in vivo embryonic expression of laminin and its involvement in cell ... · sphaerechinus granularis...

Development 101. 659-671 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

659

In vivo embryonic expression of laminin and its involvement in cell

shape change in the sea urchin Sphaerechinus granularis

ROBERT A. MCCARTHY1 * and MAX M. BURGER2

' Biocenler. University of Basel, CH-4056 Basel. Switzerland2Friedrich Miescher-lnstitme, Postfach 2543, CH-4002 Basel. Switzerland

* Present address: Department of Anatomy and Cellular Biology. Harvard Medical School. 220 Longwood Avenue. Boston, MA02115. USA

Summary

Laminin, a component of the embryonic sea urchinbasal lamina, is recognized by monoclonal antibodyBL1 (Mab BL1). Our results demonstrate that lamininis secreted into the blastcoel at the early blastula stageat a time when the blastomeres undergo a cell shapechange and are organized into an epithelium. Lamininis present on the basal surfaces of ectodermal cells andis absent or reduced on migrating primary mesen-chyme cells. Microinjection of a monoclonal antibodydirected against laminin induces a morphological

change in cell shape and a deformation of the embry-onic epithelium. Investigation of selected stages of liveembryos suggests that the distribution of laminin maybe heterogeneous within the basal lamina during earlydevelopment. The results implicate laminin as amediator of cell shape change during early morpho-genesis.

Key words: basement membranes, extracellular matrix,monoclonal antibodies, sea urchin, Sphaerechinusgranularis, laminin, cell shape.

Introduction

During embryogenesis, the interaction of cells withthe basement membrane results in changes in cellshape, cell growth and differentiation (Hay, 1981;Bernfield, Banerjee, Koda & Rapraeger, 1984, forreviews). Laminin has been shown to be an abundantprotein component of the basement membrane invertebrate embryos and adults which, in culturedcells, induces morphological changes of cell spread-ing, elongation and neurite outgrowth (Von der Mark& Kiihl, 1985, for review). As part of an extracellularmatrix (ECM) substrate of cultured cells, laminin hasalso been implicated in the maintenance of epithelialcell shape, in the regulation of cell adhesion anddifferentiation of cultured epithelial cells (Gospodar-owicz, Greenburg & Birdwell, 1978; Sugrue & Hay,1981; Greenburg & Hay, 1986; see Watt, 1986, forreview). During the development of the mouse em-bryo, laminin is one of the earliest of the ECMcomponents to appear, preceding the establishmentof an organized basement membrane. These results

have suggested a coordinating role with other ECMcomponents in the formation of basement membrane(Leivo, Vaheri, Timpl & Wartiovaara, 1980; Wu,Wan, Chung & Damjanov, 1983; Dziadek & Timpl,1985). Its expression on the surface of early mouseblastomeres also indicates that it may play a role inthe establishment of cell polarity, cell adhesion dur-ing compaction or in the mediation of cell interactionsduring preimplantation development (Dziadek &Timpl, 1985).

In the early sea urchin embryo, a thin fibrous layeror basal lamina has long been known to exist on thebasal surfaces of cells beginning at the early blastulastage (Endo & Uno, 1960; Wolpert & Mercer, 1963;Okazaki & Niijima, 1964). The basal lamina has beenthought to play a role in many morphogenetic eventsof early sea urchin development such as cell-celladhesion and cell shape change (Gustafson & Wol-pert, 1967), primary mesenchyme cell migration(Okazaki, Fukushi & Dan, 1962; Katow, Yamada &Solursh, 1982) and gastrulation (Spiegel, Burger &

660 R. A. McCarthy and M. M. Burger

Spiegel, 1980, 1983). Some evidence points to extra-cellular matrix components as mediators of theseevents in the sea urchin embryo. In sulphate-deprivedembryos, primary mesenchyme cell migration andgastrulation are blocked along with normal metab-olism of sulphated glycosaminoglycans, glycoproteinsand proteoglycans (Sugiyama, 1972; Karp & Solursh,1974; Solursh & Katow, 1982). Exposure of embryosto /5-xylosides will inhibit proteoglycan synthesis,leading to a block of gastrulation and primary mesen-chyme cell migration (Kinoshita & Saiga, 1979; Aka-saka, Amemiya & Terayama, 1980). Tunicamycin, aninhibitor of protein and lipid glycosylation, will pre-vent gastrulation (Schneider, Nguyen & Lennarz,1978; Akasaka et al. 1980) under conditions whereglycosaminoglycan synthesis is normal (Heifetz &Lennarz, 1979). Little is known, however, of themechanisms by which the extracellular matrix com-ponents influence these morphogenetic events.

