© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
RESEARCH ARTICLE
The splicing factor PQBP1 regulates mesodermal and neural
development through FGF signaling
ABSTRACT
Alternative splicing of pre-mRNAs is an important means of regulating
developmental processes, yet the molecular mechanisms governing
alternative splicing in embryonic contexts are just beginning to
emerge. Polyglutamine-binding protein 1 (PQBP1) is an RNAsplicing factor that, when mutated, in humans causes Renpenning
syndrome, an X-linked intellectual disability disease characterized by
severe cognitive impairment, but also by physical defects that
suggest PQBP1 has broader functions in embryonic development.
Here, we reveal essential roles for PQBP1 and a binding partner,
WBP11, in early development of Xenopus embryos. Both genes are
expressed in the nascent mesoderm and neurectoderm, and
morpholino knockdown of either causes defects in differentiation
and morphogenesis of the mesoderm and neural plate. At the
molecular level, knockdown of PQBP1 in Xenopus animal cap
explants inhibits target gene induction by FGF but not by BMP, Nodal
or Wnt ligands, and knockdown of either PQBP1 or WBP11 in
embryos inhibits expression of fgf4 and FGF4-responsive cdx4
genes. Furthermore, PQBP1 knockdown changes the alternative
splicing of FGF receptor-2 (FGFR2) transcripts, altering the
incorporation of cassette exons that generate receptor variants
(FGFR2 IIIb or IIIc) with different ligand specificities. Our findings may
inform studies into the mechanisms underlying Renpenning
syndrome.
KEY WORDS: Alternative splicing, FGF, FGF receptor, Mesoderm,
Neural, PQBP1, Renpenning syndrome, WBP11, Xenopus
INTRODUCTION
Polyglutamine binding protein-1 (PQBP1) is a 38 kDa nuclear
protein abundantly expressed in the adult mammalian central
nervous system (Komuro et al., 1999a; Waragai et al., 1999), and
mutations in the human PQBP1 gene cause X-linked intellectual
disability (XLID) diseases that include Renpenning, Sutherland–
Haan, Hamel cerebropalatocardiac, Golabi–Ito–Hall and Porteous
syndromes (Kalscheuer et al., 2003; Lenski et al., 2004; Stevenson
et al., 2005; Cossee et al., 2006; Lubs et al., 2006; Martinez-Garay
et al., 2007). These are collectively referred to as Renpenning
syndrome, and affected patients display unifying clinical features,
including intellectual disability, microcephaly and short stature.
However, a variety of other physical manifestations can be
observed, including midline and cardiac defects, small testes, lean
muscle and reduced body mass (Stevenson et al., 2005; Germanaud
et al., 2010). These phenotypes suggest that, in addition to brain
Department of Biochemistry and Cell Biology, Center for Developmental Genetics,
Stony Brook University, Stony Brook, NY 11794-5215, USA.
*Author for correspondence ([email protected])
Received 2 December 2013; Accepted 3 August 2014
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development and cognition, PQBP1 regulates a broader scope of
body patterning events, yet the normal developmental functions of
PQBP1 and the pathogenic mechanisms underlying Renpenning
syndrome remain largely undeciphered.
At the biochemical level, PQBP1 functions in transcription and
pre-mRNA splicing. PQBP1 interacts with the C-terminus of
activated RNA polymerase II (Okazawa et al., 2002) and with
spliceosome components, including U5-15kD (Dim1p) (Waragai
et al., 2000; Zhang et al., 2000) and the U2 snRNP component
Sf3b1 (Wang et al., 2013). Most human disease-causing PQBP1
mutations truncate the C-terminal domain of PQBP1 that is required
for interaction with the spliceosomal protein U5-15kD (Waragai
et al., 2000; Zhang et al., 2000). Knockdown of PQBP1 in mouse
embryo primary neurons reduces general splicing efficiency and
promotes the use of alternative splice sites in a variety of transcripts
(Wang et al., 2013). A missense mutation within the WW domain at
the N-terminus of PQBP1 in Golabi–Ito–Hall (GIH) patients
(Y65C) causes abnormal pre-mRNA splicing and inhibits PQBP1
binding to spliceosome components and a partner protein, WBP11
(Lubs et al., 2006; Tapia et al., 2010; Sudol et al., 2012). PQBP1
and WBP11 both associate with the BΔU1 or PRP19 spliceosomal
complexes (Makarova et al., 2004; Deckert et al., 2006) and
colocalize to nuclear speckles enriched in splicing factors (Komuro
et al., 1999b; Llorian et al., 2004, 2005; Nicolaescu et al., 2008).
A central, polar amino acid-rich domain (PRD) in mammalian
PQBP1 can bind to proteins with polyglutamine ( polyQ) tracts,
such as transcription factors Brn2 and Ataxin-1, and the
cytoplasmic transport protein Huntingtin (Htt), which is enhanced
in poly(Q)-expanded pathogenic mutants of these human proteins
(Waragai et al., 1999; Okazawa et al., 2002; Busch et al., 2003).
Although the molecular effects of PQBP1 have been studied in
cultured cells and some model animals, little is known about its
physiological function in developing embryos. Partial loss of
function of PQBP1 in mice and Drosophila causes neurobehavioral
phenotypes with no reported developmental defects (Ito et al., 2009;
Tamura et al., 2010). However, a homozygous knockout null mouse
line apparently cannot be generated, probably due to lethality,
suggesting an essential role for PQBP1 in embryonic development.
Overexpression of PQBP1 is also deleterious, promoting neuronal
cell death in mice (Okuda et al., 2003; Marubuchi et al., 2005), and
impaired long-term memory and behavioral abnormalities in
Drosophila (Yoshimura et al., 2006). A PQBP1 gene duplication
has been found in a patient with Renpenning syndrome, in which the
PQBP1 protein may be overexpressed (Flynn et al., 2011). All of
these findings suggest that maintaining PQBP1 within a restricted
concentration range is essential for its proper biological actions.
Here, we use the animal model Xenopus laevis to investigate the
embryonic expression and functions of PQBP1 and to identify
potential molecular targets. We also evaluate one of its physiological
partner proteins, WBP11, for which no developmental expression or
functional information has been reported. We have found that both
DEVELOPMENT
Yasuno Iwasaki and Gerald H. Thomsen*
genes are expressed in the developing mesoderm and nervous system,
and that loss of function (by morpholino knockdown) of either one
results in abnormal gastrulation and neurulation. We also find that
inhibiting PQBP1 or WBP11 causes similar defects in mesodermal
and neural differentiation in Xenopus embryos, particularly in FGFresponsive gene expression. Mechanically, PQBP1 knockdown
causes a shift in the alternative splicing of FGF receptor-2 (FGFR2)
pre-mRNA, and a loss of MAPK activation in response to FGF4
(eFGF). Our findings may provide clues to the cause of developmental
abnormalities associated with human PQBP1 mutations in
Renpenning syndrome, with the particular suggestion that PQBP1
mutations may result in aberrant FGF signaling in embryonic and
postnatal Renpenning patients.
