm, the membrane overlying the pole of clathrin-positive vesicles alsostaining clearly (e.g. Fig. 4h,j). Enhanced membrane staining for clathrin was alsoobserved in regions of cell apposition.

The redistribution of clathrin from homogeneous to polar pattern, and itsrelationship to the surface pole of Con A binding, are not artefacts of the in vitroculture of 2/8 pairs, as was shown by the staining patterns of blastomeres isolatedfrom precompact 8-cell and from compacted late 8-cell embryos (Fig. 41,m), inwhich a homogeneous clathrin distribution predominated in the former and apolar pattern in the latter.

(b) Clathrin in 2/16 pairs

The analysis of intracellular clathrin distribution in 2/16 pairs is complicated bythe cell heterogeneity at the 16-cell stage. Thus, two populations of cells exist thatdiffer in surface phenotype (bright or polar; dull and apolar) as well as inproperties, developmental fate and lability (reviewed Johnson, 1985). One suchproperty is the tendency after 4 to 8 h in culture of the polar cells to envelope theapolar 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 tosurface polarity

Scored cells

(1)Age I

(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

dumber

1240563658419124

* Patterns of intracellular clathrin distribution (%)

(3)H

1610531

15240

in cells with

Apolar

(4)Z

84552028121008

a surface phenotype that is

(5)P

035663339241117

(6)H

00005040

Polar

(?)Z

0003200

13

(8)P

009

3341516162

summary oi% of cells showing(9)

Surfacepoles

009

3648516575

(10)

Clathrinpoles

035756680757279

*H: homogeneous; Z: zonal; P: poles; No cells scored as ring or aggregate - see Fig. 3 forillustration of this classification.

t Group 7 provided a control for the experiment summarized in Table 2 (see text); cells wereincubated in the presence of antibody to cadherin.


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 Abinding (b,e,g,i,k,m). (a,b) 0-1 h old pair of cells both zonal for clathrin and apolar attheir surface; (c,d,e) 0-1 h old pairs of cells homogeneous for clathrin and apolar atsurface; (f,g) 3-4 h old pair of cells both apolar at surface whilst lower is polar andupper intermediate between zonal and polar for clathrin; (h,i) 5-6 h old pair of cellsupper polar for clathrin and at surface, lower polar for clathrin only; (j,k) 7-8h oldpair of cells both polar for clathrin and at surface; (l,m) all three cells polar for clathrinand at surface. (Mag. X700.)

to

Tab

le 2

. C

lath

rin

dist

ribu

tion

in

natu

ral p

airs

of

2/16

bla

stom

eres

in r

elat

ion

to s

urfa

ce p

olar

ity

da

Scor

ed c

ells

(1)

Age

1

Pol

ar C

ells

(1)

0-1

h(2

)5-6

h(3

)5-6

ht(4

)8-9

hf

Apo

lar

Cel

ls(5

) 0-

1 h

(6)5

-6h

(7)5

-6ht

(8)8

-9ht

(2)

dum

ber

74 40 30 13 68 23 23 9

Pat

tern

s of

surf

ace

Con

Adi

stri

buti

on (

%)

(3)

Bri

ght

apol

ar

92 5 0 0 0 0 0 0

(4)

Pola

r 8 95 100

100 0 0 0 0

H (5) 8 0 0 0 12 35 13 44

R (6)

69 12 13 0 47 65 70 44

* Pa

tter

ns o

f int

race

llula

r cl

athr

in d

istr

ibut

ion

(%)

A (7)

20 0 0 0 41 0 0 0

P (8) 3 88 87 100 0 0 17 12

Co.

100 97 92 92 - - - -

Cla

thri

n po

les

subc

ateg

oriz

ed

in r

elat

ion

tosu

rfac

e po

le(9

)

Op. 0 0 0 0 — - - -

Lat

.

0 3 8 8 - - - -

Adj

.

0 2 23 8 - - 75 0

in r

elat

ion

toco

ntac

t po

int

ivith

oth

er c

ell

(10)

Op

.

100 74 15 23 - — 0 0

I Lat

. 0 24 62 69 - - 25 100

fa o 3- c_ O 2, O V.

t_ T O w SO o > 2, a

* H

: hom

ogen

eous

; R: r

ing;

A: a

ggre

gate

s; P

: pol

es; C

o: c

oinc

iden

t; O

p: o

ppos

ite; L

at: l

ater

al; A

dj: a

djac

ent

(no

cells

wer

e sc

ored

as

zona

l);

for

expl

anat

ion

see

Fig.

