Baf60c is a nuclear Notch signaling component required for the establishment of left–right

asymmetry

Jun K. Takeuchi, Heiko Lickert, Brent W. Bisgrove, Xin Sun, Masamichi Yamamoto, Kallayanee
Chawengsaksophak, Hiroshi Hamada, H. Joseph Yost, Janet Rossant, and Benoit G. Bruneau
PNAS 2007;104;846-851; originally published online Jan 8, 2007;
doi:10.1073/pnas.0608118104
This information is current as of January 2007.

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Notes:
Baf60c is a nuclear Notch signaling component
required for the establishment of
left–right asymmetry
Jun K. Takeuchi*†‡, Heiko Lickert§, Brent W. Bisgrove¶, Xin Sun*†储, Masamichi Yamamoto**,
Kallayanee Chawengsaksophak†, Hiroshi Hamada**, H. Joseph Yost¶, Janet Rossant†储,
and Benoit G. Bruneau*†储††
Departments of *Cardiovascular Research and †Developmental Biology, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8;
储Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; §National Research Center for Environment and

Health, Institute of Stem Cell Research, Neuherberg 85764, Germany; ¶Huntsman Cancer Institute, Center for Children, Department of Oncological Sciences,
University of Utah, Salt Lake City, UT 84112; and **Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, 1-3
Yamada-oka, Suita, Osaka 565-0871, Japan

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 27, 2006 (received for review
September 14, 2006)

Notch-mediated induction of Nodal at the vertebrate node is a Here, we show that loss of Baf60c in mouse and zebrafish leads
critical step in initiating left–right (LR) asymmetry. In mice and to defects in the establishment of the LR asymmetry cascade. In
zebrafish we show that Baf60c, a subunit of the Swi/Snf-like BAF mouse embryos, this is because of impaired activation of Nodal
chromatin remodeling complex, is essential for establishment of LR at the node. We further show that Baf60c is an essential
asymmetry. Baf60c knockdown mouse embryos fail to activate component of nuclear Notch signaling and propose that the
Nodal at the node and also have abnormal node morphology with integration of nuclear Notch signaling components by BAF
mixing of crown and pit cells. In cell culture, Baf60c is required for complexes is a critical mechanism for transcriptional activation
Notch-dependent transcriptional activation and functions to sta- of Nodal in the mouse node, and perhaps for Notch-dependent
bilize interactions between activated Notch and its DNA-binding transcription in general.
partner, RBP-J. Brg1 is also required for these processes, suggesting
that BAF complexes are key components of nuclear Notch signal- Results
ing. We propose a critical role for Baf60c in Notch-dependent LR Defects in Smarcd3 Knockdown Embryos. In our analysis of
transcription and LR asymmetry. altered cardiogenesis in Smarcd3 short hairpin RNA (shRNA)
knockdown embryos (14), we noticed that the situs of the heart
chromatin 兩 Swi/Snf 兩 node
and direction of embryonic turning in the most severe knock-
down line (A#1) were randomized (Fig. 1 A and B). Lower