RESULTS
PQBP1 knockdown causes mesodermal and neural defects
in Xenopus embryos
Two X. laevis pqbp1 genes were revealed by BLAST searches of
Xenopus expressed sequence tag (EST) and genome databases
using mammalian pqbp1 sequences, which are homeologs that
result from an ancient interspecies hybridization event that gave rise
to the allotetraploid X. laevis lineage (Hughes and Hughes, 1993).
The two predicted proteins (PQBP1a and PQBP1b) share 94%
sequence identity, and are highly homologous to human PQBP1
(79-80% identity) except for the poly(Q)-binding PRD region,
which is less conserved among species (supplementary material
Fig. S1A).
To lay the groundwork for predicting and testing potential
developmental functions of PQBP1, we determined the
spatiotemporal expression of these genes in developing Xenopus
embryos by whole-mount in situ hybridization (WISH) and semiquantitative RT-PCR (qPCR) (Fig. 1; supplementary material
Fig. S2). During early cleavage (∼64 cells), pqbp1 transcripts were
present in animal pole blastomeres (Fig. 1A), and in the early
gastrula (stage 10.5) pqbp1 was expressed throughout the
mesoderm (Fig. 1B,C). In the late gastrula, pqbp1 expression
Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
coincided with both chordin (marking the notochord) and sox2
(marking the prospective neural plate) (Fig. 1D). At neurula stages,
pqbp1 was expressed in the neural plate and the head primordium
(Fig. 1A,E) in a region that overlapped with ncam expression but
also encompassed the cranial neural crest (Fig. 1E) and cells in the
ventrolateral neural tube (supplementary material Fig. S3). In
tailbud tadpole stages, pqbp1 was expressed in head, eyes, spinal
cord and branchial arches (Fig. 1A).
To determine which early developmental processes require
PQBP1, we performed gene knockdown in X. laevis embryos
with translation-blocking antisense morpholino oligonucleotides
(MOs) that target each individual pqbp1 homeolog (MOa and MOb
for pqbp1a and pqbp1b, respectively), or another MO predicted to
inhibit both homeologs simultaneously (MO1; Fig. 2). The ability
of MO1 to block pqbp1 mRNA translation was confirmed by
western blot (Fig. 2G). We first examined the effects of PQBP1
knockdown in the mesoderm by bilaterally injecting MOs into the
marginal zone of two-cell stage blastulae (Fig. 2). MO-injected
embryos are referred to as morphants, and MO1 morphant
phenotypes commonly included shortened anteroposterior (AP)
body axes, small heads and no tail structures (Fig. 2C). These
phenotypes were also accompanied by some cell dissociation at
neurula stages. Interestingly, embryos injected singly with either
MOa or MOb showed only mild developmental defects (Fig. 2D,E),
but when these MOs were combined the resulting morphants lacked
head and tail structures that were very similar to, but more severe
than, the defects caused by MO1 (Fig. 2F). The congruent effects of
knocking down one or both PQBP1 homeologs indicate that the
individual PQBP1 morphant phenotypes are specific to the MOs
and that both homeologs contribute similarly to normal X. laevis
development.
We next tested the effects of PQBP1 knockdown on development
of the dorsal, Spemann–Mangold organizer mesoderm and adjacent
neural tissue. MO1 was injected into the dorsal marginal zone
(DMZ) of four-cell embryos, and the resulting morphants showed
abnormal cell movements associated with gastrulation and neural
Fig. 1. pqbp1 and wbp11 genes are similarly
expressed in mesoderm and neural tissues
during Xenopus development. (A) WISH detection
of pqbp1 and wbp11 transcripts in 64-cell blastula,
neurula and tailbud tadpole; lateral views except
anterior (ant) view of neurula. (B-E) WISH on intact
(C,E) or longitudinally bisected (B,D) embryos with
probes indicated. Asterisks mark the dorsal
blastopore lip. (B) pqbp1 transcripts are present in the
animal pole ectoderm and marginal zone in early
gastrula (stage 10.5), upper panel; sense probe,
lower panel. (C) Expression of pqbp1 and chordin in
gastrulae, stage 11.5. (D) Expression of pqbp1,
wbp11, chordin and sox2 in late gastrulae, dorsalposterior views. Expression of pqbp1 overlaps with
chordin in dorsal/axial mesoderm (arrows) and with
sox2 in anterior neurectoderm. (E) Expression of
pqbp1 and ncam mRNA in the neural plate of early
neurula embryos. Note pqbp1 expression is in a
broader region than ncam. an, anterior
neurectoderm; chd, chordin; S, pqbp1 sense probe.
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
plate folding, and later stage tadpoles had reduced head and
shortened AP axial structures. The severity of the phenotypes was
dose-dependent and increased as the amount of MO1 was raised
(Fig. 2H). In some cases, open blastopores and abnormally folded
neural plates were clearly visible (Fig. 2J,K). Importantly, these
morphant defects could be partly rescued by co-injection of
synthetic pqbp1 mRNA (0.4 ng or 2.0 ng) resistant to the PQBP1
MOs, indicating that the effects of MO1 are specific (Fig. 2J,K;
supplementary material Fig. S5). Consistent with abnormal
phenotypes caused by gain of function in other species,
overexpression of PQBP1 in Xenopus embryos also caused severe
developmental defects in gastrulation and neurulation (Fig. 2I;
supplementary material Fig. S4), and those resembled the PQBP1
knockdown phenotypes (Fig. 2H).
To further characterize the developmental defects in PQBP1
morphants, we examined molecular markers of regional patterning.
