© 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 Yasuno Iwasaki and Gerald H. Thomsen*

ABSTRACT development and cognition, PQBP1 regulates a broader scope of Alternative splicing of pre-mRNAs is an important means of regulating body patterning events, yet the normal developmental functions of developmental processes, yet the molecular mechanisms governing PQBP1 and the pathogenic mechanisms underlying Renpenning alternative splicing in embryonic contexts are just beginning to syndrome remain largely undeciphered. emerge. Polyglutamine-binding protein 1 (PQBP1) is an RNA- At the biochemical level, PQBP1 functions in transcription and splicing factor that, when mutated, in humans causes Renpenning pre-mRNA splicing. PQBP1 interacts with the C-terminus of syndrome, an X-linked intellectual disability disease characterized by activated RNA polymerase II (Okazawa et al., 2002) and with severe cognitive impairment, but also by physical defects that spliceosome components, including U5-15kD (Dim1p) (Waragai suggest PQBP1 has broader functions in embryonic development. et al., 2000; Zhang et al., 2000) and the U2 snRNP component Here, we reveal essential roles for PQBP1 and a binding partner, Sf3b1 (Wang et al., 2013). Most human disease-causing PQBP1 WBP11, in early development of Xenopus embryos. Both genes are mutations truncate the C-terminal domain of PQBP1 that is required expressed in the nascent mesoderm and neurectoderm, and for interaction with the spliceosomal protein U5-15kD (Waragai morpholino knockdown of either causes defects in differentiation et al., 2000; Zhang et al., 2000). Knockdown of PQBP1 in mouse and morphogenesis of the mesoderm and neural plate. At the embryo primary neurons reduces general splicing efficiency and molecular level, knockdown of PQBP1 in Xenopus animal cap promotes the use of alternative splice sites in a variety of transcripts explants inhibits target gene induction by FGF but not by BMP, Nodal (Wang et al., 2013). A missense mutation within the WW domain at – – or Wnt ligands, and knockdown of either PQBP1 or WBP11 in the N-terminus of PQBP1 in Golabi Ito Hall (GIH) patients embryos inhibits expression of fgf4 and FGF4-responsive cdx4 (Y65C) causes abnormal pre-mRNA splicing and inhibits PQBP1 genes. Furthermore, PQBP1 knockdown changes the alternative binding to spliceosome components and a partner protein, WBP11 splicing of FGF receptor-2 (FGFR2) transcripts, altering the (Lubs et al., 2006; Tapia et al., 2010; Sudol et al., 2012). PQBP1 Δ incorporation of cassette exons that generate receptor variants and WBP11 both associate with the B U1 or PRP19 spliceosomal (FGFR2 IIIb or IIIc) with different ligand specificities. Our findings may complexes (Makarova et al., 2004; Deckert et al., 2006) and inform studies into the mechanisms underlying Renpenning colocalize to nuclear speckles enriched in splicing factors (Komuro syndrome. et al., 1999b; Llorian et al., 2004, 2005; Nicolaescu et al., 2008). A central, polar amino acid-rich domain (PRD) in mammalian KEY WORDS: Alternative splicing, FGF, FGF receptor, Mesoderm, PQBP1 can bind to proteins with polyglutamine (polyQ) tracts, Neural, PQBP1, Renpenning syndrome, WBP11, Xenopus such as transcription factors Brn2 and Ataxin-1, and the cytoplasmic transport protein Huntingtin (Htt), which is enhanced INTRODUCTION in poly(Q)-expanded pathogenic mutants of these human proteins Polyglutamine binding protein-1 (PQBP1) is a 38 kDa nuclear (Waragai et al., 1999; Okazawa et al., 2002; Busch et al., 2003). protein abundantly expressed in the adult mammalian central Although the molecular effects of PQBP1 have been studied in nervous system (Komuro et al., 1999a; Waragai et al., 1999), and cultured cells and some model animals, little is known about its mutations in the human PQBP1 gene cause X-linked intellectual physiological function in developing embryos. Partial loss of disability (XLID) diseases that include Renpenning, Sutherland– function of PQBP1 in mice and Drosophila causes neurobehavioral Haan, Hamel cerebropalatocardiac, Golabi–Ito–Hall and Porteous phenotypes with no reported developmental defects (Ito et al., 2009; syndromes (Kalscheuer et al., 2003; Lenski et al., 2004; Stevenson Tamura et al., 2010). However, a homozygous knockout null mouse et al., 2005; Cossee et al., 2006; Lubs et al., 2006; Martinez-Garay line apparently cannot be generated, probably due to lethality, et al., 2007). These are collectively referred to as Renpenning suggesting an essential role for PQBP1 in embryonic development. syndrome, and affected patients display unifying clinical features, Overexpression of PQBP1 is also deleterious, promoting neuronal including intellectual disability, microcephaly and short stature. cell death in mice (Okuda et al., 2003; Marubuchi et al., 2005), and However, a variety of other physical manifestations can be impaired long-term memory and behavioral abnormalities in observed, including midline and cardiac defects, small testes, lean Drosophila (Yoshimura et al., 2006). A PQBP1 gene duplication muscle and reduced body mass (Stevenson et al., 2005; Germanaud has been found in a patient with Renpenning syndrome, in which the et al., 2010). These phenotypes suggest that, in addition to brain 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. Department of Biochemistry and Cell Biology, Center for Developmental Genetics, Here, we use the animal model Xenopus laevis to investigate the Stony Brook University, Stony Brook, NY 11794-5215, USA. embryonic expression and functions of PQBP1 and to identify *Author for correspondence ([email protected]) potential molecular targets. We also evaluate one of its physiological partner proteins, WBP11, for which no developmental expression or