T he bodies of all vertebrates are asymmetric on the left and

right sides, resulting in distinct situs of organs, such as the
left-pointing heart. Proper regulation of left–right (LR) asym-
penetrance of looping defects was also observed for another
knockdown line. Expression of key asymmetrically expressed
regulators of the LR cascade (Lefty1, Lefty2, and Pitx2) was
metry is necessary for normal organ positioning during embry- undetectable in Smarcd3 knockdown embryos (Fig. 1 C and D,
onic development (1, 2). Strong genetic and cell biologic evi- n ⫽ 5). Nodal expression around the node is essential for lateral
dence in mammals suggests that the critical events in breaking plate mesoderm (LPM) expression of LR genes (3–6). Perinodal
symmetry take place at the node, an important organizer expression of Nodal was lost in Smarcd3 knockdown embryos
structure of the vertebrate embryo. These symmetry-breaking (Fig. 1E, n ⫽ 5). Expression of Notch1 was reduced in Smarcd3
events include the asymmetric movement of fluids across the
knockdown embryos (n ⫽ 2), whereas Delta1 (Dll1) mRNA was
node (so-called nodal flow) and the Notch pathway-dependent
increased (Fig. 1 G and I, n ⫽ 2).
activation of the secreted protein Nodal in cells surrounding the
We previously reported that Smarcd3 was cardiac-specific
node (1–7). This is followed by a cascade of secreted factors and
during development (14). Careful examination of Smarcd3
transcription factors restricted to the left side of the embryo,
establishing left-sided identity and organ situs (1, 2). mRNA distribution in the mouse embryos showed clear expres-
The Swi/Snf-like BAF chromatin remodeling complexes are sion in Nodal-expressing perinodal cells in mouse embryos (Fig.
important regulators of transcription during development (8).
BAF complexes are large multisubunit assemblies that are Author contributions: J.K.T., H.L., and B.W.B. contributed equally to this work.; J.K.T., H.L.,
characterized by polymorphic components; e.g., the core B.W.B., J.R., and B.G.B. designed research; J.K.T., H.L., B.W.B., X.S., M.Y., and K.C. performed
ATPase of the complexes can be either Brahma (Brm) or research; M.Y. contributed new reagents/analytic tools; J.K.T., H.L., B.W.B., X.S., H.H.,
Brahma-related gene 1 (Brg1) (8–10). Another example of this H.J.Y., J.R., and B.G.B. analyzed data; and J.K.T., H.L., B.W.B., and B.G.B. wrote the paper.

combinatorial assembly of BAF complex subunit composition is The authors declare no conflict of interest.

the 60-kDa subunit Baf60. Baf60 is found in most BAF com- This article is a PNAS direct submission.
plexes, although it is not essential for the chromatin remodeling Abbreviations: LR, left–right; LPM, lateral plate mesoderm; shRNA, short hairpin RNA; KV,
function of the complex. It can be represented by Baf60a, Kupffer’s vesicle; MO, morpholino; NDE, node-dependent enhancer; ASE, asymmetry
enhancer; En, embryonic day n.
Baf60b, or Baf60c, which are encoded by the Smarcd1, Smarcd2, ‡Present address: Gladstone Institute of Cardiovascular Disease and University of California,
and Smarcd3 genes, respectively (10). Baf60 proteins have been San Francisco, CA 94158.
shown to interact with transcription factors, including nuclear ††Towhom correspondence should be sent at the present address: Gladstone Institute of
receptors, the AP-1 complex, and others, and are thought to Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158. E-mail:
bridge interactions between these transcription factors and BAF [email protected].
complexes (11–14). Very little is known about the developmental This article contains supporting information online at www.pnas.org/cgi/content/full/
or physiological roles played by Baf60 proteins. Baf60c was 0608118104/DC1.
recently shown to be critical for heart development (14). © 2007 by The National Academy of Sciences of the USA

846 – 851 兩 PNAS 兩 January 16, 2007 兩 vol. 104 兩 no. 3 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0608118104
 Fig. 2. Abnormal node morphology in Smarcd3 knockdown embryos. (A–D)
Expression of Cer2 and Gdf1 in wild-type and Smarcd3 knockdown (Si) em-
bryos. Red arrowheads show decreased or missing expression. (E–K) Scanning
electron microscopy shows high magnification of the node in wild type (E),
abnormal node morphology in Smarcd3 knockdown (F and G), and Rbpsuh⫺/⫺
(H) embryos at E7.75. c, crown cells; p, pit cells. Smarcd3 knockdown results in
abnormal node morphology, with separated pit cells (F, red asterisks) and
abnormally migrated crown cells (yellow circles). Some Smarcd3 mutants also
have abnormal leftward shifted nodes in addition (G, red line shows embry-
onic midline). Abnormal crown cells were seen in Rbpsuh⫺/⫺ embryos (H,
yellow circles). (I–L) Expression of Cryptic and Foxa2 in wild-type and Smarcd3
knockdown (Si) embryos.

