ARTICLE DOI: 10.1038/s41467-017-01961-y OPEN Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes Demian Burguera1,2,3, Yamile Marquez1,3, Claudia Racioppi4,5, Jon Permanyer1,3, Antonio Torres-Méndez 1,3, Rosaria Esposito4, Beatriz Albuixech-Crespo2, Lucía Fanlo6, Ylenia D’Agostino4, Andre Gohr1,3, Enrique Navas-Perez2, Ana Riesgo7, Claudia Cuomo4, Giovanna Benvenuto 4, Lionel A. Christiaen 5, Elisa Martí6, Salvatore D’Aniello4, Antonietta Spagnuolo4, Filomena Ristoratore 4, Maria Ina Arnone4, Jordi Garcia-Fernàndez 2 & Manuel Irimia 1,3 1234567890 Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co- opted into the development of many novel traits in vertebrates. 1 Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, Barcelona 08003, Spain. 2 Department of Genetics, School of Biology, and Institut de Biomedicina (IBUB), University of Barcelona, Diagonal 643, Barcelona 08028, Spain. 3 Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain. 4 Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy. 5 Center for Developmental Genetics, Department of Biology, New York University, New York, NY 1003, USA. 6 Instituto de Biología Molecular de Barcelona, CSIC, Parc Científic de Barcelona, Baldiri Reixac 20, Barcelona 08028, Spain. 7 Department of Life Sciences, Natural History Museum of London, Cromwell Road, SW7 5BD London, UK. Yamile Marquez and Claudia Racioppi contributed equally to this work. Correspondence and requests for materials should be addressed to M.I.A. (email: [email protected]) or to J.G.-F. (email: [email protected]) or to M.I. (email: [email protected]) NATURE COMMUNICATIONS | 8: 1799 | DOI: 10.1038/s41467-017-01961-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01961-y uring embryo development, tissues proliferate and dif- different phyla. Exemplifying this split, we show that the Fgfr Dferentiate in a coordinated manner to build a whole event affecting the IgIII domain originated from a Bilateria hot- organism through a genome-guided process. Different cell spot of recurrent AS evolution that was co-opted as an Esrp target types express distinct transcriptomes to control cellular identity at the base of chordates. and physiology, and to establish differential interaction cap- abilities between embryonic tissues. Final morphology is thus achieved by cell-specific transcriptomic responses to external and Results internal stimuli within each tissue. Therefore, changes in the esrp1 and esrp2 are involved in morphogenesis in zebrafish.A genetic networks involved in morphogenesis are ultimately broad phylogenetic analysis showed that the Esrp family predates responsible for both the modification of organs and, at a mac- the origin of metazoans and that a single copy of Esrp has been roevolutionary scale, the origin of new structures1,2. maintained in most metazoan groups with the exception of the In particular, epithelial-mesenchymal interplays are essential to vertebrate lineage, in which two copies are present in all studied many organogenetic processes in vertebrates3,4. These tissues species (Supplementary Figs. 1, 2). To investigate the evolution of often interact in morphogenetic interfaces through the exchange Esrp roles across deuterostomes at various phylogenetic distances, of cells and signaling molecules5,6. Despite the great diversity of we first examined the expression and function of the two Esrp cell types across the embryo, the majority can be classified as paralogs (esrp1 and esrp2) in zebrafish. A highly dynamic showing either mesenchymal or epithelial characteristics. This expression pattern was observed for both genes during the broad distinction, which is independent of tissue origin, has also development of zebrafish using whole-mount in situ hybridiza- been shown to be reflected in the patterns of gene expression and tion (WMISH) (Fig. 1a, Supplementary Fig. 3). At early stages, alternative splicing (AS)7. Those transcriptomic programs confer both genes displayed different expression patterns. esrp1 tran- partly antagonistic morphogenetic properties to epithelial and scripts were detected in the whole epidermis at 14 h post- mesenchymal tissues by modulating certain cellular features, such