Monoclonal antibody BL1 (Mab BL1) recognizestwo proteins in the sea urchin embryo. Metabolic andembryo surface labelling studies have shown that oneprotein is restricted to the hyaline layer. The otherprotein is a sea urchin basal lamina component whichis structurally related to the vertebrate basementmembrane protein laminin (McCarthy, Beck &Burger, 1987). In the present study, we use the MabBL1 to study the expression and involvement oflaminin in epithelium formation in vivo. Microinjec-tion of fluorescently labelled Mab BL1 antibodydemonstrates that laminin is secreted on the basal cellsurface at the early blastula stage. Expression oflaminin is correlated in time with the change in cellshape of embryonic blastomeres. Injection of MabBL1 results initially in a dramatic cell shape changeand a rounding up of cells, leading, in some cases, toembryonic deformation. These results are consistentwith the hypothesis that laminin functions in vivo as acell adhesion protein of the basal lamina and that itmay have a role in the regulation of cell polarity andcell shape during early embryogenesis.

Materials and methods

Handling of embryosAdult Sphaerechinus granularis were obtained from Labora-toire Arago, 66650 Banyuls-sur-Mer, France. Gameteswere collected by electrical stimulation into Woods Holeformula artificial sea water containing Tris buffer insteadof sodium carbonate (MBLSW; Cavanaugh, 1956). Eggswere fertilized as described for other sea urchin species(McCarthy & Spiegel, 1983) and embryos were raised at16°C to the appropriate developmental stage.

Preparation of FITC-Mab BL1Monoclonal antibody IgG was prepared from hybndomasupernatant as previously described (McCarthy et al. 1987)and labelled with fluorescein isothiocyanate (FITC).Briefly, 0-5-1 mg of purified Mab IgG was mixed with6mgml~' cytochrome C in 20 ml of 0-05M-sodium carbon-ate buffer pH9-5, 015M-sodium chloride and dialysedagainst the same buffer. The dialysis tubing was placed into20 ml sodium carbonate buffer pH 9-5, 015M-sodium chlor-ide containing 0-2mgmP' FITC and dialysed 4h in thedark at room temperature. After dialysis, free FITC wasremoved from the protein mixture by chromatography on aSephadex G-25 column and the FITC-labelled Mab wasreisolated by protein-A affinity chromatography. TheFITC-Mab preparation was concentrated to 1 mg ml"1 anddialysed extensively against PBS.

MicroinjectionEmbryos at specific stages were injected using the pressureinjection method of Hiramoto (1962) with the modificationsdescribed by Kiehart (1981). Before loading into theinjection chamber, the embryos were washed three times byhand centrifugation in MBLSW. The pellet of embryos wasthen suspended in four volumes of MBLSW containing0-2 % agarose which had been melted and quickly broughtto room temperature. The suspension of embryos wasimmediately loaded into the injection chamber. The cooledagarose formed a loose gel within the microinjectionchamber which allowed the ciliated embryos to be observedover long periods. The amount of IgG injected was com-puted by calibrating the micropipette with vegetable oilinjected into the sea water.

MicroscopyEmbryos were observed and photographed with a ZeissAxiomat or ICM microscope equipped with Nomarskidifferential interference contrast (DIC) and epifluor-escence optics. Kodak Tri-X Pan film was used and pro-cessed at 1600 ASA using Ilford Microphen developer.

Results

Normal development of Sphaerechinus granularisThe blastula epithelium undergoes complex morpho-genetic changes during the early development ofSphaerechinus granularis (Fig. 1). These events in-clude polarization of the blastomeres and dynamicchanges in cell-cell contacts and cell shape. After thefifth cleavage, the embryo consists of 32 rounded cellsattached at their apical ends to the hyaline layer(Fig. 1A, arrow). A dramatic cell shape changeoccurs at approximately the ninth cleavage (Fig. IB)during which the apparent volume of the blastocoeliccavity is reduced by approximately two thirds. Thecells remain attached to the hyaline layer and take ona wine-glass shape and elongated appearance(Fig. 1C, large arrow). During the next few hours ofdevelopment as the embryo enlarges (Fig. ID), cells

r- S a.

Fig.

1.

Nor

mal

dev

elop

men

t of

S.

gran

ular

is.

Nom

arsk

i D

1C m

icro

grap

hs o

f la

rvae

rai

sed

at 1

6°C

. (A

) 32

-cel

l em

bryo

, 7h

af

ter

fert

iliz

atio

n. C

ells

are

att

ache

d to

the

hya

line

lay

er (

arro

w)

at t

heir

api

cal

surf

aces

and

are

rou

nded

on

the

basa

l su

rfac

es.

