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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, dorsal-posterior 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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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 5â² 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 two-cell stage with 50â
ng control (CT) or pqbp1 (PQ) MOs as indicated (embryos in F received 100â
ng MOs in total). (G) Translation of C-terminal myc-tagged 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, wild-type embryo (stage 26); right panel, embryos injected dorsally with pqbp1 mRNA (2â
ng) at the four-cell stage. (J,K) Gastrulation and neurulation defects are partially rescued by MO-resistant pqbp1 mRNA. PQ MO (30â
ng) co-injected dorsally with 2â
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 (2â
ng) (J). Embryos injected with PQ MO and either 0.4 or 2â
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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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 20â
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 20â
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 20â
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.
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Fig. 4. WBP11 knockdown resembles and enhances PQBP1 knockdown phenotypes. (A) WBP11 MO (blue arrow) targets the 5â² UTR of both X. laevis wbp11 homeologs. (B) Expression of endogenous WBP11 was blocked by injection of 40â
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 100â
ng control MO (CT), a mix of pqbp1 MOa and MOb (50â
ng each; PQ MO) or 50â
ng wbp11 MO (WB MO). (D) Neurula (stage 20, anterior view) embryos injected dorsally at the four-cell stage (schematic drawing) with 75â
ng control MO (CT), 25â
ng wbp11 MO (WB), a mixture of pqbp1 MOa plus MOb (25â
ng each; 50â
ng total; PQ) or a combination of wbp11 and pqbp1 MOa and MOb (25â
ng each; 75â
ng total; PQ+WB).
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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 150â
ng control (CT), 100â
ng pqbp1 (PQ), 50â
ng wbp11 (BP) or a combination of pqbp1 and wbp11 (PQ+BP) MOs (150â
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 SpemannâMangold organizer-specific markers, chordin, goosecoid, siamois and noggin. (C) Expression of early neural marker sox2.
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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 (50â
pg), BMP4 (500â
pg) and Nodal2 (Xnr2; 100â
pg) was not affected by PQBP1 knockdown (50â
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 (1â
pg) and either control MO (50â
ng), pqbp1 MO1 (50â
ng) or MOa+MOb (25â
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.
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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: 150â
ng control (CT), 100â
ng PQBP1 (PQ), 50â
ng WBP11 (BP), or combined PQBP1 and WBP11 (PQ+BP) (150â
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) Semi-quantitative 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 (100â
ng pqbp1 MO) by co-injection of 1â
ng Xenopus fgfr2IIIc (*P<0.05; Student's t-test, n=3).
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Fig. S1. PQBP1 and WBP11 sequence comparisons. A. Alignment of PQBP1 protein sequences of
human (Homo sapiens, Hs), mouse (Mus musculus, Mm), Xenopus laevis (Xl) homeologs a and b,
zebrafish (Danio rerio, Dr) and Starlet sea anemone (Nematostella vectensis, Nv). Shading indicates
homology (black 80â100%, grey 60â80%). WW: WW domain, PRD: polar amino acid rich domain,
NLS: nuclear localization signal, CTD: C-terminal conserved domain. The additional polar amino acidrich
tail highly conserved among species is marked by wavy line. B. Alignment of WBP11 protein
sequences of human (Hs), mouse (Mm), Xenopus laevis (Xl) homeologs a and b, and zebrafish (Dr).
Shading indicates homology (black 80â100%, grey 60â80%).
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Fig. S2. Expression profile of pqbp1 and wbp11 mRNA during embryogenesis. Total RNA
extracted from indicated developmental stages of X. laevis was used for qPCR analysis. The results
were normalized to ornithine decarboxylase (ODC) levels and plotted relative to the level measured in
the stage 35 embryos (set as 1.0).
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Fig. S3. Expression of pqbp1 and wbp11 in neurula stage embryos. Whole mount in situ
hybridization with indicated probes on embryos that were longitudinally sectioned (A) or crosssectioned
(B) stage 18 embryos. S: sense probe of pqbp1. Both pqbp1 and wbp11 transcripts are
localized to ventro-lateral neural tube cells (B).
