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Williams Syndrome Transcription Factor is critical for neuralcrest cell function in Xenopus laevis
Chris Barnett a, Oya Yazgan a, Hui-Ching Kuo a, Sreepurna Malakar a, Trevor Thomas a,Amanda Fitzgerald a, William Harbour a, Jonathan J. Henry b, Jocelyn E. Krebs a,*
a Department of Biological Sciences, University of Alaska Anchorage, 3101 Science Circle, Anchorage, AK 99508, USAb Department of Cell and Developmental Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL 61801, USA
A R T I C L E I N F O
Article history:
Received 6 January 2012
Received in revised form
31 May 2012
Accepted 1 June 2012
Available online 9 June 2012
Keywords:
WSTF
Neural crest
Xenopus
Williams Syndrome
BAZ1b
Chromatin remodeler
A B S T R A C T
Williams Syndrome Transcription Factor (WSTF) is one of �25 haplodeficient genes in
patients with the complex developmental disorder Williams Syndrome (WS). WS results
in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes,
characteristic ‘‘elfin’’ facial features, low muscle tone and heart defects. WSTF exists in sev-
eral chromatin remodeling complexes and has roles in transcription, replication, and
repair. Chromatin remodeling is essential during embryogenesis, but WSTF’s role in verte-
brate development is poorly characterized. To investigate the developmental role of WSTF,
we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF
mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-defi-
cient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced,
coupled with defects in a number of developing embryonic structures and organs. WSTF-
deficient embryos display defects in anterior neural development. Induction of the neural
crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaf-
fected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a
severe defect in neural crest migration and/or maintenance. Consistent with a mainte-
nance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the
tailbud stage in regions corresponding to the path of cranial neural crest migration. Our
work is the first to describe a role for WSTF in proper neural crest function, and suggests
that neural crest defects resulting from WSTF haploinsufficiency may be a major contrib-
utor to the pathoembryology of WS.
� 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The development of a multicellular organism is a period of
extensive transcriptional regulation, which is highly depen-
dent on chromatin remodeling. During development, special-
ized cells arise from the gradual loss of pluripotency in the
progeny of embryonic stem cells. Recent studies reveal the
importance of dynamically regulated chromatin structure in
the maintenance of pluripotency and in cell differentiation
(Delgado-Olguin and Recillas-Targa, 2011).
WSTF (Williams Syndrome Transcription Factor), encoded
by the gene WSCR9/BAZ1B, is one of about 25–30 contiguous
genes that are haplodeficient in individuals with Williams
Syndrome (WS). WS is an autosomal dominant disorder
0925-4773/$ - see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mod.2012.06.001
* Corresponding author. Tel.: +1 907 786 1556; fax: +1 907 786 4607.E-mail addresses: [email protected] (C. Barnett), [email protected] (O. Yazgan), [email protected] (S. Malakar),
[email protected] (T. Thomas), [email protected] (A. Fitzgerald), [email protected] (W. Harbour), [email protected] (J.J. Henry), [email protected] (J.E. Krebs).
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occurring in �1 in 8000 live births and is the result of a �1.5
megabase deletion on chromosome 7 (Lu et al., 1998). Individ-
uals with WS exhibit characteristic malformations of cranio-
facial, heart and neural structures, infantile hypercalcaemia,
developmental delays, and complex and distinctive cognitive
and behavioral profiles. The contributions of most of the de-
leted genes to the clinical outcomes of WS patients are not
clear, though a handful of genes have been assigned tentative
roles (reviewed in Osborne, 2010). Evidence from mouse mod-
els suggests that WSTF may play a critical role in heart and
craniofacial development, and WSTF hemizygosity may also
explain the hypercalcaemia observed in WS patients (Ashe
et al., 2008; Yoshimura et al., 2009).
WSTF is a subunit of three well-characterized ATP-
dependent chromatin remodelers: WINAC (WSTF including
the nucleosome assembly complex), WICH (WSTF-ISWI
chromatin remodeling complex) and B-WICH. WSTF is a
170 kDa protein in the BAZ/WAL family, characterized by
six sequential motifs, including motifs implicated in chro-
matin binding. WSTF has roles in DNA repair, replication,
transcriptional activation and repression, and also possesses
histone H2A kinase activity (recently reviewed in Barnett
and Krebs, 2011).
In humans, WSTF transcripts are detected in all sampled
adult tissues (heart, brain, placenta, skeletal muscle, and
ovary) and four fetal tissues (brain, lung, kidney, and liver)
in non-WS individuals (Lu et al., 1998). WSTF mRNA is de-
tected in Xenopus laevis oocytes and is ubiquitous until early
neurulation, when WSTF expression becomes confined to
the neural ectoderm and closing neural tube (Cus et al.
2006). As neural tissue continues to differentiate into the
early tadpole stage, WSTF mRNA localizes to specific neural
structures and cells including the neural tube, optic cup, ante-
rior brain, and migrating cranial neural crest cells (Cus et al.,
2006). WSTF also displays strong expression in the branchial
(pharyngeal) apparatus, a primordium for a number of organs
and facial structures, such as the thyroid, aorta, the maxillary
and mandibular bones, ligaments, and nerves as well as audi-
tory structures like the auditory tube, bones of the middle ear
and the auricles. This branchial arch expression appears to be
limited to a population of cells located deep to the branchial
arch ectoderm and is likely the contribution of cranial neural
crest cells that originate from a space just dorsal to the neural
tube; a number of these cells migrate to the branchial arches
and play a crucial role in the proper development of this
important primordial structure (Noden and Schneider, 2006).
The neural crest is a unique group of embryonic cells
found only in vertebrates (Gans and Northcutt, 1983). The
neural crest originates from the ectoderm of the neural plate
border that undergoes an epithelial-to-mesenchymal transi-
tion, giving rise to neural crest cells during development. This
transition requires the orchestrated activities of a network of
transcription factors that gradually lead to this transition (re-
viewed in Sauka-Spengler and Bronner-Fraser, 2008). Neural
crest cells possess migratory ability as well as the ability to
undergo extensive cellular reprogramming, which results in
an increase in differentiation potential. The pluripotency of
neural crest cells allows them to contribute to a number of
developing tissues, including the central and peripheral ner-
vous system, as well as many non-neural structures such as
bone, cartilage, connective tissue, skin (melanocytes), and
smooth muscle.
The migration and maintenance of the neural crest cells is
guided by morphogens, signaling molecules that are secreted
from surrounding tissues. Bone morphogenic protein 4
(Bmp4) is a key developmental morphogen, and both inhibi-
tion and activation of the BMP4 gene is necessary for proper
induction and maintenance of neural crest (Steventon et al.,
2009). Additionally, both overexpression and inhibition of
BMP4 alters facial development in chick, suggesting accurate
timing and expression of BMP4 is crucial to proper neural
crest function (Creuzet et al., 2002; Ruhin et al., 2003; Wawer-
sik et al., 2005). Sonic hedgehog (Shh) is another morphogen
that along with Bmp4 is critical for the proper patterning of
the neural tube during embryogenesis (sometimes playing
antagonistic roles) and is also important in craniofacial devel-
opment of chick, human and mouse (Briscoe et al., 1999;
Campuzano and Modolell, 1992; Owens et al., 2005; Roelink
et al., 1995). Shh acts as an anti-apoptotic signaling molecule
for neural crest cells (Ahlgren et al., 2002).