Fig. 1. Baf60c regulates LR asymmetry at the node. (A) Smarcd3 knockdown sion in Smarcd3 knockdown embryos; Nodal expressed from the
caused randomized heart looping. Red arrows indicated the direction of heart
transfected expression construct could be detected as strong
looping. (B) Histogram shows the percentage of control (siTbx20), Smarcd3
knockdown (siSmarcd3), and Rbpsuh⫺/⫺ mutant embryos with rightward,
punctate staining in a few cells, whereas Nodal-induced endog-
linear, and leftward heart morphologies. (C–E) Expression of LR regulators in enous Nodal was fainter and broader throughout the LPM.
wild-type (wt) and Smarcd3 knockdown (Si) embryos. Shown are Lefty1/2 (C), Because endogenous Nodal could be induced by Nodal misex-
Pitx2 (D) at E8.25, and Nodal (E) at E7.75. (F–I) Decreased expression of Notch1 pression in the right LPM of wild-type and Smarcd3 knockdown
and increased expression of Dll1 in siSmarcd3 embryos. (J–O) Smarcd3 expres- embryos (n ⫽ 6), we conclude that Baf60c is not important for
sion in perinodal cells and coexpression with Nodal by in situ hybridization on the response of the LPM to Nodal (SI Fig. 6 J–L).
consecutive sections at E7.75 (O). Red lines in K show the plane of sections
Our results together indicate that Baf60c function at the

DEVELOPMENTAL
shown in L and M.
mouse node, similar to that of Notch signaling, is critical for

BIOLOGY
the initiation of Nodal expression and thus the progression of the
1 J–O). Bilateral expression of Smarcd3 expanded beyond the LR asymmetry cascade.
node into the LPM, similar to that of Notch1 (Fig. 1J).
We tested the specificity of the Smarcd3 knockdown in Defects in Node Morphology in Smarcd3 Knockdown Embryos. Other
cultured embryos with localized Lipofectamine-mediated genes important for LR determination at the node (Cer2 and
shRNA Smarcd3 knockdown [supporting information (SI) Fig. 6 Gdf1) (18, 19) were detected in Smarcd3 knockdown embryos
B and C, n ⫽ 4] (15). Localized application of Smarcd3 shRNA- (Fig. 2 A–D, n ⫽ 3), although Cer2- and Gdf1-expressing crown
expressing plasmids resulted in loss of Nodal in transfected cells cells did not properly align around the node, reflecting abnormal
(SI Fig. 6 B and C). We further tested the specificity of the node morphology. Indeed, scanning electron microscopy showed
knockdown by examining heart situs and Nodal expression in abnormal node morphologies in Smarcd3 mutants (Fig. 2 F and
Smarcd3 knockdown embryos expressing a Baf60b-IRES-EGFP G). Abnormal crown cells were seen (Fig. 2F, yellow circles), and
transgene under control of the CMV enhancer/␤-actin promoter node pit cells were separated into noncohesive groups (Fig. 2F,
(CAGGS-Baf60b-IR ES-EGFP) (14). Although CAGGS- red asterisks). In ⬇30% of mutant embryos, the node itself was
Baf60b-IRES-EGFP is expressed at higher levels in the heart shifted to the left (Fig. 2G). Monociliated pit cells were apparent
(14), it also expresses homogeneously at lower levels throughout in Smarcd3 knockdown embryos, and by video microscopy the
the embryo (SI Fig. 6 E and H) (16). Nodal expression was largely cilia in Smarcd3 knockdown embryos (n ⫽ 4) were motile, but
rescued in Smarcd3 knockdown CAGGS-Baf60b-IRES-EGFP their motion was restricted, and flow across the node was
embryos (SI Fig. 6 F and I, n ⫽ 5), as was cardiac looping [100% severely impaired (data not shown). The midline markers Foxa2
leftward looping at embryonic day 9.5 (E9.5); n ⫽ 10]. Further- (n ⫽ 2) and Shh (n ⫽ 4) were expressed in Smarcd3 knockdown
more, ES cell–tetraploid complementation method or heart embryos (Fig. 2 I–L). We believe that these defects in node
defects did not affect the LR pathway, as ES cell-derived morphology and flow are secondary to altered crown cell
embryos lacking the cardiac transcription factor Tbx20 (17) had morphology, as has been proposed for mice lacking the Notch
normal heart situs (Fig. 1B). ligand Dll1, which also have LR defects (20). We observed
We tested whether Baf60c is important in the LPM for similar heart looping and node morphology defects in mouse
receiving Nodal signals from the node by misexpressing Nodal in embryos derived from ES cells homozygous for a deletion of
the right LPM (SI Fig. 6 J–L, n ⫽ 6). This resulted in bilateral Rbpsuh (Figs. 1B and 2H) (6), which encodes RBP-J, the nuclear
Nodal expression in wild-type embryos and right-sided expres- effector of Notch signaling (21, 22). We do not believe that the

