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Epiblast integrity requires CLASP and Dystroglycanmediated microtubule anchoring to the basal cortex
Yukiko Nakaya, Erike W. Sukowati, and Guojun Sheng
Laboratory for Early Embryogenesis, Institute of Physical and Chemical Research (RIKEN) Center for Developmental Biology, Chuo-Ku Kobe, Hyogo 650-0047, Japanepiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediatedthrough the CLASP-binding partner LL5. Live-imaging
using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilizedbasal microtubules. We further show that Dystroglycan
is localized to basolateral membrane in epiblast cells.
Basal but not lateral localization of Dystroglycan was
regulated by CLASP. We propose that epiblast–BM inter­action requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which
initiates gastrulation EMT.

Introduction
Epithelial-to-mesenchymal transition (EMT) is a phenomenon
in which cells change their shape and intercellular organizationfrom an epithelial type to a mesenchymal one. Correct executionof EMT and its reverse process MET is vital for tissue morphogenesis during animal development, and their abnormal reinitiation leads to organ fibrosis and tumor metastasis (Moreno-Bueno
et al., 2008; Kalluri and Weinberg, 2009; Thiery et al., 2009;
Kerosuo and Bronner-Fraser, 2012; Lim and Thiery, 2012). Cellbiologically, EMT has been well characterized as a multistep
process that includes dissolution of local basement membrane(BM), loss of the epithelial polarity and tight junctions (TJs),
switch of the adherens junction (AJ) subtypes, and mesenchymal cell migration. Molecularly, transcriptional factors suchas Snail, Twist, Zeb1, and Zeb2 are viewed as key mediators
between signaling input (e.g., TGF, FGF, EGF, and HGF) on
one hand and cell biological output on the other, measured mainlyby E-cadherin down-regulation and loss of epithelial polarity
markers (Moreno-Bueno et al., 2008; Kalluri and Weinberg,
2009; Thiery et al., 2009; Kerosuo and Bronner-Fraser, 2012;Lim and Thiery, 2012). Recently, control of epithelial cell–BMinteraction has been recognized as another crucial componentof EMT regulation (Levayer and Lecuit, 2008; Nakaya et al.,2008; Rowe and Weiss, 2009; Hagedorn and Sherwood, 2011;
Williams et al., 2012). However, it is unclear how this regulationis achieved molecularly and whether it utilizes the same signaling and transcriptional mediators as those involved in E-cadherin
and apicobasal polarity regulations.
Gastrulation, one of the best-known examples of EMT,
is a conserved developmental process during which the threeprincipal germ layers (ectoderm, mesoderm, and endoderm)
are generated (Nakaya and Sheng, 2008, 2013; Acloque et al.,2009; Lim and Thiery, 2012; Solnica-Krezel and Sepich, 2012).
Gastrulation EMT in amniotes (birds and mammals) involveschanges of epiblast cells from a proper epithelium with the fullarray of epithelial characteristics to mesenchymal-shaped mesoderm cells (Nakaya and Sheng, 2008). The primitive streak,a transient embryonic structure where gastrulation EMT takes
place, is composed of cells that have initiated EMT, but are stillconnected to the rest of the epiblast as a continuous sheet. Thesecells are considered to be metastable, partial-EMT cells, having

Correspondence to Guojun Sheng: sheng@cdb.riken.jp© 2013 Nakaya et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see http://
www.rupress.org/terms). After six months it is available under a Creative Commons License(Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Abbreviations used in this paper: BM, basement membrane; CLASP, CLIP-associatedprotein; DG, Dystroglycan; eGFP, enhanced GFP; EMT, epithelial-to-mesenchymaltransition; MO, morpholino; MT, microtubule; PH, pleckstrin homology.

The Rockefeller University Press  $30.00
J. Cell Biol. Vol. 202 No. 4 637–651
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THE JOURNAL OF CELL BIOLOGY

A

mniote epiblast cells differentiate into mesoderm
and endoderm lineages during gastrulation
through a process called epithelial-to-mesenchymaltransition (EMT). Molecular regulation of gastrulation EMT
is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules
to the basal cortex via CLIP-associated protein (CLASP), a
microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm
formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation

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Results
CLASP mRNAs are expressed in chicken
epiblast cells during gastrulation

Like in mammals, there are two CLASP genes in the chicken
genome, CLASP1 (available from GenBank under accession no.
XM_426599) and CLASP2 (accession no. NM_001177385).
Sequence alignment analysis (Fig. S1 A) revealed that chicken
CLASP1 and CLASP2 are orthologous to human CLASP1(accession no. NP_056097) and CLASP2 (NP_055912), respectively. Three TOG (tumor overexpressed gene) domains andregions for EB1, CLIP, and LL5 binding are highly conserved.
To see whether chicken CLASP genes are expressed during
gastrulation, we generated antisense RNA probes (Materials
and methods) and analyzed their mRNA expression patterns.
The CLASP1 gene starts to be expressed before streak formation (HH1 in Fig. 1 A). Strong expression was detected at gastrulation stages in the area pellucida, and at later stages theexpression became limited to the neural territory (HH3–4 in
Fig. 1 A and HH5–7 in Fig. S1 B). Section analysis revealed
that CLASP1 expression at stage HH4 was restricted to theepiblast (Fig. 1 A1). Medial epiblast cells within the streak
exhibited down-regulated CLASP1 levels, and mesoderm cells
showed very weak or no expression (Fig. 1 A1). Expression of
CLASP2 mRNAs was relatively weaker than that of CLASP1,
but these two genes had very similar dynamic patterns (Fig. 1 B
and Fig. S1 C). At stage HH4, CLASP2 expression was likewise detected in the epiblast, down-regulated in medial epiblast
cells, and absent in the mesoderm (Fig. 1 B1). These results suggested that both CLASP genes function in chicken epiblast cells
before they undergo gastrulation EMT.
CLASPs function to maintain BM
integrity through their MT-binding
ability during EMT