3.

t Pa

irs

wer

e cu

ltur

ed i

n pr

esen

ce o

f an

ant

ibod

y to

cad

heri

n in

ord

er t

o av

oid

the

enve

lopm

ent

of th

e ap

olar

cel

l by

the

pola

r ce

ll -

see

text

.r o


-..*

f g | h

Fig. 5. Pairs of 8-cell blastomeres derived by division of a 1/4 blastomere doublelabelled to reveal patterns of Concanavalin A binding (d,f,h) and Golgi antigendistribution (a-c,e,g) at (a,b) 0-1 h-dispersed Golgi antigen snowing some localizationat poles of spindle in B; (c,d) 6-8 h, Golgi antigen aggregated in clumps in a couplet inwhich one cell is polarized and the other non-polarized at the surface (e-h), 9—10 h, allcells 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 pheno-type more difficult. In most cases therefore we incubated the 2/16 couplets in thepresence of a monoclonal antibody to cadherin (see Materials & Methods), asurface homotypic, Ca2+-dependent, adhesion molecule; this antibody preventedcells 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 (comparelines 6 and 7 in Table 1; also Johnson, 1985).

The data summarizing the distribution of intracellular clathrin in 2/16 pairs issummarized for each cell subpopulation in Table 2. Four points emerge from thesedata. First, immediately after division, most cells regardless of surface phenotypeshowed 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


polar distribution of clathrin (Table 2, columns 4 and 8, lines 2-4). Third, in thesecells 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 theclathrin tended to cluster between the nucleus and the surface pole, but whenflattening and envelopment occurred the clathrin-positive staining was displacedlaterally as the nucleus became located closer to the surface membrane. Thesurface and clathrin poles showed no consistent relationship to the contact pointwith the other cell (Table 2, columns 9 and 10, lines 2-4; this latter result confirmsand extends a previous report; Johnson & Ziomek, 1981). Fourth, at later stages,most cells with an apolar surface phenotype did not have a polar distribution ofclathrin (Table 2, columns 5-8, lines 6-8).

L

5^,• 1

Fig. 6. Pairs of 8-cell blastomeres derived by division of 1/4 blastomeres doublelabelled to reveal patterns of surface Con A binding (b,d,f,h) and either endoplasmicreticulum (E: a,c) or lOOkD antigen distribution (L: e,g). (a,b) 2-3 h; both cellshomogeneous 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 cellshomogeneous for lOOkD protein and polar at the surface. (g,h) 9-10 h; both cells polarat the surface, upper cell zonal for lOOkD protein and the lower cell provides a rareexample of a blastomere polar for lOOkD protein. (Mag. x700.)

Membranous organelle distribution in mouse embryos

c • • c299

CFig. 7. Couplets of 2/16 blastomeres derived from division of a polarized 1/8blastomere and cultured for a varying number of hours. For each consecutive pair offigures the first is stained with antiserum to an organelle, and the second is the Con Abinding pattern. Throughout cells that are polar for Con A are indicated with a solidarrowhead. (a,b) 0-1 h, anti-clathrin (C) - note bright larger polar cell and pale smallercell. 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 ittends 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 ofGolgi antigen (open arrowhead) in each of the polar cells in this pair are located indifferent positions. In the upper cell, the Golgi antigen and the surface pole arecoincident 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 tosurface polarity are summarized in Table 3 and illustrated in Fig. 5. No clear trendtowards a polar organization of the Golgi antigen is evident. Immediately afterdivision the Golgi antigen appeared in many cells to concentrate in a single polaraggregate in association with the spindle pole (Table 3, line 1; Fig. 5a,b).However, thereafter the Golgi antigen was dispersed throughout the cell inmultiple aggregates of varying size and distribution (Fig. 5c-h).


8 ^ E E

/\

j k I

Fig. 8. Couplets of 2/16 blastomeres, derived by division of a polarized 8-cellblastomere, and cultured for a varying number of hours. For each pair of consecutivefigures the first shows the staining with antiserum to endoplasmic reticulum (E: a-d) orlOOkD lysosomal antigen (L: e-1) and the second the staining pattern observed withConcanavalin A. (a,b) 5-6h old, zonal distribution of endoplasmic reticulum, uppercell polar at surface; (c,d) 5-6h in antibody to cadherin, homogeneous distribution ofendoplasmic reticulum, upper cell polar at surface; (e,f) 5-6h old, polar cell hasenveloped the apolar cell, note intense lOOkD (lysosomal) antigen concentratedadjacent to nucleus of inner cell, and aggregates of antigen in cytoplasmic processes ofouter cells (arrowheads) distant from outer cell nucleus (arrow); (g,h) 5-6h old pair inwhich 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 oldpair incubated in antibody to cadherin, each cell with a single, polar focus of lysosomalantigen, opposite to the surface pole in the upper cell (open arrowhead); (k,l) - similarto previous pair except that lower cell shows a nonpolar surface and a homogeneousdistribution of lysosomal antigen. (Mag. xllOO.)