fertilization (h.p.f.), but its expression was restricted to the pos- as adhesion, motility and polarity. terior part of the embryo at 16 h.p.f.. On the other hand, esrp2 Of particular importance for mammalian morphogenesis is a was found only in the hatching gland rudiment at these stages. mutually exclusive exon skipping event found in members of the However, from 24 h.p.f. to 5 days post-fertilization (d.p.f.), the FGF receptor (Fgfr) gene family8. Exons IIIb and IIIc encode a expression of both genes converged in most territories. Their region of the third immunoglobulin domain (IgIII) of the FGFR1, transcription was transiently activated during the development of FGFR2, and FGFR3 proteins, and are differentially included in multiple organs, including the olfactory epithelium, otic vesicle, transcripts from epithelial or mesenchymal cells, respectively. pharynx, epidermis and notochord. esrp2 showed expression in a Importantly, their mutually exclusive inclusion has a dramatic few additional tissues, such as pronephros, hatching gland, liver, effect on the affinity of the receptors for FGF ligands9, providing and heart. epithelial cells specificity for FGF signals secreted by the Next, we used the CRISPR-Cas9 system to generate loss-of- mesenchyme, and vice versa. Consistent with the importance of function zebrafish mutant lines for esrp1 and esrp2. We targeted this regulatory system in development, disruption of the Fgfr2- single guide-RNAs to the first and third exons of esrp1 and esrp2, IIIb isoform leads to severe defects during mice organogenesis10. respectively (Fig. 1b). For esrp1, we selected a mutant allele with a These and other morphogenesis-associated AS events are 168-bp deletion and 14-bp insertion that induced the usage of an directly regulated by the Epithelial Splicing Regulatory Protein upstream cryptic splice donor, resulting in skipping of the whole (Esrp) genes in mammalian species11. Esrp1 and Esrp2 were ori- coding sequence of exon 1, including the start codon. For esrp2, ginally identified as positive regulators of IIIb exon inclusion of we selected a mutant allele with a 17-bp frame-disrupting deletion the Fgfr2 gene12. They encode RNA-binding proteins that are that produced a premature termination codon before the dynamically expressed mainly in a subset of epithelial tissues translation of any RNA-binding domain and that was predicted during mouse development13, although mesenchymal expression to trigger non-sense mediated decay (Fig. 1b). Expression of the has also been reported in chicken14. Recently, double knockout mutant alleles was highly reduced in homozygous embryos, while (DKO) mice for both Esrp genes were shown to display severe transcript levels of the other paralog were not increased as a organogenetic defects and a complete shift to exon IIIc inclusion compensatory mechanism in the single mutants, as shown by in Fgfr1, Fgfr2, and Fgfr315. In addition, many Esrp exon targets quantitative PCR (Supplementary Fig. 3m). Furthermore, western were identified in genes involved in cell–cell adhesion, cell blot assays confirmed the loss of the full-length protein for both polarity, and migration16. However, the origin and evolution of mutant alleles, although faint bands that might correspond to Esrp morphogenetic functions and its regulated AS programs shorter truncated proteins or unspecific signal were detected for remain largely unknown. esrp1 (Supplementary Fig. 3n). Therefore, to further determine Here, to investigate the evolution of Esrp functions and asso- the functional impact of the mutations, we cloned the mutant and ciated transcriptomic programs, we performed Esrp loss-of- wild-type (WT) esrp1 alleles in a pcDNA3.1 vector and transfect function or gain-of-function experiments in several deuterostome the construct into human 293T cells. Whereas expression of the species. Within bony vertebrates, Esrp genes play conserved roles WT allele of zebrafish esrp1 was able to modify the splicing in the development of numerous homologous organs. Con- pattern of previously reported endogenous targets (EXOC7, sistently, Esrp regulates a core set of homologous exons in the ARHGAP17, and FGFR1) in the same way as