Neural crest cells migrate in specific segmented regions
between the overlying ectoderm and underlying mesoderm.
The pathways of cranial neural crest cells are highly con-
served in vertebrate development and proper axial pattern-
ing and organization of the region is critical for proper
formation and migration of cranial neural crest (reviewed
in Basch and Bronner-Fraser, 2006). Migration occurs from
the forebrain and rhombomeres of the hindbrain to regions
in the head and to the branchial arches (Noden and Schnei-
der, 2006). The contributions of neural crest cells are both
unique to and critical for the proper development of all
vertebrates.
Little is known about the role of chromatin remodeling in
the formation of the neural crest and migrating neural crest
cells. One recent study has shown that Chd7 (Chromodomain
helicase DNA-binding domain member 7), an ATP-dependent
chromatin remodeler, is essential for early neural crest for-
mation in Xenopus embryos (Bajpai et al., 2010). In humans,
heterozygous mutation of CHD7 leads to the congenital devel-
opmental disorder CHARGE Syndrome, characterized by spe-
cific craniofacial features, neural defects and malformations
in the eyes, ears and heart (Pagon et al., 1981). Within the
PBAF complex (Polybromo- and BRG1-Associated Factor-con-
taining complex), Chd7 has been shown to bind in vivo to pro-
moter-distal elements of the neural crest specifier SOX9 and
the neural crest gene Twist in human neural crest-like cells
(Bajpai et al., 2010).
A number of the clinical features of WS involve structures
that are derived from the neural crest, including craniofacial
structures, heart and neural tissue. Given this observation,
and the expression of WSTF in neural crest in X. laevis, we
sought to determine the effects of WSTF knockdown in Xeno-
pus embryos, and to particularly address the effects of WSTF
knockdown in neural crest formation. We show specific de-
fects in neural crest cell migration and/or maintenance in
WSTF knockdown Xenopus embryos and propose a model in
which misregulation of BMP4 and SHH expression in WSTF
knockdowns results in the failure of normal neural crest con-
tributions during development. Our data suggest that a num-
ber of the symptoms present in WS patients could be due to
M E C H A N I S M S O F D E V E L O P M E N T 1 2 9 ( 2 0 1 2 ) 3 2 4 – 3 3 8 325
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WSTF haploinsufficiency and the downstream effects of this
multifunctional chromatin remodeler subunit.
2. Results
2.1. WSTF Morphant embryos exhibit severe defects inneural development
To determine the function of WSTF in early development,
an anti-WSTF morpholino (MO) was injected into fertilized X.
laevis eggs at the one-cell stage to inhibit the translation of
endogenous WSTF mRNA. Eggs were injected with 42 ng of
either anti-WSTF MO, or with a control MO that is the inverse
of the anti-WSTF MO sequence (INV-MO). The inverse MO-in-
jected embryos are indistinguishable from uninjected con-
trols. Anti-WTSF MO injection results in an average
reduction to �50–60% of endogenous WSTF levels, compara-
ble to the hemizygous state in WS patients (Howald et al.,
2006; Merla et al., 2006). Complete knockdown in early stages
is not achievable due to the presence of high levels of mater-
nally-deposited WSTF protein present in the developing em-
bryos. Knockdown of WSTF protein levels was confirmed by
Western blot (Supplementary Fig. 1).
WSTF knockdown embryos progress normally through the
early stages of development, at least at the gross morpholog-
ical level. However, severe abnormalities become evident
once embryos reach about stage 35, late tailbud stage
(Fig. 1A and B) (Nieuwkoop and Faber, 1967). WSTF knock-
down embryos display abnormal craniofacial development,
including a deformed cement gland, an unusually pro-
nounced indentation above the cement gland (in the region
that will develop into the olfactory organs, primary mouth
and anterior pituitary; Dickinson and Sive, 2007), and a
protruding/misshapen forehead. In addition, the developing
eyes are undersized and irregularly shaped, and the embryos
exhibit exopthalmia (protruding eyes). Finally, the WSTF
knockdown embryos show abnormal A–P axis development
and particularly strong axial curvatures. By stage 41 the
developmental abnormalities are more severe and the em-
bryos die around late tadpole stage, about stage 45. Injections
were performed in numerous independent batches of hun-
dreds of embryos, with each injection resulting in 100% pen-
etrance of this gross morphological phenotype (for example,
in a typical experiment 333/355 embryos, or 94%, injected
with anti-WSTF MO exhibit the strong WSTF phenotype,
while 22/355, or 6%, show a milder phenotype mostly charac-
terized by eye defects). Injection of the INV-MO typically re-
sulted in non-specific developmental defects in <10% of
embryos (for example, in a typical experiment 9/124, or 7%,
embryos exhibit various defects).
To obtain a more detailed understanding of the pheno-
types of the WSTF knockdown embryos, we performed histo-
logical analysis to further characterize the anterior neural
defects. Transverse sections through the anterior region of
the embryos reveal details of the defects in eye and brain
development in the WSTF knockdown embryos. Eye develop-
ment is profoundly disrupted (Fig. 1C and D). The optic cups
are generally shallow and poorly shaped, though in most
(but not all) cases the pigmented retinal epithelium is present.
The multiple layers of the neural retina completely fail to
differentiate and are typically present as a solid mass of
undifferentiated cells. The optic vesicle often retains a sub-
stantial attachment with the forebrain, without a clear optic
nerve. Furthermore, the lens is occasionally absent, or pres-
ent merely as a minimally differentiated thickening within
the surface ectoderm, though in a few cases some primary
lens fibers are detectable. The lenses invariably fail to detach
Fig. 1 – WSTF knockdowns exhibit neural defects and severely impaired eye development. A–B: Embryos (anterior to the left)
injected at the one-cell stage with either WSTF-inverse control MO (top of each panel) or WSTF MO (bottom) at stages 37 (A)
and 41 (B). The WSTF MO injected embryos exhibit reduction and malformation of neural structures, deformed eyes, and
axial defects. Asterisks indicate the pronounced concavity at the site of the presumptive primary mouth. C-F: Stained histo-
logical sections of control morpholino-injected embryos (C, E) and of WSTF knockdown embryos (D, F), all injected at the
one-cell stage. WSTF knockdowns display perturbed lens and retinal cell differentiation (D) and profound disorganization of
neural tissues (F). e: eye; cg: cement gland; l: lens; r: retina; pe: pigmented retinal epithelium; nt: neural tube; nc: notochord.
Scale bars: 1 mm (A and B) and 100 lm (C, D, E & F).
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from the ectoderm, and instead remain tightly associated
with this layer. Many lenses protrude outward from the sur-
face ectoderm.