(B)

12 h

em

bryo

show

ing

cell

elon

gati

on a

nd b

asal

cel

l-ce

ll c

onta

ct a

t th

e ti

me

corr

espo

ndin

g to

bas

al l

amin

a fo

rmat

ion.

(C

) In

the

J3h

em

bryo

som

e ce

llsta

ke o

n a

win

e-gl

ass

shap

e (l

arge

arr

ow)

and

othe

rs a

re r

ound

ed a

nd a

ttac

hed

to t

he h

yali

ne l

ayer

(sm

all

arro

w).

(D

) A

t 16

h t

he e

mbr

yobe

gins

to

incr

ease

in

volu

me

and

mos

t of

the

cel

ls t

ake

on a

win

e-gl

ass

shap

e. (

E)

The

vol

ume

of t

he e

mbr

yo i

ncre

ases

and

a w

ell-

orde

red

epit

heli

um d

evel

ops

at 2

1 h.

(F

) P

rim

ary

mes

ench

yme

cell

s m

igra

te i

nto

the

blas

toco

el (

arro

ws)

at

24 h

. T

he e

pith

eliu

m i

s sl

ight

lyth

icke

ned

at t

he s

ite

of p

rim

ary

mes

ench

yme

mig

rati

on (

G,

arro

ws)

and

mor

e pr

omin

entl

y at

the

ani

mal

pla

te (

H,

arro

ws)

. S

cale

bar

,50

/im

.

3 o •3 a-


occasionally round up and lose their wine-glass shapeand adhere closely to the hyaline layer (Fig. 1C, smallarrow).

By 21 h after fertilization (Fig. IE), a well-orderedepithelium begins to form and by 24 h the embryoconsists of cells bounded on their apical surfaces bythe hyaline layer and on their basal surfaces by thebasal lamina. At this time, the primary mesenchymecells enter the blastocoel and begin to migrate(Fig. IF, arrows). Other morphogenetic changes oc-cur in the epithelium after primary mesenchyme cellingression. These are localized thickenings of theepithelium visible in the vegetal half of the 24-30 hembryo (Fig. 1G, arrows) in which cells are oftenpresent in a wine-glass shape with apparent spacesbetween adjacent cells as in the 35-48 h embryo in theregion of the animal plate (Fig. 1H, arrows).

Expression of laminin in vivoMonoclonal antibody BL1 recognizes two large mol-ecular weight proteins: a protein restricted to thehyaline layer and a basal lamina protein, sea urchinlaminin. Sea urchin laminin has been isolated fromthe sea urchin basal lamina and is analogous to thevertebrate basal lamina protein laminin (McCarthy etal. 1987), a major component of vertebrate basementmembranes. In order to investigate the expressionand distribution of sea urchin laminin in the earlydevelopment of the sea urchin embryo, we directlycoupled FITC to Mab BL1 (FITC-Mab BL1) andmicroinjected the fluorescently labelled antibody intothe blastocoel of living embryos at various stages.

Binding of FITC-Mab BL1 is first detected asspotty fluorescence on the basal surfaces of the cells,indicating secretion and cell binding of laminin(Fig. 2B, embryo upper left). As cells begin toelongate at approximately 12 h after fertilization(Fig. 2C, white arrows, uninjected control andFig. IB), fluorescence is associated with the basal cellsurfaces in the areas where blastula cells appearelongated (Fig. 2C,D, arrow, embryo upper right).Injection of a control FITC-Mab results in diffusefluorescence (Fig. 2B,D, embryos lower left). FITC-Mab 6A4 is directed against a hyaline layer proteinand will bind to the hyaline layer if administered tothe external surface of the embryo (not shown). Thisantibody was used routinely to determine specificbinding of FITC-Mab BL1.

At 17h after fertilization, most of the cells of theembryo are wine-glass shaped (Fig. 2E). FITC-MabBL1 binds extensively to the basal cell surfaces,demonstrating the presence of laminin (Fig. 2F) and,as is the case in the mesenchyme blastula stageembryo (Fig. 2G,H), fluorescence is coincident withthe position of the basal lamina. Particularly interest-ing is the result of injection into the midgastrula-stage

embryo. Although present on most basal cell sur-faces, the fluorescence is heterogeneous with intenselabelling noted at the animal pole in a positioncorresponding to the animal plate (Fig. 2J, arrow).At the locations of increased fluorescence, the ecto-dermal cells are elongated (Fig. 21, arrow). Addition-ally, fluorescence is limited to the basal cell surface ofectodermal cells and is absent from primary mesen-chyme cells and the invaginating archenteron. At thegastrula stage, fluorescence is distributed over mostof the basal surfaces of ectodermal cells (Fig. 2L) andis additionally present on the archenteron over thebasal surface of the endodermal cells (arrow). Bind-ing is also absent or reduced from primary mesen-chyme cells.