As a combination of MOa and MOb had the strongest and most
consistent effects, this pair was applied together in these and in
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subsequent PQBP1 knockdown experiments, unless otherwise
noted. Examining general mesoderm, injection of PQBP1 MOs into
any region of the mesoderm reduced or eliminated brachyury
expression in recipient cells (Fig. 3A), which also underwent
abnormal gastrulation movements (Fig. 3B). Furthermore, although
chordin expression (marking the organizer mesoderm) was not
inhibited by PQBP1 knockdown, chordin-expressing cells spread
ventrolaterally during gastrulation, indicating abnormal
convergence extension movements (Fig. 3B). Blastopore closure
was delayed in morphants but eventually finished by the time
control embryos reached neurula stage. However, the AP axes of
PQBP1 morphants were much shorter than in controls, consistent
with insufficient extension movements (Fig. 3B). In the neural
domain, expression of the general neural marker ncam and neural
crest marker snail2 (slug) were substantially decreased in PQBP1
morphants compared with controls (Fig. 3C,D). Furthermore,
unilateral injection of PQBP1 MO1 at the two-cell stage inhibited
sox2 expression and perturbed neural folding on the injected side
DEVELOPMENT
Fig. 2. Knockdown of PQBP1 causes defective
embryo morphogenesis. (A) Design of three
pqbp1 antisense morpholino oligonucleotides
(MOs). MO1 ( purple bar) targets the ATG start
codon of pqbp1a and pqbp1b (with three nucleotide
mismatches), while MOa and MOb (blue bars)
target the 5′ UTRs of pqbp1 a and b, respectively.
Identical nucleotides between pqbp1 a and b are
indicated by black background. (B-F) Tailbud
tadpole stage embryos injected bilaterally at the twocell stage with 50 ng control (CT) or pqbp1 (PQ)
MOs as indicated (embryos in F received 100 ng
MOs in total). (G) Translation of C-terminal myctagged Xenopus PQBP1 (PQ-myc) was blocked by
co-injection of pqbp1 MO1 but not control MO (CT).
GFP mRNA co-injected as a negative control for MO
targeting and loading control. (H) Phenotypes of
tailbud stage embryos dorsally targeted with control
(CT) or the indicated amount of pqbp1 MO1.
(I) Overexpression of PQBP1 via injected mRNA
(PQ) perturbs normal development. Left panel, wildtype embryo (stage 26); right panel, embryos
injected dorsally with pqbp1 mRNA (2 ng) at the
four-cell stage. (J,K) Gastrulation and neurulation
defects are partially rescued by MO-resistant pqbp1
mRNA. PQ MO (30 ng) co-injected dorsally with
2 ng lacZ mRNA encoding β-galactosidase (β-gal),
displayed perturbed gastrulation (arrowheads point
to open blastopores) substantially rescued by
co-injection of morpholino-resistant pqbp1 (2 ng) (J).
Embryos injected with PQ MO and either 0.4 or 2 ng
of pqbp1 mRNA scored for the following phenotypes
(K): closed blastopore with complete neural folds
(closed+com NF), closed blastopore with partial
neural folds (closed+part NF), closed blastopore
without neural folds (closed) or open blastopore
(open). WT, wild type.
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
Fig. 3. The effects of PQBP1 knockdown on embryonic
mesoderm and neurectoderm. (A) Expression of the
pan-mesodermal marker brachyury (bra) in late gastrula
(stage 12.5) embryos injected with 20 ng pqbp1 MO1
into dorsal (D), ventral (V) or both (DV) blastomeres at the
four-cell stage. Note lack of bra expression wherever the
pqbp1 MO was injected (white brackets). (B) Expression of
the dorsal (axial) mesoderm marker chordin in embryos
injected dorsally with 20 ng of either control MO (CT MO)
or pqbp1 MO1 (PQ MO1) at the four-cell stage.
(C) Expression of neural marker ncam in uninjected (WT)
or pqbp1 MO1-injected embryos targeted dorsally as in
B. Lateral views with anterior to left. (D) Expression of the
neural crest marker snail2 (slug) and the neural marker
sox2 in neurula embryos (stage 19) injected in a single
two-cell stage blastomere with 20 ng pqbp1 MO1 or control
MO (CT) along with β-galactosidase lineage tracer (red).
Dorsal views with anterior down. Perturbed neural folding
is shown by differences between width of left and right
neural folds (brackets). Loosely adherent sox2-positive
cells, marked by arrowheads in the magnified view of the
boxed area. WT, wild-type control embryos.
WBP11 is essential for normal development
The spliceosome protein WBP11 is an endogenous partner of
mammalian PQBP1 (Nicolaescu et al., 2008; Tapia et al., 2010), but
despite its functional importance in pre-mRNA splicing, nothing is
known about its developmental expression or function. We identified
two homeologs in X. laevis that encode predicted proteins with 95%
(604/636) identity to each other and 74% identity to human WBP11
(supplementary material Fig. S1B). In developing Xenopus embryos,
wbp11 is expressed maternally, with maternal transcripts persisting
through blastula stages. Postzygotically, wbp11 is expressed in
ectoderm and mesoderm during gastrulation, in neural plate during
neurulation and in spinal cord and brain in tadpole stages (Fig. 1A;
supplementary material Fig. S2). Wbp11 expression closely coincides
with that of pqbp1, consistent with a physical and functional
partnership between these proteins in mammalian systems. We
validated the ability of Xenopus PQBP1 and WBP11 proteins to
physically associate by co-immunoprecipitation from mammalian
cultured cells (supplementary material Fig. S6A), and by observing
their ability to colocalize to nuclei (supplementary material Fig. S6B).
Potential embryonic functions for WBP11 have not been
evaluated in any species. Therefore, we tested whether WBP11 is
required for development by inhibiting expression of the two
X. laevis homeologs with translation-blocking MOs (Fig. 4A). We
confirmed the specificity of the WBP11 MOs by showing that they
block endogenous WBP11 translation in X. laevis embryos
(Fig. 4B). Embryos injected bilaterally with WBP11 MOs
displayed shortened AP axes, open blastopore remnants, small or
absent heads and truncated tails (Fig. 4C). These phenotypes closely
resembled those of PQBP1 morphants, so we tested whether
combined knockdown of PQBP1 and WBP11 might prove more
deleterious than individual gene knockdowns. Embryos were
targeted in the marginal zone with low doses of either the PQBP1
or WBP11 MO, which alone allowed normal gastrulation and caused
only a slight perturbation of neural folding (Fig. 4D). However,
simultaneous low-dose knockdown of PQBP1 and WBP11 blocked
gastrulation (note open blastopores) and neural folding (Fig. 4D).
These seemingly additive phenotypic effects suggest that these
proteins perform similar developmental functions.
Defective mesodermal and neural marker expression in
PQBP1 and WBP11 morphants
To better understand the PQBP1 and WBP11 morphant phenotypes,
we scored regional and tissue-specific marker gene expression by
qPCR on embryos injected bilaterally into the marginal zone at the
two-cell stage and harvested at early gastrulation (stage 10.5).