Received 2 December 2013; Accepted 3 August 2014 functional information has been reported. We have found that both DEVELOPMENT

3740 RESEARCH ARTICLE Development (2014) 141, 3740-3751 doi:10.1242/dev.106658 genes are expressed in the developing mesoderm and nervous system, coincided with both chordin (marking the notochord) and sox2 and that loss of function (by morpholino knockdown) of either one (marking the prospective neural plate) (Fig. 1D). At neurula stages, results in abnormal gastrulation and neurulation. We also find that pqbp1 was expressed in the neural plate and the head primordium inhibiting PQBP1 or WBP11 causes similar defects in mesodermal (Fig. 1A,E) in a region that overlapped with ncam expression but and neural differentiation in Xenopus embryos, particularly in FGF- also encompassed the cranial neural crest (Fig. 1E) and cells in the responsive gene expression. Mechanically, PQBP1 knockdown ventrolateral neural tube (supplementary material Fig. S3). In causes a shift in the alternative splicing of FGF receptor-2 (FGFR2) tailbud tadpole stages, pqbp1 was expressed in head, eyes, spinal pre-mRNA, and a loss of MAPK activation in response to FGF4 cord and branchial arches (Fig. 1A). (eFGF). Our findings may provide cluesto the cause of developmental To determine which early developmental processes require abnormalities associated with human PQBP1 mutations in PQBP1, we performed gene knockdown in X. laevis embryos Renpenning syndrome, with the particular suggestion that PQBP1 with translation-blocking antisense morpholino oligonucleotides mutations may result in aberrant FGF signaling in embryonic and (MOs) that target each individual pqbp1 homeolog (MOa and MOb postnatal Renpenning patients. for pqbp1a and pqbp1b, respectively), or another MO predicted to inhibit both homeologs simultaneously (MO1; Fig. 2). The ability RESULTS of MO1 to block pqbp1 mRNA translation was confirmed by PQBP1 knockdown causes mesodermal and neural defects western blot (Fig. 2G). We first examined the effects of PQBP1 in Xenopus embryos knockdown in the mesoderm by bilaterally injecting MOs into the Two X. laevis pqbp1 genes were revealed by BLAST searches of marginal zone of two-cell stage blastulae (Fig. 2). MO-injected Xenopus expressed sequence tag (EST) and genome databases embryos are referred to as morphants, and MO1 morphant using mammalian pqbp1 sequences, which are homeologs that phenotypes commonly included shortened anteroposterior (AP) result from an ancient interspecies hybridization event that gave rise body axes, small heads and no tail structures (Fig. 2C). These to the allotetraploid X. laevis lineage (Hughes and Hughes, 1993). phenotypes were also accompanied by some cell dissociation at The two predicted proteins (PQBP1a and PQBP1b) share 94% neurula stages. Interestingly, embryos injected singly with either sequence identity, and are highly homologous to human PQBP1 MOa or MOb showed only mild developmental defects (Fig. 2D,E), (79-80% identity) except for the poly(Q)-binding PRD region, but when these MOs were combined the resulting morphants lacked which is less conserved among species (supplementary material head and tail structures that were very similar to, but more severe Fig. S1A). than, the defects caused by MO1 (Fig. 2F). The congruent effects of To lay the groundwork for predicting and testing potential knocking down one or both PQBP1 homeologs indicate that the developmental functions of PQBP1, we determined the individual PQBP1 morphant phenotypes are specific to the MOs spatiotemporal expression of these genes in developing Xenopus and that both homeologs contribute similarly to normal X. laevis embryos by whole-mount in situ hybridization (WISH) and semi- development. quantitative RT-PCR (qPCR) (Fig. 1; supplementary material We next tested the effects of PQBP1 knockdown on development Fig. S2). During early cleavage (∼64 cells), pqbp1 transcripts were of the dorsal, Spemann–Mangold organizer mesoderm and adjacent present in animal pole blastomeres (Fig. 1A), and in the early neural tissue. MO1 was injected into the dorsal marginal zone gastrula (stage 10.5) pqbp1 was expressed throughout the (DMZ) of four-cell embryos, and the resulting morphants showed mesoderm (Fig. 1B,C). In the late gastrula, pqbp1 expression 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, 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. DEVELOPMENT

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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 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 two- cell 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 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 (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 2nglacZ 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.

