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RSPO2 Inhibition of RNF43 and ZNRF3 Governs Limb Development

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RSPO2 Inhibition of RNF43 and ZNRF3 Governs Limb Development LETTER https://doi.org/10.1038/s41586-018-0118-y RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6 Emmanuelle Szenker-Ravi1,17, Umut Altunoglu2,17, Marc Leushacke1,17, Célia Bosso-Lefèvre1,3, Muznah Khatoo1, Hong Thi Tran4, Thomas Naert4, Rivka Noelanders4, Amin Hajamohideen1, Claire Beneteau5, Sergio B. de Sousa6,7, Birsen Karaman2, Xenia Latypova5, Seher Başaran2, Esra Börklü Yücel8, Thong Teck Tan1, Lena Vlaeminck4,16, Shalini S. Nayak9, Anju Shukla9, Katta Mohan Girisha9, Cédric Le Caignec5,10, Natalia Soshnikova11, Zehra Oya Uyguner2, Kris Vleminckx4*, Nick Barker1,12,13*, Hülya Kayserili2,8* & Bruno Reversade1,3,8,14,15* The four R-spondin secreted ligands (RSPO1–RSPO4) act via RSPO2 (Extended Data Fig. 1b) that affects a residue conserved in all their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT R-spondin paralogues and homologues (Fig. 1c and Extended Data signalling1–3. Here we report an allelic series of recessive RSPO2 Fig. 2a). The analogous mutation p.Arg64Cys in RSPO4 was shown to mutations in humans that cause tetra-amelia syndrome, which is cause congenital anonchia15. The seven affected fetuses from families 2 characterized by lung aplasia and a total absence of the four limbs. to 5 presented with complete absence of four limbs, lung hypo/aplasia, Functional studies revealed impaired binding to the LGR4/5/6 cleft lip-palate, and labioscrotal fold aplasia, all characteristic of receptors and the RNF43 and ZNRF3 transmembrane ligases, and TETAMS (Fig. 1a, b, Extended Data Fig. 1a and Extended Data Table 1). reduced WNT potentiation, which correlated with allele severity. A p.Gln70* nonsense mutation in RSPO2 was identified in family 216 Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, (Fig. 1a, c). In family 317, array comparative genomic hybridization Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 (array-CGH) analysis identified a biallelic deletion of 154 kilobases (kb) loss-of-function phenotypes. Moreover, endogenous depletion or spanning intron 5 and exon 6 of RSPO2 (Fig. 1a, c and Extended Data addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 Fig. 1c). In family 4, exome sequencing revealed a recessive p.Glu137* cells could still affect WNT responsiveness. Instead, we found that nonsense mutation in RSPO2 (Fig. 1a, c). In family 5 with three the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was consecutive TETAMS fetuses, a homozygous RSPO2 frameshift sufficient to trigger the outgrowth of supernumerary limbs. Our p.Gly42Val fs*49 mutation was identified (Fig. 1a–c). These results estab- results establish that RSPO2, without the LGR4/5/6 receptors, serves lish a new aetiology for tetra-amelia and demonstrate the crucial involve- as a direct antagonistic ligand to RNF43 and ZNRF3, which together ment of RSPO2 in craniofacial, limb and lung development in humans. constitute a master switch that governs limb specification. These We selected the p.Arg69Cys (R69C) and p.Gln70* (Q70X) muta- findings have direct implications for regenerative medicine and tions that are responsible for HFH-RTRD and TETAMS, respectively, to WNT-associated cancers. assess whether mutant RSPO2 retained binding to its cognate receptors. Limb development is governed by a three-dimensional signalling sys- By co-immunoprecipitation analysis, only wild-type RSPO2, but not tem that defines proximodistal, anteroposterior and dorsoventral axes4. the RSPO2(F105A/F109A) mutant that specifically abrogates binding Tetra-amelia with lung hypo/aplasia syndrome (TETAMS; MIM data- to LGRs18, nor the RSPO2(R69C) or RSPO2(Q70X) mutants, was able base entry 273395) is an extreme condition, in which fetuses lack all four to be pulled down by LGR5 (Fig. 1d and Extended Data Fig. 2b, c). limbs. TETAMS without lung hypoplasia has been linked to a WNT3 Although the RSPO2(F105A/F109A) mutant could be readily co-im- nonsense mutation in humans5. The four R-spondin ligands (RSPO1– munoprecipitated by RNF43 or ZNRF3, neither RSPO2(R69C) nor RSPO4) act as enhancers of WNT signalling6–9. They bind to their cog- RSPO2(Q70X) could interact with RNF43 or ZNRF3 (Fig. 1e and nate receptors LGR4, LGR5 and LGR6 via their Furin-like 2 domain, Extended Data Fig. 2d). Similarly, only wild-type RSPO2, but not the and to the E3 ubiquitin ligases RNF43 and ZNRF3 via their Furin- RSPO2(R69C) or RSPO2(Q70X) mutants, could be retained on the like 1 domain10. This tripartite interaction prevents WNT receptor surface of HEK293T cells overexpressing LGR5 or RNF43 (Extended degradation mediated by RNF43 or ZNRF3. Rspo2 mutation in mice Data Fig. 2e). Wild-type RSPO2, and to a lesser extent RSPO2(R69C) leads