Results in Fig. 5A show that expression of several general
mesodermal markers was reduced in morphants by individual or
combined knockdown of PQBP1 or WBP11. To assess general
mesoderm specification, we evaluated brachyury and found it was
reduced by about 40% of control levels by PQBP1 knockdown,
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DEVELOPMENT
(Fig. 3D). As seen in Fig. 2C, we also observed some cell dissociation
at neurula stages, typified by sox2-positive cells detaching from the
neural plate in PQBP1 morphants (Fig. 3D, magnified).
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
Two other mesodermal markers, fgf4 and cdx4, were the most
significantly disrupted in PQBP1 morphants (Fig. 5A). Single
knockdown of PQBP1 or WBP11 resulted in 70-90% reduction of
fgf4 and cdx4, whereas combined knockdown almost completely
eliminated their expression. Expression of another mesodermal
FGF gene, fgf8, was normal in all PQBP1 or WBP11 morphants.
The specificity of PQBP1 morpholino was further confirmed
by the ability of MO-resistant pqbp1 mRNA to rescue fgf4 and
cdx4 expression in PQBP1 morphants (supplementary material
Fig. S7B), adding support to rescue experiments in whole-embryo
morphants (Fig. 2J). Specificity is also supported by results
showing that different PQBP1 morpholinos (MO1, MOa or MOb)
had similar effects (supplementary material Fig. S7A).
In the neural ectoderm, single or combined knockdown of
PQBP1 and WBP11 caused significant reduction (∼60%) in the
expression of the early neural marker, sox2 (Fig. 5C), consistent
with reduced sox2 and ncam expression in PQBP1 morphants
observed using WISH (Fig. 3C,D).
We also tested PQBP1 knockdown in related X. tropicalis
embryos, and found phenotypes similar to those of X. laevis PQBP1
morphants, with missing head and tail structures and short body
length, as well as reduced fgf4 and cdx4 expression in early gastrulae
(supplementary material Fig. S7C). The results of morphant
analyses in X. laevis and X. tropicalis are consistent with each
other and provide more general evidence that PQBP1 regulates
essential developmental functions.
Fig. 4. WBP11 knockdown resembles and enhances PQBP1 knockdown
phenotypes. (A) WBP11 MO (blue arrow) targets the 5′ UTR of both X. laevis
wbp11 homeologs. (B) Expression of endogenous WBP11 was blocked by
injection of 40 ng wbp11 MO (WB MO) but not control MO (CT MO).
Endogenous (red arrowhead) and overexpressed (black arrowhead) WBP11
were detected by western blot with anti-WBP11 antibodies. An asterisk
indicates a non-specific band that, along with β-tubulin staining, controls for
sample loading. (C) Tailbud stage embryos injected into two dorsal cells at the
four-cell stage with 100 ng control MO (CT), a mix of pqbp1 MOa and MOb
(50 ng each; PQ MO) or 50 ng wbp11 MO (WB MO). (D) Neurula (stage
20, anterior view) embryos injected dorsally at the four-cell stage (schematic
drawing) with 75 ng control MO (CT), 25 ng wbp11 MO (WB), a mixture of
pqbp1 MOa plus MOb (25 ng each; 50 ng total; PQ) or a combination of wbp11
and pqbp1 MOa and MOb (25 ng each; 75 ng total; PQ+WB).
consistent with reduced brachyury expression seen in morphants by
WISH (Fig. 3A). Combined knockdown of PQBP1 and WBP11
resulted in an even greater reduction (about 80%) of brachyury
expression, despite normal brachyury levels with WBP11
knockdown alone (Fig. 5A). Another general mesodermal marker,
zygotic vegT (apod), was reduced by about 50% of control levels by
double knockdown of PQBP1 and WBP11.
We also observed distinct differences in the regional mesodermal
marker expression in single or double PQBP1 and WBP11
morphants. Expression of the ventrolateral markers sizzled and
wnt8 were reduced by about 40 and 60%, respectively, by either
single or combined knockdown of PQBP1 and WBP11 (Fig. 5).
Several genes that mark the Spemann–Mangold organizer, chordin,
goosecoid and siamois, were expressed at near normal levels in
single or double PQBP1/WBP11 morphants, but noggin expression
was reduced by about 50% in the double morphants (Fig. 5B).
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Multiple signaling pathways regulate mesoderm and neural
induction, tissue patterning and morphogenesis in Xenopus
embryos, including FGF, Nodal/Vg1, BMP and Wnt. Inhibition
of any of these pathways might account for the phenotypes of
PQBP1 or WBP11 morphants. To delineate which signaling
pathways might be impaired in PQBP1 morphants, we examined
the effects of PQBP1 knockdown on the induction of marker genes
by FGF, Nodal, BMP or Wnt signals in Xenopus animal caps.
Growth factors (as mRNAs) together with morpholinos were
injected into animal poles of two-cell embryos, and caps were cut
and screened for induction of target genes by qPCR. We found that
PQBP1 morphant animal caps responded normally to Wnt8, Nodal2
(Xnr2) and BMP4, but response to FGF4 was significantly inhibited
(Fig. 6B). Specifically, induction of cdx4 and bra by FGF4 was
diminished in PQBP1 morphants, despite normal induction of cdx4
by Wnt8 or bra by BMP4. Furthermore, induction of fgf4 by FGF4,
induced by FGF4/Brachyury-positive feedback loops (Isaacs et al.,
1994; Fujii et al., 2008), was also blocked by PQBP1 knockdown.
We also tested whether PQBP1 might be required for normal
operation of the FGF receptor tyrosine kinase (RTK) signaling
cascade by analyzing MAPK (Erk) phosphorylation in PQBP1
morphant animal caps. Injection of 1pg fgf4 mRNA into animal
caps induced phosphorylation of MAPK, but co-injection of either
PQBP1 MO1 or MOa+MOb blocked MAPK phosphorylation
(Fig. 6C). In sum, the results of experiments performed in whole
embryos (Fig. 5) and isolated animal caps (Fig. 6) demonstrate that
responses to FGF signaling require PQBP1, whereas Nodal, BMP
and Wnt pathways do not.
PQBP1 regulates alternative splicing of an FGF receptor
One plausible explanation for the deleterious effects of PQBP1
knockdown on FGF responses is that PQBP1 acts at the level of gene
transcription or pre-mRNA splicing for FGF signaling components.
