The four R-spondin secreted ligands (RSPO1–RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1,2,3. 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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We are grateful to the families of affected fetuses whose cooperation made this study possible. We thank M. Krahn and D. Bertola for contributing more families with TETAMS for ongoing research. We thank C. Niehrs for the RSPO2-ΔC-AP-PCS2 + construct. We thank D. Virshup for HEK293T-STF cells. The TALEN plasmids were obtained from C. H. K. Cheng. We are grateful to our groups’ members for discussion and advice. E.S.-R. thanks M. Ravi for her patience and support, and is supported by a BMRC Young Investigator Grant (1613851035). S.S.N. is a recipient of Senior Research Fellowship from Indian Council of Medical Research (45/10/2015-HUM-BMS). N.B. is funded by A*STAR and an NRF Investigatorship. B.R. is a fellow of the Branco Weiss Foundation, an A*STAR Investigator, a NRF and AAA fellow and a Young EMBO Investigator. This work was partly funded by the Department of Science and Technology, India, for the project ‘Application of autozygosity mapping and exome sequencing to identify genetic basis of disorders of skeletal development’ (SB/SO/HS/005/2014); by the Research Foundation, Flanders (FWO-Vlaanderen) (grants G0A1515N and G029413N), the Concerted Research Actions from Ghent University (BOF15/GOA/01) and the Hercules Foundation, Flanders (grant AUGE/11/14), Belgium; by TUBITAK 108S412 and 112S398 (ERA-Net consortiums, CRANIRARE and CRANIRARE2), Turkey; and by a Strategic Positioning Fund on Genetic Orphan Diseases from A*STAR, Singapore.
Nature thanks C. Niehrs, C. Tabin and the other anonymous reviewer(s) for their contribution to the peer review of this work.