Microinjection of FITC-Mab BL1 results in cell shapechange in vivoThe development of individual embryos that hadbeen injected with FITC-Mab BL1 was observed inorder to investigate possible functions of lamininduring early sea urchin morphogenesis. Laminin isfirst detected over most of the basal cell surfaces at16 h after fertilization (see Fig. 2F), The cells of theembryo are wine-glass shaped and the blastocoel isdelimited by the basal cell surfaces (Fig. 3A). Withinlmin after injection, the basal cell surfaces of theseembryos become more distinct (Fig. 3C, arrows). Thesame embryo viewed with fluorescence optics after3 min demonstrates fluorescent binding of the FITC--Mab BL1 antibody to the basal cell surfaces (com-pare Fig. 3C and D). At 7min after injection, thecells begin to elongate and the apparent blastocoelvolume is reduced by 60-75 % (compare Fig. 3E and

Fig. 2. Laminin distribution in live embryos. FITC-MabBL1 was microinjected into the blastocoel of embryosand the embryos recorded in Nomarski DIC(A,C,E,G,I,K) and fluorescence (B,D,F,H,J,L)microscopy. The embryo injected 11-5 h after fertilization(A,B) with FITC-Mab BL1 shows spotty fluorescence onbasal cell surfaces (upper left) while control injectedembryo shows only diffuse fluorescence (lower left).Embryos at 12-5 h after fertilization (C,D) begin to showthickening of the blastula cells (C, white arrows,uninjected embryo). In the FITC-Mab BL1 injectedembryo, intense fluorescence is associated with those cellsthat are changing shape (CD embryo upper right,arrows). The control injected embryo shows diffusefluorescence in the blastocoel (C,D embryo lower left).Embryos injected with FTTC-Mab BL1 after 17h (E,F),24h (G,H), 36h (I,J) and 48 h (K,L) of development at16°C show fluorescence associated with the basal cellsurfaces coincident with the position of the basal lamina.Note intense fluorescence associated with the animalplate (I,J arrows) and the basal surface of thearchenteron cells (L, arrow). Scale bars in B for A-Dand in H for E-L, 50 >m.

Laminin and sea urchin morphogenesis 663

A). The cells remain elongated and by 30min theembryo has taken on the appearance of an earlierembryonic stage (compare Figs IB and 3G). Thesmall volume of the blastocoel and extensive cellelongation is similar to the 12 h control embryos at atime when laminin is first detected on the basal cell

surface. The degree of cell elongation varies, how-ever; in some areas cells are twice the length recordedbefore injection (compare Fig. 3A with F and G).After 1 h the cells of the injected embryo begin toround up and adhere to the hyaline layer (Fig. 3H,arrow).


Fig. 3. Effect of FITC-Mab BL1 injection at 16h after fertilization. Embryos viewed by Nomarski DIC(A-C,E-H,J-L) and fluorescence (D,I) microscopy. The same embryo is recorded before injection (A), at injection(B), and 1 min (C), 3min (D), 7min (E),.14 min (F), 30min (G), lh (H) after injection of FITC-Mab BL1. Notethickening of the basal cell surfaces after injection (C, arrows). The cells rapidly lengthen (E-G) and individual cellsround up (H, arrow). Embryos were injected either with FITC-Mab BL1 (I-L, upper embryo) or Mab 6A4 (I-J, lowerembryo) and recorded after 3 min (I), 5 min (J), 30 min (K) and 2h (L) after injection. Note that cells lengthen (J,K)and that after 2h some cells round up against the hyaline layer (L. arrows). Scale bar, 50f.im.

The rounding up of cells after antibody injection isshown more clearly in Fig. 3I—L. Bright fluorescenceis associated with the basal surfaces of cells afterFITC-Mab BL1 is injected (Fig. 31, upper embryo);however, only diffuse fluorescence is observed in thecase of control FITC-Mab 6A4 (Fig. 31, lower em-bryo). Nomarski DIC micrographs of an injectedembryo show little effect of the injection of FITC-Mab 6A4 over a period of 2h (Fig. 3J-L). In the case

of Mab BL1, the blastocoel volume is reduced within15 min and the cells elongate (Fig. 3J,K). After 2h,some cells lose their wine-glass shape. These cellsround up and remain closely associated with thehyaline layer (Fig. 3L, arrows). The cells return to anormal shape over a period of hours and the embryodevelops normally.