(d) Golgi in 2/16 pairs

Data for the distribution of Golgi antigen in the two cell subpopulationsidentifiable at the 16-cell stage are summarized in Table 4, from which four pointsemerge. First, with time Golgi antigen changed from a dispersed into an in-creasingly aggregated organization and ultimately into a single aggregate (desig-nated polar in Table 4; columns 8 and 9, Fig. 7g,h). Second, this concentration ofthe Golgi antigen occurred regardless of whether cells had a polar or an apolarsurface phenotype. Third, the polar aggregate was not obviously or consistentlyrelated to the contact point with the other cells (Table 4; column 11). Fourth, incells that had surface poles, the Golgi antigen was more often on the axis ofpolarity than off it (Table 4, column 10). Moreover at 5-6 h, and especially in pairsin which flattening and envelopment occurred, the Golgi was mainly locatedcoincident with the surface pole (e.g. Fig. 7g, upper cell) whereas at the later timepoint 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 atall time points and in all cell types examined (Table 5). Only two deviations fromthis pattern were observed. First, as cells flattened on each other at the 8-cell or 16-cell stage, a zone free of endoplasmic reticulum developed adjacent to the zone ofcontact 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 tocadherin, zonal clearance of antigen was reduced (Table 5, column 10 comparelines 3 and 4, also compare Fig. 8a and 8c). Second, in some polar cells the apicalcytoplasmic 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 surfacepolarity

Scored cells(1)

Age

(l)O-lh(2)2-4h(3)4-6h(4)6-8h

(2)Number

20161834

* Patterns of intracellular Golgi

(3)H

45504435

Apolar(4)Z

0385624

(5)A

35120

12

distributionphenotype that is

(6)

P

20000

(7)

H

0009

(%) in cells with

Polar(8)

Z

0003

(9)A

0009

a surface

(10)

P

0009

(5) 9-10 h 34 21 0 12 3 32 0 18 15

* H: homogeneous; Z: zonal; A: aggregates; P: poles; no cells were scored as ring stained (forillustration of classification see Fig. 3).

Tab

le 4

. G

olgi

dis

trib

utio

n

Sco

red

cells

(1)

(2)

Pat

tern

s of

surf

ace

Con

Adi

stri

buti

on (

%)

(3)

Bri

ght

Age

N

umbe

r ap

olar

Pol

ar C

ells

(1)

0-1

h(2

)5-6

h(3

)5-6

hf

(4)8

-9h

f

Apo

lar

Cel

ls(5

) 0-

1 h

(6)5

-6h

(7)5

-6h

t(8

)8-9

hf

52 27 27 26 60 22 21 15

96 19* 8 8 0 0 0 0

(4)

Pola

r

4 81 92 92 0 0 0 0

H (5)

44 4 7 0 66 45 6 13

i in

nat

ural

pai

rs o

f 2/

16 b

last

omer

es i

n re

lati

on

to

surf

ace

pola

rity

Z (6) 0 7 0 0 0 0 0 0

*

R (7)

52 37 5 4 31 14 23 0

Patte

rns

of in

trac

ellu

lar

Gol

gi d

istr

ibut

ion

(%)

A (8) 4 15 37 19 1 5 14 21

P (9) 0 37 51 77 0 36 57 66

Co. - 80 58 39 - - - -

Gol

gi p

oles

sub

cate

gori

zed

in r

elat

ion

tosu

rfac

e po

le(1

0)

Op.

L

at.

- -

10

109

3344

17 -

- - - -

in r

elat

ion

toco

ntac

t po

int

with

oth

er c

ell

(11)

Adj

. O

p.

