ARTICLESRhoA and microtubule dynamics control cell–basement
membrane interaction in EMT during gastrulation
Yukiko Nakaya1, Erike W. Sukowati1, Yuping Wu1 and Guojun Sheng1,2
Molecular and cellular mechanisms of epithelial–mesenchymal transition (EMT), crucial in development and pathogenesis,
are still poorly understood. Here we provide evidence that distinct cellular steps of EMT occur sequentially during gastrulation.
Basement membrane (BM) breakdown is the first recognizable step and is controlled by loss of basally localized RhoA activity
and its activator neuroepithelial-transforming-protein-1 (Net1). Failure of RhoA downregulation during EMT leads to BM retention
and reduction of its activity in normal epithelium leads to BM breakdown. We also show that this is in part mediated by RhoAregulated basal microtubule stability. Microtubule disruption causes BM breakdown and its stabilization results in BM retention.
We propose that loss of Net1 before EMT reduces basal RhoA activity and destabilizes basal microtubules, causing disruption ofepithelial cell–BM interaction and subsequently, breakdown of the BM.
EMT is a complex process that requires the coordination of multiple cellular events, including disruption of epithelial cell–cell junctions, lossof apicobasal polarity, breakdown of cell–BM interaction and changes
in cytoskeletal architecture1. Molecular studies have revealed that it isregulated by several major pathways2–4. Extensive crosstalk between thesepathways has been documented both in vitro and in vivo2,4,5. However, it
is unclear whether EMT is regulated as a single process or at the level of
its individual cellular events. Cadherins, which can mediate differential
adhesion, are commonly viewed as a central target6–8. The picture emerging from diverse EMT-related studies suggests that precise molecular andcellular control of EMT is complex and context-dependent9. Rho GTPases
belong to a superfamily of small GTPases, which consists of 22 members
in mammals10. Among its five major subfamilies, three (Rho-like, Cdc42like and Rac-like) have been reported to function in EMT-related cellular events, such as cytoskeletal remodelling, microtubule dynamics, themaintenance of tight junctions and cadherin-mediated interactions10–13.
The earliest EMT in development occurs during gastrulation, a process that generates mesoderm and endoderm from the ectoderm14,15. In
amniotes, the majority of cells undergoing gastrulation EMT contributeto mesoderm15,16. Coordinated cell ingression during gastrulation is crucial for separating mesoderm-fated cells from adjacent non-ingressing
neuroectoderm cells17, and for generating anterior/posterior regionalization by controlling the timing of mesoderm formation18. Pan-mesoderm
markers start to be expressed while precursor cells are still located in theepiblast, which consists of a single-cell-thick epithelial sheet and contains all ectoderm-fated cells, epithelial-shaped mesoderm precursor
cells and pre-ingression mesoderm cells that may have lost some of theirepithelial characteristics. Many mouse mutations causing gastrulation
1
2

defects do not affect expression of pan-mesoderm markers, suggestingthat mesoderm-fate specification and EMT are regulated independently.
Moreover, it has been postulated that the individual cellular events of
EMT during gastrulation may be under separate molecular controls19. A
similar concept, termed EMT phenotypic modules4 or intracellular crosstalk modules20, has been proposed for the EMT process in general. Due
to limitations of in vivo studies, such a concept has only recently started
to be tested in amniotes.
In this study we have investigated the order and regulation of EMT
cellular events during avian gastrulation. In particular, we show that
controlled BM breakdown is a crucial component of, and the first recognizable step in, gastrulation EMT. In pre-EMT epiblast cells, the BM
is maintained by basally localized RhoA activity, mediated by RhoGEF
Net1. Loss of basal RhoA activity during EMT leads to disruption of cell–
BM interaction and consequently to BM breakdown. We also provide
evidence that basal microtubule stability has a crucial function in thisprocess. Our data demonstrate an important role of BM regulation in an
essential and evolutionarily conserved developmental process.
RESULTS
Mis-expression of RhoA causes EMT defects
RhoA-expressing cells fail to contribute properly to mesoderm
lineages21,22. We investigated the timing of the defect with in vivo timelapse imaging. Using gastrulation-stage chick embryos, we electroporated control green fluorescent protein (GFP) or wild-type RhoA+GFP
at mid-streak level into mesoderm precursors located in the epiblast(Fig. 1a). In controls, 6 h after electroporation, most GFP-positive
cells completed their ingression, giving rise to individually migrating

Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.
Correspondence should be addressed to G.S. e-mail: (sheng@cdb.riken.jp)

Received 8 January 2008; accepted 23 April 2008; published online 15 June 2008; DOI: 10.1038/ncb1739

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eRhoA + DAPI

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Endoderm

ApicalApicalf

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Basal

Basal

ApicalApicalEctoderm cells
Basal

Basal

Figure 1 RhoA mis-expression causes EMT defects. (a) Schematic diagramof electroporated area in b and c. (b) GFP-expressing cells show normal
ingression and migration. (c) RhoA+GFP-expressing cells show ingression andmigration defects. (d) RhoA protein expression at HH4 embryo, sectioned

through mid-streak level. Germ layers indicated on the right. Regions ofmagnified views in e and f indicated with white rectangles. (e) Lateralepiblast cells, showing both apical and basal RhoA. (f) Medial epiblast cells,lacking basal RhoA. Scale bars are 50 µm (b, c) and 30 µm (d).

mesoderm cells (Fig. 1b; Supplementary Information, Fig. S1, Movie 1).
In contrast, many RhoA-expressing cells failed to complete EMT andaccumulated in streak midline (Fig. 1c); those able to ingress formed
aggregates (Fig. 1c; Supplementary Information, Fig. S1, Movie2). Thissuggests that RhoA has a role in the cellular events leading to mesoderm-cell ingression and early migration. Immunofluorescent staining
showed that RhoA is expressed strongly in the epiblast, but is undetectable in the mesoderm (Fig. 1d). Within individual epiblast cells, we saw
localized expression along the apicobasal axis. In medial cells, RhoA
was only detected in the apical part (Fig. 1f), whereas both apical andbasal expression was observed in cells lateral to the streak (Fig. 1e). This
led us to investigate the significance of basal RhoA downregulation in
gastrulation EMT.