Takeuchi et al. PNAS 兩 January 16, 2007 兩 vol. 104 兩 no. 3 兩 847
Fig. 3. Baf60c is sufficient to induce Nodal. Cultured embryos transfected
with an EGFP expression construct and Baf60c, activated Notch (NIC), or Nodal
expression constructs, or EGFP vector alone (control) were cultured for 15 h.
(A, D, G, and I) EGFP fluorescence, showing transfected cells. (A–C) Control
studies showed normal/bilateral nodal (B) at the node and Notch1 (C) expres-
sion around the node. (D–H) Smarcd3-transfected embryos: ectopic Nodal
induction along the midline was seen (E and H), and expansion of Notch1
expression was also observed (F, red arrowhead). (I and J) Overexpression of
NIC enhanced Nodal expression. (K) Histogram shows the length (in millime-
ters) of Nodal expression on the node between the left side (light purple,
control) and the right side (light blue, injected experiments, n ⫽ 5).

Fig. 4. Smarcd3 is required for LR axis specification in zebrafish. (A–E)

loss of Nodal-expressing cells would be the main cause of
Expression of smarcd3 (A–C and E are lateral views, anterior to left, and D is a
disruption in LR asymmetry, because the loss of Nodal mRNA caudal view, dorsal to top). (A) During gastrulation. (B) Expression in the
is much broader than the disruptions in crown cell integrity as developing tailbud. (C and D) Early somitogenesis. (E) Mid-somitogenesis.
demonstrated by loss of other perinodal markers. Thus, Baf60c Knockdown of smarcd3 randomizes direction of heart looping in embryos 40 h
regulates perinodal Nodal expression and node morphology, after fertilization (F–H). A, atrium; V, ventricle. In H, red bars show normal
similar to the Notch signaling pathway. looping and blue bars show reversed looping. Heart looping is partially
rescued by smarcd3 RNA. (I–L) Altered expression of lefty1 (lft1) in the dien-
Misexpression of Smarcd3 Expands the Field of Nodal Expression. cephalon and lefty2 (lft2) in the heart field of 22- to 24-somite-stage embryos.
(I) Normal left-sided expression. (J) Right-sided expression. (K) Bilateral ex-
Lipofectamine-mediated misexpression of Baf60c to the right of
pression. (L) Absence of expression. Expression of lft1 in the midline is unal-
the node of cultured mouse embryos resulted in expanded Nodal tered. (M–R) Quantification of alterations in asymmetric gene expression
induction along the anteroposterior axis (Fig. 3 E and H, red patterns in smarcd3 morphants. (M and P) southpaw (spaw) expression in the
arrowheads, n ⫽ 4) and slightly expanded Notch1 expression LPM (19- to 21-somite stage). (N and Q) lft1 expression. (O and R) lft2 expres-
(Fig. 3F, red arrowheads, n ⫽ 2), whereas activated Notch1 sion. L, left-sided; R, right-sided; B, bilateral; A, absent.
(NIC) misexpression resulted in enhanced Nodal induction at the
node without expansion of its expression domain (Fig. 3J, n ⫽ 5).
This indicates that Baf60c is a limiting factor in Notch-mediated beginning at later shield/70% epibody (Fig. 4A). At the end of
induction of Nodal. These in vivo misexpression studies strongly gastrulation (bud stage) smarcd3 is highly expressed in a band of
suggested that Baf60c acts as a critical Nodal inducer at the node six to eight cell diameters that surrounds the location of the
and is thus a key factor in the establishment of LR asymmetry. dorsal forerunner cells, the precursors of Kupffer’s vesicle (KV),
the ciliated organ of asymmetry (analogous to the mouse node)
Zebrafish Smarcd3 Regulates LR Asymmetry. To address conserva- in the zebrafish (Fig. 4B). During early somitogenesis (4–10 s),
tion of Baf60c function during vertebrate LR asymmetry deter- when LR patterning is being established, smarcd3 is strongly
mination, we used zebrafish (Danio rerio) embryos, because early expressed in the notochord and in cells surrounding the KV (Fig.
LR pathways are generally conserved between mammals and fish 4 C and D). At later stages smarcd3 is restricted to the KV, eye,
(1, 2, 7). A candidate for the zebrafish orthologue of Smarcd3 midbrain, and forebrain (Fig. 4E). Morpholino (MO) knock-
was identified in the region of zebrafish chromosome 7 syntenic down of Smarcd3 resulted in 36% of embryos with reverse
to the mouse Smarcd3 locus. Zebrafish smarcd3 is first expressed looping heart morphologies (n ⫽ 249; vs. 1.3% for control MO,
in a band of three to four cell diameters at the blastoderm margin n ⫽ 360) (Fig. 4 F–H); this phenotype was partly rescued (14%