Microinjection of FITC-Mab BL1 at 15-23 h afterfertilization results in the elongation of cells in the


Table 1. FITC-Mab BL1 injections of embryos*

Developmental stage(h. 16°C)

Cleavage (7-10)Blastula (11-12)Mesenchvme blastula(15-23) '

Early gastrula (S24)

* Embryos were injectedt Embryos were injected

No. ofembryos

722

28177

FITC-Mab

(Pg)

300 ±55300 ±55

300 ±55600 ± 114t300 ±55

at all stages with FITC-Mab 6A4 at equalwith two sequential injections of 300 pg.

BLbinding

012

28177

concentrations. No

Effects (no. of embryos)

Cellelongation

00

22150

effects were observed.

Epitheliumdisruption

00

050

blastula epithelium and subsequent rounding up ofcells (Table 1). At this time, most cells are part of theblastula epithelium and it is not possible to determineany polarity of the cell elongation event with respectto the animal-vegetal axis of the embryo. FITC-MabBL1 was therefore microinjected into mesenchymeblastula embryos where the primary mesenchymecells leave the vegetal epithelium and designate thevegetal pole of the embryo (Fig. 4A, arrows). Immu-nofluorescence of FITC-Mab BL1 binding demon-strates that laminin is confined to the basal surfaces ofthe ectodermal cells and absent from inwardly migrat-ing primary mesenchyme cells (Fig. 4B; Table 1).After 1 h (Fig. 4C) and 3 h (Fig. 4D), the cells of theectoderm elongate. However, there is little effect onprimary mesenchyme cells, which continue to mi-grate. Comparison of Fig. 4D and E demonstratesthat the fluorescent Mab is still associated with thebasal surfaces of the ectodermal cells and that theelongation is restricted to these cells. A hetero-geneous distribution of laminin in the early embryo issupported by the result of microinjection of FITC-Mab BL1 into the midgastrula stage where it is alsoabsent from primary mesenchyme cells and thearchenteron (see Fig. 21,J).

Normally, at the mesenchyme blastula stage, thecells of the blastula wall are of an equivalent lengthwithin the ectodermal epithelium (Fig. 4A,F; see alsoFig. 1E,F). After injection of FITC-Mab BL1, thecells at the animal pole elongate to a greater extentthan the cells lateral to the animal-vegetal axis(Fig. 4G). These injected embryos, with elongatedanimal pole cells, resemble older control embryosthat are in the process of forming the animal plate(compare Fig. 1H to 4G,D). Comparison of Fig. 41and J demonstrates that the fluorescently labelledantibody is distributed over the basal surfaces of cellsremaining in the epithelium. Microinjection of acontrol FITC-Mab 6A4 (Fig. 4K-O) has little effecton either the shape of ectodermal epithelial cells or

the normal progression of primary mesenchyme cellsduring the injection period (arrows).

FITC-Mab BL1 initiates epithelial elongation atspecific stages when injected at amounts of approxi-mately 300 pg (in 300 pi) of antibody (Table 1). Injec-tions of larger amounts of Mab BL1 can causeadditional deformation of the ectodermal epithelium(Table 1). Fig. 5 demonstrates the effect of micro-injection of approximately 600 pg FITC-Mab BL1into a mesenchyme blastula stage embryo. The bind-ing of the FITC-Mab BL1 antibody to the basal cellsurfaces is apparent after 4min (Fig. 5A) and, atlOmin, there is a slight elongation of cells (Fig. 5B).After 2h, however, major changes occur in theblastula epithelium (Fig. 5C). Cells begin to round up(Fig. 5C) and there is a general deformation of theepithelium. Cells initially elongated have flattenedand other cells have aggregated into clumps (compareFig. 5B,C), presumably over the surface of the basallamina since the overall internal shape of the blasto-coel is maintained. The embryo is able to regulate thiseffect and will go on to form a normal gastrula stageembryo (Fig. 5D). During the process of embryonicreorganization, an epithelium is reformed consistingof elongate cells. Fig. 5E-H demonstrates an ad-ditional embryo in which the binding of the antibodyto the basal cell surface (Fig. 5E) results in defor-mation of the epithelium and loss of embryonic shape(Fig. 5F,G). After 3h, however, some cells begin toreform an epithelium and are once again arranged aselongated cells (Fig. 5H, arrow).