It has been reported that CLASPs can mediate the interaction
between the cell cortex and MT plus ends in mammalian culture cells and that CLASPs together with their binding partners LL5s mediate cellular interaction with laminin-based cell
substrate (Mimori-Kiyosue et al., 2005; Hotta et al., 2010).
We asked whether chicken CLASPs play a role in epiblastcell–substrate interaction before and during gastrulation EMT.
First, we electroporated constructs either expressing control
enhanced GFP (eGFP) or expressing eGFP-CLASP1 or eGFPCLASP2 (eGFP-tagged human CLASP1 or human CLASP2,
respectively; Akhmanova et al., 2001; Mimori-Kiyosue et al.,2005) into lateral epiblast cells at the mid-streak level of stage
HH3–4 embryos. CLASP1 is the full-length isoform of CLASP1,
and CLASP2 is a short splicing isoform of CLASP2 with full
MT binding efficiency. Electroporated embryos were incubatedfor another 6–7 h followed by fixation and staining for laminin expression. In control embryos, eGFP-expressing cells ingressed normally and became mesoderm cells (Fig. 1 C, top).
The BM underneath medial epiblast cells at the primitive streak(both eGFP-positive and -negative cells) was properly broken
down (Fig. 1 C) as in normal untreated embryos (Nakaya et al.,2008). In contrast, the BM underneath medial epiblast cells

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lost the epiblast cell–BM interaction but retained apicobasal
polarity and apical cell–cell junctions. In both chick and mousemodels, the first cell-biological sign of gastrulation EMT is theinitiation of BM breakdown underneath the epiblast (Nakaya
et al., 2008; Williams et al., 2012). We have previously reported
that this BM breakdown coincides with and is regulated by
the destabilization of microtubules (MTs) at the basal cortex of
epiblast cells (Nakaya et al., 2008). Before EMT, MTs in epiblast cells are organized along the apicobasal axis with theirplus ends oriented toward the basal cell membrane. In cells
undergoing gastrulation EMT, MTs at the basal cell cortex aredestabilized, resulting in a weakening of epiblast cell–BM interaction and, consequently, BM breakdown (Nakaya et al., 2008;
Nakaya et al., 2011). This process is partially controlled by theloss of basal RhoA activity, but how basal MTs are anchoredto the basal cell cortex and how this anchoring regulates the
epiblast cell–BM interaction is not well understood.
CLIP-associated proteins (CLASPs) are evolutionarily
conserved MT plus end tracking proteins (+TIPs) known to playessential roles in local regulation of MT dynamics (Akhmanova
et al., 2001; Carvalho et al., 2003; Akhmanova and Hoogenraad,
2005; Mimori-Kiyosue, 2011). In cultured mammalian cells,
CLASPs, together with their binding partners LL5s and other+TIP proteins, mediate the interaction between distal MT ends
and the cell cortex (Akhmanova et al., 2001; Lansbergen et al.,2006; Hotta et al., 2010; Mimori-Kiyosue, 2011). But it is unclear whether, during gastrulation EMT, CLASPs are involvedin basal cortical anchoring of MTs in epiblast cells; and if so,
how CLASPs can facilitate cross-interaction between the cytoskeleton and the BM. One potential candidate for mediatingsuch cross-interaction is Dystroglycan (DG). DG was isolated
as a component of the dystrophin–glycoprotein complex andfunctions by connecting muscle BM, sarcolemma, and muscle cytoskeleton (Ervasti and Campbell, 1991; IbraghimovBeskrovnaya et al., 1992). DG is composed of  and  subunits
encoded from a single dystroglycan (DAG1) gene. -DG islocalized extracellularly and binds to laminin and other BM
components; and -DG is a transmembrane protein that interacts extracellularly with -DG and intracellularly with actin filaments and MTs (Campbell, 1995; Henry and Campbell, 1999;
Ayalon et al., 2008; Cerecedo et al., 2008; Bozzi et al., 2009;
Prins et al., 2009).
In this paper, we investigated the role of CLASPs in basal
MT stabilization and cortical anchoring during avian gastrulation EMT. We also analyzed the involvement of DG in linking
the intracellular cortical MTs with the extracellular BM. We
show that CLASPs and their binding partners LL5s stabilize
MTs in the basal cortex. Overexpression of CLASPs in streak
epiblast cells results in BM retention, and CLASP mutants lacking the MT and cortical binding ability or antisense morpholino(MO)-mediated knockdown of CLASPs and LL5s lead to premature BM breakdown in lateral epiblast cells. Furthermore,
we provide evidence that CLASPs regulate basal membrane
localization of DG, and that this localization is required forstabilizing the basal membrane–BM interaction in pre-EMT
epiblast cells. Disengagement of this stabilizing mechanism
promotes BM breakdown and initiates gastrulation EMT.

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Figure 1. Endogenous CLASP mRNA expression and the effect of exogenous CLASP overexpression on laminin in the primitive streak. (A and B) Whole-mountin situ hybridization of CLASP1 (A) and CLASP2 (B) transcripts. Both CLASP1 and CLASP2 are expressed in epiblast cells (epi) and absent in mesoderm cells(mes). Bars, 20 µm. (C–E) Effect of CLASP overexpression on laminin. (C) eGFP control overexpression. Normal laminin breakdown and normal ingressionof expressing cells. (D and E) Electroporation with eGFP-CLASP1 (D) or eGFP-CLASP2 (E) expression vectors. Arrowheads, streak midline; yellow arrows,
laminin retention in CLASP-expressing cells. ps, primitive streak. Bars, 20 µm.