Organization of tissues within the developing brain of
WSTF knockdown embryos is greatly affected as well
(Fig. 1E and F). Neural tissue in the developing brain is poorly
formed and differentiated. There appears to be a lack of cell
proliferation and absence of a clearly defined proliferative
(ventricular) zone. In some cases it appears that there is
incomplete fusion of the neural tube. The prechordal meso-
derm and notochord are frequently malformed, possibly en-
larged in diameter and poorly differentiated.
The whole mounts and sections of embryos in which
WSTF has been globally knocked down (i.e. morpholino in-
jected at the one-cell stage) clearly show that neural develop-
ment is perturbed, and neural tissues are highly disorganized.
It also appears that the amount of neural tissue is reduced in
these embryos, at least in the anterior. This apparent reduc-
tion of neural tissue formation was confirmed by analyzing
the expression of the neural-specific marker Neural Cell
Adhesion Molecule (NCAM) by whole mount in situ hybridiza-
tion (Fig. 2; representative embryos shown from a sample of
8–10 embryos for each stage). In addition to the embryos in
which WSTF expression has been globally knocked down,
we also examined embryos that were injected with anti-
WSTF MO into one cell of an embryo at the two-cell stage of
development (Fig. 2A–C). This results in an embryo in which
the MO affects the embryo unilaterally and thus only exhibits
the knockdown phenotype on the injected side, while the
other side serves as the control. The injected side is traced
by visualizing the fluorescently tagged MO (Fig. 2A).
NCAM expression is reduced in the injected side of a uni-
laterally-injected two-cell stage embryo post-neurulation at
stage 21, particularly in the anterior region and the eye
(Fig. 2B and C). NCAM expression is also reduced in embryos
where WSTF has been globally knocked down. WSTF global-
knockdown embryos at a late tailbud stage (stage 35) show
a severe reduction of NCAM expression compared to INV-
MO injected controls, especially in the brain and branchial ar-
ches (Fig. 2D and E). These data indicate that WSTF is impor-
tant for normal neural tissue development.
2.2. BMP4, SHH and MRF4 genes are misregulated inWSTF knockdown embryos
To further investigate the effects of WSTF knockdown on
neural development, we examined two genes known to regu-
late neural proliferation and differentiation during embryonic
Fig. 2 – WSTF function is required for neural tissue formation in vivo. A: Two-cell stage embryos (dorsal view, anterior at top)
were injected with carboxyfluorescein-labeled WSTF MO unilaterally into a single blastomere; fluorescence indicates the
injected side. B-C: NCAM expression analyzed by whole mount in situ hybridization at late neurulation (stage 21; B: dorsal
view, anterior at top; C: anterior view, dorsal at top). NCAM is normally expressed in the eyes and neural ectoderm. NCAM
signal is diminished in the anterior on the WSTF MO-injected side. Asterisk denotes region of greatest loss of NCAM signal. C:
Anterior view of embryo in (B), highlighting anterior reduction of NCAM expression in the eye rudiments and neural ectoderm
on the injected side. Asterisk denotes loss of NCAM signal. D-E: NCAM expression analyzed by whole mount in situ hybridi-
zation at late tailbud stage (stage 35; side views, anterior to left). D: One-cell stage embryos were injected with WSTF-inverse
MO (INV) and display normal NCAM expression in neural ectoderm, eye (e) and branchial arches (ba). E: One-cell stage
embryos injected with WSTF MO (MO) display reduced NCAM expression at late tailbud stage. Bracket marked by asterisk
indicates loss of NCAM staining in branchial arches in WSTF-MO injected embryos (E). Scale bars: 0.5 mm (A–C) and 1 mm (D
and E).
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development. Bmp4 is a signaling molecule from the TGFb/
BMP family of proteins known for neural patterning, cell dif-
ferentiation and cell death (reviewed in Hogan, 1996). The
expression domain of BMP4 is significantly expanded on the
injected side of a unilaterally-injected embryo during late
neurulation (stage 17; Fig. 3A; representative embryos shown
from a sample of �12 embryos for each stage). While normal
BMP4 expression is displayed in embryos globally injected
with inverse morpholino, in global WSTF knockdowns, BMP4
expression is markedly increased in late tailbud stage em-
bryos (stage 40; Fig. 3B), as well as in earlier tailbud embryos
(stage 37) as measured both by in situ hybridization and RT-
qPCR of whole embryos (Fig. 3E and data not shown).
Strikingly, BMP4 expression is spatially misregulated. BMP4
Fig. 3 – SHH, BMP4 and MRF4 are misexpressed in WSTF knockdown embryos. A: BMP4 expression in the neural plate analyzed
by whole mount in situ hybridization at late neurulation (stage 17; dorsal view, anterior at top) in embryos injected with WSTF
morpholino at the two-cell stage. BMP4 expression is expanded beyond its normal domain on the WSTF knockdown side (left)
compared to the uninjected control side (right). B: Whole mount in situ hybridization displaying normal expression pattern of
BMP4 compared to global WSTF knockdown embryos at late tailbud stage (stage 40; side view, anterior to left). One-cell stage
embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (INV, top). Inverse MO globally injected embryos
display normal BMP4 expression in the hindbrain, developing heart, and faintly in the otic vesicle. Global WSTF knockdowns
display decreased BMP4 anterior expression as well as aberrant expression in dorsal tissues. C: Whole mount in situ hybridi-
zation displaying normal expression of pattern SHH compared to global WSTF knockdowns at late tailbud stage (stage 36–37;
side view, anterior to left). One-cell stage embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (top).
Globally injected inverse MO embryos display SHH expression in the head and notochord. Global WSTF knockdown embryos
display decreased SHH expression throughout. D: Global WSTF knockdown embryo (right) displaying cyclopia/holoprosen-
cephaly compared to globally injected inverse MO embryo (left) at stage 45 (dorsal views of heads, anterior at top). Arrows
indicate location of eye(s). One-cell stage embryos were injected with either WSTF MO or WSTF-inverse MO. E: Real time RT-
PCR results for BMP4 and SHH graphed as a percentage of normal expression. BMP4 (stage 37) is significantly over-expressed
and SHH expression (stage 15) is significantly reduced in WSTF knockdown embryos injected at the one-cell stage. Data are the
average of 3–4 independent experiments and standard errors are shown. F: MRF4 expression in a stage 15 embryo (dorsal
view, anterior at top) injected with WSTF MO at the two-cell stage. The injected side (left) displays a significant decrease in
normal MRF4 expression in the developing somites. Scale bars: 0.5 mm (A and F) and 1 mm (B–D).
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exhibits reduced expression within its normal expression do-
main, specifically in anterior structures like the heart and
hindbrain (Martinez-Barbera et al., 1997). In addition, WSTF
morphants show a marked increase of BMP4 expression with-
in tissues around the dorsal midline that include notochord
and somitic mesoderm, tissues that do not normally express
BMP4 (Fig. 3B and Supplementary Fig. 2).