DEVELOPMENT
PQBP1 knockdown impairs responses to FGF but not other
inductive signals
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
At the ligand level, alternative splicing of fgf4 and fgf8 pre-mRNA
can generate protein variants with distinct biological activities
(Fletcher et al., 2006; Guo and Li, 2007; Mayshar et al., 2008). In
particular, two FGF8 isoforms, FGF8a and FGF8b, respectively,
induce neural and mesodermal tissues in Xenopus embryos
(Fletcher et al., 2006). However, we observed no differences in
the splicing patterns of fgf4 or fgf8 transcripts in PQBP1 and/or
WBP11 morphants compared with controls (data not shown).
PQBP1 or WBP11 knockdown defects could also be explained
by mis-splicing of the FGF receptors. Although only four genes
encode FGF receptors, alternative splicing results in numerous
isoforms with different ligand specificity (Ornitz et al., 1996;
Eswarakumar et al., 2005; Zhang et al., 2006). FGF receptors 1, 2
and 3 incorporate exons 8a or 8b through alternative splicing of their
pre-mRNAs, resulting in receptor isoforms IIIb or IIIc, respectively.
These isoforms possess distinct ligand specificity resulting from
unique residues in the immunoglobulin-like loop III of the
extracellular, ligand-binding domain encoded by exon 8. Among
mammalian receptors, the IIIb isoforms of FGFR1-3 preferably bind
FGF3, FGF7 and FGF10, whereas IIIc isoforms prefer FGF4 and
FGF8 (Eswarakumar et al., 2005; Holzmann et al., 2012). FGFR4
does not undergo alternative splicing of exon8, but binds FGF4 and
FGF8b (Blunt et al., 1997).
In order to determine whether expression or splicing of FGF
receptors was modified, we surveyed the levels of FGFR1-3 IIIb or
IIIc isoform transcripts, as well as the total expression levels of all
four FGF receptors, in control (control MO-injected), PQBP1 or
WBP11 morphant Xenopus embryos at early gastrulation (stage
10.5). To assay IIIb and IIIc isoforms, we used primers that targeted
exon8a or exon8b on the upstream side, and thus amplified only
spliced transcripts that contained one of these alternative exons in
fgfr1-3 (as illustrated for fgfr2 in Fig. 7C,D). In wild-type or control
embryos, qPCR indicated the presence of all potential receptor
isoforms, but relative isoform levels varied (Fig. 7; supplementary
material Fig. S8). We observed a dramatic change in the isoform
ratio of fgfr2 IIIb and IIIc transcripts, but no change in the total level
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DEVELOPMENT
Fig. 5. Effects of PQBP1 and WBP11
knockdown on embryonic gene
expression. qPCR performed on early
gastrula (stage 10.5) morphant embryos
injected bilaterally at the two-cell stage with
150 ng control (CT), 100 ng pqbp1 (PQ), 50 ng
wbp11 (BP) or a combination of pqbp1 and
wbp11 (PQ+BP) MOs (150 ng total). Values
were plotted relative to the control cap signal
and shown as mean±s.e.m of n=3 with
Student’s t-test to control embryos (CT)
(*P<0.05 or **P<0.01). (A) Expression of
general mesodermal markers brachyury (bra),
antipodean (apod), sizzled, wnt8, fgf4, cdx4
and fgf8. (B) Expression of Spemann–
Mangold organizer-specific markers,
chordin, goosecoid, siamois and noggin.
(C) Expression of early neural marker sox2.
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
(Fig. 7; supplementary material Fig. S8). We did not observe an
increase in unspliced transcripts resulting from knockdown of either
PQBP1 or WBP11 (data not shown), which potentially could have
happened if there was a general failure in pre-mRNA splicing,
which appears unlikely.
As FGFR2IIIc, but not FGFR2IIIb, can respond to FGF4 or FGF8b
ligands, the reduced levels of fgfr2IIIc transcripts in Xenopus PQBP1
morphants might be contributing to lowered FGF signaling and
abnormal phenotypes. To address this possibility, we tested whether
reduced expression of endogenous FGF response genes in PQBP1
morphants could be rescued by ectopic expression of an FGFR2IIIc
isoform (Fig. 7E). To do this, we co-injected a synthetic mRNA
encoding the X. tropicalis FGFR2IIIc isoform together with PQBP1
MOs and found that it partially rescued endogenous fgf4 and cdx4
gene expression. This result indicates that altered splicing of fgfr2
transcripts due to PQBP1 knockdown has biological consequences
for embryogenesis.
DISCUSSION
of all frfgr2 transcripts were observed (Fig. 7B-D). In control
embryos, fgfr2IIIc transcripts (containing exon 8b) were more
abundant than those of fgfr2IIIb (containing exon 8a); this ratio was
reversed, however, in PQBP1 morphants, as measured by qPCR
(Fig. 7C) or by gel-based RT-PCR (Fig. 7D).
The other FGF receptors showed only minor shifts in isoform
splicing and total levels in PQBP1 morphants (supplementary
material Fig. S8). Knockdown of WBP11 alone had no significant
effects on the relative level of these fgfr2 splice isoforms, nor did
splicing enhance changes in PQBP1/WBP11 double morphants
3746
PQBP1 and WBP11 are required for normal mesodermal and
neural development
Despite the recognized importance of pqbp1 mutations in the
etiology of human XLID syndromes, information about the
embryonic expression and function of PQBP1 or WBP11 is
limited to a description of mouse pqbp1 expression (Qi et al.,
2005). Our analysis of Xenopus embryos shows that pqbp1 and
wbp11 genes are maternally expressed, present throughout
cleavage late blastula stages, then zygotically expressed in the
mesoderm during gastrulation and the developing neural plate and
nervous system of tadpoles.
The expression of pqbp1 in tailbud tadpoles appears homologous
to that reported for embryonic and postnatal mice, where pqbp1
mRNA and protein are predominantly expressed in the central
nervous system and neuronal stem cells (Waragai et al., 1999; Qi
et al., 2005; Wang et al., 2013). The neural expression of pqbp1 in
Xenopus and mouse is consistent with the fact that human pqbp1
mutations cause mental retardation and microcephaly. Moreover,
DEVELOPMENT
Fig. 6. Effects of PQBP1 knockdown on animal cap response to growth
factors. (A,B) Two-cell embryos were injected into the animal pole with growth
factor mRNAs and MOs, animal caps were cut at mid-blastula and harvested at
the equivalent of early gastrula stage, followed by qPCR, as depicted (top).