expressing eGFP-CLASP1 or eGFP-CLASP2 remained tobe laminin positive (Fig. 1, D and E, arrows), which suggested
that endogenous CLASPs function to maintain BM integrity
during gastrulation.
Previous studies showed that MT plus end and corticalbinding ability of CLASPs are necessary for MT stabilizationin the leading edge of fibroblast cells (Akhmanova et al., 2001;
Mimori-Kiyosue et al., 2005). We next examined whether MT
plus end and cortical binding of CLASP2 is involved in laminin
retention in primitive streak cells. The CLASP2-MC mutant
has its EB1-binding domain in the middle and CLIP-binding
domain in the C terminus deleted (Fig. 2 A), and lacks theMT plus end binding activity in COS cells (Akhmanova et al.,
2001; Mimori-Kiyosue et al., 2005). No laminin retention wasobserved when eGFP-CLASP2-MC mutant was expressedin the primitive streak (Fig. 2 B, arrows), which suggests thatthe laminin retention phenotype seen with eGFP-CLASP2

requires the MT plus end and cortical binding ability of CLASP2.
Interestingly, we saw ectopic laminin breakdown when eGFPCLASP2-MC was expressed in lateral epiblast cells wherelaminin should be present (arrows in Fig. 2 C and control in
Fig. 2 D and Fig. S2 A). A similar effect in lateral epiblast
cells was also seen with CLASP2-C, a CLASP2 mutant with
the C-terminal domain deleted (Fig. 2, A and D; and Fig. S2 B).
CLASP2-M, a CLASP2 mutant with the EB1 binding domain deleted, caused only a very mild disruption of laminin
expression in the lateral epiblast cells (Fig. 2, A and D; andFig. S2 C). These results suggest that the C-terminal domain
is important for laminin maintenance in lateral epiblast cells
before EMT. To confirm the involvement of CLASPs in mediating the cell–BM interaction in lateral epiblast cells, we used
specific antisense MOs to knock down endogenous levels of
CLASPs. Although CLASP2 MOs showed only a weak pheno­
type (Fig. 2 G), CLASP1 MOs caused potent laminin breakdown
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Figure 2. CLASPs maintain BM integrity during EMT through their MT-binding ability. (A) Schematic diagram of human CLASP1 and human CLASP2,and deletion mutants of human CLASP2 used in this study. (B and C) Effect of eGFP-CLASP2-MC overexpression. White arrowheads in B indicate thestreak midline. White arrows in B indicate normal BM breakdown in streak midline cells. White arrows in C indicate that CLASP2-MC–overexpressing
cells in lateral epiblast region have premature laminin breakdown. (D) Percentage of expressing cells showing laminin breakdown. Phenotypes are classified into three categories: no breakdown, mild breakdown, and complete breakdown. MC (77.6%, n = 134 cells from six embryos) and C (78.1%,
n = 105 cells from four embryos) showed severe phenotypes. M mutant showed a mild phenotype (35.6%, n = 90 cells from four embryos) in comparison
with control GFP (14.3%, n = 105 cells from five embryos). (E) Knockdown of CLASP1 protein by CLASP1-specific MOs caused laminin breakdown. White
arrows indicate ectopic BM breakdown in lateral epiblast cells. (F) 5mis-control MO does not affect laminin expression. Yellow arrows indicate normal BM
levels in 5mis-CLASP1 control MO–receiving cells. (G) Percentage of MO-containing cells showing laminin breakdown with different MOs. CLASP1 MO,
88.8% (n = 107 cells from five embryos); CLASP2 MO, 51.8% (n = 112 cells from three embryos); CLASP1 + CLASP2 MOs, 75.6% (n = 86 from threeembryos); 5mis CLASP1 control MO, 14.8% (n = 115 from three embryos). Bars, 20 µm.

in lateral epiblast cells (Fig. 2, E and G, arrows). 5-mis con­trolof CLASP1 MOs had no effect on laminin expression (Fig. 2,
F and G). These data suggested that maintenance of the cell–BM640

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interaction in chicken epiblast cells requires the C-terminal
domain of CLASP proteins, and that CLASP down-regulation
leads to BM breakdown during gastrulation EMT.

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Figure 3. CLASP proteins are localized to the basal cortex in lateral epiblast cells. (A and B) When eGFP-CLASP1 (A) or eGFP-CLASP2 (B) is electroporated into lateral epiblast cells at low concentrations, GFP signals are detected at the basal side. Arrowheads indicate basal localization of CLASP1 (A) and
CLASP2 (B). (C) Endogenous CLASP protein signals detected by cCLASP1 antibody are localized to the basal side. ps, primitive streak. (D and E) Validation of cCLASP1 antibody specificity. In standard MO-electroporated cells (E, yellow arrowheads), basal cCLASP1 antibody signals are not diminished. In
CLASP1 MO–electroporated cells, basal cCLASP1 antibody signals are diminished. Yellow arrowheads in D show the normal level of cCLASP1 signals incells not receiving CLASP1 MO. White arrowheads indicate loss of basal CLASP1 in CLASP1 MO receiving cells. Bars, 20 µm.

CLASP proteins are localized to the basalside of lateral epiblast cells before EMT

We next asked whether subcellular localization of chicken
CLASPs is consistent with their putative role in mediating epiblast cell–BM interaction. When low concentrations of either
eGFP-CLASP1– or CLASP2-expressing constructs wereintroduced into lateral epiblast cells, both were found to be localized mainly to the basal side (Fig. 3, A and B, arrowheads).
To investigate endogenous CLASPs’ subcellular localization,
we generated antibodies against the chicken CLASP1 (cCLASP1
antibody), which was produced using a polypeptide corresponding to amino acid residues 189–325 of chicken CLASP1.
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Figure 4. CLASPs and LL5s cooperate in maintaining BM integrity in lateral epiblast cells.
(A and C) Electroporated at low concentrations, eGFP-LL5 (A) and eGFP-LL5(C) havea basal cortical localization (white arrows).
(B and D) Overexpression of eGFP-LL5 (B) andeGFP-LL5 (D) in streak cells causes ectopic
laminin expression (yellow arrows). ps, primitive streak. (E) Quantification of laminin breakdown when LL5s or LL5s + CLASP1 are knocked
down with MOs in lateral epiblast cells. LL5
and CLASP1 knockdown leads to strong laminin breakdown phenotypes. (F–I) Examples ofphenotypes caused by LL5 and  double MO(F), LL5 (G), or LL5 (H) single MO, standard
control MO (I), and LL5 and CLASP1 triple MO(J) knockdown. White arrows in F–H and J indicate premature BM breakdown. Yellow arrows in
I indicate normal BM in standard MO-receiving
cells. Arrowheads indicate the streak midline.
Bars, 20 µm.