to limb truncations reminiscent of tetra-amelia11–14, but a role but not RSPO2(Q70X), could enhance WNT3A-mediated activation for its receptors has not been substantiated, to our knowledge, during of SUPERTOPFLASH (STF) luciferase (Fig. 1f and Extended Data limb morphogenesis. Fig. 2f). These in vitro results indicate that the R69C and Q70X muta- Here, we describe five families with eleven affected individuals that tions diminish the ability of RSPO2 to bind to LGRs, RNF43 or ZNRF3, display severe developmental limb defects. In family 1, four affected and to amplify β-catenin-dependent WNT signalling. These signalling fetuses presented with radial ray deficiency with humeral involvement, defects correlate with the severity of the fetuses’ phenotypes—the non- absence of tibiae with or without femoral deficiency, and absence of digits sense Q70X mutation (responsible for TETAMS) behaving as a null on the preaxial side (Fig. 1a, b, Extended Data Fig. 1a and Extended mutation, and the R69C mutation (responsible for HFH-RTRD) as a Data Table 1). We propose to name this severe dysostosis humero- hypomorphic allele. femoral hypoplasia with radio-tibial ray deficiency (HFH-RTRD). In mice, Rspo2 is expressed in the apical ectodermal ridge of the Exome sequencing identified a homozygous p.Arg69Cys mutation in growing limb and in the lung mesenchyme11. Accordingly, Rspo2 1Institute of Medical Biology, A*STAR, Singapore, Singapore. 2Medical Genetics Department, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey. 3Department of Paediatrics, National University of Singapore, Singapore, Singapore. 4Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. 5CHU Nantes, Service de Génétique Médicale, Nantes, France. 6Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal. 7University Clinic of Genetics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. 8Medical Genetics Department, Koç University School of Medicine (KUSOM), Istanbul, Turkey. 9Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, India. 10INSERM, UMR1238, Bone Sarcoma and Remodeling of Calcified Tissue, Université Bretagne Loire, Nantes, France. 11Institute of Molecular Biology (IMB) gGmbH, Mainz, Germany. 12Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan. 13Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK. 14Institute of Molecular and Cellular Biology, A*STAR, Singapore, Singapore. 15Reproductive Biology Laboratory, Academic Medical Center (AMC), Amsterdam-Zuidoost, The Netherlands. 16Present address: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium. 17These authors contributed equally: Emmanuelle Szenker-Ravi, Umut Altunoglu, Marc Leushacke. *e-mail: [email protected]; [email protected]; [email protected]; [email protected] NATURE| www.nature.com/nature © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. RESEARCH LETTER a F1 HFH-RTRD b F1-II:5 1.5° 2.5° I:3 I:4 I:1 I:2 26 gw +/– +/– +/– +/– II:1 II:2 II:3 II:4 II:5 II:6 II:7 +/– +/– –/– –/– –/– c.205C>T; p.Arg69Cys F2 TETAMS F3 TETAMS 1° ? I:1 I:2 I:1 I:2 +/– +/– F5-II:1 II:1 II:2 II:3 II:4 II:1 20 gw +/– +/– –/– c.208C>T; p.Gln70* 154 kb deletion (Ex. 6) F4 TETAMS F5 TETAMS 1° 1° I:1 I:2 I:1 I:2 +/– +/– +/– II:1 II:1 II:2 II:3 –/– –/– –/– c.409G>T; p.Glu137* c.123delG; p.Gly42Val fs*49 c Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 1 94 95 283 284 427 428 616 617 732 RSPO2 5′UTR 3′UTR 154 kb deletion c.123delG c.205C>T c.208C>T c.409G>T chr8: 108,809,266–108,963,256 p.Gly42Val fs*49 p.Arg69Cys p.Gln70* p.Glu137* 1 22 37 84 91 132 145 204 243 RSPO2 SP Furin-like 1 Furin-like 2 TSR-1 C-term – Heparin+ Heparin – Heparin – Heparin+ Heparin – Heparin d e f *** RSPO2-ΔC-AP RSPO2-ΔC-AP 40 * A A 30 ** F105 F105 AP aloneWT Q70X WT R69C WT F109A AP aloneWT Q70X WT R69C WT F109A 20 AP AP 75 75 Input Input 10 SUPERTOPFLASH relative to AP alone AP AP 75 75 0 RSPO2 WT 75 Flag Flag R69C Q70X 25 WNT3A Transfection IP: LGR5-ECD-Flag IP: RNF43-ECD-Flag Fig. 1 | Identification of RSPO2 mutations in fetuses presenting with (ΔC; RSPO2-ΔC-AP) with Flag-tagged extracellular domains (ECD) of severe limb defects. a, Pedigrees of family 1 (F1) with HFH-RTRD and LGR5 (LGR5-ECD-Flag). WT, wild-type. e, Co-immunoprecipitation of families 2–5 (F2–F5) with TETAMS, and RSPO2 germline mutation status RSPO2-ΔC-AP with RNF43-ECD-Flag. Asterisks indicate non-specific for available family members. b, Pictures and radiographs illustrating limb bands. Experiments in d and e were repeated three times. defects in a fetus with HFH-RTRD, and complete absence of limbs and f, SUPERTOPFLASH assay in HEK293T-STF cells transfected with lungs in a fetus with TETAMS. gw, gestational weeks. c, RSPO2 genomic WNT3A and the indicated RSPO2 constructs. n = 3 biological replicates. (top) and protein (bottom) structures with identified mutations. Ex., exon. Data are mean ± s.e.m.
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