Results were analyzed and plotted as per Fig. 5. (A) Marker gene induction
by Wnt8 (50 pg), BMP4 (500 pg) and Nodal2 (Xnr2; 100 pg) was not affected
by PQBP1 knockdown (50 ng MO). There was no statistically significant
difference between CT and PQBP1 MO-injected caps treated with each ligand
(Student’s t-test, n=3). (B) Marker gene induction by FGF4 was significantly
reduced by PQBP1 knockdown (*P<0.05 or **P<0.01; Student’s t-test,
triplicate biological replicates). Animal caps were injected with fgf4 mRNA (1 pg)
and either control MO (50 ng), pqbp1 MO1 (50 ng) or MOa+MOb (25 ng each).
(C) Phosphorylation of MAPK (Erk) was induced in fgf4-injected animal caps,
but blocked by co-injection of pqbp1 MO, either MO1 or MOa+MOb. The levels
of β-tubulin and total MAPK protein did not change among these samples.
PQBP1 and one of its binding proteins, WBP11, are implicated in
RNAPII-dependent transcription and pre-mRNA splicing, but
neither protein has clearly delineated roles or gene targets in
developing embryos. Here, we have shown that during
development of Xenopus embryos, PQBP1 and WBP11 are coexpressed in nascent mesodermal and neural tissues, and loss of
function of these genes causes defects in mesodermal and neural
patterning accompanied by abnormal gastrulation and neurulation,
as well as by reduced mesodermal gene expression. At the
molecular level, we find that PQBP1 is required for MAPK
activation, target gene expression and mesoderm induction by
FGF4 ligand, but not for responses to other mesodermal inducers
and patterning agents, including Nodal2, BMP4 or Wnt8.
Furthermore, we find that PQBP1 regulates alternative splicing
of FGF receptor-2 transcripts, influencing the relative abundance
of two FGFR2 isoforms (IIIb and IIIc) that have different binding
specificities for FGF ligands. Our results reveal important roles for
PQBP1 in vertebrate embryonic development, and those may be
relevant to the molecular mechanisms of human developmental or
neural/cognitive defects caused by pqbp1 mutations. Although
WBP11 is not implicated in human disease or birth defects, our
study is the first to indicate a normal developmental role for
WBP11 in any embryo, raising the possibility that wbp11
mutations could contribute to birth defects.
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Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
Fig. 7. Alternative splicing pattern of fgfr2
exon 8a/b is altered by PQBP1 knockdown.
(A) Alternative splicing incorporates either exon
8a or 8b into fgfr2 transcripts, which generates
two isoforms of FGFR2: IIIb and IIIc,
respectively (see text). Intron sizes (kb)
indicated. (B,C) Sum total of qPCR
measurements of fgfr2IIIb/c transcripts (B), and
the relative levels of each splicing variant (C),
on total RNA extracted from early gastrula
(stage 10.5) morphant embryos bilaterally
injected at the two-cell stage with MOs: 150 ng
control (CT), 100 ng PQBP1 (PQ), 50 ng
WBP11 (BP), or combined PQBP1 and WBP11
(PQ+BP) (150 ng total). Samples were
prepared and analyzed as per Fig. 5 (*P<0.05
or **P<0.01; Student’s t-test, n=3). (D) Semiquantitative RT-PCR amplification products of
alternative spliced exon 8a or 8b from embryos
injected with morpholinos (as above).
(E) Partial rescue of fgf4 expression in pqbp1
morphants (100 ng pqbp1 MO) by co-injection
of 1 ng Xenopus fgfr2IIIc (*P<0.05; Student’s
t-test, n=3).
PQBP1 is required for FGF signaling and regulates splicing of
FGF receptor transcripts
Our work further shows that the molecular and morphological defects
observed in PQBP1 morphant embryos appear to be the result of
aberrant FGF signaling. Direct tests of mesoderm induction in
animal caps showed PQBP1 knockdown is required for signaling
triggered by FGF4 but not Nodal, BMP or Wnt ligands. The loss of
RTK signal transduction in particular indicates defects upstream of
FGF-responsive gene transcription, and not some general defect in
transcription or mRNA splicing of FGF target genes. FGF receptors,
of which there are four, were foremost among candidates because their
transcripts can undergo alternative splicing in various biological
systems to generate receptor isoforms with different ligand-binding
specificities. Our survey of fgfr expression and splicing shows that
among the four receptors, fgfr2 showed drastically altered levels of
alternatively spliced transcripts encoding isoforms IIIb and IIIc in
PQBP1 morphants (supplementary material Fig. S8). Normally, early
gastrula stage embryos express approximately 50% more fgfr2IIIc
transcripts than fgfr2IIIb transcripts. We found that PQBP1
knockdown reverses this ratio, lowering fgfr2IIIc transcript levels to
about half the level of fgfr2IIIb transcripts. As the FGFR2IIIc, but not
IIIb, receptor isoform binds to FGF4 and FGF8, we postulate that the
lowered abundance of FGFR2IIIc reduces the ability of the embryo to
respond to mesoderm-inducing FGF4/8 ligands.
3747
DEVELOPMENT
the co-expression of pqbp1 and wbp11 in the same tissues of
Xenopus embryos is consistent with the known physical and
functional links between the two proteins (Komuro et al., 1999b;
Llorian et al., 2004, 2005; Nicolaescu et al., 2008). Whether these
proteins function together mechanistically to govern the same
biochemical or embryonic processes remains to be determined. At
neurula stages, PQBP1 or WBP11 morphants display incomplete
neural tube closure accompanied by reduced expression of neural
marker genes, underscoring their requirement for normal neural
development.
The neural expression of pqbp1 in Xenopus embryos might have
been anticipated, but our finding that both pqbp1 and wbp11 are
expressed and required for normal development of mesoderm is new
and reveals a broader role for these genes in early developmental
processes, particularly in non-neural tissues. Knockdown of either
gene causes abnormal cell migration during gastrulation, notably a
failure of dorsal mesodermal convergence-extension movements.
These defects in gastrulation are probably linked to reduced
brachyury expression in PQBP1 morphants, as this gene is
essential for mesodermal cell motility as well as differentiation.
Some physical manifestations observed in Renpenning syndrome
patients, including midline and cardiac defects, lean muscle and
short stature, may be analogous to defects in mesoderm observed in
PQBP1 morphants.
