© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850

RESEARCH ARTICLE RNA splicing regulated by RBFOX1 is essential for cardiac function in zebrafish Karen S. Frese1,2,*, Benjamin Meder1,2,*, Andreas Keller3,4, Steffen Just5, Jan Haas1,2, Britta Vogel1,2, Simon Fischer1,2, Christina Backes4, Mark Matzas6, Doreen Köhler1, Vladimir Benes7, Hugo A. Katus1,2 and Wolfgang Rottbauer5,‡

ABSTRACT U2, U4, U5 and U6) and several hundred associated regulator is one of the major mechanisms through which (Wahl et al., 2009). Some of the best-characterized the proteomic and functional diversity of eukaryotes is achieved. splicing regulators include the -arginine (SR)-rich family, However, the complex nature of the splicing machinery, its associated heterogeneous nuclear ribonucleoproteins (hnRNPs) proteins, and splicing regulators and the functional implications of alternatively the Nova1 and Nova2, and the PTB and nPTB (also known as spliced transcripts are only poorly understood. Here, we investigated PTBP1 and PTBP2) families (David and Manley, 2008; Gabut et al., the functional role of the splicing regulator in vivo using the 2008; Li et al., 2007; Matlin et al., 2005). The diversity in splicing is zebrafish as a model system. We found that loss of rbfox1 led to further increased by the location and nucleotide sequence of pre- progressive cardiac contractile dysfunction and heart failure. By using mRNA enhancer and silencer motifs that either promote or inhibit deep-transcriptome sequencing and quantitative real-time PCR, we splicing by the different regulators. Adding to this complexity, show that depletion of rbfox1 in zebrafish results in an altered isoform regulating motifs are very common throughout the genome, but are expression of several crucial target , such as actn3a and hug. not necessarily functional (Barash et al., 2010; Black, 2003; This study underlines that tightly regulated splicing is necessary for Fairbrother et al., 2002; Wang et al., 2004; Zhang and Chasin, 2004). unconstrained cardiac function and renders the splicing regulator Defective splicing of transcripts has been related to disturbed rbfox1 an interesting target for investigation in human heart failure embryonic development, cellular de-differentiation and the and cardiomyopathy. pathogenesis of several diseases (Ars et al., 2000; Creemers et al., 2006; Davis et al., 2002; Disset et al., 2006; Kalsotra et al., KEY WORDS: , Genetics, Zebrafish, Deep 2008; Kong et al., 2010; Pasumarthi and Field, 2002; Tang et al., sequencing, Splicing 2012, 2009). Although such alterations in the splicing machinery have mainly been related to cancer or mental disorders (Cork INTRODUCTION et al., 2008; French et al., 2007; Klinck et al., 2008), they also Alternative transcript initiation, alternative splicing and alternative play a role in the development and progression of heart diseases, polyadenylation of pre-mRNAs are considered to be key such as heart failure and cardiac hypertrophy. For example, mechanisms for generating the huge proteomic diversity in aberrant splicing of cardiac is linked to the progression eukaryotic species (Griffith et al., 2010). At least >95% of human of cardiomyopathies and heart failure (Biesiadecki et al., 2002; genes are estimated to be alternatively spliced in a tissue- or cell- Biesiadecki and Jin, 2002; Philips et al., 1998). Expression of an type-dependent manner (Johnson et al., 2003; Pan et al., 2008; alternatively spliced variant of the sarcomere (TTN)has Wang et al., 2008), allowing the cell to act in a certain environment been associated with progression of chronically ischemic heart or adopt distinct tissue-specific functions. Regulation of alternative failure and differentially spliced MYH7 and FLNC genes are splicing builds upon a complex splicing machinery that joins both found at high copy numbers in failing or hypertrophied hearts constitutive and regulated exons within nuclear pre-mRNA (Davis et al., 2002; Kong et al., 2010; Neagoe et al., 2002). molecules. Removal of from pre-mRNAs is carried out by Recently, Guo et al. have also shown that the splice factor a large macromolecular machine that is known as the spliceosome, RBM20 is strongly expressed in cardiac tissue, and transcriptome which includes five small nuclear ribonucleoproteins (snRNPs; U1, analysis in humans and rats has revealed RBM20-mediated regulation of alternative splicing events in a set of relevant cardiac genes (Guo et al., 2012), demonstrating the importance 1Department of Medicine III, University of Heidelberg, D-69120 Heidelberg, Germany. 2German Centre for Cardiovascular Research (DZHK), partner site of stoichiometrically correct isoform expression. Reduced Heidelberg/Mannheim, D-69120 Heidelberg, Germany. 3Chair for Clinical activity of RBM20 results in an altered isoform expression of Bioinformatics, Saarland University, D-66123 Saarbrücken, Germany. 4Department of Human Genetics, Saarland University, D-66123 Saarbrücken, Germany. cardiac proteins crucial for proper heart structure and function 5Department of Medicine II, University of Ulm, D-89081 Ulm, Germany. (Carboni et al., 2010). 6Comprehensive Biomarker Center, D-69120 Heidelberg, Germany. 7EMBL, Recent studies have shown that members of the so-called European Molecular Biology Laboratory, Genomics Core Facility, D-69117 Heidelberg, Germany. RBFOX family, such as rbfox1-like and rbfox2, also play an *These authors contributed equally to this work important role in mRNA splicing (Gallagher et al., 2011). Using