plate folding, and later stage tadpoles had reduced head and subsequent PQBP1 knockdown experiments, unless otherwise shortened AP axial structures. The severity of the phenotypes was noted. Examining general mesoderm, injection of PQBP1 MOs into dose-dependent and increased as the amount of MO1 was raised any region of the mesoderm reduced or eliminated brachyury (Fig. 2H). In some cases, open blastopores and abnormally folded expression in recipient cells (Fig. 3A), which also underwent neural plates were clearly visible (Fig. 2J,K). Importantly, these abnormal gastrulation movements (Fig. 3B). Furthermore, although morphant defects could be partly rescued by co-injection of chordin expression (marking the organizer mesoderm) was not synthetic pqbp1 mRNA (0.4 ng or 2.0 ng) resistant to the PQBP1 inhibited by PQBP1 knockdown, chordin-expressing cells spread MOs, indicating that the effects of MO1 are specific (Fig. 2J,K; ventrolaterally during gastrulation, indicating abnormal supplementary material Fig. S5). Consistent with abnormal convergence extension movements (Fig. 3B). Blastopore closure phenotypes caused by gain of function in other species, was delayed in morphants but eventually finished by the time overexpression of PQBP1 in Xenopus embryos also caused severe control embryos reached neurula stage. However, the AP axes of developmental defects in gastrulation and neurulation (Fig. 2I; PQBP1 morphants were much shorter than in controls, consistent supplementary material Fig. S4), and those resembled the PQBP1 with insufficient extension movements (Fig. 3B). In the neural knockdown phenotypes (Fig. 2H). domain, expression of the general neural marker ncam and neural To further characterize the developmental defects in PQBP1 crest marker snail2 (slug) were substantially decreased in PQBP1 morphants, we examined molecular markers of regional patterning. morphants compared with controls (Fig. 3C,D). Furthermore, As a combination of MOa and MOb had the strongest and most unilateral injection of PQBP1 MO1 at the two-cell stage inhibited consistent effects, this pair was applied together in these and in sox2 expression and perturbed neural folding on the injected side DEVELOPMENT

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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 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.

(Fig. 3D). As seen in Fig. 2C, we also observed some cell dissociation confirmed the specificity of the WBP11 MOs by showing that they at neurula stages, typified by sox2-positive cells detaching from the block endogenous WBP11 translation in X. laevis embryos neural plate in PQBP1 morphants (Fig. 3D, magnified). (Fig. 4B). Embryos injected bilaterally with WBP11 MOs displayed shortened AP axes, open blastopore remnants, small or WBP11 is essential for normal development absent heads and truncated tails (Fig. 4C). These phenotypes closely The spliceosome protein WBP11 is an endogenous partner of resembled those of PQBP1 morphants, so we tested whether mammalian PQBP1 (Nicolaescu et al., 2008; Tapia et al., 2010), but combined knockdown of PQBP1 and WBP11 might prove more despite its functional importance in pre-mRNA splicing, nothing is deleterious than individual gene knockdowns. Embryos were known about its developmental expression or function. We identified targeted in the marginal zone with low doses of either the PQBP1 two homeologs in X. laevis that encode predicted proteins with 95% or WBP11 MO, which alone allowed normal gastrulation and caused (604/636) identity to each other and 74% identity to human WBP11 only a slight perturbation of neural folding (Fig. 4D). However, (supplementary material Fig. S1B). In developing Xenopus embryos, simultaneous low-dose knockdown of PQBP1 and WBP11 blocked wbp11 is expressed maternally, with maternal transcripts persisting gastrulation (note open blastopores) and neural folding (Fig. 4D). through blastula stages. Postzygotically, wbp11 is expressed in These seemingly additive phenotypic effects suggest that these ectoderm and mesoderm during gastrulation, in neural plate during proteins perform similar developmental functions. neurulation and in spinal cord and brain in tadpole stages (Fig. 1A; supplementary material Fig. S2). Wbp11 expression closely coincides Defective mesodermal and neural marker expression in with that of pqbp1, consistent with a physical and functional PQBP1 and WBP11 morphants partnership between these proteins in mammalian systems. We To better understand the PQBP1 and WBP11 morphant phenotypes, validated the ability of Xenopus PQBP1 and WBP11 proteins to we scored regional and tissue-specific marker gene expression by physically associate by co-immunoprecipitation from mammalian qPCR on embryos injected bilaterally into the marginal zone at the cultured cells (supplementary material Fig. S6A), and by observing two-cell stage and harvested at early gastrulation (stage 10.5). their ability to colocalize to nuclei (supplementary material Fig. S6B). Results in Fig. 5A show that expression of several general Potential embryonic functions for WBP11 have not been mesodermal markers was reduced in morphants by individual or evaluated in any species. Therefore, we tested whether WBP11 is combined knockdown of PQBP1 or WBP11. To assess general required for development by inhibiting expression of the two mesoderm specification, we evaluated brachyury and found it was

X. laevis homeologs with translation-blocking MOs (Fig. 4A). We reduced by about 40% of control levels by PQBP1 knockdown, DEVELOPMENT

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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.