- -

0 80

42

1615

20

- -

25

2558

9

20

10

Lat

. - 20 42 65 - 50 33 70

302 B > O E <—t o X V) o 25 C/J

•«—

1

HH VJ o WrS w5O 25 O >

*H:

hom

ogen

eous

; Z

: zo

nal;

R:

ring

; A

: ag

greg

ates

; P

: po

les;

Co:

coi

ncid

ent;

Op

: opp

osit

e; L

at:

late

ral;

Ad

j: a

djac

ent

(see

Fig

. 3

for

illu

stra

tion

of

cate

gori

es).

t Pa

irs

wer

e cu

ltur

ed i

n pr

esen

ce o

f an

ant

ibod

y to

cad

heri

n in

ord

er to

inhi

bit t

he e

nvel

opm

ent

of th

e ap

olar

cel

l by

the

pola

r ce

ll -

see

text

.$

In tw

o-th

irds

of

thes

e ca

ses,

cel

ls w

ere

enve

lopi

ng a

n ap

olar

cel

l, du

ring

whi

ch p

roce

ss b

righ

t Con

A b

indi

ng s

prea

ds o

ver

the

who

le s

urfa

ce -

see

Zio

mek

& J

ohns

on,

1982

and

Fig

. 8e

,f.

r o c


(f) lOOkD membrane antigen in 2/8 pairs

The distribution of the lOOkD antigen is recorded in Table 6. As for the Golgiantigen, little evidence of redistribution to a focal, polar state was evident duringthe 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 relationto surface polarity

* Patterns of endoplasmic reticulum distribution (%) in cells with asurface phenotype that is

Scored cells Apolar Polar(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Stage Age Number H Z R A P H Z R A P

2/8(1)(2)

2/16(3)(4)

2-3 h9-10 h

5-6 h5-6 hf

2834

5424

1135

4446

756

08

00

00

70

00

70

00

044

1342

015

434

00

00

00

00

00

00

*H: homogeneous; Z: zonal; R: ring; A: aggregates; P: poles (see Fig. 3 for illustration ofpatterns).

t Pairs were cultured in presence of antibody to cadherin in order to avoid the envelopment ofthe apolar cell by the polar cell - see text.

Table 6. lOOkD protein distribution in 2/8 natural pairs of blastomeres in relation tosurface polarity

Scored(1)

Age

(l)O-lh(2)3-4h(3)6-7h(4) 9-10 h(5)9-10ht

cells(2)

Number

2220584746

* Patterns of intracellular :(95

(3)

H

8670341728

lOOkD protein distribution?) in cells with a surface phenotype that is

Apolar(4)

Z

142016232

(5)

P

00000

(6)

H

00

382852

Polar(7)

Z

0103

2311

(8)P

00997

*H: homogeneous; Z: zonal; P: poles; no cells scored as ring or aggregate (see Fig. 3 forillustration of classification).

t Control for 2/16 experiment: pairs were cultured in presence of antibody to cadherin - seetext.


(g) lOOkD membrane antigen in 2/16 pairs

The distributions of lOOkD antigen in the two cell subpopulations identifiable atthe 16-cell stage are summarized in Table 7. Four points emerge from thesedata. First, with time the lOOkD antigen concentrated into a single aggregate(designated polar in Table 7, column 9; Fig. 8g-l). Second, this concentrationoccurred regardless of the cell surface phenotype. Third, the polar aggregate wasnot 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 antigentended to lie along the axis of polarity (Table 7; column 10). In the 5-6 h groupthere was a particularly high incidence of flattening and envelopment and thelysosomes tended to concentrate in the 'arms' of the outer cell processes thatextend round the apolar cell (see Fig. 8e). In pairs cultured in the presence of theantiserum to cadherin the lysosomal antigen was concentrated initially betweenthe nucleus and the pole, but at the later time point had shifted to the opposite orbasal side of the nucleus in many polar cells scored (Table 7, lines 3 and 4, column10; Fig. 8i,e,k).

DISCUSSION

The process of de novo polarization of blastomeres in the mouse early embryo isof central importance to the generation of cell diversity in the blastocyst (Johnson,1985) and of considerable interest as a cell biological phenomenon. The polar-ization process is oriented by contact signals from other cells (Ziomek & Johnson,1980), and is initiated at a characteristic stage of development. The acquisition ofpolarized features by the cell occurs progressively, new polar features beingacquired, and established polar features being elaborated, at successive 8-, 16- and32-cell stages (see Johnson, 1985; Fleming etal 1984; Fleming & Pickering, 1985).In this paper we have examined the changing distribution with time of fourmembranous organelles, as inferred from antigenic distribution, and havedetected three distinctive patterns of change.