Table 1 is a compilation of many injections ofFITC-Mab BL1 into embryos of various stages.Basal cell surface binding becomes evident in em-bryos injected at 11-12 h after fertilization. Since atthis stage the blastula cells undergo shape changesand the blastocoel is quite small, no effect on cellelongation could be attributed to the antibody. Cellelongation in response to Mab BL1 injection is firstnoted at approximately 15 h after fertilization and canbe detected until the mesenchyme blastula stage. In

I a a. a; is

Fig.

4.

Ger

m-l

ayer

-spe

cifi

c bi

ndin

g an

d an

imal

-veg

etal

pol

arity

of

cell

elon

gatio

n du

e to

FFT

C-M

ab B

L1

inje

ctio

n. E

mbr

yos

wer

e ra

ised

at 1

6°C

for

21 h

and

inj

ecte

d w

ith e

ither

FIT

C-M

ab B

Ll

(A-J

) or

Mab

6A

4 (K

-O).

The

inj

ecte

d em

bryo

s w

ere

reco

rded

by

Nom

arsk

iD

IC (

A.C

-D.F

-I.K

-N)

or f

luor

esce

nce

(B,E

,J,O

) m

icro

scop

y at

1 m

in (

A,F

,K),

lO

min

(B

,G,L

), 1

h (

C,H

,M)

and

3h (

D,E

,I,J

,N,O

)af

ter

inje

ctio

n. F

ITC

-Mab

BL

l bi

nds

to t

he b

asal

sur

face

s of

cel

ls w

ithin

the

epi

thel

ium

(B

) bu

t is

red

uced

or

abse

nt f

rom

pri

mar

ym

esen

chym

e ce

lls (

A,

arro

ws)

. D

ue t

o a

slig

ht r

otat

ion

of t

he e

mbr

yo,

the

anim

al-v

eget

al a

xis

is m

arke

d by

lin

es o

n th

e m

icro

grap

hs(F

-I).

The

ani

mal

pol

e is

in a

ll ca

ses

indi

cate

d by

the

upp

er l

ine.

Not

e th

at t

he a

nim

al p

ole

cells

elo

ngat

e to

a g

reat

er e

xten

t th

an t

hece

lls l

ater

al t

o th

e an

imal

-veg

etal

axi

s (F

.G).

In

the

cont

rol

inje

cted

em

bryo

(K

-O),

arr

ows

deno

te t

he i

ngre

ssin

g pr

imar

y m

esen

chym

ece

lls.

Scal

e ba

r, 5

0j.im

.


•

5A

\

\K

X

ft

C

•

•

\ •

Fig. 5. Deformation of the embryonic epithelium by FITC-Mab BLJ. Embryos (21 h after fertilization) were injectedwith 600 pg FITC-Mab BLl and recorded in fluorescence (A.E) or Nomarski DIC (B-D,F-H) at 4min (A), lOmin(B),2h (C),21h (D), 5min (E), 8min (F), 1-5 h (G) and 3-5 h (H) after injection. Note that injection of FITC-MabBLl results in deformation of the embryos (B,C and F,G). Arrow (H) indicates where cells are reorganized into ablastula-stage-like epithelium (see Fig. IB). These animals recover to form normal-looking gastrula-stage embryos (D).Scale bar, 50/«n.

early gastrula stage embryos, FITC-Mab BLl bind-ing could be detected; however, no effects of thebinding on cell shape were noted. Embryos from allembryonic stages were injected with an equivalentamount and volume of FITC-Mab 6A4 IgG. Noeffects could be attributed to these injections.

Discussion

Expression and function of laminin in vivoIn this study, we have microinjected a fluorescentlylabelled monoclonal antibody, which recognizes seaurchin laminin, into living sea urchin embryos and

demonstrate a correlation between the time of lam-inin expression on the basal surfaces of embryonicblastomeres and a change in the shape of the blasto-meres. Laminin is expressed on the basal cell surfacejust prior to the organization of the blastomeres intoan epithelium. During the formation of an epitheliumthe blastomeres elongate and a very thin basal laminais observed by electron microscopy (Gibbins, Tilney& Porter, 1969). The appearance of laminin at earlystages in mouse embryoic development (Leivo et al.1980; Wu etal. 1983; Dziadek & Timpl, 1985) and thefact that it is a major component of adult basementmembranes (Timpl etal. 1979) has led to the idea thatlaminin may be involved in the organization of thecytoskeleton and establishment of epithelia. Our


experiments support the idea that the secretion andappearance of laminin on the basal cell surface is animportant step in the organization of blastomeres intoan epithelium during early development.