PQBP1 knockdown impairs responses to FGF but not other inductive signals 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 Fig. 4. WBP11 knockdown resembles and enhances PQBP1 knockdown PQBP1 or WBP11 morphants. To delineate which signaling phenotypes. (A) WBP11 MO (blue arrow) targets the 5′ UTR of both X. laevis wbp11 homeologs. (B) Expression of endogenous WBP11 was blocked by pathways might be impaired in PQBP1 morphants, we examined injection of 40 ng wbp11 MO (WB MO) but not control MO (CT MO). the effects of PQBP1 knockdown on the induction of marker genes Endogenous (red arrowhead) and overexpressed (black arrowhead) WBP11 by FGF, Nodal, BMP or Wnt signals in Xenopus animal caps. were detected by western blot with anti-WBP11 antibodies. An asterisk Growth factors (as mRNAs) together with morpholinos were indicates a non-specific band that, along with β-tubulin staining, controls for injected into animal poles of two-cell embryos, and caps were cut sample loading. (C) Tailbud stage embryos injected into two dorsal cells at the and screened for induction of target genes by qPCR. We found that 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 PQBP1 morphant animal caps responded normally to Wnt8, Nodal2 20, anterior view) embryos injected dorsally at the four-cell stage (schematic (Xnr2) and BMP4, but response to FGF4 was significantly inhibited drawing) with 75 ng control MO (CT), 25 ng wbp11 MO (WB), a mixture of (Fig. 6B). Specifically, induction of cdx4 and bra by FGF4 was pqbp1 MOa plus MOb (25 ng each; 50 ng total; PQ) or a combination of wbp11 diminished in PQBP1 morphants, despite normal induction of cdx4 and pqbp1 MOa and MOb (25 ng each; 75 ng total; PQ+WB). by Wnt8 or bra by BMP4. Furthermore, induction of fgf4 by FGF4, induced by FGF4/Brachyury-positive feedback loops (Isaacs et al., consistent with reduced brachyury expression seen in morphants by 1994; Fujii et al., 2008), was also blocked by PQBP1 knockdown. WISH (Fig. 3A). Combined knockdown of PQBP1 and WBP11 We also tested whether PQBP1 might be required for normal resulted in an even greater reduction (about 80%) of brachyury operation of the FGF receptor tyrosine kinase (RTK) signaling expression, despite normal brachyury levels with WBP11 cascade by analyzing MAPK (Erk) phosphorylation in PQBP1 knockdown alone (Fig. 5A). Another general mesodermal marker, morphant animal caps. Injection of 1pg fgf4 mRNA into animal zygotic vegT (apod), was reduced by about 50% of control levels by caps induced phosphorylation of MAPK, but co-injection of either double knockdown of PQBP1 and WBP11. PQBP1 MO1 or MOa+MOb blocked MAPK phosphorylation We also observed distinct differences in the regional mesodermal (Fig. 6C). In sum, the results of experiments performed in whole marker expression in single or double PQBP1 and WBP11 embryos (Fig. 5) and isolated animal caps (Fig. 6) demonstrate that morphants. Expression of the ventrolateral markers sizzled and responses to FGF signaling require PQBP1, whereas Nodal, BMP wnt8 were reduced by about 40 and 60%, respectively, by either and Wnt pathways do not. single or combined knockdown of PQBP1 and WBP11 (Fig. 5). Several genes that mark the Spemann–Mangold organizer, chordin, PQBP1 regulates alternative splicing of an FGF receptor goosecoid and siamois, were expressed at near normal levels in One plausible explanation for the deleterious effects of PQBP1 single or double PQBP1/WBP11 morphants, but noggin expression knockdown on FGF responses is that PQBP1 acts at the level of gene was reduced by about 50% in the double morphants (Fig. 5B). transcription or pre-mRNA splicing for FGF signaling components. DEVELOPMENT

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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 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.

At the ligand level, alternative splicing of fgf4 and fgf8 pre-mRNA mammalian receptors, the IIIb isoforms of FGFR1-3 preferably bind can generate protein variants with distinct biological activities FGF3, FGF7 and FGF10, whereas IIIc isoforms prefer FGF4 and (Fletcher et al., 2006; Guo and Li, 2007; Mayshar et al., 2008). In FGF8 (Eswarakumar et al., 2005; Holzmann et al., 2012). FGFR4 particular, two FGF8 isoforms, FGF8a and FGF8b, respectively, does not undergo alternative splicing of exon8, but binds FGF4 and induce neural and mesodermal tissues in Xenopus embryos FGF8b (Blunt et al., 1997). (Fletcher et al., 2006). However, we observed no differences in In order to determine whether expression or splicing of FGF the splicing patterns of fgf4 or fgf8 transcripts in PQBP1 and/or receptors was modified, we surveyed the levels of FGFR1-3 IIIb or WBP11 morphants compared with controls (data not shown). IIIc isoform transcripts, as well as the total expression levels of all PQBP1 or WBP11 knockdown defects could also be explained four FGF receptors, in control (control MO-injected), PQBP1 or by mis-splicing of the FGF receptors. Although only four genes WBP11 morphant Xenopus embryos at early gastrulation (stage encode FGF receptors, alternative splicing results in numerous 10.5). To assay IIIb and IIIc isoforms, we used primers that targeted isoforms with different ligand specificity (Ornitz et al., 1996; exon8a or exon8b on the upstream side, and thus amplified only Eswarakumar et al., 2005; Zhang et al., 2006). FGF receptors 1, 2 spliced transcripts that contained one of these alternative exons in and 3 incorporate exons 8a or 8b through alternative splicing of their fgfr1-3 (as illustrated for fgfr2 in Fig. 7C,D). In wild-type or control pre-mRNAs, resulting in receptor isoforms IIIb or IIIc, respectively. embryos, qPCR indicated the presence of all potential receptor These isoforms possess distinct ligand specificity resulting from isoforms, but relative isoform levels varied (Fig. 7; supplementary unique residues in the immunoglobulin-like loop III of the material Fig. S8). We observed a dramatic change in the isoform extracellular, ligand-binding domain encoded by exon 8. Among ratio of fgfr2 IIIb and IIIc transcripts, but no change in the total level DEVELOPMENT