The levels of the endoplasmic reticulum antigen detected appear to increaseafter the 1-cell stage but otherwise the antigen was distributed uniformly through-out the cytoplasm at all stages examined except adjacent to contact zones withother cells. A similar distribution is observed in differentiated cells (Louvard et al.1982). Exclusion of actin (Johnson & Maro, 1984) and myosin (Sobel, 1983) fromcontact zones has also been described previously, and in this study intracellularclathrin was likewise excluded from contact zones.

Cytoplasmic clathrin also showed a dispersed distribution (other than in contactzones) 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 waslocated immediately underneath the surface pole. Moreover, this polarity ofclathrin preceded by several hours the occurrence of detectable polarity at the

Tab

le 7

. lO

OkD

pro

tein

dis

trib

utio

n in

nat

ural

pai

rs o

f 2/1

6 bl

asto

mer

es i

n re

lati

on to

sur

face

pol

arit

y

Scor

ed c

ells

(1)

(2)

Age

N

umbe

rPo

lar

Cel

ls(1

) 0-

1 h

(2)5

-6h

(3)5

-6h

f(4

)8-9

hf

Ano

lar

Cel

ls(5

) 0-

1 h

(6)5

-6h

(7)5

-6h

t(8

)8-9

ht

49 31 36 65 35 29 22 37

Pat

tern

s of

surf

ace

Con

Adi

stri

buti

on (

%)

(3)

Bri

ght

apol

ar

92 42* 5 2 0 0 0 0

(4)

Pola

r

8 58 95 98 0 0 0 0

H (5)

36 3 0 6 51 18 0 13

Z (6) 0 6 0 0 0 0 0 0

* Pa

ttern

s of

intr

acel

lula

r lO

OkD

pro

tein

dis

trib

utio

n

R (7)

49 4 11 12 49 34 23 32

A (8)

14 13 16 5 0 0 14 12

P (9) 0 74 72 77 U 48 63 43

(%)

lOO

kD p

oles

sub

cate

gori

zed

in r

elat

ion

tosu

rfac

e po

le(1

0)

Co.

O

p.

Lat

.

_ _

_17

70

13

84

8 8

29

49

22

_ _

__

_ _

_ _

__

_ _

Adj

. - 53 27 18 - 50 50 25

in r

elat

ion

toco

ntac

t po

int

with

oth

er c

ell

(11)

Op. - 20 8 24 - 28 8 19

1 Lat

. - 27 65 58 - 22 42 56

I s © § ©" S' 3 © to O

S 9*H

: ho

mog

eneo

us;

Z:

zona

l; R

: ri

ng;

A:

aggr

egat

es;

P:

pole

s; C

o: c

oinc

iden

t; O

p: o

ppos

ite;

Lat

: la

tera

l; A

dj:

adja

cent

(se

e Fi

g. 3

for

illu

stra

tion

of

clas

sifi

catio

n).

t Pa

irs

wer

e cu

ltur

ed in

pre

senc

e of

an

antib

ody

to c

adhe

rin

in o

rder

to a

void

the

enve

lopm

ent

of th

e ap

olar

cel

l by

the

pola

r ce

ll -

see

text

.$

In 8

5 %

of

case

s po

lar

cells

had

env

elop

ed a

pola

r ce

lls (

see

Fig.

8e,

f),

and

so h

ave

an o

vera

ll br

ight

phe

noty

pe.

1 o


surface. The redistribution of intracellular clathrin coincided with that reportedfor endosomes (Fleming & Pickering, 1985) and for filamentous, cytoplasmic actin(Johnson & Maro, 1984), both of which also polarized in advance of the cellsurface and which also colocalized with clathrin. It seems probable that these threeevents are linked either causally to each other or via some underlying mechanismthat affects each. The reorganization of clathrin, endosomes and actin thusprovides 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, atmitotic (or meiotic) division, the clathrin redistributed from its polar location tothe spindle; an association between microtubules or tubulin and coated vesicles orclathrin has also been observed in other mitotic and interphase cell types (Imhofet al. 1983; Kelly et al. 1983; Louvard & Reggio, 1981; Louvard et al. 1983;Pfeffer, Drubin & Kelly, 1983). The association between the spindle and clathrinpresumably ensures that the latter is distributed to each daughter cell in a 2/16couplet, in which it is then relocated in a polar distribution only in the progeny cellsthat 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 non-polar cells of 2/16 couplets, clathrin remained distributed throughout the cell withno obvious polar cluster. In a few cells, a polar cluster did form after 5-9 h at the16-cell stage but only in cells treated with antibody to cadherin. Under suchconditions, envelopment of the apolar cell is prevented, and it is known fromprevious work that non-enveloped apolar 1/16 cells will start to develop elementsof polarity at about this time (Ziomek & Johnson, 1982). The polar clustering ofclathrin could therefore represent the earliest manifestations of this regulativepolarization.