848 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0608118104 Takeuchi et al.

 DEVELOPMENTAL
BIOLOGY
Fig. 5. Baf60c potentiates transcription by promoting interaction within nuclear Notch complexes and BAF complexes. (A) Baf60c activates the Nodal-NDE-
luciferase reporter construct and synergizes with activated Notch (NIC). Baf60c or NIC do not activate Nodal-mutNDE-luciferase (black bars), which has mutated
RBP-J binding elements. ⫹, 250 ng of expression construct; ⫹⫹, 500 ng of expression construct. (B) Depletion of Brg1 (siBrg1) or Baf60c (siBaf60c) by RNAi
abrogates the NIC-dependent activation of Nodal-NDE-luciferase. siTbx20 is a control for nonspecific effects of RNAi. (C) Baf60c potentiates transcriptional
activation of Hes1-luciferase with NIC. (D) Baf60c does not enhance Nodal-ASE, Lefty2-ASE, or Pitx2-ASE luciferase reporters, which are stimulated by activated
TGF␤ signaling and Fast2. (E) Coimmunoprecipitation of Flag-RBP-J or Myc-NIC, alone or with a Baf60c expression construct, followed by Baf60c immunodetection
shows that Baf60c interacts weakly with NIC and strongly with RBP-J. (F) GST pull-down of 35S-labeled RBP-J, NIC, Tbx5, or Smad4. GST-Baf60c could pull down
RBP-J and Tbx5 but not NIC or Smad4. Fifty percent input is shown below the pull-down. (G) Coimmunoprecipitation of Flag-RBP-J or Myc-NIC, cotransfected
with Baf60c, followed by immunodetection of Brg1 shows that Brg1 can interact with NIC but not with RBP-J. The Brg1/RBP-J interaction depends on Baf60c,
and Brg1/NIC/RBP-J interaction is strengthened by Baf60c. (H) Interaction of NIC with RBP-J is enhanced by Baf60c. Depletion of endogenous Baf60c (siSmarcd3)
or Brg1 (siBrg1) destabilized the interaction of RBP-J with NIC in 10T1/2 cells. (I) Model for the integration of Notch signaling by Baf60c.

reverse looping, n ⫽ 138) by coinjection of smarcd3 mRNA (Fig. for spaw using Smarcd3 MO) their expression was completely
4H). Similarly, knockdown of Smarcd3 using a splice-blocking lost (Fig. 4 M–R), similar to the response in Smarcd3 shRNA
MO (Smarcd3 SpMO) resulted in 27% of embryos with reversed knockdown mouse embryos. Midline markers including lft1 (Fig.
heart looping (n ⫽ 159; vs. 1% for uninjected controls, n ⫽ 173). 4 I–L), ptc1, shh, and ntl (data not shown) were intact.
Expression of left-side-specific genes [lefty1 (lft1), lefty2 (lft2),
and southpaw (spaw)] (23, 24) was also altered (Fig. 4 I–R). In Baf60c Functionally and Physically Interacts with Nuclear Notch Com-
smarcd3 morphants, expression of lft1, lft2, and spaw was abnor- ponents. The Nodal node-dependent enhancer (NDE) relies on two
mally right-sided or bilateral, and in ⬇30–50% of embryos (10% RBP-J binding elements for its activity in the node (5, 6). We