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(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 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 co- expressed 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 Fig. 6. Effects of PQBP1 knockdown on animal cap response to growth neural/cognitive defects caused by pqbp1 mutations. Although factors. (A,B) Two-cell embryos were injected into the animal pole with growth WBP11 is not implicated in human disease or birth defects, our factor mRNAs and MOs, animal caps were cut at mid-blastula and harvested at study is the first to indicate a normal developmental role for the equivalent of early gastrula stage, followed by qPCR, as depicted (top). WBP11 in any embryo, raising the possibility that wbp11 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 mutations could contribute to birth defects. by PQBP1 knockdown (50 ng MO). There was no statistically significant difference between CT and PQBP1 MO-injected caps treated with each ligand PQBP1 and WBP11 are required for normal mesodermal and (Student’s t-test, n=3). (B) Marker gene induction by FGF4 was significantly neural development reduced by PQBP1 knockdown (*P<0.05 or **P<0.01; Student’s t-test, Despite the recognized importance of pqbp1 mutations in the triplicate biological replicates). Animal caps were injected with fgf4 mRNA (1 pg) etiology of human XLID syndromes, information about the pqbp1 and either control MO (50 ng), MO1 (50 ng) or MOa+MOb (25 ng each). embryonic expression and function of PQBP1 or WBP11 is (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 limited to a description of mouse pqbp1 expression (Qi et al., of β-tubulin and total MAPK protein did not change among these samples. 2005). Our analysis of Xenopus embryos shows that pqbp1 and wbp11 genes are maternally expressed, present throughout of all frfgr2 transcripts were observed (Fig. 7B-D). In control cleavage late blastula stages, then zygotically expressed in the embryos, fgfr2IIIc transcripts (containing exon 8b) were more mesoderm during gastrulation and the developing neural plate and abundant than those of fgfr2IIIb (containing exon 8a); this ratio was nervous system of tadpoles. reversed, however, in PQBP1 morphants, as measured by qPCR The expression of pqbp1 in tailbud tadpoles appears homologous (Fig. 7C) or by gel-based RT-PCR (Fig. 7D). to that reported for embryonic and postnatal mice, where pqbp1 The other FGF receptors showed only minor shifts in isoform mRNA and protein are predominantly expressed in the central splicing and total levels in PQBP1 morphants (supplementary nervous system and neuronal stem cells (Waragai et al., 1999; Qi material Fig. S8). Knockdown of WBP11 alone had no significant et al., 2005; Wang et al., 2013). The neural expression of pqbp1 in effects on the relative level of these fgfr2 splice isoforms, nor did Xenopus and mouse is consistent with the fact that human pqbp1 splicing enhance changes in PQBP1/WBP11 double morphants mutations cause mental retardation and microcephaly. Moreover, DEVELOPMENT

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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: 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) 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 (100 ng pqbp1 MO) by co-injection of 1 ng Xenopus fgfr2IIIc (*P<0.05; Student’s t-test, n=3).

the co-expression of pqbp1 and wbp11 in the same tissues of PQBP1 is required for FGF signaling and regulates splicing of Xenopus embryos is consistent with the known physical and FGF receptor transcripts functional links between the two proteins (Komuro et al., 1999b; Our work further shows that the molecular and morphological defects Llorian et al., 2004, 2005; Nicolaescu et al., 2008). Whether these observed in PQBP1 morphant embryos appear to be the result of proteins function together mechanistically to govern the same aberrant FGF signaling. Direct tests of mesoderm induction in biochemical or embryonic processes remains to be determined. At animal caps showed PQBP1 knockdown is required for signaling neurula stages, PQBP1 or WBP11 morphants display incomplete triggered by FGF4 but not Nodal, BMP or Wnt ligands. The loss of neural tube closure accompanied by reduced expression of neural RTK signal transduction in particular indicates defects upstream of marker genes, underscoring their requirement for normal neural FGF-responsive gene transcription, and not some general defect in development. transcription or mRNA splicing of FGF target genes. FGF receptors, The neural expression of pqbp1 in Xenopus embryos might have of which there are four, were foremost among candidates because their been anticipated, but our finding that both pqbp1 and wbp11 are transcripts can undergo alternative splicing in various biological expressed and required for normal development of mesoderm is new systems to generate receptor isoforms with different ligand-binding and reveals a broader role for these genes in early developmental specificities. Our survey of fgfr expression and splicing shows that processes, particularly in non-neural tissues. Knockdown of either among the four receptors, fgfr2 showed drastically altered levels of gene causes abnormal cell migration during gastrulation, notably a alternatively spliced transcripts encoding isoforms IIIb and IIIc in failure of dorsal mesodermal convergence-extension movements. PQBP1 morphants (supplementary material Fig. S8). Normally, early These defects in gastrulation are probably linked to reduced gastrula stage embryos express approximately 50% more fgfr2IIIc brachyury expression in PQBP1 morphants, as this gene is transcripts than fgfr2IIIb transcripts. We found that PQBP1 essential for mesodermal cell motility as well as differentiation. knockdown reverses this ratio, lowering fgfr2IIIc transcript levels to Some physical manifestations observed in Renpenning syndrome about half the level of fgfr2IIIb transcripts. As the FGFR2IIIc, but not patients, including midline and cardiac defects, lean muscle and IIIb, receptor isoform binds to FGF4 and FGF8, we postulate that the short stature, may be analogous to defects in mesoderm observed in lowered abundance of FGFR2IIIc reduces the ability of the embryo to