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Immunohistochemistry analysis showed that the cCLASP1 antibody signals were localized to the basal side of lateral epiblast
cells (Fig. 3 C, C2), and no signal was observed on the basalside in medial epiblast cells (Fig. 3 C, C1). We next validated
the specificity of cCLASP1 antiserum using CLASP1 MO. In lateral epiblast cells expressing CLASP1 MO, cCLASP1 antibody
signals were reduced prominently (Fig. 3 D, white arrowheads),

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whereas standard MO-receiving cells showed no change in
CLASP1 basal expression (Fig. 3 E, yellow arrowheads). Theseobservations are consistent with our hypothesis that basally localized CLASP proteins contribute to lateral epiblast cell–BM interactions and that dissociation of CLASPs from the basal cortex
in medial streak cells contributes to BM breakdown during gastrulation EMT.

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Figure 5. Localization of LL5 and CLASP1 after nocodazole and cytochalasin D treatment. (A) eGFP-LL5 localization after control DMSO (left), nocodazole(middle), and cytochalasin D treatment for 2 h. (B) Endogenous CLASP1 localization after control DMSO (left), nocodazole (middle), and cytochalasin D
treatment for 1.5 h. Broken lines indicate the apical limit of the epiblast. Bars, 20 µm.

LL5s play an inter-linking role with CLASPs
in the basal cortex of epiblast cells tomaintain BM integrity

It has been reported that LL5 and -, also known as pleckstrin
homology (PH)-like domain family B member 1 and 2 (PHLDB1
and -2), respectively, are localized adjacent to laminin-5 deposition in mammary epithelial cells (Hotta et al., 2010). Functionally, LL5 and - are CLASP binding partners, and it has been
hypothesized that CLASP–LL5 complex anchors MT plus ends
to the site where deposited laminin interacts with its receptor integrins (Hotta et al., 2010; Mimori-Kiyosue, 2011). In chicken
gastrula, transcripts of both LL5 and LL5 were detected in
epiblast cells (Fig. S3). We first investigated subcellular localization of LL5s in lateral epiblast cells and found that when either
eGFP-LL5 or eGFP-LL5 was expressed, they were predominantly localized to the basal cortex (Fig. 4, A and C, arrows).
Interestingly, when high concentrations of the LL5 or LL5gene were overexpressed, we observed laminin retention in
streak cells, a phenotype similar to that of CLASP overexpression (arrows in Fig. 4, B and D). When LL5 and - were simultaneously knocked down in lateral epiblast cells, premature BM

breakdown was observed (71.7% with n = 230 cells; Fig. 4,
E and F), whereas MOs against either LL5 (30.3% with n =119 cells; Fig. 4, E and G) or LL5 (31.5% with n = 108 cells;
Fig. 4, E and H) had a weaker, but still detectable effect. The
combi­nation of anti-LL5 and anti-CLASP1 MOs also resulted
in a clear effect on laminin expression (76.7%, n = 206 cells; vs.
control 15.6%, n = 237 cells; Fig. 4, E, I, and J). The severity
caused by triple MOs (LL5s and CLASP1) was however slightly
less than that by single CLASP1-MO knockdown (88.8% in
Fig. 2 G), possibly due to dilution of MO concentrations in
triple mix. We next asked whether subcellular localization of
LL5s depends on cortical MT stability. When embryos weretreated with nocodazole, an MT-disrupting drug, the basal cortical localization of LL5 was only mildly affected (Fig. 5 A,
middle), although nocodazole’s effect on basal MT dynamics
was strong as revealed by acetylated tubulin and laminin staining (Fig. S4, A and B). Control DMSO or cytochalasin D treatment did not affect LL5 localization (Fig. 5 A, left and rightpanels, respectively). In contrast, CLASP1 protein became diffuse and localized away from the basal cortex after nocodazole
treatment (Fig. 5 B). In LL5 MO cells, basal cortical localization
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Published August 12, 2013of CLASP1 was only mildly affected (Fig. S4 C). These results
indicated that LL5 and  are localized to the basal cortex in an
MT-independent fashion. Their interaction with CLASPs could
help stabilize basal MTs and maintain the integrity of extracellular laminin in lateral epiblast cells.
CLASPs and LL5s are required foranchoring MTs to the basal cell cortex
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How can the intracellular interplay between MTs, CLASPs,
and LL5s promote cell–BM interactions and extracellular BM
integrity? Bioinformatics data mining of our primitive streak
transcriptomic analyses (Alev et al., 2010; Nakaya et al., 2011)
suggested two categories of genes, encoding transmembraneproteins—integrins and DGs, respectively—as potential medi­
ators. Integrins, through adaptor proteins such as talin and-actinin, are known to mediate extracellular matrix and intra­
cellular F-actin interaction. Their role in MT-mediated cell–
BM interaction is less clear. We have previously shown that
integrins are expressed and likely play a role in gastrulation
EMT (Nakaya et al., 2008). But the presence of multiple  and integrin genes in the primitive streak makes functional analy­
sis difficult. DG, however, is known to interact with both theF-actin and MTs, and its two subunits, -DG and -DG, areencoded by a single DAG1 gene. In this work, we focused ourinvestigation on DG as a potential link between the BM andcortically stabilized MTs. During gastrulation, DG expression
is restricted to the epiblast cells (Nakaya et al., 2011). Subcellularly, DG is localized to the basolateral membrane in lateralepiblast cells; and in streak epiblast cells, basal membrane
local­ization of DG is lost and lateral membrane localization is
weakly detectable (Fig. 7 A). When wild-type DG was overexpressed in streak epiblast cells, laminin retention was observed(Fig. 7 C, arrows; with control shown in Fig. 7 B). Knockdownof DG in lateral epiblast cells with MOs resulted in abnormalities in cell shape and BM integrity (Fig. 7, D–F). In contrast
to control MOs, cells receiving translation blocking type MO(TB) or TB + splicing blocking type MO (TB+SB) showedpremature breakdown of laminin (TBMO, 81.7% with n =149 cells; TB+SBMOs, 74.5% with n = 153 cells; Fig. 7,
D and E, arrows; and Fig. 7 F). Overall, these results indicatedthat basally localized DG plays a role in mediating the epiblastcell–BM interaction during EMT.
CLASP affects basal DG localization
during EMT