Shh is known for its role in midline signaling and the
splitting of bilateral eye fields, and is essential for survival
of a number of neural cell types during embryogenesis
(Chiang et al., 1996; Prykhozhij, 2010). SHH transcripts are
significantly decreased in both the anterior regions and neu-
ral tube in global WSTF knockdown embryos compared to
controls (Fig. 3C). Mutations in SHH are linked to holoprosen-
cephaly, the failure or incomplete division of the two hemi-
spheres of the forebrain, and cyclopia, the failure or
incomplete separation of the eye fields (Dubourg et al.,
2007). Consistent with the reduced SHH expression we ob-
serve, WSTF global knockdown embryos that survive well
into the tadpole stage exhibit a severe anterior phenotype
characterized by cyclopia (Fig. 3D). Misregulation of SHH (at
stage 15) and BMP4 (at stage 37) was also confirmed by real
time RT-PCR (Fig. 3E).
SHH is expressed in the notochord, a mesoderm-derived
tissue that defines the axis of all chordates (Yamada et al.,
1993). To further investigate the effect of WSTF depletion on
mesoderm development we assayed the expression of MRF4,
a marker for somites, also a mesoderm-derived tissue. Injec-
tion of anti-WSTF MO into one cell of two-cell stage embryos,
results in embryos in which only half of the embryo is af-
fected. MRF4 expression in WSTF unilateral-knockdown em-
bryos is reduced on the injected side during neurulation
(stage 15; Fig. 3F).
These results indicate that in WSTF knockdowns, both
ectoderm- and mesoderm-derived tissues show perturbed
gene expression, including overexpression and spatially aber-
rant expression of BMP4, and reduced expression of meso-
derm-derived tissue markers SHH and MRF4.
2.3. WSTF knockdown embryos exhibit perturbed PAX2,EPHA4, and SOX2 expression
While the reduced expression of NCAM and the sections
of WSTF knockdown embryos clearly indicate a reduction
in neural tissue in these embryos, we wished to further
examine the differentiation of anterior neural tissues. The
Paired box 2 (PAX2) gene encodes a Pax family transcription
factor with important roles in vertebrate organogenesis (Car-
roll and Vize, 1999; Dressler et al., 1990). PAX2 expression is
crucial for development of the midbrain hindbrain boundary
(MHB) in vertebrates, which is required for neuronal differ-
entiation and patterning of the midbrain and anterior hind-
brain (Li Song and Joyner, 2000). We examined PAX2
expression via whole mount in situ hybridization to investi-
gate midbrain and cerebellum development in our WSTF
knockdown embryos. Fertilized embryos were injected with
anti-WSTF MO at the one cell stage, resulting in global
knockdown of WSTF. WSTF knockdown embryos at tailbud
stage display an almost complete loss of PAX2 expression
within the hindbrain and a severe reduction at the MHB
when compared to controls (Fig. 4A–C; representative em-
bryos shown from a sample of 5 control and 7 knockdown
embryos). Additionally, at this stage, expression of PAX2 is
vital to the formation of the pronephros, the nascent yet
functional embryonic kidney (Heller and Brandli, 1997).
PAX2 signal is also reduced when compared to controls, par-
ticularly at the anterior end of this structure, suggesting that
normal kidney development may be perturbed in our WSTF
morphant embryos (Fig. 4A and B).
Eph receptors are a family of receptor tyrosine kinases
that bind a family of ephrin ligands involved in numerous
developmental processes. EPHA4 is expressed in a number
of tissues during Xenopus development including the fore-
brain, hindbrain, pronepheros, and the olfactory and otic
placodes (Park et al., 2004; Winning and Sargent, 1994; Xu
et al., 1995). In Xenopus, EPHA4 is involved in neural tissue
development in the forebrain and the MHB, and plays a cru-
cial role in neural crest cell migration (Smith et al., 1997; Xu
et al., 1995). We examined EPHA4 expression by whole
mount in situ hybridization to further investigate its role in
neural development in our WSTF knockdown embryos. Fer-
tilized embryos were injected with anti-WSTF MO at the
one cell stage, resulting in global knockdown of WSTF in
the developing embryos. Consistent with PAX2 expression,
knockdown embryos at the tailbud stage display a severe
reduction of EPHA4 expression within the forebrain and
MHB when compared to controls, further indicating failure
of neural tissue differentiation at this stage (Fig. 4). Normal
expression of EPHA4 in rhombomeres 3 and 5 is also reduced
in WSTF knockdown embryos (Fig. 4D–F; representative em-
bryos shown from a sample of 4 control and 8 knockdown
embryos) (Smith et al., 1997). Rhombomere formation is cru-
cial for the segmentation and proper migration of neural
crest cells (Smith et al., 1997). This reduction in EPHA4 could
result in underdeveloped rhombomeres and improper neural
crest migration in our WSTF morphant embryos. Addition-
ally, as with PAX2, expression of EPHA4 in the pronephros
may be reduced, and the pronephros is smaller and de-
formed compared to the controls (Fig. 4E).
Sox2 is a transcription factor well known for its role in
maintaining the pluripotency of embryonic stem cells (Heng
et al., 2010). In addition, Sox2 in combination with different
binding partners plays numerous roles in embryonic develop-
ment (Archer et al., 2011; Kondoh and Kamachi, 2010), includ-
ing development of the CNS and the retina and lens of the
eye. In the developing brain, Sox2 has dual roles in maintain-
ing proliferative neural stem cells as well as being required for
specific neuronal differentiation (Agathocleous et al., 2009;
Cavallaro et al., 2008; Pevny and Nicolis, 2010). As above, we
examined SOX2 expression by whole mount in situ hybridiza-
tion in our WSTF knockdown embryos. In this case, fertilized
embryos were injected with anti-WSTF MO at the two cell
stage, resulting in unilateral knockdown of WSTF in one side
of the developing embryos. Again consistent with the other
neural markers, the MO-injected side at the early tailbud
stage displays a severe reduction of SOX2 expression through-
out the brain, eye, otic vesicle, lateral line placodes, and bran-
chial arches when compared to the uninjected control side
(Fig. 4 G–J; representative embryos shown from a sample of
4–6 embryos for each stage).
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2.4. WSTF is crucial for proper neural crest cell functionin vivo
WSTF mRNA expression is detected in both the migratory
neural crest as well as branchial arches during Xenopus
embryogenesis (Cus et al., 2006). This expression, and the re-
duced expression of SHH and EPHA4, genes known to play
roles in neural crest function, suggest that WSTF knockdown
might impact the neural crest. In the anterior, neural crest
cells originate from just above the neural tube and migrate
to the branchial arches, after which they serve as a critical
component for the formation of a number of subsequent
anterior structures that originate from the branchial arches.