RhoA inhibits laminin breakdown in medial streak
We next analysed the effect of RhoA on these markers. RhoA mis-expression caused a marked retention of laminin in medial epiblast (Fig. 3c)and nascent mesoderm cells (Fig. 3b; Supplementary Information,
Fig. S3a). Control GFP had no effect on laminin (Fig. 3a). No obvious
change in aPKC, ZO-1 or adherens junction markers was observed with
RhoA expression (Supplementary Information, Fig. S2a–c and data notshown). The effect on laminin is cell-autonomous, as retention was seen
to be associated with individual newly ingressed mesoderm cells expressing RhoA (Fig. 3e). Lateral epiblast cells with exogenous RhoA did not
upregulate normal laminin expression (Fig. 3d), and in situ analysis withlaminin γ1, part of the laminin1-trimer, showed no detectable transcripts
in mesoderm aggregates produced by RhoA (data not shown). This suggests that the effect of RhoA on laminin is due mainly to the failure of BM
breakdown. Furthermore, analysis of integrin expression revealed thatα6β1 is the major integrin isoform mediating the epiblast cell–BM interaction (Fig. 3f, g; Supplementary Information, Fig. S3b, d). Both chainsare downregulated in medial epiblast cells during normal gastrulation(Fig. 3f, g; Supplementary Information, Fig. S3b, d) and mis-expressionof RhoA caused retention of both in nascent mesoderm aggregates(Fig. 3h, i; Supplementary Information, Fig. S3c, e). This suggests thatBM retention caused by RhoA is an indirect consequence of failure to
disrupt integrin-mediated epiblast cell–BM interaction.
As the presence of RhoA protein may not reflect the distribution ofactive RhoA, we used eGFP–rGBD as a sensor for the active form of
RhoA23 to detect the subcellular distribution of its activity. eGFP–rGBD
was electroporated into the lateral epiblast and embryos were cultured
until the electroporated cells began to ingress (Fig. 3j; Supplementary
Information, Fig. S4). In ingressed mesoderm cells, eGFP–rGBD was seen
throughout the cytoplasm (Fig. 3j1), indicating a lack of subcellular localization and consistent with the absence of RhoA protein in these cells. In

BM breakdown is the first recognizable step of EMT
The temporal sequence of cellular events during gastrulation EMT
has not been carefully investigated in amniotes. We first characterized
expression patterns of several EMT-related markers: aPKC (apical),ZO-1 (tight junction), E-cadherin and N-cadherin (adherens junction)and fibronectin and laminin (BM). Both aPKC and ZO-1 (Fig. 2a, b)
were expressed in all epiblast cells and were undetectable in mesoderm
cells. E-cadherin (Fig. 2c) was expressed in epiblast and nascent mesoderm cells, with a gradual shift to N-cadherin in more lateral mesoderm
cells (Fig. 2d). Fibronectin and laminin (Fig. 2e, f) were detected beneaththe epiblast cells lateral to the streak. Their degradation was seen at a
distance of 5–10 cells away from streak midline (Figs 2e, f). These data
indicate that BM breakdown is the earliest cellular event leading to EMT
during chick gastrulation. Tight junctions and apicobasal polarity areboth maintained throughout epiblast cells and are lost immediately after
ingression. In contrast to this sharp transition, change of adherens junction markers was gradual and did not correlate with ingression.
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A RT I C L E SaaPKCaPKC + DIC

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E-cadherin + DIC

N-cadherin

E-cadherin + N-cadherin

Fibronectin

Fibronectin + DIC

LamininLaminin + DIC

b

c

d

e

f

Figure 2 Expression of EMT-related markers. All panels show mid-streaklevel section of stage HH4 embryo. (a) aPKC; (b) ZO-1; (c) E-cadherin;(d) N-cadherin; (e) fibronectin; (f) laminin. Right panel in d shows
merged E-cadherin and N-cadherin. Scale bars are 30 µm.

the epiblast, we detected apical localization in all cells expressing eGFP–
rGBD (Fig.3J2–4), whereas basal eGFP–rGBD was observed only in morelateral cells (Fig. 3j4, compared with 3j2); this was most obvious in thetransition region (Fig. 3j3). Laminin co-staining revealed that the reduction in basal activity coincides with BM breakdown (Fig. 3j, green).

(Supplementary Information, Fig. S5a); at gastrulation stages, expression
became restricted to the future neuroepithelium (Fig. 4b, c; Supplementary
Information, Fig. S5b). No expression was seen in the mesoderm at thesestages (Fig. 4b1, c1,2). In medial epiblast cells, Net1 transcripts were downregulated or absent (Fig. 4b1, c2). In lateral epiblast cells, its transcripts wereseen to be restricted basally (Fig. 4b1, c1,2). To assess its protein localization,
we generated a chick Net1 antibody and western blot analysis showedthat it recognizes a specific band of expected size in cells transfected witha cNet1-expressing construct (Fig. 4d). Immunofluorescent staining
revealed a basally restricted localizaton of Net1 protein (Fig. 4e), similar
to that of integrin α6 (Fig. 4f), suggesting that Net1 is involved in regulating RhoA activity in the basal cortex or membrane of the epiblast cells. To
test this, we generated an expression construct using mouse Net1 (ref. 28)
and analysed its effect on laminin expression. Similarly to RhoA, Net1 misexpression resulted in laminin retention in medial epiblast cells (Fig. 4g)
and in mesoderm aggregation (data not shown).

Net1, a RhoA GEF, is basally restricted in lateral epiblast anddownregulated before EMT
RhoA cycles between the inactive GDP-bound and active GTP-boundstates10. A family of GEFs catalyse the transition from the inactive toactive state. We therefore investigated the role of RhoGEFs during gastrulation EMT. In a screen for genes differentially expressed in medial
versus lateral epiblast (data not shown), we found that Net1, a RhoGEF,
was specifically downregulated in medial epiblast cells. Net1 was first
identified as an oncogene in human neuroepithelioma24 and was shownto have RhoA-specific GEF activity25–27.
The overall structure of chick Net1 is similar to that of other vertebrate
Net1s (Fig. 4a). In situ analysis revealed a specific and dynamic patternof Net1 transcripts (Fig. 4b, c; Supplementary Information, Fig. S5a–d).
At pre-streak stages, Net1 expression was weak in the area pellucida

Reducing RhoA activity causes premature BM breakdown
Prominent localization of RhoA in the apical cortex suggests that it
has other roles in addition to BM maintenance. RhoA mis-expression,