The distributions of the lOOkD (acidic organelle/lysosome) and the Golgiantigens are similar to each other but differ from that of clathrin, an observation instriking contrast to the situation in fully differentiated cells in which clathrin andGolgi antigen tend to colocalize (Louvard & Reggio, 1981; Louvard et al. 1983),and in which the acid vesicle antigen can be detected in endosomes and coatedvesicles (Reggio et al. 1984). These differences presumably relate to the relativeimmaturity 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 increasinglyaggregated into fewer, larger clumps. During mitosis, the antigens distributed toboth poles of the spindle, thereby presumably ensuring transmission of eachorganelle to both progeny. During the 16-cell stage, each antigen becameorganized into a single (polar) clump, but did so regardless of the surfacephenotype of the cell. Thus, unlike the focal and polar distribution of clathrin, thatof the Golgi/lysosomal antigens did not appear to be related to the development ofpolarity but more to the maturation of endocytic and secretory function in thecells. Analysis of the maturing endocytic pathway at this time supports the viewthat major changes in the organization of and capacity for lysosomal processing


occur during the 16-cell stage (Fleming & Pickering, 1985), with the first appear-ance of secondary lysosomes. However, although the concentration of Golgi/lysosomal antigens into a single aggregated focus represents a maturational changein both polar and apolar cell types at the 16-cell stage, the location at which the fociof antigen developed in cells with polar surface phenotypes may be related to theaxis of polarity of each cell in this subpopulation. Thus, in antibody-treated polarcells there was the suggestion of a shift from an initially mainly apical to a latermainly basal location. In non-antibody-treated pairs there is a suggestion that,unlike clathrin, the lysosomal and Golgi antigens localize in the enveloping armsof the polar cells away from the nuclei. In intact embryos, the aggregates ofantigen appear to locate basally. Independent cytochemical evidence also suggeststhat at the late 16- and early 32-cell stages lysosomal-like bodies and the Golgiapparatus locate basally (Fleming & Pickering, 1985) or in the enveloping arms ofpolar, trophectoderm cells (Fleming etal. 1984).

Alignment of the Golgi apparatus along the axis of polarization through the cellhas been observed in differentiated cells, in which an association with themicrotubule organizing centre is also reported (Carpen, Virtanen & Saksela, 1982;Kupfer, Dennert & Singer, 1983). However, in late 8-cell mouse blastomeres theMTOC is located apically (unpublished observations by B. Maro and S. J.Pickering), again stressing that early embryonic cells may not have established therange of interorganelle associations characteristic of more mature cells. It is alsonoteworthy, that in embryonic chick corneal epithelium the Golgi apparatus shiftsfrom an apical to basal position during two periods each correlating in time withthe appearance of an acellular collagenous matrix beneath the epithelium(Trelstad, 1970). Significantly, in this regard, in mouse embryos, all three poly-peptide subunits of laminin are first synthesized and secreted basolaterally fromthe 16-cell stage (Leivo, Vahari, Timpl & Wartiovaara, 1980; Cooper &MacQueen, 1983).

Thus, a temporal sequence for the development of polarity in mouse earlyblastomeres may be emerging. Actin, endosomal and clathrin redistribution areevident early in the 8-cell stage. The surface polarization becomes evident later inthe 8-cell stage. Golgi and lysosomal bodies align on the polar axis during the 16-cell stage at the same time as endocytic processing pathways mature. In theaccompanying paper (Johnson & Maro, 1985) we describe experiments to disruptselectively elements of this sequence of maturation, and as a result we propose amodel for the process of polarization.

We wish to thank our research colleagues and Professor S. J. Singer for their critical adviceand reading of the manuscript, Gin Flach, Ian Edgar and Sheena Glenister for their technicalassistance, and Dr M. Takeichi for supplying the antibody to cadherin. The work was supportedby grants to M. H. Johnson and P. R. Braude from the Medical Research Council and theCancer Research Campaign. B. Maro is a visiting EMBO Research Fellow.


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{Accepted 19 June 1985)

changes in the distribution of membranous organelles during

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