Brg1 governs distinct pathways to direct multiple
aspects of mammalian neural crest cell development
Wei Lia,1, Yiqin Xionga,1, Ching Shanga, Karen Y. Twua, Calvin T. Hanga, Jin Yanga, Pei Hana, Chieh-Yu Lina,
Chien-Jung Lina, Feng-Chiao Tsaib, Kryn Stankunasa, Tobias Meyerb, Daniel Bernsteinc, Minggui Pana,
and Ching-Pin Changa,2
aDivision of Cardiovascular Medicine, Department of Medicine,bDepartment of Chemical and Systems Biology, andcDivision of Pediatric Cardiology,
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved December 12, 2012 (received for review October 18, 2012)
Development of the cerebral vessels, pharyngeal arch arteries
(PAAs). and cardiac outflow tract (OFT) requires multipotent neural
crest cells (NCCs) that migrate from the neural tube to target tissue
destinations. Little is known about how mammalian NCC develop-
ment is orchestrated by gene programming at the chromatin level,
however. Here we show that Brahma-related gene 1 (Brg1), an
ATPase subunit of the Brg1/Brahma-associated factor (BAF) chro-
matin-remodeling complex, is required in NCCs to direct cardiovas-
cular development. Mouse embryos lacking Brg1 in NCCs display
immature cerebral vessels, aberrant PAA patterning, and shortened
OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating
kinase 1 (Ask1), and a cell cycle inhibitor, p21cip1, to inhibit apoptosis
cell reservoir at the neural crest. Brg1 also supports Myosin heavy
chain 11 (Myh11) expression to allow NCCs to develop into mature
vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1
partners with chromatin remodeler Chromodomain-helicase-DNA-
binding protein 7 (Chd7) on the PlexinA2 promoter to activate Plex-
inA2, which encodes a receptor for semaphorin to guide NCCs into
the OFT. Our findings reveal an important role for Brg1 and its
downstream pathways in the survival, differentiation, and migra-
tion of the multipotent NCCs critical for mammalian cardiovascular
embryo, where they differentiate into a variety of local cells,
including cardiovascular tissues (1). NCCs that emigrate from
the neural crest of rhombomere 6–8 to pharyngeal arches and
the heart are essential for the patterning of pharyngeal arch
arteries (PAAs) and the cardiac outflow tract (OFT) (2, 3).
These NCCs also differentiate into vascular smooth muscle cells
(SMCs) of PAAs and the muscular septum of the aorta and
pulmonary trunk (4, 5). In contrast, NCCs from the cephalic
neural tube migrate to the face and forebrain to form craniofa-
cial bones, as well as SMCs of facial and forebrain vessels (6).
Thus, NCCs are critical for the formation of cardiac OFT and
vascular supplies of large areas of the body.
Disruption of NCC development, either directly or indirectly,
results in many forms of human birth defects with cardiovascular
malformations, including Alagille, Carpenter, Ivemark, Leopard,
Williams, DiGeorge, and CHARGE syndromes (7). These syn-
dromes involve defects in PAAs or cardiac OFT, such as co-
arctation of the aorta, interrupted aortic arch, pulmonary artery
stenosis, double-outlet right ventricle, tetralogy of Fallot, or
persistent truncus arteriosus. During PAA and OFT de-
velopment, NCCs are regulated by numerous transcription fac-
tors, including Pax3, Pbx1/2/3, Tbx1/2/3/20, Msx1/2, Hand2, AP-
2a, Cited2, Pitx2, Sox4, Foxc1/c2/d3/h1, Fog2, Gata3/4/6, and
Notch/NICD (8). Such extensive involvement of transcription
factors indicates the importance of gene programming in NCC,
PAA, and OFT development.