In the early sea urchin blastula, the cells are not yetorganized into a differentiated cell epithelium. Thebasal surfaces are attached to a newly formed basallamina. However, the cells do not yet have a colum-nar shape typical of an epithelium but instead have awine-glass shape (Gibbins et al. 1969) as if undertension. In this regard, the cells may be considered tobe in the very early stages of epithelium formation.Since it is at this time that laminin is detected on mostbasal cell surfaces, it is interesting to speculate thatthe organization and cell binding of the basal laminacomponents, including laminin, may be sufficient tocause temporally regulated changes in cell shape earlyin development which would not occur after a morehighly structured basement membrane is present.

Microinjection of FITC-Mab BL1 results in aninitial elongation, a rounding up of cells and loss ofepithelial morphology. In the mesenchyme blastula,the elongation is asymmetric with longer cells associ-ated with the animal pole ectoderm. These changes incells and in the overall form of the embryo mimicthose that occur in the normal development of theblastula and of an animal plate ectoderm.

The mechanism by which Mab BL1 accomplishesits effect is open to interpretation. If we assume thatthe effect of the antibody is due to its interaction withlaminin, several interesting possibilities exist whichpertain to basal lamina structure. As laminin ispresent over most of the basal surfaces of the blastulacells, microinjection of the bivalent antibody maycrosslink the laminin, reducing the intermoleculardistances of laminin molecules within the basal lam-ina. Cell elongation could then occur either directly ifthe cells are attached to laminin or indirectly since thecells are attached to the basal lamina. Cell roundingand epithelium disruption may occur if the cellsrelease from the basal lamina. Alternatively, theantibody may affect the ability of laminin to provide astructural support for the newly forming basal laminaby interfering with the interaction of laminin withcells or with other extracellular matrix components. Itis known that laminin interacts with other structuralcomponents of basement membranes (Martin &Timpl, 1987). Elimination of structural constraints inthe basal lamina imposed by laminin may have theresult of disrupting the basal lamina and affecting themolecular interactions of other components.

Traditionally, cell shape change has been thoughtto involve extracellular matrix interactions with thecytoskeleton (Gospodarowicz et al. 1978; Hay, 1983).It is possible that the antibody binding affects the

cytoskeleton by the induction of microtubule polym-erization (Porter, 1966; Burnside, 1971) or inter-ference with microfilaments or cell junctions. It isinteresting, in this regard, that concentrations ofcolchicine that disrupt microtubules have little effecton the shape of the blastula ectoderm and onlytreatments that disrupt cell-cell contacts result inepithelium disruption (Tilney & Gibbins, 1969).

We have recently been able to isolate smallamounts of laminin from embryonic sea urchins andassess its structure at the electron microscopic level(McCarthy etal. 1987). We are initiating experimentsin order to identify the binding site of the Mab BL1on the laminin molecule and to study the effect ofMab binding on the interaction of laminin with otherextracellular matrix components in vitro. Such studiesshould provide relevant information with which toevaluate the effects of the antibody in vivo.

Laminin and primary mesenchyme cellsBinding of FITC-Mab BL1 is limited to the ecto-dermal cells being absent or reduced on primarymesenchyme cells. This is also observed in indirectimmunofluorescence using Mab BL1 on paraffinsections of embryos (McCarthy etal. 1987). Immuno-logical assays are limited in their ability to determinethe presence of an antigen and a lack of signal mayrepresent a masking of the Mab BL1 antigenic site oflaminin by another extracellular molecule. In thisregard, a mesenchyme-specific molecule has beendescribed which appears on the cell surface of mesen-chyme cells upon the entry of these cells into theblastocoel (Wessel & McClay, 1985). The presentresults are, however, consistent with studies on thedistribution of laminin in vertebrate embryos andadult tissues which show that laminin is most oftenassociated with epithelial cells and absent from mes-enchymal cells (Wartiovaara, Leivo & Vaheri, 1980;Foidartef al. 1980).

Electron microscopic evidence demonstrates thatpresumptive primary mesenchyme cells initiallyelongate and bind to the blastula basal lamina (Gib-bins etal. 1969). After the primary mesenchyme cellshave migrated into the blastocoel, their cell surfacechanges. The cells lose their ability to bind thehyaline layer protein, hyalin, and gain an affinity forfibronectin (Fink & McClay. 1985; Venkatasubram-anian & Solursh, 1984) and for the lectin, wheat germagglutinin (DeSimone & Spiegel, 1986). Katow et al.(1982) have also demonstrated that primary mesen-chyme cells bind a polyclonal antibody directedagainst vertebrate fibronectin and increase their mi-gration speed in vitro on surfaces coated with ver-tebrate fibronectin and dermatan sulphate but not onlaminin or collagen IV (Katow, 1986). In vivo, pri-mary mesenchyme cells change in their ability to bind


the anti-laminin Mab BL1 upon entry into the blasto-coel. The above results suggest a possible mechanismfor primary mesenchyme ingression and migration.The cessation of laminin synthesis or masking oflaminin by other extracellular molecules may inter-fere with the cells' ability to bind to the basal lamina.A decreased affinity for hyalin and an increasedaffinity for fibronectin may allow the cells to leave theepithelium and to migrate.