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c HA–RhoA

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RhoA

LamininLamininLaminin

GFP + Laminin

RhoA +
Laminin

RhoA + Laminind

RhoA + Laminin + DIC

RhoA + Laminin

RhoA + Laminin + DAPIeRhoA + Laminin + DIC

RhoA + Laminin

RhoA + Laminin + DAPI

f

Integrin α6hIntegrin α6iIntegrin β1

Normal embryog

Integrin β1

RhoA +
Integrin β1

RhoA +
Integrin α6

Normal embryoj

eGFP–rGBD
2

rGBD

rGBD + Laminin + DAPI

Ectoderm

j3

j2

3

2

3 4
1
Mesoderm

j1

Laminin

4

1
Ectoderm

Ectoderm
Apicalj4

ApicalApical

Basal

Basal

Basal

Figure 3 Effect of RhoA on laminin and distribution of active RhoA. (a–c)
Electroporated regions are shown in the top panels (dotted rectangle).
(a) GFP does not affect normal laminin expression. (b) RhoA causes
cell aggregation with ectopic laminin (white arrows). (c) RhoA prevents
laminin degradation in medial epiblast (white arrows). (d) Individual
RhoA-expressing cells in the lateral epiblast have normal laminin levels.
(e) Individual RhoA-expressing cells in medial streak region are seen
associated with ectopic laminin (arrows; dotted rectangles in d and e
are the regions magnified in the right panels. (f, g) Protein expression of
integrin α6 (f) and β1 (g). (h, i) Ectopic RhoA induces ectopic α6(h, arrows) and β1 (i, arrows) expression. (j) Localization of active RhoA
shown with eGFP–rGBD. Magnified views of indicated areas shown in j 1–4.
In mesoderm cells (j1), eGFP–rGBD (red) is distributed throughout thecytoplasm. In lateral epiblast cells (j2), eGFP–rGBD is seen in both apicaland basal sides. Basal eGFP–rGBD colocalizes with laminin (green), seen
as merged yellow colour. In epiblast cells undergoing BM degradation(j3), loss of basal eGFP–rGBD coincides with loss of laminin. In medial
epiblast cells (j4), no basal eGFP–rGBD or laminin was detected. Apical
localization of eGFP–rGBD is still prominent. Scale bars are 20 µm (d, e)
and 30 µm (a–c, f–j). Arrowheads indicate streak midline.

however, had a marked effect only on BM markers. To confirm therole of basal RhoA in BM maintenance, we investigated the effect of
reducing RhoA activity on gastrulation and laminin expression. We

first used C3 exoenzyme, a potent Rho pathway inhibitor29. As describedearlier, RhoA caused nascent mesoderm cells to form aggregates becauseof the failure in BM breakdown (Fig. 5a1, arrows). This was rescued by768

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A RT I C L E Sa

NLS1 NLS2DHPH

542 aa (80%)

100% 78%92%85%

541 aa (80%)

100% 78%

92%

90%

Chick Net1

540 aaHuman Net1
Mouse Net1

b

c

b1

c1
b1

c1

c2
c2

Net1 mRNA

HH4

d

HH5

Net1 mRNA

DF1 cells
Mr(K) cNet1 eGFP
75

HEK 293 cells
Mr(K) cNet1 eGFP
75
WB: cNet1

WB: cNet1
50

50

e

Net1 protein

Net1 protein + DIC

f

Net1 protein + Integrin α6g

Myc–Net1

Integrin α6

Net1 + Integrin α6

Net1

DAP I + Net1 + Integrin α6

Myc–Net1 + Laminin

LamininLamininMyc–Net1 + Laminin

Primitive streak

Figure 4 Net1 expression pattern and its effect on laminin. (a) Diagramof chick Net1 and its homology to mammalian Net1 (NLS: nuclear
localization sequence; DH: Dbl homology domain; PH: pleckstrin
homology domain). (b, c) Whole-mount in situ of Net1 transcripts atstage HH4 (b) and HH5 (c). Black lines: section levels. (d) Specificity

of anti-Net1 antibody shown with exogenously introduced cNet1 in DF1(left) and HEK293 (right) cells. (e) Immunostaining with chick anti-Net1
antibody. (f) Co-staining of Net1 and integrin α6. (g) Mouse-Net1 protein
expressed in medial epiblast cells retains laminin (arrows). Scale bars are
30 µm.

co-expression of C3 and RhoA (Fig. 5a2, arrows). In contrast to RhoAexpressing cells (Fig. 3b), these cells did not have laminin (Fig. 5b,arrows; Supplementary Information, Fig. S3f). C3-expressing mesoderm cells migrated individually (Fig. 5a3, arrows), although epiblast
cell ingression was perturbed (Fig. 5a3; Supplementary Information,
Fig. S2d), possibly due to a marked effect of C3 on both apical and basal
RhoA activities. In epiblast cells away from the streak, the effect of C3
on BM integrity was clearly seen (Supplementary Information, Fig. S2e),leading to ectopic EMT (Supplementary Information, Fig. S2e). Thesecells showed premature breakdown of the BM, and either ingressed as innormal EMT (white arrows) or egressed into the apical space above theepiblast (Supplementary Information, Fig. S2e, yellow arrows).

Because the effect of C3 may be broader than inhibiting only RhoA activity, we next used antisense morpholino oligonucleotides (MOs) against
RhoA and Net1 to reduce RhoA activity. Both RhoA- and Net1-MOs wereable to specifically reduce transcript and protein levels (Supplementary
Information, Fig. S6a–d for transcripts, Fig. S6e–j for proteins). Control
MOs did not affect laminin expression (Fig. 5c). Cells receiving RhoAMOs or Net1-MOs remained in the epiblast but showed premature breakdown of laminin (RhoA-MOs: 58% with n = 79 cells; Net1-MOs: 72%
with n = 78) (Fig. 5d, e, arrows). A similar effect on BM breakdown wasseen with a construct expressing dominant-negative RhoA (DN-RhoA;
Supplementary Information, Fig. S5e). Additional RhoA- or Net1-specificMOs and their 5-mismatch controls confirmed the above observations

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A1

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RhoA + C3

Control MO

DAPI + C3 + Laminin

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C3 + Laminin

RhoA
MO + Laminind

RhoA-MOe

Net1-MOf

Net1-MO +eGFP–rGBD

DAPI + MO + Laminin

DAPI + MO + Laminin

MO + eGFP-rGBD + DIC

MO + Laminin

MO + Laminin

MO

LamininLaminineGFP–rGBD

* *

* *

* *

*

*

*

*

*

*

*

*

*

*

*

*

Figure 5 Inhibition of RhoA signalling causes premature BM breakdown
and rescues mesoderm aggregation. Diagrams in b–h indicate regions
shown in section. White arrowheads: midline. (a) C3 rescues RhoAinduced cell aggregation. RhoA alone causes mesoderm cell aggregation(A1, arrows); C3 rescues aggregation caused by RhoA (A2, arrows); C3
alone does not cause mesoderm cell aggregation (A3, arrows). (b) Section
shows C3+RhoA-expressing cells do not form aggregates or express

laminin (arrows). (c–f) Reducing RhoA activity by RhoA or Net1 MOs
causes laminin breakdown in lateral epiblast cells. (c): Control MOs do
not affect laminin. (d): RhoA-MOs cause BM breakdown (arrows).
(e) Net1-MOs cause BM breakdown (arrows). (f) Net1 MOs reduce basal
RhoA activity (yellow asterisks), whereas apical RhoA activity was notaffected (white asterisks). Scale bars are 50 µm (a), 30 µm (b) and10 µm (c–f).

(Supplementary Information, Fig. S6k, l). These additional MOs showedcomparable efficiency in causing BM breakdown (RhoA–TB2: 72%
n = 87; RhoA–TB1+TB2: 62% n = 93; Net1–TB2+SB2: 78% n = 82). The
specificities of translation-block MOs were more clearly demonstrated ina Xenopus laevis assay system (Supplementary Information, Fig. S6k1, l1).
Furthermore, confirming the basal specific activity of Net1 protein, Net1MOs in epiblast cells resulted specifically reduced basal RhoA activity, as
revealed by co-introduced eGFP–rGBD localization (59%, n = 70; Fig. 5f;
yellow asterisks), whereas apical RhoA activity was not affected, with similar levels in cells that do or do not have MO present (white asterisks).