Furthermore, whereas PQBP1, FGF4 and FGF8 morphants share
similar mesodermal, neural and gastrulation defects (Fisher et al.,
2002; Fletcher et al., 2006; Isaacs et al., 2007), PQBP1 morphant
phenotypes are more severely affected, yet rather similar to those
generated by a dominant negative FGF receptor (XFD) or FGFR
inhibitor SU5402, which block all FGF signaling, including that
stimulated by FGF4 and FGF8b (Amaya et al., 1993; Delaune et al.,
2005; Fletcher and Harland, 2008). This greater similarity between
PQBP1 knockdown and wholesale loss of FGF signaling is
potentially explained by the reduction of the FGFRIIIc isoform in
PQBP1 morphants, but we cannot exclude the possibility that
PQBP1 inhibition affects expression of other essential embryonic
gene targets.
FGFR2 isoform switching via alternative splicing has been
observed in multiple contexts, including normal epithelialmesenchymal transition (Warzecha and Carstens, 2012), embryonic
development (Eswarakumar et al., 2002; Rice et al., 2003; Takeuchi
et al., 2005; Liu et al., 2011), human birth defects (Hajihosseini et al.,
2001; Teebi et al., 2002; Ibrahimi et al., 2005), cancer and various
pathologies (Katoh, 2008, 2009; Holzmann et al., 2012; Kelleher
et al., 2013). Whether PQBP1 regulates fgfr2 alternative splicing in
these normal and disease situations will be worth investigating. Our
preliminary results also show changes in relative levels of alternative
transcripts for fgfr1-3, but changes in fgfr2 transcript splicing were
greater than fgfr1 and fgfr3 upon PQBP1 knockdown (supplementary
material Fig. S8). The time and place of expression of all FGF
receptors and their splicing variants in Xenopus embryos has not been
well delineated, so further investigation is required to understand their
precise roles in mesoderm and neural induction by FGFs and how
pqbp1 impacts these functions.
In addition to our findings, previous studies have identified other
factors regulating fgfr2 pre-mRNA splicing. Epithelial splicing
regulatory proteins 1 and 2 (ESRP1 and ESRP2) govern alternative
splicing of fgfr2 and other transcripts in a human epithelial cell line,
and depletion of ESRP1 and ESRP2 causes splice switching from
an epithelial IIIb form to a mesenchymal IIIc form (Warzecha et al.,
2009). Fused in sarcoma (FUS) has also been shown to regulate
fgfr2 transcript splicing in Xenopus embryos (Dichmann and
Harland, 2012), and its knockdown causes a complete loss of
fgfr2IIIc transcripts but has no effect on fgfr2IIIb transcript levels.
The basis of FUS and PQBP1 target gene specificity is unknown,
but FUS can directly bind RNA sequences, whereas direct
interaction between PQBP1 and endogenous RNA was not
evident by crosslinking immunoprecipitation (Wang et al., 2013).
Whether FUS, ESRP1/2, PQBP1 and/or WBP11 interact or
function together in the splicing of fgfr2 or other gene transcripts
awaits investigation.
Potential implications for XLID
Besides cognitive defects, Renpenning patients have physical
abnormalities that suggest underlying defects in the development
of mesodermal as well as neural tissues (Stevenson et al., 2005;
Germanaud et al., 2010). The details of human tissue pathologies
associated with PQBP1 mutations, particularly those of non-brain
tissue, are not well described, but the cardiac and skeletal muscular
defects and reduced height prevalent in XLID patients may echo the
mesodermal defects we observe in Xenopus PQBP1 morphants.
FGF signaling is essential not only for development of mesodermal
and neural tissues in vertebrate embryos, but also for the ongoing
normal functions of adult tissues derived from these germ layers,
including brain functions (Reuss and von Bohlen und Halbach,
2003; Iwata and Hevner, 2009; Mudo et al., 2009; Turner et al.,
3748
Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
2012). Our findings raise the hypothesis that abnormal FGF
signaling may underlie body, brain, and possibly cognitive, defects
in Renpenning patients.
One aspect of PQBP1 function suggested by human and
animal studies, and reinforced by our work, pertains to the dosage
effects of PQBP1 on development and disease. Viable human
PQBP1 mutations encode single residue changes, or frame-shifted
proteins with C-terminal truncations that are probably partly
functional (hypomorphic). No complete deletions of the PQBP1
gene or entire loss of its coding region have been described in
PQBP1-linked XLIDs. Mouse gene knockout and overexpression
models to investigate PQBP1 developmental functions have not
been very successful, which seems to indicate a requirement for
PQBP1 protein expression within a critical range: too little or too
much protein is apparently lethal (Okuda et al., 2003). Our own
studies here support that notion, as we observed dose-dependent
embryonic defects in both loss- and gain-of-function tests.
The highest doses of pqbp1 morpholinos or injected mRNA
inhibit gastrulation and neurulation (Fig. 2; supplementary
material Fig. S4), whereas moderate morpholino doses cause
mild, dose-dependent phenotypes.
Finally, the PQBP1 partner protein WBP11 has not been
implicated in the etiology of Renpenning or other XLIDs, or
human diseases in general, but as these proteins appear to have
partially overlapping functions in Xenopus development, it will be
valuable to evaluate whether altered WBP11 expression or activity
might contribute to Renpenning or other human diseases. Further
investigation of PQBP1 and WBP11 target genes and molecular
mechanisms using Xenopus embryos should contribute to a more
complete understanding of how these genes regulate normal
vertebrate developmental processes and, when mutated, contribute
to human developmental and intellectual disabilities.
MATERIALS AND METHODS
Embryo manipulations
Xenopus laevis embryos were obtained by standard in vitro fertilization,
de-jellied in 2% cysteine pH 8.0, microinjected and incubated for several
hours in 3% Ficoll+0.5× MMR+10 μg/ml gentamycin and grown in 0.1×
MMR+10 μg/ml gentamycin thereafter. All stages are according to
Nieuwkoop and Faber (1994). Animal caps were excised at stage 8 and
cultured in 0.5× MMR +10 μg/ml gentamycin until the sibling embryos
reached stage 10.5.