DNA is tightly packed by chromatin, and the access of tran-
scription factors to genomic loci depends on the chromatin
eural crest cells (NCCs) originate from the neural crest of
the dorsal neural tube and migrate to many regions of the
structure. Chromatin thus acts as a major controller of gene
expression. Chromatin structure can be altered by covalent his-
tone modifications through histone-modifying enzymes or by
changes in nucleosome position and composition through ATP-
dependent chromatin-remodeling factors. Despite the importance
of chromatin regulation and NCC-related human diseases, little is
known about how NCCs are programmed at the chromatin level
for cardiovascular development (8–10). Our studies demonstrate
a cell-autonomous function of a chromatin remodeler, Brg1, in
NCCs and downstream pathways to orchestrate NCC development
Brahma-related gene 1 (Brg1) is an essential ATPase subunit
of the Swi/Snf-like BAF chromatin-remodeling complex in ver-
tebrates (11). Brg1 hydrolyzes ATP to drive the chromatin
remodeling activity of the BAF complex. A recent study in-
directly linked Polybromo-BAF (PBAF) (containing Brg1) to the
pathogenesis of CHARGE syndrome (12), characterized by
coloboma, heart defects, atresia choanae, retarded growth and
development, genital hypoplasia, and ear abnormalities/deaf-
ness. CHARGE syndrome is caused by haploinsufficiency of a
chromodomain chromatin-remodeling factor, Chromodomain-
Helicase-DNA-binding protein 7 (CHD7) (13), and includes
cardiovascular defects in PAA and OFT (14). Although Chd7
knockdown in frog embryos causes abnormal OFT positioning,
and Chd7 associates with PBAF in frog embryos (12), there is no
direct evidence of the need for Brg1 in NCCs for PAA and OFT
development in frogs or mice.
Through tissue-specific deletion of Brg1 in NCCs, our studies
demonstrate a cell-autonomous role of Brg1 in NCCs for the
development of cerebral vessels, PAAs, and cardiac OFT in
mice. In addition, we identified molecular pathways downstream
of Brg1 that control cell apoptosis, proliferation, differentiation,
and migration of NCCs.
Deletion of Brg1 in NCCs Results in Embryonic Lethality. To test Brg1
function in NCCs, we deleted Brg1 by crossing mice carrying
a loxP-flanked allele of Brg1 (Brg1f) (15) with mice harboring
Wnt1Cre, whose Cre is active in NCCs (4, 16). To confirm Brg1
deletion in NCCs, we immunostained Brg1 of embryonic day (E)
10 Wnt1Cre;Brg1f/fembryos and found that Brg1 proteins were
absent in NCCs and NCC-derived tissues, including dorsal root
Author contributions: W.L., Y.X., and C.-P.C. designed research; W.L., Y.X., C.S., K.Y.T., C.T.H.,
J.Y., P.H., C.-Y.L., C.-J.L., F.-C.T., and K.S. performed research; J.Y., P.H., F.-C.T., and T.M.
contributed new reagents/analytic tools; W.L., Y.X., T.M., D.B., M.P., and C.-P.C. analyzed
data; and W.L., Y.X., D.B., M.P., and C.-P.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1W.L. and Y.X. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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| vol. 110
| no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1218072110
dynamically programmed for to perform a multitude of develop-
Chromatin regulators interact with transcription factors to
control gene expression. Gata6, a transcription factor with muta-
tions identified in patients with OFT defects, is essential for the
activation of PlexinA2 in NCCs (42, 43). Other transcription fac-
tors, such as Pax3 and Pbx1, are required to program NCCs for
PAA/OFT development (16, 44–46). Determining how the Brg1–
Chd7 complex interacts with these transcription factors will be
crucial to elucidating the gene programming mechanism in NCCs
for cardiovascular development.
Our findings provide direct evidence in mammalian heart de-
NCC, and CHARGE syndrome. However, many cells besides
NCCs are required for cardiac OFT development, including
second heart field progenitor cells, endocardial cells, and myo-
cardial cells (8). Further investigation of whether Brg1 interacts
with Chd7 in those non-NCC tissues for heart development will
provide a better understanding of the interactions of chromatin
remodelers relevant to CHARGE syndrome. These studies will
also elucidate how those cells are programmed by chromatin
remodelers to non–cell-autonomously influence NCC survival,
differentiation, or migration during embryonic development.
Materials and Methods
Immunostaining, RNA in situ hybridization, qRT-PCR, ChIP, and reporter
assays have been described previously (47), as have Brg1f/f(15), Wnt1Cre (16),
and R26R (48) mice. The date on which a vaginal plug was observed in the
mice was set as E0.5. Animal care and handling were in accordance with the
regulations of Administrative Panel for Laboratory Animal Care at Stanford
University and guidelines of the National Institutes of Health. Detailed in-
formation on materials and experimental procedures is provided in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. P. Chambon for providing the Brg1f/f
mice. C.-P.C. was supported by the March of Dimes Foundation, the CHARGE
Syndrome Foundation, an American Heart Association Established Investiga-
tor Award, the National Institutes of Health (NIH), and the California Insti-
tute of Regenerative Medicine. W.L. and Y.X. were supported by the Oak
Foundation and Stanford Child Health Research Institute. Y.X. was also sup-
ported by the American Heart Association and C.S. by an NIH fellowship
and the March of Dimes Foundation.
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