PQBP1 morphants. respond to mesoderm-inducing FGF4/8 ligands. DEVELOPMENT

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Furthermore, whereas PQBP1, FGF4 and FGF8 morphants share 2012). Our findings raise the hypothesis that abnormal FGF similar mesodermal, neural and gastrulation defects (Fisher et al., signaling may underlie body, brain, and possibly cognitive, defects 2002; Fletcher et al., 2006; Isaacs et al., 2007), PQBP1 morphant in Renpenning patients. phenotypes are more severely affected, yet rather similar to those One aspect of PQBP1 function suggested by human and generated by a dominant negative FGF receptor (XFD) or FGFR animal studies, and reinforced by our work, pertains to the dosage inhibitor SU5402, which block all FGF signaling, including that effects of PQBP1 on development and disease. Viable human stimulated by FGF4 and FGF8b (Amaya et al., 1993; Delaune et al., PQBP1 mutations encode single residue changes, or frame-shifted 2005; Fletcher and Harland, 2008). This greater similarity between proteins with C-terminal truncations that are probably partly PQBP1 knockdown and wholesale loss of FGF signaling is functional (hypomorphic). No complete deletions of the PQBP1 potentially explained by the reduction of the FGFRIIIc isoform in gene or entire loss of its coding region have been described in PQBP1 morphants, but we cannot exclude the possibility that PQBP1-linked XLIDs. Mouse gene knockout and overexpression PQBP1 inhibition affects expression of other essential embryonic models to investigate PQBP1 developmental functions have not gene targets. been very successful, which seems to indicate a requirement for FGFR2 isoform switching via alternative splicing has been PQBP1 protein expression within a critical range: too little or too observed in multiple contexts, including normal epithelial- much protein is apparently lethal (Okuda et al., 2003). Our own mesenchymal transition (Warzecha and Carstens, 2012), embryonic studies here support that notion, as we observed dose-dependent development (Eswarakumar et al., 2002; Rice et al., 2003; Takeuchi embryonic defects in both loss- and gain-of-function tests. et al., 2005; Liu et al., 2011), human birth defects (Hajihosseini et al., The highest doses of pqbp1 morpholinos or injected mRNA 2001; Teebi et al., 2002; Ibrahimi et al., 2005), cancer and various inhibit gastrulation and neurulation (Fig. 2; supplementary pathologies (Katoh, 2008, 2009; Holzmann et al., 2012; Kelleher material Fig. S4), whereas moderate morpholino doses cause et al., 2013). Whether PQBP1 regulates fgfr2 alternative splicing in mild, dose-dependent phenotypes. these normal and disease situations will be worth investigating. Our Finally, the PQBP1 partner protein WBP11 has not been preliminary results also show changes in relative levels of alternative implicated in the etiology of Renpenning or other XLIDs, or transcripts for fgfr1-3, but changes in fgfr2 transcript splicing were human diseases in general, but as these proteins appear to have greater than fgfr1 and fgfr3 upon PQBP1 knockdown (supplementary partially overlapping functions in Xenopus development, it will be material Fig. S8). The time and place of expression of all FGF valuable to evaluate whether altered WBP11 expression or activity receptors and their splicing variants in Xenopus embryos has not been might contribute to Renpenning or other human diseases. Further well delineated, so further investigation is required to understand their investigation of PQBP1 and WBP11 target genes and molecular precise roles in mesoderm and neural induction by FGFs and how mechanisms