RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6

Nature. 2018 May;557(7706):564-569. doi: 10.1038/s41586-018-0118-y.

Emmanuelle Szenker-Ravi ^# ¹, Umut Altunoglu ^# ², Marc Leushacke ^# ¹, Célia Bosso-Lefèvre ¹ ³, Muznah Khatoo ¹, Hong Thi Tran ⁴, Thomas Naert ⁴, Rivka Noelanders ⁴, Amin Hajamohideen ¹, Claire Beneteau ⁵, Sergio B de Sousa ⁶ ⁷, Birsen Karaman ², Xenia Latypova ⁵, Seher Başaran ², Esra Börklü Yücel ⁸, Thong Teck Tan ¹, Lena Vlaminck ⁴ ⁹, Shalini S Nayak ¹⁰, Anju Shukla ¹⁰, Katta Mohan Girisha ¹⁰, Cédric Le Caignec ⁵ ¹¹, Natalia Soshnikova ¹², Zehra Oya Uyguner ², Kris Vleminckx ¹³ ¹⁴, Nick Barker ¹⁵ ¹⁶ ¹⁷, Hülya Kayserili ¹⁸ ¹⁹, Bruno Reversade ²⁰ ²¹ ²² ²³ ²⁴

Affiliations

1 Institute of Medical Biology, A*STAR, Singapore, Singapore.
2 Medical Genetics Department, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey.
3 Department of Paediatrics, National University of Singapore, Singapore, Singapore.
4 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
5 CHU Nantes, Service de Génétique Médicale, Nantes, France.
6 Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.
7 University Clinic of Genetics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
8 Medical Genetics Department, Koç University School of Medicine (KUSOM), Istanbul, Turkey.
9 Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
10 Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, India.
11 INSERM, UMR1238, Bone Sarcoma and Remodeling of Calcified Tissue, Université Bretagne Loire, Nantes, France.
12 Institute of Molecular Biology (IMB) gGmbH, Mainz, Germany.
13 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. [email protected].
14 Center for Medical Genetics, Ghent University, Ghent, Belgium. [email protected].
15 Institute of Medical Biology, A*STAR, Singapore, Singapore. [email protected].
16 Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan. [email protected].
17 Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK. [email protected].
18 Medical Genetics Department, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey. [email protected].
19 Medical Genetics Department, Koç University School of Medicine (KUSOM), Istanbul, Turkey. [email protected].
20 Institute of Medical Biology, A*STAR, Singapore, Singapore. [email protected].
21 Department of Paediatrics, National University of Singapore, Singapore, Singapore. [email protected].
22 Medical Genetics Department, Koç University School of Medicine (KUSOM), Istanbul, Turkey. [email protected].
23 Institute of Molecular and Cellular Biology, A*STAR, Singapore, Singapore. [email protected].
24 Reproductive Biology Laboratory, Academic Medical Center (AMC), Amsterdam-Zuidoost, The Netherlands. [email protected].
# Contributed equally.

PMID: 29769720 DOI: 10.1038/s41586-018-0118-y

Abstract

The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify Wnt signalling^1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced Wnt potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect Wnt responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.

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