We next investigated whether there is a functional relationshipbetween CLASPs and DG. In normal epiblast cells, -DG is localized to the basolateral cell membrane. The basal membrane
domain has relatively stronger expression than the lateral one(Fig. 7 A). Knockdown of CLASP levels in lateral epiblast cells
led to a reduction in basal -DG expression (Fig. 8, A and E).
In contrast, 5mis-CLASP1-MOs (Fig. 8, D and E) or LL5 MOs(Fig. S5 C) had no effect on the subcellular localization of -DG
in lateral epiblast cells. A similar effect on -DG local­ization wasobserved when the dominant-negative eGFP-CLASP2-MC
was electroporated (Fig. 8, B, C, and E), and wild-type eGFPCLASP2 overexpressing medial streak cells retained strong
DG expression (Fig. 8 F, arrows). These results strongly suggested that CLASPs regulate basal DG localization in epiblast
cells before EMT. We asked whether there is a physical interaction between CLASPs and DG. HEK293T cell lysates of either
CLASP or control transfectants were examined for coimmunoprecipitation of cotransfected -DG. We found that anti–-DG

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We therefore examined whether CLASPs and LL5s can stabilize
and anchor MTs to the basal cortex in lateral epiblast cells. For
this purpose we performed live epiblast cell imaging of EB1
comets, which mark the growing MT plus ends (Fig. 6 A). Timelapse movie sequences (1 s per frame for 1 min) of GFP-fusedEB1-expressing epiblast cells were acquired by using spinningdisk confocal microscopy. When images were taken from theapical side, EB1 fluorescence signals at the apical cortex werestrong and could be observed to move toward the lateral rims ofthe cell in a radial fashion (Fig. 6 B and Video 1, taken from theapical side). But signals from the basal cortex were very weak.
We therefore decided to image from the basal side after physical removal of mesoderm and endoderm tissues. With this method
we were able to visualize EB1 comets at both apical and basal
cortices of epiblast cells (Fig. 6 A). We attempted to analyze EB1
comets in CLASP1 MO–receiving cells. However, maximal
concentration of CLASP1 MO led to severe defects in epithelial
polarity (Fig. 2 E), which was not an appropriate condition for
tracking EB1 dynamics. When we reduced the concentration of
CLASP1 MO so that the epithelial polarity was maintained, no
obvious difference was seen between control MO– and CLASP1
MO–treated cells. However, when we adjusted the triple MO(CLASP1 and LL5s) concentrations, a reduction in basal MT
dynamics in triple MO receiving was observed compared with
control MO–receiving cells. In control MO–treated cells, themovement of EB1 comets at the basal side was not as dynamic
as that at the apical side, but was still readily detectable (Fig. 6 C,
top; and Video 2). This is in agreement with the presence of
MTs at the basal cell cortex, which we had reported previously
using electron microscopy and biochemical methods (Nakaya
et al., 2008). In contrast, when CLASP1 and LL5s were knocked
down, EB1 signals were reduced at the basal side (Fig. 6 C, bottom; and Video 3). The difference between these two types ofcells in MT distribution was also seen in 3D-reconstituted images (compare the top-right panel in Fig. 6 C and Videos 4 and 5with the bottom-right panel in Fig. 6 C and Videos 6 and 7).
This suggested that MTs in the basal region were destabilizedin triple MO–receiving cells that had a broken BM (Fig. S4 D)
and reduced levels of basal CLASP1 (Fig. S4 D) and basal MT(Fig. S5, A and B). Quantification analysis of basal EB1-GFP
signals relative to total EB1-GFP signals (Fig. 6 E) in control
MO cells versus triple MO cells exhibiting mild phenotypes, inwhich measurement of basal EB1-GFP signals was still possible(Fig. 6 D), supported this hypothesis (Fig. 6, F and G). Collec­
tively, although the epistatic relationship between MT stability
and cortical localization of CLASPs/LL5s awaits further clarification, these data suggested that they mutually reinforce each other,and together they regulate the epiblast cell–BM interaction.

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antibody can coprecipitate with either HA-CLASP1 or HACLASP2, and that anti-HA antibody can also coprecipitate
with -DG (Fig. 8 G). Results from proximity ligation assays

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Figure 6. CLASP and LL5s are involved inregulating basal MT dynamics. (A) Diagram of
eGFP-EB1 electroporation and live-embryo imaging. (B) Imaging of lateral epiblast cells fromthe apical side. eGFP-EB1 molecules show
dynamic and radial movement from the center toward the rim of the cell. (C) Imaging oflateral epiblast cells from the basal side. EB1
signals in the apical, middle, and basal planes
in control MO cell (top) or CLASP1 and LL5s
MO cell (bottom). In the latter cell, EB1 signals
are relatively weaker basally. Right-most panels are examples of 3D-reconstituted images
showing the difference in eGFP-EB1 signals
between control and CLASP/LL5s knockdown
cells. (D–F) Quantification of relative basal
eGFP-EB1 signal intensity in frozen sections.
(D) Examples of cellular morphology with electroporated control MOs (left) and triple MOs(middle, mild phenotype; right, severe phenotype). Only triple MO cells with mild phenotype
are used in quantification. Broken lines outline
individual epiblast cells. (E) Relative eGFPEB1 intensity is calculated as a ratio between
basal cortical signals and total cellular signals.
(F) Mean relative basal eGFP-EB1 signal intensity in cells (n = 50) without MO, with standardMO, or with triple knockdown MOs. (G) Relative intensities are shown as proportions of cellswith their basal eGFP-EB1 signals representing
the 10th, 20th, 30th, and 40th percentiles of
total eGFP-EB1 signals. Error bars indicate one
standard deviation. Bars, 5 µm.