Nocodazole causes BM breakdown and taxol causes BM
retention
Net1 is expressed and basally localized only in cells with basal RhoA activity(Fig. 4). We next investigated how this activity can maintain extracellularlaminin. Data shown in Fig. 3f–i and Fig. 4e, f indicate that integrin-mediated cell–BM interaction may be a more direct target, possibly through RhoA
regulation of actin cytoskeletal or microtubule dynamics10 at the basal cortex
of epiblast cells. HH4 embryos were treated with cytochalasin D or nocodazole to determine whether chemicals disrupting either actin filaments or
microtubules, respectively, can affect laminin expression. Embryos treated770

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A RT I C L E SaLaminin + aPKC

bLaminin + aPKC

cLaminin + aPKC

Laminin + aPKC + DIC

DMSO
Laminin + aPKC + DIC+ Nocodazole
Laminin + aPKC + DIC+ TaxoldLateral Medial

e1

Lateralef1Medial

f
e2
e1

f2
f1

Apicale2

f2

Basolateral

Figure 6 Effect of nocodazole and taxol on laminin and distribution of
microtubules during EMT. (a–c) Section of HH4 embryo treated with DMSO(a), nocodazole (b) or taxol (c) for 2 h. White arrowheads: midline; green:
laminin; red: aPKC. Nocodazole causes BM breakdown in lateral epiblast(b) and taxol causes BM retention in medial epiblast (c). (d) EM overview of

HH4 embryo at mid-streak level. Locations of cells in e and f are indicatedby arrows. (e) Lateral cell. (f) Medial cell. Regions shown in e1–2 and f1–2 areindicated in d. (e1, f1) Apical area. Arrows: microtubule. (e2, f2) Basal area.
Microtubules (arrows) seen in lateral (e2), not in medial cell (f2). Scale bars
are 30 µm (a–c), 20 µm (d) and 0.5µm (e).

with nocodazole showed prominent BM breakdown (Fig. 6b compared with
Fig. 6a). Cytochalasin D treatment caused no obvious BM breakdown, butinstead had a more prominent effect on apical integrity (data not shown).
This indicates that basal RhoA activity may control BM maintenance byregulating microtubule stability at the basal side of epiblast cells. The effectof nocodazole, however, may be general and its effect on BM integrity secondary. We therefore tested the effect of taxol, a chemical that stabilizes
dynamic microtubules. Taxol treatment had no effect on laminin expression
in lateral epiblast cells, whereas in medial epiblast cells it caused prominent
BM retention (Fig. 6c), suggesting that microtubule stability in epiblast cells
is directly involved in BM maintenance.

microtubules were seen throughout the cell along the apicobasal axis
(Fig. 6e1,2). In medial cells (Fig. 6f), they were seen in apical regions withsimilar abundance, as in lateral cells (Fig. 6f1), but were rarely detectable inthe basal or basolateral region (Fig. 6f2). This was confirmed by counting
microtubules detectable under the electron microscope (Supplementary
Information, Fig. S7j) and by immunohistochemical staining with antibodies against pan β-tubulin (Supplementary Information, Fig. S7a–f) andtyrosinated α-tubulin (Supplementary Information, Fig. S7g), strongly suggesting that destabilization of basal microtubules is important for initiating
BM breakdown during EMT. Furthermore, antibodies against acetylated
and detyrosinated α-tubulin, which are commonly thought to recognize
more stable microtubules, showed a primarily apical staining throughout
the epiblast (Supplementary Information, Fig. S7h, i), suggesting that basal
and apical microtubules may have some intrinsic difference.

Basal microtubule loss coincides with BM breakdown
If RhoA activity regulates microtubule stability in epiblast cells, we would
expect destabilization of microtubules to be limited to the basal side of
medial epiblast cells, as prominent apical RhoA protein and activity wereobserved in both lateral and medial epiblast cells (Figs 1d, 3j). To test this,we examined the abundance of microtubules in lateral and medial epiblast
cells by electron microscopy (Fig. 6d, overview). In lateral cells (Fig. 6e),Modified tubulin at the basal cortex is a potential mediator in
RhoA-regulated BM maintenanceOur data demonstrate the importance of both RhoA and microtubule
dynamics in regulating epiblast cell–BM interaction but it is not clear

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a

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Embryos

DF1 Cells

Embryos

DF1 Cells

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Embryos

6G7 + RhoA

c

100
75

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50
37
IB: 6G7de

IB:β-tubulin
IB:β-tubulin(150 min exposure) (5 min exposure)α-tubulin

6G7

α-tubulin + 6G7 + DAPI

α-tubulin

Net1

α-tubulin + Net1 + DAPI

Figure 7 6G7 recognizes a modified β-tubulin localized to the basal cortex inlateral cells and absent in medial cells. (a) Section of HH4 embryo stainedfor 6G7. Arrowheads: midline. (b) RhoA mis-expression causes ectopic
retention of 6G7 in medial epiblast (arrows). (c) Immunoblotting reveals that
6G7 recognizes several minor bands (one prominent minor band indicatedby arrowheads), but not the major tubulin band (arrows) in pan-β-tubulin

blot. The position of the minor band suggests a modified β-tubulin insteadof a tubulin isoform. (d) Punctate localization of 6G7 to microtubules in
DF1 cells. (e) Punctate localization of endogenous Net1 along microtubules
in DF1 cells. Although 6G7 punctae are seen on microtubule tracts, Net1
punctae are seen more abundantly, and sometimes do not colocalize withmicrotubules. Scale bars are 30 µm (a, b) and 10 µm (d, e).

how they are linked. Microtubule stability can be a direct target for
RhoA activity30,31, and recent data suggest that Rho GTPases can regulate cell shape and polarity through modifying proteins associated withmicrotubule ends32–35. In addition, RhoGEFs have been reported to bindmicrotubules36,37 and regulate microtubule-tip dynamics by controlling
RhoA activity37. To find end-modifying proteins or modified formsof tubulins with localized expression in the epiblast, we found that
6G7, a chick β-tubulin antibody38, recognizes an antigen with strikingly restricted expression (Fig. 7a). Immunoblotting showed that
6G7 recognizes a post-translationally modified β-tubulin (Fig. 7c). In
lateral epiblast cells, 6G7 antigen was localized close to the basal cell
membrane; whereas it was absent in medial epiblast cells. Moreover,when RhoA was mis-expressed in medial epiblast cells, 6G7 antigen wasprominently retained (Fig. 7b, arrows). Although the precise nature of

the modification awaits further study, immunostaining with cultured
DF1 cells (chick fibroblast cells) revealed a punctate pattern of incorporation of 6G7-specific β-tubulin into microtubules (Fig. 7d). A similar
pattern, although more abundant, was observed with endogenous Net1
along microtubules (Fig. 7e). In contrast, exogenously introduced Net1
was seen mainly in the nucleus (data not shown). These data suggestthat special microtubule modifications at the basal cortex in normal
epiblast cells may be an important target for Net1- and RhoA-regulated
microtubule stabilization (Fig. 8).