SNAIL and SLUG are two members of the Snail family that
function in the epithelial-to-mesenchymal transition cas-
cade. SLUG is expressed in the premigratory neural crest cells
in both chick and X. laevis (Mayor et al., 1995, 1999; Nieto et al.,
1994; Sefton et al., 1998). In Xenopus, SNAIL is expressed in
ectoderm, premigratory neural crest, and the branchial ar-
ches (Mayor et al., 1993). In many vertebrates, SNAIL and SLUG
Fig. 4 – PAX2, EPHA4, and SOX2 expression are reduced in WSTF knockdown embryos. A: PAX2 expression in the midbrain
hindbrain boundary (MHB), the otic vesicle, optic stalk and the pronephros analyzed by whole mount in situ hybridization in
control embryo at tailbud stage (Stage 33; lateral view, anterior at left) B: Tailbud stage embryo injected with WSTF morpho-
lino at the one-cell stage. PAX2 expression is reduced in all expression domains. C: Side-by-side dorsal view of control
embryo (left) with WSTF knockdown embryo (right) at tailbud stage, showing reduced expression of PAX2 in the MHB and the
otic vesicle of WSTF knockdown embryo compared to control (Stage 33; dorsal view, anterior at top). D: EPHA4 expression in
the forebrain, hindbrain, rhombomeres 3 and 5 and in the pronephros analyzed by whole mount in situ hybridization in
control embryo at tailbud stage (Stage 33; lateral view, anterior at left) E: Tailbud stage embryo injected with WSTF morpho-
lino at the one-cell stage. EPHA4 expression is reduced in all expression domains. F: Side-by-side dorsal view of control
embryo (left) with WSTF knockdown embryo (right) at tailbud stage showing reduced expression of EPHA4 in the forebrain,
hindbrain and rhombomeres 3 and 5 of WSTF knockdown embryo compared to control (stage 33; dorsal view, anterior at top).
G-J: Tailbud stage embryos injected with WSTF morpholino at the two-cell stage. G: SOX2 expression in the brain, eye, otic
vesicle, lateral line placodes and branchial arches analyzed by whole mount in situ hybridization on the uninjected side at
tailbud stage (Stage 32; lateral view, right side of embryo). H: SOX2 expression is reduced in all expression domains on the
injected side (left side of embryo). I and J: Dorsal views of two embryos injected at the two-cell stage (WSTF MO injected into
the left side in both cases). Embryo in I is at stage 28, embryo in J is stage 32. Both show reduced expression of SOX2 in all
expression domains on the injected side. STD: Standard control morpholino; MO: WSTF morpholino; os: optic stalk; br: brain;
pd: pronephric duct; hb: hindbrain; pn: pronephros, MHB: mid-hindbrain boundary; ov: otic vesicle; r3, r5: rhombomeres 3
and 5; op: olfactory placode; cp: cranial placodes [otic and lateral line]; ba: branchial arches; e: eye. Asterisks indicate areas of
reduced expression relative to controls. Scale bars: 0.5 mm (A, B, D, E, G, H), 0.1 mm (C, F, I, J).
330 M E C H A N I S M S O F D E V E L O P M E N T 1 2 9 ( 2 0 1 2 ) 3 2 4 – 3 3 8
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play important and seemingly redundant roles in the initia-
tion of neural crest formation (Aybar et al., 2003). However,
in Xenopus, SNAIL appears to be an initiator of the neural crest
genetic cascade and has been shown to induce the expression
of SLUG as well as a number of other important neural crest
genes (Aybar et al., 2003).
To investigate whether neural crest cell formation is per-
turbed in WSTF knockdown embryos, expression of SNAIL
and SLUG were analyzed by whole mount in situ hybridiza-
tion in unilateral WSTF knockdown embryos (Fig. 5; repre-
sentative embryos shown from a sample of 5–6 embryos
for each stage and probe). Expression of both SNAIL and
SLUG is unaffected on the injected side of unilaterally-in-
jected embryos during early neurulation (stage 15; Fig. 5A
and E), suggesting that neural crest induction is unaffected
by WSTF depletion. In contrast, both SNAIL and SLUG
expression patterns within the branchial arches are severely
perturbed in WSTF-MO unilaterally-injected embryos later
in development (Fig. 5B and C, F and G). This suggests that
some neural crest cells either fail to successfully migrate to
the branchial arches, or are not successfully maintained fol-
lowing migration. Histological sections of the branchial ar-
ches of unilateral WSTF knockdown embryos highlight the
decreased expression of both SNAIL and SLUG on the WSTF
knockdown side (particularly in the interior structures of
the arches), and also reveal significant morphological mal-
formations of the branchial arches on the injected side
(Fig. 5D and H). These malformations include changes in
shape, size and number of branchial arches in the WSTF
MO-injected side, including the apparent loss or fusion of
Fig. 5 – WSTF knockdown embryos display proper neural crest induction, but fail to maintain normal neural crest in later
development. A–H: Two-cell stage embryos were injected with WSTF MO unilaterally into a single blastomere and analyzed
by whole mount in situ hybridization at neural groove stage (stage 15) to display expression pattern of neural crest genes
SNAIL and SLUG. Both genes are expressed at the neural crest boundary. WSTF knockdown has no effect on the stage 15
expression of neural crest genes SNAIL (A) and SLUG (E) (dorsal views, anterior at top). Identically treated embryos analyzed
by whole mount in situ hybridization at tailbud stage (stage 30; side views of heads, anterior at top) display perturbed
expression of neural crest genes SNAIL (B and C) and SLUG (F and G) on the WSTF-knockdown side (C and G). 60-lm sections
through the branchial arches of unilateral WSTF knockdown embryos (anterior at top) display diminished expression of
SNAIL (D) and SLUG (H) on the injected side, particularly in the internal structures (light blue staining). Arches are labeled in B–
D and F–H. Asterisks indicate the relative position of missing/fused arch number 3 on the sides derived from the WSTF MO-
injected blastomeres. Dashed lines in B and F indicate the plane of sectioning of D and H, respectively. Scale bars: 1 mm (A, E),
0.5 mm (B, C, F, G) and 0.1 mm (D, H).
M E C H A N I S M S O F D E V E L O P M E N T 1 2 9 ( 2 0 1 2 ) 3 2 4 – 3 3 8 331
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arch number 3 in 5/6 sectioned embryos, and the sixth ani-
mal with no detectable arches on the injected side at all
(Fig. 5C and D, G and H, and data not shown). These results
strongly suggest that while neural crest induction is intact
in WSTF knockdowns, the resulting neural crest cells exhi-
bit either abnormal migration or maintenance, leading to a
cascade of morphological defects.
2.5. WSTF knockdown embryos display a specific patternof increased apoptosis
The results described above indicate failures of CNS
development and neural crest migration or survival in WSTF
knockdowns. To determine whether the net loss of neural
tissues and neural crest might be due to increased apoptosis
in these tissues in our WSTF knockdown embryos, we per-
formed a whole mount Terminal deoxynucleotidyl transfer-
ase dUTP nick end-labeling (TUNEL) assay to visualize
apoptosis in situ. Anti-WSTF MO was injected at the one cell
stage post-fertilization, resulting in global knockdown of
WSTF within developing embryos. The TUNEL assay reveals
an increase in apoptosis in specific regions of the WSTF
knockdown embryos when compared to the controls
(Fig. 6; representative embryos shown from a sample of 15
control embryos and 18 knockdown embryos at stages 31–
38). We observe increased staining at the olfactory placode
and a striking increase in regions flanking the anterior dor-
sal midline at early tailbud stage (stage 33). This aberrant
increase of apoptosis within the hindbrain is the same re-
gion where cranial neural crest cells begin their migration
to the branchial arches (Smith et al., 1997), and the mid-
hindbrain boundary is also a region that exhibits particu-
larly robust WSTF expression beginning at stage 32 (Cus
et al., 2006). A transverse section of the affected region dis-
plays punctate staining in the peripheral tissues, lateral and
ventral to the hindbrain and appears to be beneath the
overlying epidermal ectoderm (Fig. 6D). This punctate stain-
ing follows a pattern that is strikingly similar to the path of
cranial neural crest migration and may offer an explanation
for the decrease of neural crest cells present within the
branchial arches of our WSTF knockdown embryos (Smith
et al., 1997).