It is not known whether primary mesenchyme cellsactively degrade the basal lamina components uponentry into the blastocoel or whether a developmen-tally regulated switch in the synthesis or binding ofextracellular matrix components, such as laminin,would result in local disruptions of the basal lamina.We are currently investigating the developmentalexpression of laminin by primary mesenchyme cells.Whatever the mechanism of primary mesenchymeingression, our results demonstrate that primary mes-enchyme cells change in their ability to bind the anti-laminin Mab BL1 during the epithelial-mesenchymaltransformation.

Heterogeneity of the basal lamina during earlydevelopment

FITC-Mab BL1 binding suggests that laminin ispresent over the basal surfaces of cells upon secretionat the blastula stage. Differences in FITC-Mab BL1binding within the basal lamina are noted at themidgastrula stage where more intense fluorescence isapparent on the basal surfaces of ectodermal cellswhich comprise the animal plate in both living em-bryos and tissue sections (McCarthy, unpublisheddata). Heterogeneity of sea urchin cell surface com-ponents has been previously noted using fluor-escently labelled lectins as probes (Spiegel & Burger,1982; Katow & Solursh, 1982; DeSimone & Spiegel,1986). In the midgastrula embryo, specific bindingof Concanavalin A is detected at the region ofthe animal plate, the site of attachment of thesecondary mesenchyme cells (Spiegel & Burger,1982). Microinjection of Concanavalin A, proteasesor collagenase break adhesions of attached secondarymesenchyme cells and interfere with gastrulation(Spiegel & Burger, 1982). The molecular basis for thiseffect is not known; however, collagenase treatmentstrongly implies that collagen is involved. Theseresults suggest that not only the individual com-ponents but also their distribution within the basallamina may be important during morphogenesis.

The apparent heterogeneity of the basal lamina forMab BL1 binding may be explained by either sequen-tial assembly of extracellular matrix components, asin the mouse embryo (see Wartiovaara etal. 1980, forreview), or differential expression of laminin byepithelial cells. Support for sequential assembly of

extracellular matrix components in the sea urchinembryo has been obtained using indirect immunoflu-orescence (Wessel, Marchase & McClay, 1984). Inthe above study, however, cross-reactive antigenswere not identified as bonafide sea urchin extracellu-lar matrix proteins. Nevertheless, the differences inlaminin distribution at the midgastrula stage mayrepresent the addition of extracellular matrix com-ponents which interfere with the FITC-Mab BL1binding in specific regions of the basal lamina.Alternatively, the observed fluorescent binding pat-tern may reflect differences in synthesis or binding oflaminin by subsets of epithelial cells. The presentresults do demonstrate, however, that heterogen-eities are present in the distribution of sea urchinbasal lamina components on epithelial and primarymesenchyme cells and are correlated with differencesin cell shape.

Clearly, many components are involved in thecomplex interactions of cells with the extracellularmatrix during early embryogenesis. The present datademonstrate a method by which extracellular matrixcomponents may be characterized and their functionassayed in vivo. This represents a new approach thatoffers major advantages over traditional localizationtechniques involving tissue sections. First, the micro-injection experiments permit spatial and temporallocalization of the laminin molecule in the livingembryo and allow one to correlate the expression oflaminin with important developmental events.Second, because the antibody interferes with normaldevelopment, but only when injected into specificstages, it suggests a specific role for laminin inembryonic epithelium formation and cell shapechange.

We thank Katrina Saladin for excellent technical assist-ance and Drs D. Smith, K. Beck, D. DeSimone, J. Engel, J.Fessler and L. Fessler for stimulating discussions during thecourse of this work. We also thank Dr H. J. Marthy for hiskindness and support in providing us with animals fromLaboratoire Arago. The work was supported by SwissNational Science Foundation Grant No. 3.269-0.82. and3.169-0.85.

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(Accepted 7 September 1987)

in vivo embryonic expression of laminin and its involvement in cell ... · sphaerechinus granularis...

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