Microinjection of MO and mRNA
Xenopus embryos were microinjected with MO and/or synthetic mRNA at
two-cell or four-cell stages. All MOs were designed and synthesized by Gene
Tools. The control MO was the standard scrambled MO, 5′-CCCTTACCTCAGTTACAATTTATA-3′. pqbp1 MO1 is 5′-CGCTA-AAGGGAGAGGCATCCTGGC-3′. pqbp1a MO (MOa) is 5′-AGCTCTT-GTTCTAACTCCCCGCCGT-3′. pqbp1b MO (MOb) is 5′-ACCGACACG-CTCCTGCTCCTACTCT-3′. wbp11 MO is 5′-ATGTCCTCTCTCCACC-AATATCAGA-3′,
X. tropicalis pqbp1 MO is 5′-CGGCATCTTGGGCG-GCCAACCACAC-3′.
Capped mRNAs were synthesized using AmpliCap SP6 High Yield Message
Maker (Epicentre Biotechnologies) or mMESSAGE mMACHINE (Ambion)
from the following linearized plasmids: pCS2-pqbp1, pSPYS-chordin, pCS2bmp4, pCS2-wnt8, pSP64-fgf4, pCS2-wbp11. In some cases, lacZ mRNA
encoding β-galactosidase was co-injected as a linage tracer, the activity of
which was visualized in fixed embryos using Red-Gal (5-Bromo-6-chloro3-indolyl β-D-galactopyranoside; Sigma-Aldrich).
X. laevis cDNA cloning and plasmid construction
A TBLASTN search identified two incomplete X. laevis cDNA clones
(gi:8320261 and 8319534) homologous to mammalian PQBP1. Based on
these EST sequences, the full-length pqbp1 clone was amplified by PCR from
Xenopus cDNA (blastula) with the following primers: pqbp1 forward,
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
5′-GATCGTTGGCGTCATCAACAGG-3′ and pqbp1 reverse, 5′-GCACAAGCAACTGTACAGCAGT-3′. The sequence of this clone completely
matched to another X. laevis EST clone (NM_001091714) except for
its shorter 5′ untranslated region (UTR) end. Identical sequences were
identified in X. laevis genome scaffolds (Xenbase gbrowse laevis 6.0:
Scaffold1707:1728996-1729209). MO-resistant Xenopus pqbp1 cDNA was
amplified with the primers forward 5′-CCTCGAGATGCCGTTGCCTTTAGCGCTTCAGGCT-3′, reverse 5′-CAATCAATAGGGGGCAGCAATG3′, and subcloned into pCS2+ for in vitro transcription. This resulted in nine
nucleotide mismatches at the pqbp1 morpholino recognition site, but normal
amino acid coding was retained. C-terminal myc-tagged Xenopus PQBP1
was made by PCR cloning into the myc/pRK5-SK vector. Full-length
X. laevis wbp11 was obtained by RT-PCR with the following primers: 5′-CCCATCGATATGGGGCGGCGATCCACTTCG-3′ and 5′-CGAATCGATGAAAAGTTGCTTAGAATTAGCCG-3′ (restriction enzyme sites are
underlined) with design based on an EST (BC057737). All cDNA identities
were verified by DNA sequencing, and subcloned into pCS2+ unless otherwise
noted. Full-length X. tropicalis fgfr2IIIc was a gift from Dr Richard Harland
(University of California, Berkeley, USA).
Development (2014) 141, 3740-3751 doi:10.1242/dev.106658
antibodies. Transfected and endogenous cellular control proteins were
visualized on the blots, as above, with mouse anti-β-tubulin (Santa Cruz,
sc-58884; 1:500), mouse anti-GFP (Santa Cruz, sc-9996; 1:500) and antiWBP11 rabbit polyclonal antibodies prepared by our lab using a Xenopus
WBP11-GST fusion protein. For immunofluorescence assays, HA-pqbp1 and
myc-wbp11 were transfected into COS-1 cells and detected with anti-myc
mouse monoclonal 9E10 and anti-HA rabbit polyclonal antibodies, detected
with Alexa 488 (Molecular Probes, A-11029, 1:1000) goat anti-mouse and
Alexa 594 goat anti-rabbit (Molecular Probes, A-11037, 1:1000) secondary
antibodies. COS-1 cells were grown in Dulbecco’s modified Eagle’s medium
(Gibco) supplemented with 10% fetal bovine serum.
Acknowledgements
We thank Dr R. Harland (University of California, Berkeley, USA) for the fgfr2c
plasmid and members of the G.H.T. lab for experimental advice and comments on
the manuscript. We acknowledge Xenbase for facilitating gene analyses, and the
National Xenopus Resource (NXR) and European Xenopus Resource Center
(EXRC) for supplying animals and reagents.
Competing interests
The authors declare no competing financial interests.
In situ hybridization
In situ hybridization was performed with digoxigenin-labeled probes
as previously described (Alexandrova and Thomsen, 2006). BM Purple was
used as a chromogenic substrate (Roche). Sagittal or cross sections were
performed during MEMPFA fixation. Template plasmids for making probes
were pBS-ncam, pBS-sox2, pGEM4Z-chordin, pXT1-brachyury, pMXsnail2, pCS2-pqbp1 and pCS2-wbp11.
Author contributions
Y.I. and G.H.T. conceived and designed experiments, Y.I. carried out all
experiments, and Y.I. and G.H.T. wrote the manuscript.
Funding
This research was supported partially by the U.S. National Institutes of Health
[grants R03HD064908 and R01HD032429 to G.H.T.]. Deposited in PMC for release
after 12 months.
Quantitative RT-PCR
Protein analysis, immunoprecipitation and immunofluorescence
For detection of phosphorylated MAPK, animal caps were lysed in 10 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40,
together with a proteinase and phosphatase inhibitor cocktail (Roche).
Solubilized proteins were separated by 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose
membrane (Bio-Rad) and stained with anti-phospho-p44/42 MAPK (Cell
Signaling, 4370; 1:2000), anti-p44/42 MAP kinase (Cell Signaling, 4695P;
1:1000) and anti-β-tubulin (Accurate Chemical and Scientific Corporation,
BYA6068-1; 1:5000) antibodies (Suga et al., 2006). Signals were visualized
by Alexa dye-conjugated goat anti-mouse and anti-rabbit antibody (Molecular
Probes), and visualized using the Odyssey Infrared Imager (LI-COR). For coimmunoprecipitation, HA-pqbp1 was co-transfected with myc-wbp11 into
COS-1 cells using linear polyethyleneimine transfection reagent. Twenty-four
hours after transfection cells were lysed with PBS containing 1% Triton X-100
and complete protease inhibitors (Roche). The lysate was incubated with antiHA rabbit polyclonal (Y-11, Santa Cruz, sc-805) or anti-myc mouse
monoclonal (ATCC, 9E10 hybridoma) antibodies for 2 h at 4°C, then
incubated with protein A or G agarose beads for 1 h at 4°C. Agarose beads
were spun and washed with cold lysis buffer several times, then boiled in
SDS sample buffer before loading onto SDS-PAGE gels. Proteins were
immunoblotted and stained with anti-myc (1:1000) or anti-HA (1:500)
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.106658/-/DC1
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