‡ the zebrafish (Danio rerio) as a model system to study vertebrate Author for correspondence ([email protected]) development, biological processes and disease mechanisms, we This is an Open Access article distributed under the terms of the Creative Commons Attribution investigated the functional role of the splice factor rbfox1 in the License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. vertebrate heart. By RNA sequencing and real-time PCR validation, we detected and delineated a functional role for

Received 3 December 2014; Accepted 10 May 2015 new rbfox1 splicing targets in vivo. Journal of Cell Science

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Fig. 1. Expression analysis of rbfox1 in zebrafish. (A) Amino acid sequence alignments of human, mouse and zebrafish rbfox1 demonstrating the high cross- species homology. Black background, amino acid identity; gray background, amino acids with similar chemical properties. (B,C) RNA antisense in situ hybridization against rbfox1 demonstrates a specific mRNA expression in neuronal, heart cells and at 48 hpf (V, ; A, ). wt, wild-type. (D) Relative expression analysis of rbfox mRNAs shows that rbfox1 is highly expressed in the heart in comparison to the other rbfox family members. WF, whole fish. (E) qRT-PCR analysis of rbfox1 in different tissues of adult zebrafish reveals the highest expression in brain and eye tissue, and an equal expression in heart, skeletal muscle (SKM), intestine and tissue. Data are shown as mean±s.d. (pooled tissue of adult zebrafish n=8, two replicates each). ΔCt values compared to elfa1 as a reference gene.

RESULTS alternative splicing positively or negatively in a position- The RNA-splicing regulator rbfox1 is highly expressed in the dependent manner (Jin et al., 2003; Nakahata and Kawamoto, zebrafish heart 2005; Underwood et al., 2005; Zhang et al., 2008). In detail, they The rbfox1 gene encodes a 373-amino-acid containing an promote exon inclusion when binding to the downstream RNA-binding domain (RBD) motif that is highly conserved from an alternative cassette exon, and exon skipping when among RNA-binding proteins. It is one of four rbfox paralogs in binding to the upstream intron (Auweter et al., 2006; Tang et al., zebrafish, which also include rbfox1l (also known as fox-1, Ch16; 2009). Gene ID 406569), rbfox2 (also known as , Chr6; The protein sequence identity between the zebrafish and human Entrez Gene ID 407613) and rbfox3 (Chr3; Entrez Gene ID ortholog is ∼84% (Fig. 1A). Characterization of rbfox1 gene LOC559412). Each member of the RBFOX protein family expression by whole-mount in situ hybridization and quantitative specifically binds to (U)GCAUG elements and regulates real-time PCR (qRT-PCR) revealed that rbfox1 was expressed in all Journal of Cell Science

3031 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850 tissues at 48 hours post fertilization (hpf, Fig. 1B), consistent with antisense oligonucleotides directed against either the translational previous studies (Kurrasch et al., 2007; Thisse, 2004; Gallagher start-site (MO1) or splice donor site of intron-5–exon-6 (MO2) into et al., 2011). Using whole-mount in situ hybridization, qRT-PCR one-cell-stage zebrafish embryos. To monitor the effect of the splice and RNA-Seq we could show that there was considerable expression morpholino, we performed cDNA splice-site analysis and found of rbfox1 in the zebrafish heart at 48 hpf (Fig. 1C–E). Interestingly, that blockage of the splice donor site of exon 6 of rbfox1 resulted in rbfox1 transcripts were, in comparison to the other rbfox paralogs, a complete exclusion of exon 6 (Fig. 2F). Absence of exon 6 leads to rbfox1l, rbfox2 and rbfox3, more abundant in heart tissue at 48 hpf a frameshift of the coding sequence and to a predicted truncated compared to whole-fish tissue expression (Fig. 1D). qRT-PCR protein due to a premature stop within exon 7 (Fig. 2G). As a result analysis further showed that rbfox1 was expressed in the adult in all of the rbfox1 knockdown, 85% of the zebrafish embryos injected investigated tissues (Fig. 1E). with MO1 (n=159; Fig. 2D) and 72% of those injected with MO2 (n=100, Fig. 2E) developed progressive heart failure due to Loss of zrbfox1 leads to impaired cardiac function continually decreasing ventricular contractility as measured by A cardiac role for rbfox1 had not been demonstrated, hence our aim fractional shortening (Fig. 2B,C; supplementary material Movie 2). was to investigate the functional role of this new splicing regulator In detail, the ventricular contractility of morphants decreased in vivo by using the zebrafish as a model organism. To do so, we first progressively from 28±3.2% at 48 hpf to 18±3.9% (mean±s.d.) at inactivated zebrafish rbfox1 by injecting morpholino (MO) 72 hpf (Fig. 2I). By ∼96 hpf, both chambers collapsed and became

Fig. 2. Knockdown of rbfox1 leads to cardiomyopathy and heart failure. (A–C) Lateral view of MO- control- and MO1- and MO2-rbfox1- injected embryos. After injection of 3 ng MO1-rbfox1, 85% (D) and MO2- rbfox1, 72% (E) of morphants embryos develop cardiomyopathy and heart failure. Results are mean± s.d. (n=3). (F) cDNA analysis of rbfox1 morphants after injection of MO2-rbfox1 shows skipping of exon 6 (198-bp product) compared to a product including exon 6 (259 bp product) in control-treated embryos. Sanger sequencing reveals that exon 6 is completely excluded, predictably leading to a frame shift of the coding sequence and premature stop in exon 7. (H,I) Fractional shortening (FS) of the ventricular chamber of MO-control- and MO1-rbfox1-injected embryos measured at the indicated developmental stages. Fractional shortening is significantly reduced in rbfox1 morphants after 48 hpf and further declines at 96 hpf. Results are mean±s.d. (n=10). Journal of Cell Science