(Fig. 9, A–E) further supported the idea that there is a physicalassociation between the CLASPs and DG in epiblast cells undergoing gastrulation EMT.
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Discussion
EMT involves dissociation of cell–cell and cell–matrix inter­actions in very different physiological and pathological contexts.
In many epithelia, apicobasal MT arrays important for generating
epithelial polarity are anchored to the basal cortex, where theyalso play a role in maintaining the epithelial cell–BM inter­action (Bacallao et al., 1989; Müsch, 2004; Nakaya et al., 2008;
Hotta et al., 2010). Using gastrulation EMT as a model, this work
demonstrated that before epiblast cells initiate EMT, MT +TIP
proteins CLASP1 and CLASP2, and their binding partners LL5s,
regulate MT anchoring to the basal cortex (Fig. 10). Furthermore,
we identified DG as a molecular link between the intracellular
cortical MTs and extracellular BM, with its membrane-spanning subunit interacting with cortical CLASPs (Fig. 10).
In our CLASP mutant analysis, overexpression of eitherC or MC mutant caused an ectopic breakdown of laminin in lateral epiblast cells where BM should be intact (Fig. 2,
C and D; and Fig. S2 B), which suggests that the C-terminal region of CLASP is important to maintain the BM integrity. Thisregion in human CLASP2 was shown to interact with CLIP-170
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and CLIP-115 (Akhmanova et al., 2001), as well as LL5 (MimoriKiyosue et al., 2005). The same region in human CLASP1 wasreported to mediate CLASP dimerization, and its deletion resulted in a dominant-negative phenotype on MT polymerization (Patel et al., 2012). Cls1p, the Schizosaccharomyces pombe
CLASP, also uses this region for dimerization (Al-Bassam et al.,2010). It is therefore likely that the phenotype we observedwith C-terminal deletion mutants was caused by a disruption ofmonomer/dimer balance of CLASPs or the binding with theirpartners such as LL5s, or both.
Our previous work (Nakaya et al., 2008) indicated thatthe cortically enriched small GTPase RhoA’s activity playsa critical role in stabilizing MTs anchored at the basal cellcortex during gastrulation EMT. We have yet to investigate
whether and how RhoA activity modulates DG-CLASPs–LL5sMT interactions. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) that catalyze GTP loading. Net1,
a RhoA-specific GEF, is expressed in epiblast cells and can regulate RhoA activity at the basal cortex (Nakaya et al., 2008). Inactive Net1 protein is sequestered in the nucleus, and its basal
cortical localization when activated has been proposed to be

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Figure 7. DG mediates basal membrane–BM interaction in epiblast cells. (A) -DG localization in epiblast cells. (B and C) Electroporation with control
2A-eGFP or wild-type (WT)-DG-2A-eGFP expression vector. No obvious change in laminin expression is observed in control 2A-eGFP–expressing cells (B).
WT-DG–receiving cells (C) show ectopic laminin expression (yellow arrows). (D and E) DG-specific MOs cause premature laminin breakdown (whitearrows). (D) Translation blocking (TB) MO. (E) TB-MO + splicing blocking (SB) MO. ps, primitive streak. Arrowheads indicate the streak midline. (F) Percentage
of cells showing laminin breakdown. TB-MO cells: 81.7%, n = 149 cells. TB-MO + SB-MO cells: 74.5%, n = 153 cells. 5-mis TB-MO: 9.7%, n = 165. 5mis
TB-MO + SB-MO cells: 10.0%, n = 171. Bars, 20 µm.

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Figure 8. CLASP affects basal DG localization during EMT. (A) CLASP1 + CLASP2 MOs reduce basal DG localization. (B) Standard control MO does notaffect DG subcellular localization. (C) CLASP2-MC reduces basal DG localization. (D) 5mis-CLASP1 MO–expressing cells show no change in DG localization. Arrowheads indicate reduction of basal DG localization. (E) Percentage of cells showing a reduction in basal DG. CLASP1 + CLASP2 MOs cells:
77.9%, n = 86 cells from five embryos. 5mis CLASP1 control MO cells: 19.5%, n = 82 cells from three embryos. eGFP-expressing control cells: 19.4%,
n = 93 cells from three embryos. eGFP-MC expressing cells: 62.6%, n = 91 cells from four embryos. (F) eGFP-CLASP2–expressing cells retain strong
DG expression in the medial streak. Arrows indicate mislocalization of DG in CLASP2–expressing cells. (G) Immunoprecipitation with either anti–-DG antibodies or anti-HA antibodies from HEK 293 cells transfected with human DG and either HA-CLASP1, HA-CLASP2, or mock treatment. Bars, 20 µm.

under the regulation of MTs extending to the basal cortex(Schmidt and Hall, 2002; Nakaya et al., 2008). Although further evidence is needed, a possible scenario is that CLASPs–
LL5s-MT interactions regulate basally targeted growth of MTs
and transport of Net1, resulting in basally enriched RhoA activation. Our preliminary data suggested that RhoA overexpression affects endogenous DG localization in streak cells, andwe showed in this work that -DG can be coprecipitated with
CLASPs in vitro (Fig. 8 G) and interacts with CLASPs in vivo
(Fig. 9). It is therefore likely that in a positive feedback loop,
RhoA activity controls basal cortical MT dynamics by influencing
interactions among components of the DG-CLASPs–LL5s-MTcomplex, as has been implicated by its ability to positively regulate +TIPs EB1 and APC (Wen et al., 2004).
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Figure 9. Proximity ligation analysis reveals an interaction between CLASP and DG in vivo. (A) Interaction between CLASP1 and DG in epiblast cells
revealed by using the Duolink proximity ligation assay. (B and C) CLASP1/Laminin and GM130/DG are used as negative controls. (D) Laminin–DG
interaction is used as a positive control. Broken lines indicate the tissue outline (top, apical limit of the epiblast; bottom, apical limit of the endoderm). (E) In
epiblast cells electroporated with CLASP1 MO, CLASP1–DG interaction signals are greatly reduced (arrowheads in E1). Bars, 20 µm.

BM maintenance involves balancing the synthesis and degradation of its components. BM disassembly (breakdown) in
streak cells is accompanied by a reduction in component gene
expression and an increase in transcripts for many metalloproteinases (Alev et al., 2010; Nakaya et al., 2011; unpublished data).
It is unclear whether these transcript-level changes are regulated directly by basal MT dynamics. A more likely scenario isthat down-regulation of CLASPs and LL5s, and the resulting MT
destabilization, would negatively affect basally targeted secretionof BM proteins and transport of membrane molecules involved in
BM–basal membrane interaction. This hypothesis is supported by
the fact that secretion and transport of many basolateral membrane
and BM proteins are regulated by MT dynamics (RodriguezBoulan and Nelson, 1989; De Almeida and Stow, 1991; Teng et al.,
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JCB • VOLUME 202 • NUMBER 4 • 20132005; Meng et al., 2008; Wickström et al., 2010), and by our ob­
servation that basal DG localization was reduced when CLASP’s
function or MTs were disrupted (Fig. 8, A and E; Nakaya et al.,2011). Another possible scenario is that the activity of metalloproteinases is negatively regulated by basal MT dynamics.
Metalloproteinases are known to play a role in cancer EMTs.
In gastrulation EMT, several matrix metalloproteinases and
ADAM family metalloproteinases are up-regulated in the primitive streak. Further studies will be needed to show whether
their activities (release, activation, etc.) are regulated by changes
in basal cortical MTs and basal membrane–BM interaction.
In addition to DG, at least three  integrin (4, 6, andV) and three  integrin (1, 3, and 5) genes are known to be
expressed in or around the primitive streak (Nakaya et al., 2011).