Fig. 6 – WSTF knockdown embryos display increased apoptosis of specific stages of development. A: TUNEL assay with a
representative control embryo at tailbud stage showing no detectable levels of apoptosis (Stage 33; lateral view, anterior at
left). B: A representative tailbud stage embryo injected with WSTF morpholino at the one-cell stage displaying a spatial
increase in apoptosis in the hindbrain, olfactory placode and lateral anterior tissues. C: Side-by-side dorsal view of control
embryo (left) with WSTF knockdown embryo (right) at tailbud stage, showing an increase in apoptosis in the hindbrain of the
WSTF knockdown embryo compared to control (Stage 33; dorsal view, anterior at top). D: Transverse section of WSTF
knockdown embryo (at a plane indicated by the dashed line in B) displays punctate staining in the peripheral tissues, lateral
and ventral from the hindbrain, beneath the overlying epidermal ectoderm (Stage 33; sectional view, anterior at top). STD:
standard control morpholino; MO: WSTF morpholino; nt: neural tube; nc: notochord. Scale bars: 0.5 mm (A and B), 0.1 mm (C
and D).
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3. Discussion
We have examined the specific roles of WSTF during devel-
opment in order to better characterize the contribution of
WSTF haplodeficiency to the clinical manifestations of WS.
We show that WSTF has a crucial role in neural development
and proper neural crest cell function in X. laevis. WSTF knock-
downs display severe defects in neural development, and re-
duced expression of the neural-specific marker NCAM and
reduction of PAX2 and EPHA4 within the mid and hindbrain.
The expression of WSTF in neural crest cells has been well
characterized (Cus et al., 2006). A number of genes have been
implicated in the complex sequence of neural crest cell for-
mation and function. In Xenopus, the neural crest-specific
transcription factor SNAIL has been shown to precede SLUG
and to induce its expression, as well as the expression of a
number of other neural crest markers (Aybar et al., 2003).
We show that knockdown of WSTF does not affect the expres-
sion of either SNAIL or SLUG during the initial stages of neural
crest formation. This indicates that normal levels of WSTF are
not required for neural crest induction, but does not rule out
the possibility that some WSTF is essential for this process.
Since some WSTF (likely maternally deposited) remains in
our knockdowns, this could provide a sufficient level of WSTF
for neural crest induction. However, WSTF knockdown em-
bryos do display decreased expression of both SNAIL and
SLUG within the branchial arches at early tailbud stage, sug-
gesting WSTF is required for the normal migration and/or
maintenance of the neural crest cells.
Strikingly, WSTF knockdown results in significant misre-
gulation of two signaling molecules crucial in neural develop-
ment. The first of these is Bmp4, a mitogen required for the
proper formation of many neural structures that are also
dependent on Shh signaling. Bmp4 and Shh act in an antago-
nistic fashion to pattern and shape the developing neural
tube, vertebrate eye and brain (Bakrania et al., 2008; Hu
et al., 2004; Muller et al., 2007). BMPs, and Bmp4 in particular,
inhibit ectoderm from assuming a neural tissue fate. Bmp4
not only suppresses neural tissue formation from ectoderm,
but can also induce epidermal tissue determination (Hem-
mati-Brivanlou and Thomsen, 1995; Wilson and Hemmati-
Brivanlou, 1995).
Shh is a developmental mitogen essential for proper neu-
ral patterning (reviewed in Patten and Placzek, 2000). Shh is
believed to facilitate normal neural tissue formation by three
main processes. First, Shh is known for its role in the ventral
patterning of the neural tube (reviewed in Patten and Placzek,
2000). Second, Shh promotes cell differentiation by facilitat-
ing cell cycle exit (Agathocleous et al., 2007). Lastly, Shh is
an essential survival factor for a number of neural cells by
inhibition of the p53-dependent apoptotic pathway (Ahlgren
and Bronner-Fraser, 1999; Ahlgren et al., 2002; Prykhozhij,
2010). In addition to its role in patterning of neural tissue, a
classic example of Shh’s role in neural crest function is holo-
prosencephaly, a developmental syndrome characterized in
humans by head and facial malformations, including cyclo-
pia, resulting from haplodeficiency of SHH (reviewed in Mur-
doch and Copp, 2010). Reduced SHH results in decreased cell
proliferation in the neural tube and neural crest, as well as
smaller head size, defining a role for SHH in the survival of
cranial neural crest (Ahlgren and Bronner-Fraser, 1999; Ahl-
gren et al., 2002). We show that WSTF knockdown results in
a reduction of SHH expression, as determined by both in situ
hybridization and realtime RT-PCR at a number of develop-
mental stages. Additionally, a whole mount TUNEL assay re-
veals that WSTF knockdowns exhibit an increase in
apoptosis within the hindbrain and along a path that closely
mimics the initial route of cranial neural crest migration to
the branchial arches (Smith et al., 1997). Shh’s role in cranial
neural crest survival (Ahlgren et al., 2002) could explain our
results in which both SNAIL and SLUG expression is unaf-
fected during neural crest induction, but is then severely de-
creased later in development in our WSTF knockdowns,
consistent with a failure in maintenance/survival of neural
crest.
The increased and aberrant expression of BMP4 in our
WSTF knockdowns, along with the absence of WSTF expres-
sion within the notochord of normally developing embryos,
suggests that the effect on SHH expression is likely to be
downstream of the altered BMP4 expression. In two classic
experiments aimed to investigate the function of BMP4,
BMP4 was overexpressed in the animal hemisphere of fertil-
ized Xenopus embryos (Dale et al., 1992; Jones et al., 1996). In
these studies WSTF knockdowns display reduced expression
of specific neural markers, including loss of PAX2 in the
MHB, EPHA4 in rhombomeres 3 and 5, and SOX2 throughout
the brain, eye and lateral line placodes. WSTF knockdowns
also show reduction of the ear1y neural development and
neural ectoderm marker NCAM. These neural defects could
be due to an increase in ectodermal BMP4 expression in our
WSTF knockdowns and may result in secondary malforma-
tions of the developing mesoderm from which the SHH-pro-
ducing notochord and somites (expressing MRF4) are derived.
Whether the effect of WSTF on BMP4 or SHH is direct or
indirect remains to be determined. However, previous work
in our lab has shown that ISWI, WSTF’s binding partner in
the WICH complex, is present at the BMP4 gene during Xeno-
pus embryonic development (Dirscherl et al., 2005). In addi-
tion, ISWI knockdowns exhibit BMP4 and SHH expression
levels similar to those in WSTF morphants (Dirscherl et al.,
2005). Taken together, these results suggest a role for the
WICH complex as a transcriptional repressor of BMP4.