3032 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850 almost silent. Consequently, blood flow completely arrested. (Fig. 3D). Furthermore, transmission electron microscopy (TEM) Embryos injected with a control morpholino (MO-control) revealed apparently normally developed cardiac and skeletal muscle did not show any morphological or functional abnormalities sarcomeres (Fig. 3E–H) in rbfox1 morphant embryos. Specifically, (Fig. 2A,H; supplementary material Movie 1). In addition to the we found well-organized , interconnected by Z-disks observed cardiac defect, we only noticed the development of a and aligned in discernible AI- and M-bands. slightly brain edema. In summary, we find no structural or developmental To evaluate further the causative mechanism responsible for abnormalities that sufficiently could explain the development of the severe heart failure phenotype observed in rbfox1 morphants, heart failure in rbfox1 morphants. Hence, rbfox1 deficiency leads to we next analyzed their cardiac morphology and ultrastructure. a functional defect rather than structural abnormalities. Therefore, Histological analysis showed that the cardiac chambers of the rbfox1- we hypothesized that rbfox1 proteins regulate an alternative splicing morphant hearts were well defined and separated by the atrio- program that is essential for normal heart function during zebrafish ventricular ring (Fig. 3A,B), and that the endocardial and myocardial development and that a reduced activity of rbfox1 might result in an layers of both chambers had developed properly with a multilayered altered isoform expression of proteins essential for maintenance of ventricular myocardium. Occasionally, we also observed morphants cardiac function. displaying atrio-ventricular-canal (avc) malformations, potentially due to the pronounced dilation of the atrium. On the molecular level, Characterization of the cardiac transcriptome in the atrial and ventricular cardiomyocytes of rbfox1-morphants expressed zebrafish by RNA deep-sequencing heavy chains in the typical heart-chamber-specific pattern, To gain insights into heart-specific splicing, we first analyzed the suggesting that there was normal molecular chamber specification global exon expression of genes in zebrafish hearts compared to their

Fig. 3. rbfox1 deficency does not influence heart morphology. (A,B) H&E-stained sagittal histological sections of MO-control and MO1-rbfox1 morphant hearts at 72 hpf. Morphants display normal heart morphology with distinct endocardial and myocardial cell layers in the atrium (A) and ventricle (V), and a clear differentiation and demarcation of the atrium and ventricle by the atrio- ventricular ring (AV). OFT, outflow tract. (C,D) Atrial- and ventricle-specific myosin heavy chains are expressed normally, also suggesting that there is normal molecular chamber specification [green, antibody against atrial-specific myosin (S46); red, antibody against ventricular and atrial myosin (MF20)]. (E–H) Ultrastructural analysis of heart and skeletal muscle cells of MO-control- and MO1-rbfox1-injected embryos at 48 hpf showing organized sarcomere units (SU) with thin and thick myofilaments in well-aligned bundles and discernible AI-, M- and Z-bands. Journal of Cell Science