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Figure 10. A model for the regulation of epiblast cell–BM interaction by CLASP and DG-mediated cortical anchoring of basal MTs during gastrulation EMT.
In epiblast cells before undergoing gastrulation EMT, basal MTs are anchored to and stabilized at the basal cortex through the CLASP–LL5 complex. DG
is distributed at the lateral and basal cell membrane, and its basal localization is dependent on basal MT stability and its interaction with the CLASP–LL5
complex. Basal cortical localization of LL5s is largely MT independent, and its interaction with basal membrane proteins like DG and integrins (not depicted)
awaits further clarification. It is hypothesized that LL5 basal localization helps enrich and stabilize basal CLASPs and consequently basal MTs. This positive
regulatory loop is disrupted as epiblast cells move toward the streak midline, resulting in the loss of basal membrane–BM interaction and consequently
BM breakdown.

Integrins, although not being the focus of this work, have also
been reported to mediate MT–BM connection through CLASP
and LL5 in human mammary epithelial cells (Hotta et al., 2010).
A likely scenario during gastrulation EMT is that DG and integrins cooperate in the regulation of basal membrane–BM interaction. Laminin 5, the major  laminin in chicken epiblasts,
has been reported to bind 1 integrin through its laminin G–likedomains 1–3 and to -DG through its laminin G–like domains
4 and 5 (Yu and Talts, 2003), thus bringing DG and integrins
into close physical proximity on the basal membrane. Molecular components associated with each receptor complex likely
differ, but their shared intracellular and extracellular interaction
partners suggest that functional cross-regulation between these
two receptors deserves careful investigation in the future.
By combining eGFP-EB1 electroporation and spinningdisk confocal microscopy, we could visualize in this work both
apical and basal cortical MT dynamics in live epiblast epithelium. Imaging of live epithelial tissue is often complicated byunique 3D architecture and physiological requirement, and its
resolution is generally poor. The chicken epiblast is well-suited
for such studies because it is a simple, flat epithelial structure
within an embryo of only 4–5 cell-layer thickness. Our timelapse movies revealed that apical cortical MTs are primarily
organized as centrosomal arrays and are dynamic. But most ofthese MTs appear to terminate at the apical regions of lateral
membrane. Basal cortical MTs appear to originate from lateral
membrane cortex, which suggests that lateral membrane structures, such as zonula adherens, may serve as noncentrosomal
MTOCs for basally targeted MTs. Technical improvement in
the future may bring us insight into the architecture and realtime behavior of basal MTs, and on whether they play a rolein targeted transport.

Materials and methods
Embryology, microscopy, and immunohistochemistry
Fertilized hens’ eggs were purchased from Shiroyama Farm. Embryos werecollected from incubated eggs and electroporated using an electroporator (TSS20 Ovodyne; Intracel) with the following parameters: 7 V, 50 ms
width, 200 ms internal, three pulses. Embryos were then further grown at
38.5°C using the standard modified New culture method (Nakaya et al.,2008; Alev et al., 2013). Embryos were then analyzed by live imaging
or immunofluorescence staining. A laser scanning confocal microscope(FV1000 with BX61WI upright; Olympus) was used for fluorescencemicroscopy with UPlan-SApochromat 60×/1.2 NA, 40×/0.9 NA, or
20×/0.75 NA objective lenses and Fluoview (Olympus) as the image
acquisition software. Olympus SZX12 (for whole-mount) and Olympus
BX51 (for sections) microscopes equipped with a DP70 digital camera
with a UPlan-SApochromat 20×/0.75 NA lens and DP Controller image
acquisition software were used for nonfluorescent imaging. All images
were taken at room temperature. For immunohistochemistry, the following
primary antibodies were used; Laminin (1:200, laminin-1; 3H11 [Developmental Studies Hybridoma Bank]; L9393 [Sigma-Aldrich]), GFP (1:1,000,
A-11122; Invitrogen), Fluorescein/Oregon green–Alexa Fluor 488 conjugate (1:50, A11090; Invitrogen), tetramethylrhodamine (1:50, A6397;
Invitrogen), -DG (1:100; 43DAG1/8D5, NCL-b-DG; Leica), pan–-tubulin(1:1,000, clone DM1A, T9026; Sigma-Aldrich), and acetylated tubulin(1:1,000, T6793; Sigma-Aldrich). Alexa Fluor 488, 568, 594, or 647
secondary antibodies (1:300 for whole-mount embryo staining, 1:500 forfrozen section staining) were used for double or triple color detection. For
chemical treatment, embryos were grown in standard New culture setting
and treated with Nocodazole (T7402; Sigma-Aldrich) or Cytochalasin D(C8273; Sigma-Aldrich) dissolved in thin albumen at a final concentrationof 5 µg/ml. A Duolink proximity ligation assay (#92102; Olink Bioscience; Söderberg et al., 2006) was performed on frozen sections of stage
HH4 embryos according to the protocol suggested by the manufacture. A
rabbit polyclonal antibody for -DG (1:100, sc-28535; Santa Cruz Biotechnology, Inc.) was used for this experiment.
Expression constructs and antisense MO oligos
Constructs encoding human CLASP1, human CLASP2 and its mutants(MC, M, and C), and human LL5 and human LL5 with eGFP tag
were provided by Y. Mimori-Kiyosue (Institute of Physical and Chemical
Research [RIKEN] Center for Developmental Biology, Kobe, Japan) andA. Akhmanova (Utrecht University, Utrecht, Netherlands; Mimori-Kiyosue