Pluripotent embryonic stem cells are characterized by an
abundance of dynamic euchromatin that undoubtedly re-
quires an enormous amount of input and coordination of
chromatin remodelers for proper differentiation or mainte-
nance of pluripotency (Fazzio and Panning, 2010; Serra
et al., 2007). Neural crest cells are extremely versatile progen-
itor cells that must maintain their pluripotency well into
embryonic development. WSTF expression has been detected
within a number of neural progenitor cells during Xenopus
morphogenesis (Cus et al., 2006). Our study is the first to sug-
gest that neural crest formation is initiated normally in em-
bryos with depleted WSTF levels, however, these neural
crest cells are not able to maintain their proper function or
contribute normally to the formation of the branchial arches
later in development. Here we show the misregulation of a
number of well-characterized developmental morphogens
and tissue markers and propose a mechanism in which aber-
rant BMP4 expression leads to a disruption of mesoderm
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differentiation which severely affects neural crest cell migra-
tion and/or maintenance.
This effect on neural crest and mesoderm formation could
account for a number of the symptoms that occur in WS pa-
tients. Development of the branchial arches, which are highly
dependent on proper neural crest function, gives rise to many
structures that, if malformed, could explain a number of the
symptoms present in WS. WSTF deletions in mice have reca-
pitulated both the craniofacial and the heart defects seen in
WS patients, further implicating WSTF haplodeficiency in
the development of WS (Ashe et al., 2008; Yoshimura et al.,
2009). Mice that are homozygous for a WSTF mutation that re-
sults in a single amino acid change (L733R) and reduced sta-
bility of the mouse WSTF protein exhibit altered skull
shape, including a protruding forehead, shorter snout, flat-
tened nasal bone, and upward curvature of the nasal tip (Ashe
et al., 2008). This craniofacial development is strikingly simi-
lar to many of the facial features of WS patients and suggests
that WSTF deficiency may contribute to the characteristic ‘‘el-
fin’’ features seen in individuals with WS. WSTF knockout
mice (wstf–) are small, show significant heart defects and
die just days after birth. 10% of heterozygous WSTF+/– neona-
tal mice also display specific heart defects similar to those
seen in WS patients, such as altered structure, atrial and ven-
tricular septal defects, and hypertrophy of ventricles (Yoshim-
ura et al., 2009).
This result strongly correlates with the malformation of
the branchial arches in our morphant embryos, particularly
branchial arches three and four, from which a number of car-
diac structures affected in WS are derived (Hutson and Kirby,
2007; Safa and El Ghazal, 2011). This result could also explain
the infantile hypercalcaemia observed in WS patients. The
branchial apparatus contributes to development of the thy-
roid and the ultimobranchial bodies, both of which produce
calcitonin to regulate blood calcium levels (Varga et al.,
2008). Defects in these structures could contribute to the in-
creased blood calcium levels seen in WS individuals, this is
also consistent with the increased incidence of hypoplasia
of the thyroid gland in WS patients (Manley and Capecchi,
1998; Selicorni et al., 2006). Neural crest cells also contribute
to the generation of tissues of the eyes including the cornea
endothelium, stroma, and muscles of the iris (Grocott et al.,
2011; Iwao et al., 2008). Many WS individuals do exhibit ocular
defects, including small eye openings, a stellate iris pattern,
puffiness around the eyes, higher occurrence of blue pigmen-
tation, strabismus or esotropia (cross-eyes), and abnormal
extraocular muscle anatomy (Ali and Shun-Shin, 2009; Winter
et al., 1996). Although it is almost certainly independent of
neural crest, we also show that the pronephric kidney is
reduced and poorly developed in our knockdowns, with
accompanying reduced expression of PAX2, which adds to
the number of effected mesoderm derived structures in these
knockdowns. Interestingly, WS patients display a significantly
high incidence of renal and urinary abnormalities including
the presences of cysts, narrowing of the renal arteries and
hypoplasia (Biesecker et al., 1987; Pober et al., 1993; Sugayama
et al., 2004). We note that WSTF knockdowns in Xenopus result
in phenotypes that are more severe than those observed in
human WS patients or in heterozygous WSTF+/wstf- mice
(homozygous deletions in mice are lethal shortly after birth),
suggesting that redundant mechanisms may partially allevi-
ate the effects of WSTF haploinsufficiency in mammals (Ashe
et al., 2008; Yoshimura et al., 2009). Intriguingly, two other
genes contained in the WS deleted region, GTF2I and
GTF2IRD1, are also expressed in neural crest-derived tissues
and heterozygous deletions of either gene in mice results in
craniofacial defects (Enkhmandakh et al., 2009). This suggests
other genes in the region may also play a role in neural crest
function and have additive effects on the complexity of the
WS phenotype. Additional studies will be needed in order to
better comprehend the role of WSTF during vertebrate devel-
opment. This study furthers our understanding of the role of
ATP-dependent chromatin remodeling complexes during ver-
tebrate development as well as offer a better understanding of
a number of WS symptoms that may be attributable to
haplodeficiency of the WSTF gene.
4. Materials and methods
4.1. Generation of Xenopus embryos
Female Xenopus were induced to ovulate by injection of
600 ll of human chorionic gonadotropin (1000 USP units/ml,
Intervet, Millsboro, DE) into the dorsal lymph sac. Testes were
obtained by surgical excision from a male frog euthanized in
0.06% benzocaine for 45 min. Eggs were squeezed from in-
duced females 12–18 h after induction of ovulation. Freshly
laid eggs were immediately exposed to dispersed Xenopus tes-
tis in 0.1X Marc’s modified Ringer’s (MMR) (standard protocols
as per Sive et al., 2007). Fertilized embryos were then stripped
of jelly coat by exposure to 2% L-cysteine for 3–5 min.
All husbandry and handling of adult X. laevis was per-
formed in compliance with our approved IACUC #181339–4.
4.2. Microinjection of morpholino oligonucleotides
Single-stranded oligonucleotides (morpholinos) were gen-
erated by Gene Tools (Philomath, Oregon). These morpholinos
are complementary to the 5 0 untranslated region of WSTF
messenger RNA upstream of the translation start site, and
are labeled with 3 0-carboxyfluorescein dye. As a negative con-
trol for injection, an inverse sequence or a standard control
morpholino was used. The corresponding morpholino
sequences are: WSTF-MO: 5 0GCTTCTCGTGGGATGATAGTCCC
GC3 0, inverse control MO (INV-MO): 5 0CGCCCTGATAGTAGGGT
GCTCTTCG3 0, standard control MO (STD-MO): 5 0CCTCTTACC
TCAGTTACAATTTATA3 0 (all MOs have a 3 0 carboxyfluorescein
label).
Xenopus embryos were obtained and dejellied as described
in Section 4.1 and transferred to mesh bottom Petri dishes
filled with 6% Ficoll in 0.1X MMR solution. 5 nL of a 1 mM mor-
pholino solution was injected into either 1 or 2 cell stage
dividing embryos (42 ng of morpholino per cell, resulting in
either global, or unilateral WSTF morphant embryos) with
glass needles made using a Flaming/Brown micropipette pull-
er (Sutter Instruments, Novato, CA) and a KITE-L micromanip-
ulator (World Precision Instruments, Sarasota, Florida).