3033 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850 expression in the whole fish. For this purpose, we generated and ten increased exclusion, whereas three showed no difference. In sequencing libraries from pooled control embryos at 48 hpf. In whole-fish tissue, we identified 13 differentially spliced transcripts, detail, we isolated RNA from n=70 zebrafish embryos and n=400 with most of these transcripts showing exon skipping. For the 17 isolated purified embryonic zebrafish hearts. Next, we applied differentially spliced transcripts identified in both heart and in SOLiD deep sequencing and mapped the resulting 50-bp reads to the whole-fish tissue, we partially observed a discordant exon usage zebrafish reference genome. The normalized reads were quantified, behavior (Fig. 4, middle panel). For instance, for flot2a,we and a mean expression was calculated for each gene. We found observed an increased level of exon inclusion in heart tissue after several transcripts that were highly abundant in the zebrafish heart but rbfox1 knockdown, whereas in whole-fish tissue we detected an not in the whole fish and vice versa (supplementary material Fig. almost complete exclusion of this exon. S1A). Supplementary material Fig. S1B,C exemplarily shows two clusters of genes that were highly expressed genes in whole fish (blue) Validation of differential splicing in new rbfox1 target and/or heart tissue (red), which include genes such as tmp4, nppa and transcripts myl7. To integrate the information from the complex deep sequencing For the validation experiments, we assayed 22 of the 26 identified data at a functional level, we next performed a gene set enrichment transcripts from heart tissue using qRT-PCR. Here, we also focused analysis for the differentially expressed genes based on the Kyoto on alternative exon usage (inclusion or exclusion). To do so, we Encyclopedia of Genes and Genomes (KEGG) definition. We applied two strategies to assess the splicing pattern. First, we found that genes with functions in mitogen-activated protein performed a flanking qRT-PCR, which uses primers that hybridize kinase (MAPK) signaling, Ca2+ signaling, focal adhesion and to the constitutive exons flanking the predicted alternative spliced insulin signaling tended to be more highly represented in hearts, exon. Second, we carried out a PCR with primers targeting the whereas those with functions in metabolic pathways, oxidative flanking constitutive exons as well as the alternative spliced exon. , ribosome, spliceosome, proteasome were less For normalization, constitutive exon quantification was applied. represented (supplementary material Table S1). The sensitivity and the specificity of this procedure were controlled To characterize the diversity of the zebrafish transcriptome, we by a spike-in control. For quantification, three biological replicates next investigated all sequencing reads of the heart and whole body were used, with n=400 dissected embryonic hearts each. All that non-ambiguously matched predicted exon–exon junctions (for analyses were performed at the 48 hpf stage. details, see the Materials and Methods section). Among the detected From the subset of 22 cardiac transcripts, five genes with genes in the whole fish, 86% have only one isoform, 11% two alternative exon usage events were concurrently validated by the isoforms, and 4% three or more isoforms (supplementary material qRT-PCR and gel electrophoresis (Fig. 5A–E). These genes were Fig. S1D), delineating the transcriptomic diversity caused by huG, actn3a, ptpla, camk2g1 and ktn1, which were among the genes alternative splicing mechanisms. All identified genes that undergo with strongest differences in the PSI-based RNA sequencing alternative splicing are listed in supplementary material Table S5. analysis. In detail, we found an increased exon inclusion in huG and actn3a in heart tissue of rbfox1 morphants. Here, qRT-PCR RNA-sequencing analysis identifies a specific set of rbfox1- analysis showed a 1.5-fold (huG transcript) and 1.6-fold (actn3a dependent splicing events in zebrafish hearts transcripts) increased inclusion of the alternative spliced exon in We next asked whether defective mRNA splicing could be rbfox1-depleted zebrafish hearts in comparison to the control- responsible for the functional defects observed in rbfox1-deficient injected hearts. For ptpla, camk2g1 and ktn1, we detected the embryos. To do so, we evaluated the effect of reduced rbfox1 opposite, namely an increased exon exclusion, with kinectin (ktn1) activity on mRNA processing on a global scale. mRNA deep- showing the largest observed effect. sequencing of heart and whole-fish tissue from MO-control- and MO1-rbfox1-injected embryos at the 48 hpf stage was performed as Functional delineation of disturbed mRNA splicing in rbfox1 described above. Owing to the predicted nature of rbfox1-mediated morphants splicing, we analyzed alternative splicing changes on a single-exon To address the question of whether the differentially spliced usage basis. This means that splicing events lead to single exon transcripts identified in heart tissue after depletion of rbfox1 could inclusion or exclusion in the mature mRNA. The stringent analysis partially explain the observed phenotype, we analyzed the of transcript variants between control and rbfox1-depleted whole functional role of two candidates by a reverse genetic approach. fish and heart tissue revealed 56 alternative splicing events showing For this purpose, we designed specific antisense oligonucleotides differential exon usage. In detail, we found 26 differentially spliced targeting the alternative spliced exon of huG and actn3a to disturb transcripts exclusively in heart tissue, 13 in whole-fish tissue and 17 their regular isoform expression. Injection of 3 ng of MO-huG in in both tissues (Fig. 4). zebrafish embryos led to a similar but weaker cardiac phenotype to To determine internal exon inclusion levels, we next relied on the that observed after depletion of rbfox1. Specifically, MO-huG- percentage splicing index (PSI or Ψ) approach as described injected embryos developed late-onset heart failure with dilation of previously (Wang et al., 2008), where the fraction of mRNA that the both chambers and pericardial edema (Fig. 6A; supplementary contains an exon, the ‘percentage spliced in’ value, can be estimated material Movie 3). Fractional shortening measurements showed a as the ratio of the density of inclusion reads to the sum of the significantly reduced ventricular contractility, decreasing from densities of inclusion and exclusion reads. A PSI value of 100% 42±4% at 48 hpf to 20±3% (mean±s.d.) at 96 hpf (Fig. 6B). In means that the exon is fully included and, accordingly, a value of 0% addition to their cardiac phenotype, huG morphants displayed a means that the exon is never included. slight swelling of the fore-, mid- and hind-brain. The conducted Fig. 4 gives the identified transcripts in heart and whole-fish cDNA splice-site analysis showed that there was exclusion of exon 3 tissue and the corresponding PSI values for MO-control and MO1- in the morphants. Nevertheless, loss of exon 3 led to a reduced rbfox1. In heart tissue, we found 26 differentially spliced transcripts cardiac function. after rbfox1 depletion, showing PSI ratios between 2.62 to 0.06. For actn3a, we found that induction of splice-site blockage at

Among the 26 identified transcripts, 13 showed increased inclusion exon-6–intron-6 led to mild cardiac dysfunction. actn3a-depleted Journal of Cell Science

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Fig. 4. RNA sequencing analysis of MO-control- and MO-rbfox1-injected zebrafish embryos. Venn diagram showing differentially spliced exon triplets in heart and whole-fish tissue comparing MO-control and MO1-rbfox1 morphants based on an inclusion and exclusion scoring algorithm (1). Next (2), alternative exon usage is presented as PSI values (colored bar graphs) in control- and rbfox1-depleted zebrafish. In addition, the PSI ratio is given (PSI MO1-rbfox1:MO- control). Bar graphs at the bottom detail the proportion of exon inclusion and skipping. zebrafish embryos developed heart failure with dilation of the Movie 4). In addition, actn3a-morphants showed a reduction of atrium and reduced ventricular contractility beginning at the 72 h blood flow and pericardial blood congestion as a consequence of the developmental stage (fractional shortening was 35±3% at 72 hpf reduced cardiac function. Beside cardiac functional defects, we saw and 20±3% at 96 hpf) (Fig. 6C,D; supplementary material a slightly impairment of skeletal muscle function as assessed by a Journal of Cell Science