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Fig. S4. Over-expression of PQBP1 inhibits normal specification and movement of dorsal
mesoderm. Embryos at the end of gastrulation, stage 13, that had been dorsally injected at the 2-cell
stage with 1 ng pqbp1 mRNA, were analyzed by in situ hybridization with brachyury (bra) and chordin
(chd) probes.
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Fig. S5. Partial rescue of neural folding defects in PQBP1 morphants by pqbp1 mRNA. A. PQ
MO1 (40 ng) was dorsally injected with either 2 ng b-galactosidase (b-gal) or pqbp1 (PQ) mRNA. B.
Fraction of embryos displaying specific phenotypes: closed, partially closed or open neural folds (NF).
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Fig. S6. Xenopus WBP11 interacts with PQBP1. A. Co-immunoprecipitation (co-IP) of PQBP1 and
WBP11 from COS1 cells transfected with HA-tagged PQBP1 (HA-PQ) and myc-tagged WBP11 (myc-
WB). Cell lysates were immunoprecipitated (IP) with an anti-myc antibody, followed by
immunoblotting with an anti-HA antibody to detect PQBP1 bound to WBP11, or vice versa. TCL: total
Development | Supplementary Material
cell lysate. B. Co-immunostaining of myc-tagged WBP11 (myc-WB) and HA-tagged PQBP1 (HA-PQ)
expressed in COS1 cells. Nuclei were stained with DAPI.
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Fig. S7. Validation of pqbp1 knockdown effects on fgf4 and cdx4 expression in X. laevis and X.
tropicalis embryos. Results were analyzed and plotted as described in Fig. 5. A-C. Expression of fgf4
and cdx4 was analyzed by qPCR. A. Either of the PQBP1 MOs (at 50 ng) reduced the expression of
fgf4 and cdx4 in X. laevis morphant embryos, *P < 0.05 or **P < 0.01, with comparisons to control
embryos (CT). B. Reduced fgf4 and cdx4 expression in embryos injected with 30 ng pqbp1 MOb at the
4-cell stage into the VMZ was rescued by co-injection of MOb-resistant PQBP1 mRNA (0.4 ng or 2 ng)
but not control b-gal mRNA (-); *P < 0.05 or **P < 0.01, with comparisons to MO-only injected
embryos (-). C. Analysis of PQBP1 knockdown in X. tropicalis embryos. X. tropicalis PQBP1 MO (20
ng) was bilaterally injected into embryos at the 2-cell stage, and phenotypes of stage-matched wild type
(WT) embryo (left panel) and MO-injected embryos (middle panel) are shown. Expression of fgf4 and
cdx4 were evaluated in WT and pqbp1 morphant X. tropicalis at the gastrula stage (right panel), as
described in Fig. 5; *P < 0.05 or **P<0.01, with comparisons to control embryos (CT).
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Fig. S8. Expression profile of different FGF receptors in Xenopus embryos. A. Total RNA was
extracted from early neurula stage embryos, followed by Lightcycler qPCR (27 cycles) with each
receptor-specific primer set, and amplification products were visualized by gel electrophoresis. B.
Expression profile of FGF receptors in morphant gastrula stage embryos by qPCR. Samples were
prepared and analyzed as per Fig. 5. Relative fgfr expression levels were calculated from the âcrossing
pointâ (CP) of qPCR cycle numbers for each primer set, using a common standard curve generated from
fgfr1IIIb control embryo cDNA dilution series, and normalized to levels of odc transcripts. Bar graphs
indicate the relative expression level of receptor transcripts in embryos injected with 150 ng control
(CT), 100 ng pqbp1 (PQ), 50 ng wbp11 (BP), and combined pqbp1 and wbp11 (PQ+BP) MOs. Mean
values for triplicate biological experiments are plotted; bars indicate standard error. Note, although
expression levels of fgfr1IIIc and fgfr3IIIb transcripts appear nearly zero, they were detected in gelbased
and qPCR.
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pqbp1 (polyglutamine binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7, lateral view, animal up.
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pqbp1 (polyglutamine binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
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pqbp1 (polyglutamine binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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wbp11 (WW domain binding protein 11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7, lateral view, animal up.
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wbp11 (WW domain binding protein 11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
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wbp11 (WW domain binding protein 11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
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