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DISCUSSIONEMT consists of a number of events that require precise coordination.
In the context of gastrulation, we report here that these events takeplace in a spatially and temporally separable sequence (Figs 2, 8). Lossnature cell biology volume 10 | number 7 | JULY 2008

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of cell–BM interaction and breakdown of the BM take place first, whenmost cell–cell junctions are still intact. Loss of tight junctions occurs
next, at the time when cells leave an integral epithelial sheet. The growthof early avian embryos requires epiblast tension generated by edgecell migration in the area opaca and osmotic pressure build-up in thesubgerminal cavity39. The maintenance of tight junctions in mesoderm
precursor cells ensures epiblast integrity in the primitive streak. After
ingression, cadherins shift gradually from epithelial to mesenchymal
type. We suggest that this shift reflects the post-EMT migratory behaviour of most mesoderm cells. E-cadherin generally mediates more stable
cell–cell adhesions in the epithelium, whereas N-cadherin mediates amore dynamic forming and breaking of cell–cell contacts40.
During avian gastrulation, fibroblast growth factors (FGFs) induce
BM breakdown poorly, despite being potent inducers of the mesoderm
marker brachyury. However, another mesoderm inducer, Nodal, causes
marked BM breakdown (data not shown). At present, we do not know
how signals linked to the FGF, TGF-β, Wnt and Notch pathways, allcrucial for proper gastrulation to take place, exert their influence on
RhoA activity and its subcellular distribution. Initiation of expression
in the epiblast of the mesoderm inducer Brachyury and EMT regulator
Slug, both at the transcript and protein levels, precedes the initiationof BM breakdown (data not shown), suggesting that these transcription regulators may also regulate epiblast cell–BM interaction during
EMT. Among Rho family GTPases, an obvious effect on gastrulation
was only observed with RhoA mis-expression; whereas Rac and Cdc42,
which have prominent roles during mesenchymal to epithelial transition in somitogenesis, do not perturb the gastrulation process whenmis-expressed41. Chickens have three Rho-encoding genes, rhoA, rhoB
and rhoC, sharing 83–93% identities at the protein level42. Although
our study focuses on RhoA and null mutations of either rhoB or rhoC
do not affect mouse development43,44, some of our results may not be
specific for RhoA and we cannot exclude the possible involvement of
RhoB and/or RhoC.
Rho GTPases are important in multiple cellular events; however,studies linking specific cellular functions with localized Rho activity
have emerged only recently37,45–48. Our data add to this growing list by
revealing a link between basal RhoA activity and the maintenance ofcell–BM interaction during gastrulation (Fig. 8). We also observed astrict basal localization of Net1 transcripts and protein product, suggesting that basal RhoA activity is mediated by the localization of its
activator. In cultured cells, mouse Net1 protein has a mainly nuclear
localization; cytoplasmic localization is tightly controlled and correlateswith its transforming ability27. RhoA, however, is also enriched on theapical side of ectoderm cells, possibly mediated by other apically localized GEFs. This seems to be the mechanism regulating apical RhoA
activity during posterior spiracle formation in Drosophila melanogaster47. In this case, apical localization of RhoGEF64C and RhoGEF2, andbasolateral localization of RhoGAP88C (crossveinless-c) control apical
RhoA activity. Apical localization of RhoGEF64C transcripts was also
reported47, and together with our observations, suggests that RhoGEF
mRNA localization is important in mediating Rho activity. Targeted
transport of RhoA mRNA itself has also been reported to contribute to
localized RhoA activity48.
How does basal RhoA activity regulate cell–BM interaction? Here
we provide evidence supporting the involvement of microtubules inthis process. Immunohistochemistry and electron microscopy analyses

Apical

RhoA

RhoA

Basal

RhoA

RhoA

RhoA

RhoA

Net1
Net1
RhoA

RhoA
RhoA

RhoA

Lateral

RhoA

RhoA

RhoA
RhoA

RhoA

RhoARhoA
RhoARhoA

Net1

MedialActomyosin ring

Basement membrane

Tight junction

MT end modification

Adherens junction

Basal cortex

Microtubule

Integrin α6 and β1

Figure 8 A model for the regulation of cell–BM interaction by basal RhoA
activity and microtubule dynamics. The breakdown of BM precedes the lossof tight junctions and change in adherens junctions type. This is mediatedby loss of basal Net1 and RhoA activity in medial epiblast cells. This resultsin disruption of basal microtubules and integrin-mediated epiblast cell–BMinteraction and subsequently, breakdown of BM.

show that microtubules are rarely detected in the basal compartmentof medial epiblast cells undergoing BM breakdown. In lateral epiblast
cells, the dynamics of microtubules are different in apical (acetylated
and detyrosinated tubulins; Supplementary Information, Fig. S7h, i)and basal (6G7; Fig. 7a) compartments. Treatment of embryos withchemicals affecting microtubule dynamics causes prominent effects
on the BM. However, our analyses also show that nocodazole has amore immediate effect on basal RhoA activity and on basal integrin,
than on BM integrity (Supplementary Information, Fig. S8a, b), suggesting that the epistatic relationship between basal RhoA and basal
microtubules is not straightforward. On one hand, microtubules can beused to retain basal RhoA activity and the real target for active RhoA
may be the basal cortical actin cytoskeleton. Loss of basal microtubules
may be an indirect consequence of the loss of cell–BM interaction. On
the other hand, RhoA activity can directly regulate basal microtubule
stability; destabilization of these microtubules caused by loss of basal
RhoA activity leads to the breakdown of cell–BM interaction. Thishypothesis is supported by our observation that experimental destabilization of microtubules leads to rapid breakdown of the BM in normal
epithelium, whereas actin destabilization does not cause obvious BM
defects. Furthermore, RhoA-induced mesoderm cell aggregation canbe rescued by nocodazole treatment (Supplementary Information,
Fig. S8c-f). Our observations on the basal cortical localization of a
specialized β-tubulin recognized by 6G7 and its retention by RhoA
overexpression further support this model. A recent report, using
cultured cells, showed that GEF–H1, is localized at the tips of cortical
microtubules and functions in the regulation of microtubule dynamics
through localized RhoA activation37.
In summary, our data show that during the EMT process in gastrulation, basally localized RhoA activity and basal microtubule dynamics
are essential for maintaining epiblast cell–BM interaction. Disruptionof either one leads to the disruption of the other and subsequently to theloss of the BM. Further studies are required to understand how this isnature cell biology volume 10 | number 7 | JULY 2008
© 2008 Macmillan Publishers Limited. All rights reserved.