using Xenopus embryos should contribute to a more pqbp1 impacts these functions. complete understanding of how these genes regulate normal In addition to our findings, previous studies have identified other vertebrate developmental processes and, when mutated, contribute factors regulating fgfr2 pre-mRNA splicing. Epithelial splicing to human developmental and intellectual disabilities. regulatory proteins 1 and 2 (ESRP1 and ESRP2) govern alternative splicing of fgfr2 and other transcripts in a human epithelial cell line, MATERIALS AND METHODS and depletion of ESRP1 and ESRP2 causes splice switching from Embryo manipulations an epithelial IIIb form to a mesenchymal IIIc form (Warzecha et al., Xenopus laevis embryos were obtained by standard in vitro fertilization, 2009). Fused in sarcoma (FUS) has also been shown to regulate 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× fgfr2 transcript splicing in Xenopus embryos (Dichmann and MMR+10 μg/ml gentamycin thereafter. All stages are according to Harland, 2012), and its knockdown causes a complete loss of Nieuwkoop and Faber (1994). Animal caps were excised at stage 8 and fgfr2IIIc transcripts but has no effect on fgfr2IIIb transcript levels. cultured in 0.5× MMR +10 μg/ml gentamycin until the sibling embryos The basis of FUS and PQBP1 target gene specificity is unknown, reached stage 10.5. but FUS can directly bind RNA sequences, whereas direct interaction between PQBP1 and endogenous RNA was not Microinjection of MO and mRNA evident by crosslinking immunoprecipitation (Wang et al., 2013). Xenopus embryos were microinjected with MO and/or synthetic mRNA at Whether FUS, ESRP1/2, PQBP1 and/or WBP11 interact or two-cell or four-cell stages. All MOs were designed and synthesized by Gene Tools. The control MO was the standard scrambled MO, 5′-CCCTTACCT- function together in the splicing of fgfr2 or other gene transcripts ′ ′ awaits investigation. CAGTTACAATTTATA-3 . pqbp1 MO1 is 5 -CGCTA-AAGGGAGAGGC- ATCCTGGC-3′. pqbp1a MO (MOa) is 5′-AGCTCTT-GTTCTAACTCCC- CGCCGT-3′. pqbp1b MO (MOb) is 5′-ACCGACACG-CTCCTGCTCCTA- Potential implications for XLID CTCT-3′. wbp11 MO is 5′-ATGTCCTCTCTCCACC-AATATCAGA-3′, Besides cognitive defects, Renpenning patients have physical X. tropicalis pqbp1 MO is 5′-CGGCATCTTGGGCG-GCCAACCACAC-3′. abnormalities that suggest underlying defects in the development Capped mRNAs were synthesized using AmpliCap SP6 High Yield Message of mesodermal as well as neural tissues (Stevenson et al., 2005; Maker (Epicentre Biotechnologies) or mMESSAGE mMACHINE (Ambion) Germanaud et al., 2010). The details of human tissue pathologies from the following linearized plasmids: pCS2-pqbp1,pSPYS-chordin,pCS2- associated with PQBP1 mutations, particularly those of non-brain bmp4,pCS2-wnt8, pSP64-fgf4,pCS2-wbp11.Insomecases,lacZ mRNA tissue, are not well described, but the cardiac and skeletal muscular 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-chloro- defects and reduced height prevalent in XLID patients may echo the β mesodermal defects we observe in Xenopus PQBP1 morphants. 3-indolyl -D-galactopyranoside; Sigma-Aldrich). FGF signaling is essential not only for development of mesodermal X. laevis cDNA cloning and plasmid construction and neural tissues in vertebrate embryos, but also for the ongoing A TBLASTN search identified two incomplete X. laevis cDNA clones normal functions of adult tissues derived from these germ layers, (gi:8320261 and 8319534) homologous to mammalian PQBP1. Based on including brain functions (Reuss and von Bohlen und Halbach, these EST sequences, the full-length pqbp1 clone was amplified by PCR from