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Fig. 5. Validation of differential splicing of huG, actn3a, ptpla, camk2g1 and ktn1. (A–E) Left panels show the PSI value from RNA sequencing of MO-control and MO1- rbfox1 morphants. Middle panels show the relative expression of differentially spliced exons measured by qRT-PCR. Relative expression analysis in heart tissue indicates downregulation (red lines) or upregulation (black lines) of exon– exon junctions after depletion of rbfox1. Right panels show gel electrophoresis showing the PCR products (ctrl, control; KD, MO1- rbfox1 morphant). Green and red boxes indicate forms with exon inclusion or exon skipping, respectively.

reduced touch response (data not shown). cDNA splice-site analysis been found to be particularly prevalent in neurodegenerative of actn3a morphants revealed incorrect spliced actn3a transcripts, diseases and cancer (Cartegni et al., 2002, 2006; Cartegni and with partial inclusion of intron 6. Krainer, 2002; Cooper et al., 2009; Kashima and Manley, 2003; Talbot and Davies, 2001). For instance, neurofibromatosis type I is DISCUSSION caused by mutations in the neurofibromin 1 gene (NF1), and in 50% RNA splicing is crucial for many biological processes and is of the patients the identified mutations result in splicing alterations regulated by complex mechanisms involving numerous RNA- (Ars et al., 2000). In the heart, physiological changes during the binding proteins. To date, a large number of human diseases have postnatal remodeling require extensive transcriptional and been found to result from disturbed RNA splicing, most often posttranscriptional changes, including alternative splicing caused by intronic or exonic mutations that disrupt splicing sites or mechanisms (Kalsotra et al., 2008; Olson, 2006). This has led to splice-factor binding patterns. Such splicing abnormalities have speculations that splicing also contributes to maladaptive Journal of Cell Science

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Fig. 6. Correctly balanced huG and actn3a isoform expression is essential for proper heart function. (A,B) Lateral view of a MO-huG-injected (MO-zhug) zebrafish embryo at 48 hpf. Fractional shortening (FS) shows a progressive reduction of cardiac contractility. Inset, cDNA splice-site analysis of huG morphant. (C,D) Splice-site blockage in actn3a leads to mild cardiac dysfunction. actn3a-depleted zebrafish embryos develop heart failure with dilation of the atrium and reduced ventricular contractility beginning at the 72 h developmental stage. Inset, cDNA splice-site analysis of actn3a morphant. Results in B and D are mean±s.d. (n=10). remodeling during different heart diseases. As such, mutations in 2011). To circumvent these limitations, we applied a two-step the splice factor RBM20 have recently been associated with human approach. First, we identified isoforms by deep sequencing and dilated cardiomyopathy (Brauch et al., 2009; Li et al., 2010; Refaat then quantified their expression by qRT-PCR. For the latter, et al., 2012). To gain further insights into the relevant mechanisms, Vandenbroucke et al. proposed the usage of boundary-spanning Guo et al. used deep transcriptome sequencing to identify sets of primers to quantify isoforms that strongly differ in abundance genes with conserved splicing regulation between humans and rats, (Vandenbroucke et al., 2001). By this stepped approach, we could rendering animal models suitable to investigate human splicing detect five genes with substantially changed mRNA splicing, all of defects (Carboni et al., 2010). Consequently, other RNA-binding which could explain the observed cardiac phenotype after depletion proteins such as CELF-3, CELF-5, RBM24, RBM25 and LUC7L3 of rbfox1. have been linked to the development of cardiomyopathies (Brauch The splicing factor rbfox1 is a member of the RBFOX-protein et al., 2009; Gao et al., 2011; Li et al., 2010; Musunuru, 2003; Poon family, which is characterized by a common evolutionary highly et al., 2012; Refaat et al., 2012). In the present study, we conserved RNA recognition motif (RRM), flanked by less conserved successfully applied a similar approach in zebrafish to dissect the N- and C-terminal domains unique for this proteins. In mammals, role of rbfox1 in cardiac splicing. Our results render Rbfox1, as well there are three RBFOX paralogs: RBFOX-1 (also known as ataxin2- as its validated targets, new candidates for involvement in human binding protein 1), RBFOX-2 (also known as RNA-binding motif 9, cardiomyopathy and heart failure. RBM9) and RBFOX-3 (also known as hexaribonucleotide-binding The transcriptome sequencing conducted in the present study protein 3, HRNBP3, and NeuN) (Kuroyanagi, 2009). Each member indicates that 14% of all genes expressed in the heart undergo of the RBFOX protein family specifically binds to (U)GCAUG alternative splicing by single exon skipping or inclusion. This elements and regulates alternative splicing positively or negatively in amount of alternative splicing is significantly lower than predicted a position-dependent manner (Jin et al., 2003; Nakahata and in humans (Johnson et al., 2003; Wang et al., 2008). However, we Kawamoto, 2005; Underwood et al., 2005; Zhang et al., 2008). In only evaluated splicing events that target ‘an exon in the middle’. detail, they promote exon inclusion when binding to the intron Hence, the actual diversity of the zebrafish transcriptome will be downstream from an alternative cassette exon, and exon skipping considerably higher. A major problem in quantifying mRNA levels when binding to the upstream intron (Auweter et al., 2006; Tang by RNA sequencing is that shorter reads do not always map et al., 2009). Several human mutations have previously been mapped uniquely to a single gene and the depth of coverage considerably to this , with patients exhibiting severe neuro-developmental influences the relative quantification performance (Tarazona et al., phenotypes, including mental retardation, epilepsy and autism Journal of Cell Science