773

A RT I C L E S
regulated by multiple signalling pathways active during EMT and how
the breakdown of epithelial cell–BM interaction is coordinated with thedisruption of cell–cell junctions for the proper execution of EMT.

Cell culture. DF1 chicken fibroblast cells were used for 6G7, Net1 and α-tubulin
staining. DF1 and HEK293 cells were used for Net1 western blot analysis. Standard
protocols were used for cell culture and transfection.

METHODSElectron microscopy. Electron microscopy studies were performed in the EM
facility of RIKEN CDB with the help of S. Yonemura from the Laboratory for
Cellular Morphogenesis. Samples were fixed with 2% fresh formaldehyde and2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 2 h at room temperature and then post-fixed with 1% OsO4 in the same buffer for 2 h on ice. They
were rinsed with water, stained with 0.5% uranyl acetate overnight at room temperature, dehydrated with ethanol and embedded in Polybed 812 (Polyscience).
Ultra-thin sections were cut, double-stained with uranyl acetate and lead citrate,
and then examined with a JEM 1010 transmission electron microscope (JEOL)
operated at an accelerating voltage of 100 kV.

Embryology, imaging and immunohistochemistry. Fertilized hens’ eggs wereobtained from Shiroyama Farm (Kanagawa, Japan). Embryos were electroporated and cultured using standard methods17. All fluorescence microscopy
images were taken with an Olympus FV1000, and fluorescent whole-mountimages and non-fluorescent whole-mount or section images with Olympus
BX51. For immunohistochemistry, the following primary antibodies wereused: E-cadherin (1:500; BD transduction Lab, 610181), N-cadherin (1:1000;
gift from M. Takeichi, RIKEN, Kobe, Japan), aPKC (1:60; Santa Cruz, sc-216),
ZO-1 (1:100; Zymed, 40-2200), RhoA (1:100; LuLu51, gift from S. Yonemura49),
fibronectin (1:100; VA13, DSHB), laminin (1:200; laminin-1, 3H11, DSHB;SIGMA, L9393), tyrosinated α-tubulin (1:100; Chemicon, MAB1864), acetylated
α-tubulin (1:1000; Sigma, T6793), detyrosinated α-tubulin (1:100; Synaptic
Systems, no. 302011), β-tubulin (1:100; 6G7, DSHB), HA-tag (1:100; 12CA5,
Roche, 666 878), Myc-tag (1:300; MBL, 562; 9E10, Santa Cruz, sc-40), pan-βtubulin (1:500; Sigma, T4026), Integrin α6 (1:100; P2C62C4, DSHB), Integrin
β1 (1:300; Chemicon, MAB13443) and GFP (1:1000; Invitrogen, A-11122).
Alexa Fluor secondary antibodies (1:300; Invitrogen) were used for multicolour
detection. Net1 in situ analysis was performed using a standard protocol17 with
antisense digoxygenin probes against the last 0.5 kb of chick Net1 (NCBI no.
NM_001030648). Net1 antibody was generated against an N-terminal peptide(residues 19–35) by MBL (1:100, Nagoya, Japan). New cultures were treatedwith nocodazole (Sigma, M1404) at a concentration of 10 µg ml–1 dissolved inalbumen, and taxol (Sigma, T7402) at 5 µg ml–1. Cytochalasin D (Sigma, C8273)
treatment was carried out at 4 µg ml–1.
Expression constructs. DNA fragments were cloned into pCAGGS expression vector. Wild-type RhoA and DN-RhoA (T19N) with 3×HA tag were
provided by Y. Takahashi (NAIST, Nara, Japan); cDNAs encoding mouse
Net1 by J. Frost28; C3 by A. Hall (Memorial Sloan-Kettering Cancer Center,NY) and K. Kaibuchi (University of Nagoya, Japan). A tag of 6 Myc epitopes
was added to Net1 and C3. eGFP–rGBD DNA was provided by W. Bement45.
Fluorescein-tagged RhoA translation-block MO (RhoA–TB1; –22→+3 of translation initiation junction) 5´-CATAGCTGAAACACAAGAGGAC AAC-3´,
E1I1 splicing-block MO (RhoA-SB1; –15→+10 of E1I1 junction) 5´-GTATACATACCTGCTTTCCATCCAC-3´, Net1 translation-block MO (Net1-TB1;–1→+24 of translation initiation junction) 5´-CCCGAGCTCATCGTGAGC
AACCATG-3´, I1E2 splicing-block MO (Net1-SB1; –13→+12 of I1E2 junction) 5´-ATTGCTTGGCTCCTGTAAATAAAGC-3´ and standard control MO
were purchased from Genetools, and revealed with anti-fluorescein immunostaining (Invitrogen, no. A11090). Additional MOs used to confirm thespecificities are: RhoA–TB2 (–54→–30 in 5´UTR) 5´-TCCTCCCATTGGAGCTTTTAATCGA-3´; 5-mismatch for RhoA–TB1 5´-CATCGCTCAAAGACAAGACGAGAAC-3´; 5-mismatch for RhoA-TB2 5´-TCGTCGCATTGG
ACCTTTAAATGGA-3´; 5-mismatch for RhoA–SB1 5´-GTAAACATAGCT
CCTTTCGATCGAC-3´; Net1–TB2 (–6→+18 of translation initiation junction) 5´-CTCATCGTGAGCAACCATGGCGAA-3´; Net1–SB2 (–5→+20 of
I1E2 junction) 5´-ACCCGTTTATTGCTTGGCTCCTGTA-3´; 5-mismatchfor Net1 TB1 5´-CCGGACCTCATCCTGAGGAACGATG-3´; 5-mismatchfor Net1 SB1 5´-ATTCCTTCGCTGCTCTAAATAATGC-3´; 5-mismatch for
Net1–TB2 5´-CTGATCCTGAGCAACGATCGCCAA-3´; 5-mismatch for
Net1–SB2 5´-ACACGTTTATTCCTTCGCTGCTCTA-3´. Specificity test oftranslation-block MOs in Xenopus were carried out according to a publishedprotocol50. Briefly, 5´UTR region of cRhoA (–60→–1) and translation initiation
junction region of cNet1 (–10→+24) were cloned in front of mCherry using
pCS2 vector. In vitro transcribed mRNA (10 pg per cell for Net1-mCherry;
40 pg per cell for RhoA-mCherry) and indicated MOs (12.5 ng per cell for Net1TB and 25 ng per cell for RhoA TB) were injected into four animal blastomeresat 8-cell stage. Animal cap explants were prepared at mid-blastula stage andcollected at early gastrula, following by western blot analysis using an antiDsRed antibody (Clontech, no. 32496). Net1 sequences used for phylogenetic
comparison have following NCBI accession numbers: NM_001030648 (chick),BC004699 (mouse) and BC010285 (human).
774