2003; Iwata and Hevner, 2009; Mudo et al., 2009; Turner et al., Xenopus cDNA (blastula) with the following primers: pqbp1 forward, DEVELOPMENT

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5′-GATCGTTGGCGTCATCAACAGG-3′ and pqbp1 reverse, 5′-GCACAA- antibodies. Transfected and endogenous cellular control proteins were GCAACTGTACAGCAGT-3′. The sequence of this clone completely visualized on the blots, as above, with mouse anti-β-tubulin (Santa Cruz, matched to another X. laevis EST clone (NM_001091714) except for sc-58884; 1:500), mouse anti-GFP (Santa Cruz, sc-9996; 1:500) and anti- its shorter 5′ untranslated region (UTR) end. Identical sequences were WBP11 rabbit polyclonal antibodies prepared by our lab using a Xenopus identified in X. laevis genome scaffolds (Xenbase gbrowse laevis 6.0: WBP11-GST fusion protein. For immunofluorescence assays, HA-pqbp1 and Scaffold1707:1728996-1729209). MO-resistant Xenopus pqbp1 cDNA was myc-wbp11 were transfected into COS-1 cells and detected with anti-myc amplified with the primers forward 5′-CCTCGAGATGCCGTTGCCTT- mouse monoclonal 9E10 and anti-HA rabbit polyclonal antibodies, detected TAGCGCTTCAGGCT-3′, reverse 5′-CAATCAATAGGGGGCAGCAATG- with Alexa 488 (Molecular Probes, A-11029, 1:1000) goat anti-mouse and 3′, and subcloned into pCS2+ for in vitro transcription. This resulted in nine Alexa 594 goat anti-rabbit (Molecular Probes, A-11037, 1:1000) secondary nucleotide mismatches at the pqbp1 morpholino recognition site, but normal antibodies. COS-1 cells were grown in Dulbecco’s modified Eagle’smedium amino acid coding was retained. C-terminal myc-tagged Xenopus PQBP1 (Gibco) supplemented with 10% fetal bovine serum. 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′-CCC- Acknowledgements ATCGATATGGGGCGGCGATCCACTTCG-3′ and 5′-CGAATCGATGA- We thank Dr R. Harland (University of California, Berkeley, USA) for the fgfr2c AAAGTTGCTTAGAATTAGCCG-3′ (restriction enzyme sites are plasmid and members of the G.H.T. lab for experimental advice and comments on underlined) with design based on an EST (BC057737). All cDNA identities the manuscript. We acknowledge Xenbase for facilitating gene analyses, and the were verified by DNA sequencing, and subcloned into pCS2+ unless otherwise National Xenopus Resource (NXR) and European Xenopus Resource Center (EXRC) for supplying animals and reagents. noted. Full-length X. tropicalis fgfr2IIIc was a gift from Dr Richard Harland (University of California, Berkeley, USA). Competing interests The authors declare no competing financial interests. In situ hybridization In situ hybridization was performed with digoxigenin-labeled probes Author contributions as previously described (Alexandrova and Thomsen, 2006). BM Purple was Y.I. and G.H.T. conceived and designed experiments, Y.I. carried out all used as a chromogenic substrate (Roche). Sagittal or cross sections were experiments, and Y.I. and G.H.T. wrote the manuscript. performed during MEMPFA fixation. Template plasmids for making probes Funding were pBS-ncam, pBS-sox2, pGEM4Z-chordin, pXT1-brachyury, pMX- This research was supported partially by the U.S. National Institutes of Health snail2, pCS2-pqbp1 and pCS2-wbp11. [grants R03HD064908 and R01HD032429 to G.H.T.]. Deposited in PMC for release after 12 months. Quantitative RT-PCR Total RNA was extracted from ten animal caps or three embryos and Supplementary material used for cDNA synthesis as previously described by Callery et al. Supplementary material available online at (2005). Quantitative RT-PCR using the LightCycler System and SYBR http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.106658/-/DC1 Green reagent (Roche Applied Science) according to Kalkan et al. (2009) using primers listed in supplementary material Tables S1 and S2. References Alexandrova, E. M. and Thomsen, G. H. (2006). Smurf1 regulates neural Relative amounts of PCR product were determined based on standard patterning and folding in Xenopus embryos by antagonizing the BMP/Smad1 curves derived from 1:1, 1:10 and 1:100 dilutions of the cDNA from pathway. Dev. Biol. 299, 398-410. control embryos, with target gene expression normalized to the relative Amaya, E., Stein, P. A., Musci, T. J. and Kirschner, M. W. (1993). FGF levels of ornithine decarboxylase (odc) transcripts. Most primers were signalling in the early specification of mesoderm in Xenopus. Development designed to have annealing temperature of 60°C, and product sizes 118, 477-487. between 100-200 bp; most PCR conditions were annealing at 55°C, Blunt, A. G., Lawshé, A., Cunningham, M. L., Seto, M. L., Ornitz, D. M. and elongation 12 s at 72°C, denaturation at 94°C for 10 s. cDNA from three MacArthur, C. A. (1997). Overlapping expression and redundant activation of or more biological replicate experiments were amplified in most Light mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands. J. Biol. Chem. 272, 3733-3738. Cycler reactions (see text) and statistical analyses were conducted using Busch, A., Engemann, S., Lurz, R., Okazawa, H., Lehrach, H. and Wanker, E. E. ’ Student s t-test. (2003). Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington’s disease. Protein analysis, immunoprecipitation and immunofluorescence J. Biol. Chem. 278, 41452-41461. 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Waragai, M., Junn, E., Kajikawa, M., Takeuchi, S., Kanazawa, I., Shibata, M., Yoshimura, N., Horiuchi, D., Shibata, M., Saitoe, M., Qi, M.-L. and Okazawa, H. Mouradian, M. M. and Okazawa, H. (2000). PQBP-1/Npw38, a nuclear protein (2006). Expression of human PQBP-1 in Drosophila impairs long-term memory binding to the polyglutamine tract, interacts with U5-15kD/dim1p via the carboxyl- and induces abnormal courtship. FEBS Lett. 580, 2335-2340. terminal domain. Biochem. Biophys. Res. Commun. 273, 592-595. Zhang, Y.-Z., Lindblom, T., Chang, A., Sudol, M., Sluder, A. E. and Golemis, Warzecha, C. C. and Carstens, R. P. (2012). Complex changes in alternative pre- E. A. (2000). Evidence that dim1 associates with proteins involved in pre-mRNA mRNA splicing play a central role in the epithelial-to-mesenchymal transition splicing, and delineation of residues essential for dim1 interactions with hnRNP F (EMT). Semin. Cancer Biol. 22, 417-427. and Npw38/PQBP-1. Gene 257, 33-43. Warzecha, C. C., Shen, S., Xing, Y. and Carstens, R. P. (2009). The epithelial Zhang, X., Ibrahimi, O. A., Olsen, S. K., Umemori, H., Mohammadi, M. and splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse Ornitz, D. M. (2006). Receptor specificity of the fibroblast growth factor family. The types of alternative splicing events. RNA Biol. 6, 546-562. complete mammalian FGF family. J. Biol. Chem. 281, 15694-15700. DEVELOPMENT

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