Takeuchi et al. PNAS 兩 January 16, 2007 兩 vol. 104 兩 no. 3 兩 849
examined the role of Baf60c in the transcriptional activation of the Nodal and Lefty diffusion has been proposed to explain the robust
Nodal-NDE by transient luciferase reporter assay in 10T1/2 cells. establishment of left-sided gene expression (28). This model pre-
Nodal-NDE-luciferase was activated dose-dependently by NIC as dicts that slight differences in left-sided expression or diffusion of
well as Baf60c (Fig. 5A), and the combination of Baf60c and NIC Nodal from the node would disrupt the normal LR pattern; it is
synergistically activated Nodal-NDE-luciferase. Activation by NIC possible that in the Smarcd3 zebrafish morphants spaw expression
or Baf60c depended on intact RBP-J binding sites, suggesting that surrounding the KV is partly reduced, thus still leading to disrupted
Baf60c and NIC interact to directly activate the NDE. Depletion of LR patterning via an imbalance in the self-enhancement/lateral
endogenous Baf60c or Brg1 by shRNAs greatly reduced the re- inhibition system, albeit in a pattern that differs from the mouse
sponse of Nodal-NDE-luciferase to NIC (Fig. 5B), indicating a embryos in which Nodal expression was eliminated. Regardless of
critical requirement for Baf60c and the BAF complex. Partial the precise mechanism, it is certain that Baf60c has important roles
inhibition by Baf60c RNAi may indicate compensation by other in regulating the LR pathway in both species. Interestingly, a
Baf60 proteins. Hes1-luciferase, a well characterized target of Notch portion of the Smarcd3 knockdown hearts did not loop at all,
that also relies on RBP-J sites (25), was also synergistically activated reminiscent of hearts in which the left-sided regulator of organ
by Baf60c and NIC (Fig. 5C). LPM expression of Nodal, Lefty1/2, sidedness Pitx2 has been misexpressed (29). This may be an
and Pitx2 (which are absent in Smarcd3 knockdown embryos) indicator of a linkage of Baf60c and cell-autonomous organ
depends on asymmetry enhancers (ASE) that are activated by orientation.
TGF␤/FoxH1 (26, 27). TGF␤/FoxH1-dependent activation of Nod- We also found that Smarcd3 knockdown mouse embryos had
al-ASE, Lefty2-ASE, and Pitx2-ASE luciferase constructs was not defects in node morphology, including defective arrangement of
affected by Baf60c (Fig. 5D). This finding supports the Nodal perinodal crown cells and nodal pit cells. Zebrafish smarcd3
misexpression data indicating that Baf60c is not involved in medi- knockdown did not result in any anomalies in the KV in terms
ating TGF␤-mediated induction of genes in the LPM. These results of morphology or acetylated tubulin staining of cilia (data not
therefore reveal that Baf60c is a critical mediator of Notch signaling shown). The abnormalities in node morphology may contribute
and that RBP-J or its DNA binding element is necessary for Baf60c to the defective LR patterning in Smarcd3 knockdown embryos,
function. but the primary defect appears to be the absence of Nodal
Immunoprecipitation experiments in transfected cells re- expression in perinodal cells.
vealed a strong interaction of Baf60c with RBP-J and a much
weaker interaction with NIC (Fig. 5E). This finding was con- Baf60c and the BAF Complex Functionally Interact with Notch Signal-
firmed by GST pull-down assays (Fig. 5F). We tested whether ing. Notch signaling at the mouse node is a primary regulator of
Baf60c potentiated interactions between NIC or RBP-J and Brg1 Nodal transcription via two conserved RBP-J binding sites (5, 6).
by coimmunoprecipitation: Brg1 interacted with NIC whether or Notch signaling also regulates Nodal at the node in chick embryos
not Baf60c was coexpressed; however, Baf60c was necessary for (30) and has been implicated in LR asymmetry in zebrafish as well
an association of Brg1 and RBP-J (Fig. 5G). Furthermore, (6, 31). In the mouse it is likely that the function of Baf60c in node
Baf60c could enhance an interaction between Brg1 and NIC/ morphogenesis is also partly related to Notch signaling, because we
RBP-J in this system. Interestingly, the interaction between NIC found similar defects in Rbpsuh⫺/⫺ embryos, and similar abnormal