Note: Supplementary Information is available on the Nature Cell Biology website.
Acknowledgements
We thank Fujio Toki for the initial analyses of fibronectin, laminin and 6G7;
Yoshiko Takahashi for bringing the phenotype of RhoA cells to our attention; Kathy
Joubin for sharing Net1 information; Cantas Alev for generating the Net1 probe;
Shigenobu Yonemura and Kazuyo Misaki for help with EM; Masatoshi Takeichi,
Yoshiko Takahashi, William Bement, Jeffrey Frost, Alan Hall, Kozo Kaibuchi and
Shigenobu Yonemura for sharing reagents; Noriaki Sasai and Yoshiki Sasai forhelp with Xenopus experiments. Claudio Stern, Donald Newgreen, Kozo Kaibuchi,
Masatoshi Takeichi, Fumio Matsuzaki, Shigeo Hayashi and Shigenobu Yonemura
for critical comments on the manuscript.
Author contributions
Y.N. and G.S. designed the experiments and analysed the data; Y.N, E.W.S. and Y.W.
performed the experiments; G.S wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturecellbiology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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00:00

00:40

01:20

02:00

00:00

00:40

01:20

02:00

Figure S1 Time-lapse images of Fig.1b (top) and Fig.1c (bottom), showing the formation of aggregates (arrows and arrowheads) in RhoA+GFP-expressing
embryo. Total duration: 2 hours.

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s u p p l e m e n ta r y i n f o r m at i o n

a

b

c

RhoA

RhoA

RhoA

E-cadherin

aPKC

RhoA+E-cadherin

RhoA+aPKC

E-cadherin

N-cadherin

RhoA+E-cadherin+N-cadherin

d DAPI+C3+LaminineC3+Laminin

C3+Laminin

Figure S2 a-c: RhoA mis-expression does not cause obvious effect on
Cadherins or aPKC. RhoA does not affect E-cadherin or N-cadherin
expression in mesoderm cell aggregates (a), or E-cadherin in epiblast cells(b). RhoA does not affect aPKC expression in epiblast cells (c). d,e: Effectof C3 on epiblast and mesoderm cells. C3-expressing mesoderm cells do not

2

DAPI+C3+Laminin

form aggregates or express Laminin (white arrows in d). Some cells extruded
apically are also Laminin negative (yellow arrows in d). C3 in lateral epiblast
causes premature BM-breakdown and ectopic “EMT” (bracket in e). Somecells ingress into mesoderm layer (white arrows in e), others egress from
epiblast (yellow arrows in e). None expresses Laminin. Scale bar: 30 µm.

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s u p p l e m e n ta r y i n f o r m at i o n

a

RhoA

RhoA+Laminin

Laminin

RhoA+Laminin+DAPI

Integrin a6bIntegrin b1dNormal embryo

Normal embryo

Integrin a6cIntegrin b1

e

RhoA+
Integrin b1

RhoA+
Integrin a6

f RhoA+C3
DAPI+C3+Laminin

C3+Laminin

RhoA

Figure S3 a: Magnified view of panels in Fig.3b. b-e: Magnified view of panels in Fig.3f-i. f: Magnified view of panels in Fig.5b. Scale bar: 30 µm.

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s u p p l e m e n ta r y i n f o r m at i o n

DIC

rGBD

rGBD
+DAPI

LamininLaminin
+DAPI

rGBD+
LamininrGBD+
Laminin+DAPI
rGBD+
Laminin+DAPI+DIC

Figure S4 Left panels: images in Fig.3j shown with wider field of view in various overlay combinations. Right panels: magnified view of indicated area on left
(arrow in DIC). Scale bar: 30 µm.

4

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s u p p l e m e n ta r y i n f o r m at i o n

a

c

b

d
d1

d2
C1
d3

St.3

St.1

d1

St.7

St.8

d2

d1
d3

e

f

RhoA-DN
RhoA-DN+Laminin

f-1

Mr (K)Mr (K)

150

150

100

100

75

f-2

75

50

Laminin

50

37

Mr (K)

f-3

Mr (K)

100

100

75

75

50

50

37

37

f-4

37

25

25

25

25
IB: DsRed

IB: Lulu51

IB: DsRed

IB: cNet1

Figure S5 Net1 in situ at additional stages, DN-RhoA effect and full-scan gels.
Net1 expression at pre-streak (a), HH3 (b), neural plate (c) and early somite (d)stages. Basal localisation of Net1 transcripts is not seen at later stages (c1,d1-3).

e: DN-RhoA causes BM-breakdown in lateral epiblast cells (arrowheads). Scalebar: 10 µm. f: Full-scan gels of those shown in Fig.S6g (f1), Fig.S6j (f2), Fig.
S6k1 (f3) and Fig.S6l1 (f4) with specific detected bands indicated by arrowheads.

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s u p p l e m e n ta r y i n f o r m at i o n

DAPI+RhoA+Laminin

RhoA MO(TB1+SB1)

RhoA protein

5MisTB1+SB1

TB1+5Mis SB1

Fluorescein+Laminin

Fluorescein+Laminin

LamininLamininLaminin

40

60

DAPI+Net1

40

28.1

Std MODAPI+Net1

0

Net1 MO(TB1+SB1)

Net1 protein

no changed
down regulated

20
RhoA MO(TB1+SB1)

Std MOl

5MisTB1+5MisSB1

5MisTB1+SB1
Fluorescein+Laminin

Fluorescein+Laminin

TB1+TB2

Std MO+ cNet1

LamininLamininLaminin--+

Net1 MO

Std MO

-

80
60
40
20
0

IB:
DsRed
IB:
Actin

5misTB1+5misTB2

Fluorescein+Laminin

Fluorescein+Laminin

Fluorescein+Laminin

Fluorescein+Laminin

LamininLamininLamininLamininFigure S6 Specificity of RhoA and Net1 morpholinos. Both RhoA MOs(TB1+SB1) (a) and Net1 MOs (TB1+SB1) (c) can specifically reducetranscript levels. Standard control morpholinos do not affect transcriptlevels (b for RhoA and d for Net1). e-g: Effect of RhoA morpholinos on
RhoA protein level is analysed by two methods. Immunohistochemical
analysis (section in e and statistics in f) shows 67.3% RhoA-MO cells
have reduced RhoA protein level (white arrowheads), compared to 10.4%
seen with standard control MO cells. Alternatively, western blot of isolated
epiblast tissue pieces electroporated with RhoA-MOs shows a 40% reduction
compared to standard control MO electroporated tissue due to mosaic natureof epiblast cells receiving MOs. h-j: Effect of Net1-MOs on Net1 protein
level. Immunohistochemical analysis (section in h and statistics in i) shows
84.8% Net1-MO cells have reduced Net1 protein level (white arrowheads),
compared to 28.1% in standard control MO cells. Western blot of isolated
epiblast tissue pieces electroporated with Net1-MOs and cNet1-expressing