Morpholino was delivered to embryos by applying 30 ms
bursts of 8 psi to needles using a MPPI-2 pressure injector
(Applied Scientific Instrumentation, Eugene, OR). After
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injections, embryos were transferred to 0.1X MMR. At �17 or
32 h post-fertilization, gross morphology of the embryos
was observed and photographed. Within 24 h post fertiliza-
tion, injected embryos were identified by fluorescence illumi-
nation of injected morpholinos and uninjected embryos were
discarded. Embryos were collected at specified times and used
for either total RNA or protein extractions or were fixed in
MEMFA (0.1 M Mops pH 7.5, 2 mM EGTA, 1 mM MgSO4, 3.7%
formaldehyde) for in situ hybridization or sectioning.
4.3. In situ hybridization and TUNEL assay
DNA templates were linearized with restriction enzymes,
and digoxigenin-labeled sense and antisense RNA probes
were transcribed in vitro from templates using Ambion Mega-
script in vitro transcription kits (Megascript T7TM, Megascript
Sp6TM, Applied Biosystems/Ambion, Austin, Texas). The proto-
col was altered in the following ways: all Megascript dNTPs
were diluted to 10 lM concentrations and half of the
suggested RNA polymerase concentration was used.
Xenopus embryos were generated as described in Sec-
tion 4.1. Embryos were collected at indicated stages and fixed
in MEMFA according to Sive et al. (2007). Whole mount em-
bryos were then hybridized with in situ hybridization probes
according to Sive et al. (2007); however, for some probes the
RNaseA step was omitted. BM purple (Roche, Mannheim Ger-
many) or NBT/BCIP (Promega, Madison, Wisconsin) were used
as the substrate for the alkaline phosphatase conjugated to
an anti-digoxigenin antibody (Roche, Mannheim Germany).
For the TUNEL assays, albino embryos were collected at
indicated stages and fixed in MEMFA for two hours. A whole
mount TUNEL procedure from the Harland Lab was followed
(detailed in Hensey and Gautier, 1998). Briefly, embryos were
bleached for 1 h under light and washed in PBS. Terminal
ends were labeled with Digoxygenin-dUTP (Roche, Mannheim
Germany) using Terminal Deoxynucleotidyl Transferase (TdT)
(Invitrogen, Carlsbad, California) overnight at room tempera-
ture. The reaction was stopped with 65 �C washes in PBS con-
taining 1 mM EDTA. Anti-digoxygenin AP antibody incubation
was done in MAB (100 mM Maleic acid, 150 mM NaCl) contain-
ing 2% BMB blocking reagent (Roche, Mannheim Germany)
overnight at 4 �C. Following extensive washes in MAB, the col-
or reaction was performed using NBT/BCIP (Harland, 1991).
4.4. Histology
Embryos were fixed in MEMFA and stored at �20 �C in 100%
methanol. Embryos were gradually rehydrated by incubating
in 1X PBS containing 75%, 50%, 25% methanol and in 1X PBS
for 5 min each. They were then embedded in 15% gelatin
(Bloom �300, Sigma–Aldrich, St. Louis, Missouri) in PBS
(135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4,
pH 7.2) and fixed in 10% neutral buffered formalin (3.7% form-
aldehyde) (BDH, West Chester, PA) for 36–48 h at 4 �C. The gel-
atin blocks were rinsed in PBS and 60 lm sections were cut
using a Leica VT1000P vibratome (Richmond, Illinois).
Other fixed embryos were dehydrated though a graded ser-
ies of ethanol and xylene washes, embedded in Paraplast Plus
(Oxford Labware, St Louis, MO) and serially sectioned at a
thickness of 7 lm. Sections were collected on albumin-subbed
slides (Mayer’s fixative) (Humason, 1972). Sections were
stained using Ehrlich’s hematoxylin and counterstained in
Eosin following the protocols of Humason (1972). Coverslips
were applied using Permount (Fisher Scientific, Pittsburgh,
PA). Color images were captured using a Spot digital camera
(Diagnostic Instruments, Inc., Sterling Heights, MI).
4.5. Total RNA extraction and RT-qPCR analysis
Total RNA samples were isolated from injected embryos at
various times post fertilization. Ten whole embryos were
homogenized in 200 ll Trizol reagent (Invitrogen, Carlsbad,
California) using a mortar and pestle and the RNA was iso-
lated following manufacturer’s recommendations. RNA pel-
lets were re-suspended in 100 ll RNase-free water. Turbo
DNase (Ambion, Austin, Texas) was used to remove any geno-
mic DNA contamination in the RNA preparations. cDNA gen-
eration and qPCR were performed using the one-step SYBR�Green Quantitative RT-PCR Kit (Sigma–Aldrich, St. Louis, Mis-
souri) in a Smart Cycler (Cepheid, Sunnyvale, California)
system.
4.6. Total protein extraction and Western blot analysis
10–20 embryos were collected and homogenized via mor-
tar and pestle in Laemmli sample buffer. Homogenate was
heated at 95 �C for 5 min, centrifuged at 13,000 rpm for
1 min and the supernatant was stored at �80 �C. Total protein
extracts were separated by 10% SDS polyacrylamide gel elec-
trophoresis, blotted onto a nitrocellulose membrane (What-
man, Dassel, Germany) and blocked in Odyssey blocking
buffer (Licor, Lincoln, Nebraska). The membrane was probed
with the primary antibody in Odyssey blocking buffer at room
temperature overnight. Primary antibody against Xenopus
WSTF was generously provided by Dr. Paul Wade (NIEHS). A
b-actin monoclonal antibody (Abcam ab8224, Cambridge,
Massachusetts) was used for a loading control. IR-dye conju-
gated (800CW) goat anti-mouse secondary antibodies (Licor,
Lincoln, Nebraska) were used for secondary antibodies. The
Odyssey infrared imager (Licor, Lincoln, Nebraska) was used
to visualize the probed membranes.
Acknowledgements
Template DNAs for SLUG and SNAIL were generously provided
by Peter Walentek in Dr. Martin Blum’s laboratory (University
Hohenheim, Stuttgart, Germany). BMP4 and SHH DNA tem-
plates were generously provided by Dr. Richard Harland (Uni-
versity of California, Berkley), and fully transcribed MRF4
probe was provided by Dr. Tim Hinterburger (University of
Alaska Anchorage). The NCAM and SOX2 RNA probes were
developed at the Cold Spring Harbor course on Developmen-
tal Biology of Xenopus. Primary antibody against Xenopus
WSTF was generously provided by Dr. Paul Wade (NIEHS).
This work was supported by NIH EY016029 and Whitehall
Foundation 2007-08-79 to JEK, and NIH EY09844 to JJH. CB
was also supported by NIH P20RR016466.
This paper is dedicated to the memory of Dr. Marietta
Dunaway (1952-2011).
M E C H A N I S M S O F D E V E L O P M E N T 1 2 9 ( 2 0 1 2 ) 3 2 4 – 3 3 8 335
Author's personal copy
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.mod.2012.06.001.
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