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In situ hybridization
Probes used for whole-mount in situ hybridization were obtained by RTPCR using the following primers: chick CLASP1, 5-ATGGAGCCCAGTATGGAGTA-3 (forward) and 5-TCCCAGTCGTGTTTATCATCA-3 (reverse),
1,016 bp; chick CLASP2, 5-TGGAATAGCAGACCTACGAGCA-3 (forward) and 5-AAGGGTTCCAAAAGTTGCGTGT-3 (reverse), 1,037 bp;
chick LL5 (also known as PH-like domain family B, member1 [PHLDB1];
available from GenBank under accession no. XM_417927), 5-TCGCCTGATAACATGTCCAGT-3 (forward) and 5-TGACGATGACGTCCATCCAGA-3 (reverse), 706 bp; and chick LL5 (also known as PHLDB2;
accession no. XM_416632), 5-AGCTTCAGCTATCAGATGA-3 (forward)
and 5-TTACACTGATATAGCCCTCT-3 (reverse) 1,050 bp. Whole-mountin situ hybridization was performed as described previously (Stern, 1998),
and detailed protocol for in situ hybridization can be found elsewhere(Alev et al., 2013). In brief, embryos were fixed in 4% paraformaldehyde
at 4°C overnight and stored in 100% methanol at 20°C before in situ hybridization. Embryos in methanol were rehydrated and treated with 10 µg/ml
Proteinase K for 15–30 min at room temperature, post-fixed for 30–60 min,prehybridized at 68°C for at least 2 h, and hybridized with gene-specificantisense DIG-labeled probes at 68°C overnight. After hybridization, embryos were washed in prehybridization solution a few times at 68°C and in
TBST a few times at room temperature, followed by 2 h of blocking at room
temperature, overnight incubation at 4°C in anti-DIG antibody solution,
washing, and color development.
Cell culture and immunoprecipitation
The HEK293 cell line was provided by M. Takeichi (RIKEN Center for Developmental Biology). HEK293 cells were cultured in DMEM with 10% FBS
and 1% penicillin/streptomycin (Pen Strep; Life Technologies) supplement.
Cells were transfected with expression plasmids using FuGENE HD Transfection Reagent (Roche). Immunoprecipitation was performed using Immunoprecipitation kit Dynabeads Protein G (100.07 D; Invitrogen) according to
the manufacturer’s instructions. In brief, HEK293 cells were lysed in thelysis buffer (50 mM Hepes, pH 7.4, containing 150 mM NaCl, 2 mM
EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton
X-100). Lysates, precleared with protein A/G PLUS Agarose beads (Santa
Cruz Biotechnology, Inc.), were incubated for 1 h with anti-HA antibody(16B12, ab24779; Abcam) or anti–-DG Ab (Leica) that had been coupled to Dynabeads protein G for 1 h. The beads were subsequently washedthree times in the wash buffer.
Time-lapse imaging
eGFP-labeled EB1 expression vector only or eGFP-labeled EB1 expression
vector plus the mixture of MOs of LL5s and CLASP1 were electroporated into
lateral epiblast cells. For time-lapse imaging of EB1 movement, embryos

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JCB • VOLUME 202 • NUMBER 4 • 2013

with mesoderm cells removed by gentle scratching were put on a glassbottom dish (3960-035; Iwaki), and covered with a cover-glass to avoid
drying. Time-lapse imaging was performed using a spinning-disc laser confocal unit CSU-X1 (Yokogawa) with a UPLS-Apochromat 100×/1.40 NA
lens and a cooled charge-coupled device camera (ORCA-R2; Hamamatsu
Photonics). Images were taken every 1 s for 1 min. Images were reconstituted using MetaMorph software (Molecular Devices).
Online supplemental material
Fig. S1 shows levels of homology between chicken and human CLASPs
and the mRNA expression pattern of chicken CLASPs at additional developmental stages. Fig. S2 shows the effects of CLASP2 deletion mutants onlaminin expression. Fig. S3 shows mRNA expression of LL5 and LL5 atstage HH4. Fig. S4 shows nocodazole’s effect on MT dynamics and MO’s
effect on CLASP levels and distribution. Fig. S5 shows MO’s effect ontubulin and DG distributions. Video 1 shows EB1 dynamics at the apicalside of lateral epiblast cells. Video 2 shows EB1 dynamics at the basal sideof a control MO cell. Video 3 shows EB1 dynamics at the basal side of a
triple MO cell. Videos 4 and 5 show a z-axis sequential view (4) and a 3Dview (5) of EB1 signals in a control MO cell. Videos 6 and 7 show a z-axis
sequential view (6) and a 3D view (7) of EB1 signals in a triple MO cell.
Online supplemental material is available at http://www.jcb.org/cgi/
content/full/jcb.201302075/DC1. Additional data are available in the
JCB DataViewer at http://dx.doi.org/10.1083/jcb.201302075.dv.
We thank Drs. Y. Mimori-Kiyosue (RIKEN Center for Developmental Biology)
and A. Akhmanova (Utrecht University) for sharing human CLASP 1, CLASP2,its deletion mutants, and LL5 and LL5 expression vectors; Dr. W. Weng
(RIKEN Center for Developmental Biology) for sharing 2A-eGFP expressing
vector; and Drs. M. Takeichi, S. Hayashi, S. Yonemura and Y. Mimori-Kiyosue
for critical comments of the manuscript.
This work was supported by RIKEN.

Submitted: 14 February 2013
Accepted: 8 July 2013

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et al., 2005). All fusion proteins were cloned in pEGFP-C1 vector andhad an eGFP tag fused at the C terminus. CLASP1 contains amino acidresidues 1–1,538 of human CLASP1. CLASP2 contains amino acid residues 1–1,294 of human CLASP2. CLASP2-M contains amino acid residues 36–340 and 581–1,294 of human CLASP2. CLASP2-C contains
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5-GCTGCGGGACACATCCAACAGTCAT-3; DG splicing block MO(DG-SB; 9 → 16 of EI2 junction), 5-CTTCCCAGCTTCGGAGACCTGGACT-3; Lissamine-tagged CLASP1 translation block MO (1 → 25 of translation initiation junctions), 5-AGCAGTACTCCATACTGGGCTCCAT-3;
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