6

Net1 MO+ cNet1

+

100

l-1

IB:
Actin

5mis TB2++

TB1+5Mis SB1

Fluorescein+Laminin

IB:
DsRed

TB2

-

IB: Actin

84.8

20

+

IB: Net1

71.9

60

0

80

k-1
uninjection

Fluorescein+Laminin

80

-

+

Standerd MO

100

10.4

Std MO

Std MOcNet1

15.2

5mis Net1-MO(TB2)

67.3

20

100

Net1-MO(TB2)

40

Net1 MO(TB1+SB1)

Net1

Net1

j

i

Fluorescein+Net1

5mis Net1-MO(TB1)

IB: Actin

89.6

no changed
down regulated

k 5MisTB1+5MisSB1

Standard control MO
Cell number (%)

60

0

DAPI+RhoA+Laminin

Net1 MO (TB1+SB1)
Fluorescein+Net1

32.7

80

Standerd MO

RhoA

h

IB: RhoA

Normalised RhoA level (%)

Cell number (%)

RhoA

Std MO

Normalised Net1 level (%)(TB1+SB1)

Net1-MO(TB1)

RhoA MO

Net1 mRNA + Fluorescein

Net1 mRNA

Controlg
100Standard control MO

dNet1 mRNA + Fluorescein

uninjectionf

5mis RhoA-MO(TB2)Standard control MO
Fluorescein+RhoA

Net1 mRNA

RhoA-MO(TB2)

RhoA MO (TB1+SB1)
Fluorescein+RhoA

Net1 MO (TB1+SB1)

c

RhoA mRNA + Fluorescein

RhoA mRNA

5mis RhoA-MO(TB1)eStandard control MOb

RhoA-MO(TB1)

RhoA mRNA + FluoresceinControl

RhoA MO (TB1+SB1)

a
RhoA mRNA

TB2+SB2

5MisTB2+5MisSB2

5MisTB2+SB2

TB2+5MisSB2

Fluorescein+Laminin

Fluorescein+Laminin

Fluorescein+Laminin

Fluorescein+Laminin

LamininLamininLamininLamininconstruct (endogenous Net1 expression is too low to be quantified inwestern blot) shows a 30% reduction compared to standard control. k,l:Additional MO (both specific and 5-mismatch control) experiments show
MO specificity. Green: MOs. Red: laminin. k) Additional RhoA-MOs. Top
panels: three combinations of 5-mismatch RhoA TB1 and SB1 MOs fail tocause BM-breakdown. Bottom panels: a second translation block MO (TB2)
has a stronger effect, leading to potent BM-breakdown with TB2 alone or in
combination with TB1; whereas its 5-mismatch controls do not. Specificityof translational block MOs is additionally demonstrated in Xenopus system(k-1) (see materials and methods). l) Additional Net1 MOs. Top panels: threecombinations of 5-mismatch Net1 TB1 and SB1 MOs fail to cause BMbreakdown. Bottom panels: a similar specificity is seen with combination of
two new Net1 specific MOs (TB2+SB2); whereas their 5-mismatch controls
fail to cause BM-breakdown. Specificity of Net1 TB1 and TB2 MOs is
additionally demonstrated in Xenopus system (l-1). Scale bar: 10 µm.

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s u p p l e m e n ta r y i n f o r m at i o n

a

b
d,f

c

d

e

c,e

f

g
epiblast

Tyrosinated a-tubulin mesoderm

+DIC

h

+ DAPI

epiblast

Acetylated a-tubulin

mesoderm+ DAPI+DIC

i

epiblast
mesoderm

Detyrosinated a-tubulin

j
11
10

Number of MT / mm2

9
8
7
6
5
4
3
2
1
0
M

(n=54)

L(n=63)

ApicalM(n=18)

L(n=21)

Basolateral

Figure S7 Tubulin staining and microtubule quantification. a-f: Mid-streaklevel section of HH4 embryo stained with pan-β-tubulin (a-f) and Laminin(e,f). Indicated regions in b are shown with high magnification in c andd. Co-staining with Laminin shows a similar loss of basal microtubules inmedial epiblast cells (e,f). g-i: Immunofluorescence staining with antibodies
against tyrosinated α-tubulin (g); acetylated α-tubulin (h) and detyrosinatedα-tubulin (i). Right panel in g shows magnified view of indicated area on
the left. Red arrowheads: basal staining in lateral epiblast cells. WhiteM(n=36)

L(n=42)

Basal

arrowheads: lack of basal staining in medial epiblast cells. Right panels in hand i show a lack of basal staining, with DAPI-staining outlining the epiblast
layer. White arrowheads in h indicate mesoderm staining. j: Numbers of
microtubules per µm2 are counted based on 80nm EM sections of a stage
HH4 embryo. M: medial epiblast cells; L: lateral epiblast cells. Apical: apicalthird area. Basolateral: basal third but avoiding basal cortex. It is calledbasolateral here because central part of this region is often occupied by the
nucleus. Basal: basal cortex area. Scale bar: 30 µm.

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s u p p l e m e n ta r y i n f o r m at i o n

a

DMSO

Nocodazole(10mg/ml)

60min

60min

120minLaminin+DIC

b1 Integrin

b1 Integrin+Laminin+DAPI

Lamininb1 Integrin+Laminin

b

DMSO

Nocodazole(10mg/ml)

60min

60min

rGBD(GFP) + Laminin* ** *rGBD(GFP) +Laminin+DIC* ** *cd

WT-RhoA

Primitive streak

DMSO or
Nocodazole

Electroporation


WT-RhoADMSO (control)

Culturefor 6 hours

Further culture
for 3 hours

Beforee

20 mg/ml

Nocodazole

3 hours later

Figure S8 Effect of nocodazole treatment. a,b: Nocodazole effect on Laminin,
integrin β1 and rGBD localization. a) 2 hour nocodazole treatment causes
prominent loss of both laminin and integrin β1. 1 hour treatment causes
less strong effect on both, but with more cells starting to lose integrin β1
than Laminin. White arrowheads indicate cells with intact Laminin but lost
integrin β1 staining. b) rGBD distribution shows a more rapid response to
nocodazole. White arrowheads: control treated cells retain basal rGBD signal.

8

40 mg/ml Nocodazole
WT-RhoA

WT-RhoA

Beforef

3 hours later

Before3 hours later

Asterisks: nocodazole treated cells lose basal rGBD signal. c-f: Nocodazole
effect on RhoA caused cell aggregation. c) Schematic diagram of experimental
procedure. d) Control treated embryo has prominent mesoderm cellaggregation. e) 20 µg/ml nocodazole treated embryo has some RhoA-expressing
mesoderm cells migrating as individual cells, but still has aggregates formed.
f) 40 µg/ml nocodazole treated embryo has most RhoA-expressing mesoderm
cells migrating as individual cells. Scale bar: 30 µm (a); 10 µm (b).

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s u p p l e m e n ta r y i n f o r m at i o n

Supplementary Movie Legends

Movie S1 Time-lapse movie (2-hour duration) of control GFP electroporated embryo.
Movie S2 Time-lapse movie (2-hour duration) of RhoA+GFP electroporated embryo.

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