3037 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850 spectrum disorder (Barnby et al., 2005; Bhalla et al., 2004; Martin (morphant) zebrafish were evaluated for defects in heart and muscular et al., 2007; Sebat et al., 2007). Downregulation of rbfox1 during development compared to control-injected embryos from the same embryo cardiac hypertrophy leads to altered splicing events in mice hearts clutch at 48 hpf, using fluorescence microscopy for visualization of EGFP (Park et al., 2011). Here, we found that loss of rbfox1 does not result and dsRed1. To confirm that the injected morpholinos specifically induced in a completely splicing failure, but rather alters the splicing pattern the defective phenotypes of morphants, we analyzed the MO-targeting region by PCR and a subsequent sequencing step using the morphant and of specific target genes. On a functional level, we found that rbfox1 is control cDNA as template. Morpholino-modified antisense necessary for cardiac contractility, potentially by regulating the oligonucleotides used to generate specific splicing isoforms are listed in correct splicing of the here identified downstream targets. However, supplementary material Table S2. although several targets were mis-spliced it still remains difficult to pinpoint the precise effect of each of them. Hence, we analyzed the Functional assessment functional role of two candidates by a reverse genetic approach. Functional assessment of cardiac contractility was carried out as described Using this approach, we showed that actn3a and huG might be before (Rottbauer et al., 2005). Fractional shortening of heart chambers was downstream functional effectors and that mis-splicing caused by calculated with help of the zebraFS software (www.benegfx.de). Results are rbfox-depletion results in a cumulative phenotypic effect due to expressed as mean±s.d. Analyses were performed using an unpaired ’ several splicing abnormalities. In case of huG there was, however, Student s t-test. A value of P<0.05 was accepted as statistically significant. a difference in the splicing pattern after rbfox1-depletion compared RNA isolation and quantitative real-time PCR to the splice-site blocking antisense probe, suggesting that a Total RNA was isolated from 48 hpf control-injected and MO-injected quantitatively correct isoform expression is necessary for zebrafish embryos and hearts using Trizol and chloroform and treated with unconstrained cardiomyocyte function. Taken together with results DNaseI (Invitrogen, Carlsbad, CA) to digest contaminating genomic DNA. from previous reports, this highlights the importance of balanced 60 ng of total RNA was reverse transcribed using a mixture of mRNA splicing in the heart and represents intriguing opportunities oligonucleotide (dT) and dNTPs and the SuperScript III first strand for novel therapeutic approaches (Bauer et al., 2010; Hammond and cDNA synthesis kit (Invitrogen, Carlsbad, CA). qRT-PCR was carried out Wood, 2011; Wood et al., 2010). according to standard protocols with the SYBR-Green method (Thermo The potential impact of RNA splicing in human disease is Scientific, Waltham, MA) using an ABI 7000 system (ABI). Primers were undoubtable. It will be crucial to leverage our understanding of the designed using NCBI primer blast and synthesized by Metabion biological role of alternative splicing to develop novel diagnostic (Martinsried, Germany). The optimum annealing temperature for each primer set was determined prior to the analysis of experimental samples. and therapeutic strategies. Genetic therapies will be needed to The specificity of each primer set was monitored by dissociation curve correct RNA mis-splicing, and early studies in this area have analysis to ensure only a single product was amplified and that no primer progressed from the cell culture to promising clinical trials (Wood dimers were formed. Supplementary material Table S3 details the genes et al., 2010). validated by qRT-PCR and assay conditions. Threshold cycle (CT) values were recorded in the linear phase of amplification and the data were MATERIALS AND METHODS analyzed using the ΔCT method. elfa was used as the internal reference to Zebrafish strains further normalize the data. Results are presented as relative expression Care and breeding of zebrafish (Danio rerio) were as described previously compared to control-MO-injected embryos. (Meder et al., 2011). The present study was performed under institutional approvals, which conform to the Guide for the Care and Use of Laboratory cDNA library preparation next generation sequencing Animals published by The US National Institute of Health (NIH Publication For library preparation, 4 to 6 µg of total RNA of the embryonic zebrafish No. 85-23, revised 1996). heart samples and 9 to 25 µg of the zebrafish samples were enriched with the Ambion® MicroPoly (A)Purist™ Kit to obtain all mRNA transcripts (refer Whole-mount in situ hybridization, histology and transmission to supplementary material Table S4). 500 ng of each sample were used electron microscopy for the fragmentation with RNase III. Purification of the fragmented RNA Embryos were fixed in 4% paraformaldehyde or DENT’s fix solution, was performed by the RiboMinus™ Concentration Module (Invitrogen, respectively. For histological analysis 5-µm sections were cut, dried and Carlsbad, CA). SOLiD adaptors were ligated by adding the amounts stained with hematoxylin and eosin (H&E) (Rottbauer et al., 2001). Whole- listed in supplementary material Table S4 to the enriched fraction. After mount in situ hybridization of zebrafish embryos was conducted as ligation, mRNAs were transcribed into cDNA with Array Script™ previously described (Thisse and Thisse, 2008) using a digoxigenin-labeled Reverse Transcriptase. cDNA fragments between 150 and 250 nt rbfox1 antisense probe. For TEM, embryos were fixed as described (mRNAs plus adaptors) were isolated from a 6% TBE Urea Gel (Novex- previously (Majumdar and Drummond, 1999), embedded in Epon 812 System, Invitrogen, Carlsbad, CA). RNA from gel slices was amplified with (Polysciences) and sectioned. Thin sections were cut on a Reichert Ultracut 16 PCR cycles using the same 5′-primer for each sample and six different 3′- E ultramicrotome and collected onto Formar-coated slot grids. Sections primers including the barcode sequences (SOLiD Multiplexing Barcoding were poststained with uranyl acetate and lead citrate and viewed in a Philips Kit 01-16). A total of six purified and barcoded DNA libraries were CM10 electron microscope at 80 keV. analyzed with a HS-DNA Chip in the Agilent Bioanalyzer 2100 and subsequently pooled in equimolar amounts. Antisense-mediated knockdown Morpholino-modified antisense oligonucleotides (Gene Tools, Philomath, Next generation sequencing OR) used were as follows: zebrafish rbfox1-MO1 (ENSDARG00000014746) The pooled libraries were diluted to a concentration of 60 pg/µl. The DNA targeting the start site +1 to +28 (5′-ATGGAGGAAAAAGGGAGCAA- was amplified monoclonally on magnetic beads in an emulsion PCR. GATGGTGG-3′), and rbfox1-MO2 targeting the splice-donor site of exon6 Emulsions were broken with butanol and the remaining oil was washed off (5′-AAATgtaagaacaagctccctt-3′, lowercase letters represent intronic the templated double-stranded beads. DNA on the bead surface was sequence). Morpholinos and standard control oligonucleotide (MO- denatured to allow hybridization of the enrichment beads to the single- control) were injected into one-cell stage zebrafish embryos as stranded DNA. Using a glycerol cushion, the null beads can be separated previously described (Meder et al., 2011). Embryos were derived from from the templated beads. After centrifugation, the enriched magnetic beads mating transgenic (Tg) zebrafish containing a fluorescent EGFP reporter were in the supernatant. The enrichment-beads were separated from the cassette driven by the heart-specific myl7 promoter (myl7:EGFP Tg) and magnetic beads by denaturation. The 3′-end was enzymatically modified for endothelial-cell-specific promotor fli ( fli:dsRed Tg). MO-targeted deposition on the sequencing slide. Seven hundred million beads were Journal of Cell Science