and RBP-J in10T1/2 cells was also strongly enhanced by Baf60c, nodes are seen in mice lacking the Notch ligand Delta-1 (5, 20). The
and, most importantly, depletion of endogenous Baf60c desta- clear involvement of Notch signaling in Nodal regulation and node
bilized the NIC/RBP-J interaction, as did depletion of Brg1 (Fig. morphogenesis prompted us to examine a possible functional
5H). We conclude from these results that Baf60c is a critical relationship between Baf60c and Notch. Our combined biochem-
nuclear factor in Notch signaling that promotes interactions ical data strongly suggest that the BAF complex interacts with
between activated Notch and its nuclear partner RBP-J and NIC/RBP-J and that this interaction is promoted by Baf60c. Inter-
enhances the interaction of the NIC/RBP-J complex with the estingly, the stability of the interaction between NIC and RBP-J was
BAF complexes. We propose that Baf60c interacts preferentially critically dependent on the presence of endogenous Baf60c, sug-
with RBP-J, thus creating a bridge between RBP-J and the BAF gesting not only that there was an interaction between BAF
complex. Furthermore, because NIC interacts with Brg1 (and complexes and NIC/RBP-J, but that the recruitment of the BAF
thus presumably the BAF complex), we propose that four complex is absolutely critical for the stability of the NIC/RBP-J
interactions are important for stabilization of the nuclear Notch interaction. Thus, we propose that Baf60c and the BAF complex are
signaling complex: (i) NIC with RBP-J, (ii) NIC with Brg1, (iii) integral mediators of Notch signaling in the nucleus via stabilization
Baf60c with Brg1, and (iv) Baf60c with RBP-J (Fig. 5I). The of NIC/RBP-J interaction and possibly remodeling of chromatin at
aggregate effect of these interdependent interactions is the sites bound by NIC/RBP-J.
formation of a stable complex at RBP-J binding sites that The finding that stabilization of NIC and RBP-j interactions is
includes the BAF chromatin remodeling complex. mediated by Baf60c and the BAF complex is perhaps unexpected
considering that, to date, no requirement for a stabilizing protein in
Discussion nuclear Notch signaling has been demonstrated. Biochemical data
Baf60c Regulate LR Asymmetry at the Node. We have shown that demonstrate strong interactions between NIC and RBP-j in vitro,
embryos with a specific knockdown of Smarcd3, encoding Baf60c, and they are readily isolated together from cells by coimmunopre-
have defects in the establishment of the LR asymmetry pathway due cipitation (22, 25, 32–34). However, these data collectively do not
to impaired induction of Nodal at the mouse node. Nodal expres- exclude a requirement for a stabilizing effect of other proteins,
sion in perinodal cells is essential for the initiation of the down- because in vitro data solely demonstrate that two proteins can
stream cascade of gene expression that establishes the left side of interact sufficiently well under experimental conditions, and im-
the embryo as distinct from the right side, including the expression munopurification from cells by default includes other potential
of Nodal itself in the LPM (3, 4). Our data show that the role of cellular components. In the case of the BAF complex, compelling
Baf60c in regulating LR asymmetry is conserved between mouse evidence in other organisms suggests that this function may be a
and zebrafish, suggesting a similar mode of action. However, general feature of Notch signaling and may extend to invertebrates:
whereas in mouse we consistently observed a loss of left-sided gene in Caenorhabditis elegans, ZK1128.5, encoding a Baf60c ortholog,
expression, in zebrafish we also noted bilateral and reversed gene genetically interacts with Notch signaling (35), and in a screen for
expression. Clear differences exist between species in the regulation genetic interactions with Drosophila Brahma, encoding the Brg1/
of the LR pathway from the node or its orthologous structures (1, Brm homolog, genes encoding Notch signaling components were
2). Recently, a self-enhancement/lateral inhibition model based on the predominant class of mutations identified (36). Taken together