3038 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3030-3040 doi:10.1242/jcs.166850 loaded onto a Full Slide and sequenced on a SOLiD 4 analyzer (Applied Funding Biosystems, Carlsbad, CA). This work was supported by grants from the ‘Bundesministerium für Bildung und Forschung’ [grant numbers NGFN-plus01GS0836, NGFN-transfer 01GR0823]; and the German Center for Cardiovascular Research (DZHK); the University of Mapping reads to zebrafish genome and annotated genes Heidelberg (Innovationsfond FRONTIER); and the European Union (FP7 The reads obtained from sequencing were mapped to the zebrafish genome INHERITANCE and BestAgeing). Deposited in PMC for immediate release. (Danio rerio zv8) using the BioScope software (Life Technologies, Darmstadt, Germany). In addition to the genomic sequence, the reference Supplementary material taken for mapping contained ribosomal and transfer RNA sequences from Supplementary material available online at zebrafish, as well as sequences of the SOLiD adaptors and homopolymer http://jcs.biologists.org/lookup/suppl/doi:10.1242/ jcs.166850/-/DC1 sequences (poly-A, poly-T) were also used in order to filter reads matching these sequences. For the identification of reads covering exon-exon References (inter-exon) junctions, sequences were added to the reference representing Ars, E., Serra, E., Garcıa,́ J., Kruyer, H., Gaona, A., Lázaro, C. and Estivill, X. all possible combinations of two exons from each gene based on the (2000). Mutations affecting mRNA splicing are the most common molecular zebrafish annotation (zv8; UCSC Genome Browser). defects in patients with neurofibromatosis type 1. Hum. Mol. Genet. 9, 237-247. Auweter, S. D., Fasan, R., Reymond, L., Underwood, J. G., Black, D. L., Pitsch, S. and Allain, F. H.-T. (2006). Molecular basis of RNA recognition by the human Determination and normalization of levels alternative splicing factor Fox-1. EMBO J. 25, 163-173. Expression levels of exons were determined according to their coverage by Barash, Y., Calarco, J. A., Gao, W., Pan, Q., Wang, X., Shai, O., Blencowe, B. J. the sequencing reads. To enable cross sample comparison, coverages were and Frey, B. J. (2010). Deciphering the splicing code. Nature 465, 53-59. normalized in terms of reads per kilobase of exon model per million mapped Barnby, G., Abbott, A., Sykes, N., Morris, A., Weeks, D. E., Mott, R., Lamb, J., Bailey, A. J. and Monaco, A. P. (2005). Candidate-gene screening and reads (RPKM) (Mortazavi et al., 2008). association analysis at the autism-susceptibility locus on 16p: evidence of association at GRIN2A and ABAT. Am. J. Hum. Genet. 76, 950-966. Determination of alternative splicing using RNA-seq Bauer, R., Katus, H. A. and Müller, O. J. (2010). Exon skipping with morpholino Splicing events were determined by the analysis of reads covering exon- oligomers: new treatment option for cardiomyopathy in Duchenne muscular exon junctions as identified by mapping to one of the two exon dystrophy? Cardiovasc. Res. 85, 409-410. Bhalla, K., Phillips, H. A., Crawford, J., McKenzie, O. L. D., Mulley, J. C., Eyre, H., combinations added to the reference. The presence of splicing junctions Gardner, A. E., Kremmidiotis, G. and Callen, D. F. (2004). The de novo in a sample was represented as the fraction of reads covering a particular translocations of two patients with abnormal phenotypes (mental exon-exon junction (parts per million; ppm) and related to the number of all retardation and epilepsy) disrupt the A2BP1 gene. J. Hum. Genet. 49, 308-311. reads found to map on exon junctions in that sample. Biesiadecki, B. J. and Jin, J.-P. (2002). Exon skipping in cardiac T of For the identification of splicing events removing single exons in only one turkeys with inherited dilated cardiomyopathy. J. Biol. Chem. 277, 18459-18468. of two samples (test or reference sample), we applied a function to each exon Biesiadecki, B. J., Elder, B. D., Yu, Z.-B. and Jin, J.-P. (2002). Cardiac to calculate a score for its removal. The scoring was based on the coverages variants produced by aberrant splicing of multiple exons in animals with high instances of dilated cardiomyopathy. J. Biol. Chem. 277, 50275-50285. of exon-exon junctions (CoverageA, CoverageB, coverages of exon Black, D. L. (2003). Mechanisms of alternative pre-messenger RNA splicing. Annu. junctions in test or reference sample; i, exon number) as below: Rev. Biochem. 72, 291-336. Brauch, K. M., Karst, M. L., Herron, K. J., de Andrade, M., Pellikka, P. A., Rodeheffer, R. J., Michels, V. V. and Olson, T. M. (2009). Mutations in ðCoverageAÞ ðCoverageBÞ þðCoverageBÞ ribonucleic acid binding protein gene cause familial dilated cardiomyopathy. ¼ ði;iþ2Þ ði;iþ1Þ ðiþ1;iþ2Þ : Score ð Þ ð Þ ð Þ J. Am. Coll. Cardiol. 54, 930-941. CoverageB ði;iþ2Þ CoverageA ði;iþ1Þ CoverageA ðiþ1;iþ2Þ Carboni, N., Porcu, M., Mura, M., Cocco, E., Marrosu, G., Maioli, M. A., Solla, E., Tranquilli, S., Orrù, P. and Marrosu, M. G. (2010). Evolution of the phenotype in a family with an LMNA gene mutation presenting with isolated cardiac In order to identify splice-out events in both samples, the scoring function involvement. Muscle Nerve 41, 85-91. was applied twice after swapping the coverages for the two samples. Splice- Cartegni, L. and Krainer, A. R. (2002). Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence out events were then selected for further examination based on the calculated of SMN1. Nat. Genet. 30, 377-384. score. Cartegni, L., Chew, S. L. and Krainer, A. R. (2002). Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. Pathway analysis 3, 285-298. To test whether genes with differential expression patterns were Cartegni, L., Hastings, M. L., Calarco, J. A., de Stanchina, E. and Krainer, A. R. significantly accumulated in specific biochemical pathways, we applied (2006). Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2. Am. J. Hum. Genet. 78, 63-77. the Gene Set Enrichment Analysis (GSEA) method for the differentially Cooper, T. A., Wan, L. and Dreyfuss, G. (2009). RNA and disease. Cell 136, expressed genes in heart and whole-fish tissue based on the Kyoto 777-793. Encyclopaedia of Genes and Genomes (KEGG) definition. GSEA produces Cork, D. M. W., Lennard, T. W. J. and Tyson-Capper, A. J. (2008). Alternative an ordered list of genes as input and evaluates whether the genes on top of splicing and the progesterone receptor in breast cancer. Breast Cancer Res. 10, the list are enriched for participants in the respective pathways. To this end, 207. we sorted all genes according to the absolute value of the median distances Creemers, E. E., Sutherland, L. B., Oh, J., Barbosa, A. C. and Olson, E. N. (2006). Coactivation of MEF2 by the SAP domain proteins myocardin and in heart and whole-fish tissue and uploaded the respective gene list to MASTR. Mol. Cell 23, 83-96. GeneTrail (Keller et al., 2008), a comprehensive web-based tool for various David, C. J. and Manley, J. L. (2008). The search for alternative splicing regulators: pathway analysis (http://genetrail.bioinf.uni-sb.de). GeneTrail incorporates new approaches offer a path to a splicing code. Genes Dev. 22, 279-285. all available KEGG pathways for Danio rerio and outputs significantly Davis, F. J., Gupta, M., Pogwizd, S. M., Bacha, E., Jeevanandam, V. and Gupta, enriched pathways with respect to our input list. M. P. (2002). Increased expression of alternatively spliced dominant-negative isoform of SRF in human failing hearts. Am. J. Physiol. Heart Circ. Physiol. 282, H1521-H1533. Competing interests Disset, A., Bourgeois, C. F., Benmalek, N., Claustres, M., Stevenin, J. and The authors declare no competing or financial interests. Tuffery-Giraud, S. (2006). An exon skipping-associated nonsense mutation in the gene uncovers a complex interplay between multiple antagonistic Author contributions splicing elements. Hum. Mol. Genet. 15, 999-1013. K.S.F. and B.M. designed the study and experiments. K.S.F. performed the Fairbrother, W. G., Yeh, R.-F., Sharp, P. A. and Burge, C. B. (2002). Predictive experiments and analyzed data together with A.K., C.B., M.M., V.B. and B.M. K.S.F. identification of exonic splicing enhancers in human genes. Science 297, and B.M. wrote the manuscript. J.H., S.F., D.K., B.V., H.A.K. and W.R. edited the 1007-1013. paper. K.S.F., B.M., S.F., J.H. and W.R. revised the paper and performed additional French, P. J., Peeters, J., Horsman, S., Duijm, E., Siccama, I., van den Bent, experiments. M. J., Luider, T. M., Kros, J. M., van der Spek, P. and Sillevis Smitt, P. A. Journal of Cell Science

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