850 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0608118104 Takeuchi et al.

these results suggest that Baf60c and the BAF complex is a previously described (14). Nodal, Pitx2, and Lefty2 enhancers (5, 26,
conserved key cellular nuclear component of Notch signaling in 27) were subcloned into pGL3 to generate luciferase reporter
several contexts, including the establishment of LR asymmetry in constructs. Anti-Brg1 (Upstate Biotechnology, Lake Placid, NY),
mammals. Our findings may have important implications for inter- anti-FLAG (Sigma, St. Louis, MO), anti-HA (Sigma), and anti-myc
action of the Notch pathway and Baf60c in cardiogenesis, because (Santa Cruz Biotechnology, Santa Cruz, CA) antisera were com-
Baf60c is a critical factor for heart morphogenesis (14), and Notch mercially obtained; anti-Baf60c antiserum (12) was kindly provided
signaling is important for cardiac differentiation in Xenopus (37), in
by J. Auwerx (Institut de Génétique et de Biologie Moléculaire et
ES cells (38), and as a causative factor in human congenital heart
defects (39). Our findings overall suggest a mechanism for Cellulaire, Illkirch, France).
transcriptional potentiation of critical signaling pathways in
development. GST Pull-Down Assay. GST and GST-Baf60c were expressed in
BL21 Escherichia coli. GST protein was purified from cell lysates
Methods with glutathione Sepharose 4B beads (Amersham). Proteins
Mouse in Vivo RNAi and Transgenesis. In vivo RNA interference for were translated in vitro and labeled with [35S]methionine by using
Smarcd3 and tetraploid aggregations was performed as previously a reticulocyte lysate system (Promega). Labeled proteins were
described (14). Embryo transfection was performed on E7–E7.5 incubated with GST or GST-Baf60c beads overnight at 4°C. The
embryos by using Lipofectamine 2000 as previously described (15). beads were washed, mixed with SDS loading buffer, and heated
Rbpsuh⫺/⫺ ES cells were generated from Rbpsuh⫹/⫺ ES cells (40) to 100°C. The bound proteins were analyzed by SDS/PAGE. The
selected in medium containing high levels of G418. Nodal flow was gel was dried and exposed to autoradiograph film overnight.
assessed in mouse embryos as described in ref. 41.
We thank S. McMaster (Mount Sinai Hospital Research Institute,
MO Knockdown of Smarcd3 Function. Translation-blocking (0.6 ng) University of Toronto, Toronto, ON, Canada) for tetraploid aggrega-
and splice-blocking (4 ng) MO antisense oligonucleotides (Gene tions; D. Holmyard for scanning electron microscopy; J. N. Campbell and
Tools, Philomath, OR) were used to knock down the function of B. L. McMahan for technical assistance; T. Honjo (Kyoto University,
smarcd3: smarcd3 MO is complementary to a region immedi- Kyoto, Japan) for Rbpsuh⫹/⫺ ES cells; and A. Israel (Institute Pasteur,
ately upstream of the translation start site 5⬘-TTCCCTCCGCT- Paris, France), R. Kopan (Washington University, St. Louis, MO), and
TCTCCTGCCTTTTG-3⬘. smarcd3 SpMO is complementary to J. Wrana (Mount Sinai Hospital Research Institute) for expression and
the splice donor site of exon 3, 5⬘-TCAGATCTCTTACTCAC- reporter constructs. This research was funded by the Canadian Institutes
CCTTTGTG-3⬘. The MO phenotype was rescued by coinjection of Health Research (B.G.B. and J.R.), the Heart and Stroke Foundation
of smarcd3 MO with 75 pg of in vitro synthesized smarcd3 RNA of Ontario (B.G.B.), March of Dimes Birth Defects Foundation Grant
containing the ORF of Smarcd3, with the 5⬘ UTR truncated to 1-FY05-117 (to B.G.B.), an Emmy Noether fellowship of the Deutsche
remove 18 bp of sequence recognized by the MO. Forschungsgemeinschaft (to H.L.), and a Human Frontiers Science
Program long-term fellowship (to J.K.T.). J.R. is a Canadian Institutes
Transactivation Assays and Immunoprecipitation. Transactivation of Health Research Distinguished Scientist. B.G.B. held a Canada
assays and coimmunoprecipitation experiments were performed as Research Chair in Developmental Cardiology.

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Takeuchi et al. PNAS 兩 January 16, 2007 兩 vol. 104 兩 no. 3 兩 851