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

An Author Correction to this article was published on 05 July 2018

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Abstract

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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Fig. 1: Identification of RSPO2 mutations in fetuses presenting with severe limb defects.
Fig. 2: Mouse Lgr4/5/6 triple-knockout embryos do not recapitulate the Rspo2 and Rspo3 phenotypes.
Fig. 3: Exogenous and endogenous RSPO2/3 signal in Lgr4/5/6 triple-knockout mouse embryonic fibroblasts.
Fig. 4: Frogs mutant for both rnf43 and znrf3 display complete limb duplications.

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  • 05 July 2018

    In this Letter, the surname of author Lena Vlaminck was misspelled ‘Vlaeminck’. This error has been corrected online.

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Acknowledgements

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.

Reviewer information

Nature thanks C. Niehrs, C. Tabin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations

Authors

Contributions

The recruitment of families 1 and 2 and the initial discovery of RSPO2 mutations were made by the team of H.K. with the help of U.A. and Z.O.U. Further patient data and samples were collected by H.K. from S.B.d.S., S.S.N., A.S. and K.M.G., who provided TETAMS families 3 and 5. C.B., X.L. and C.L.C. independently identified a RSPO2 mutation in family 4. E.S.-R., M.L., K.V., N.S. and B.R. designed functional studies. N.B. provided single Lgr4, Lgr5 and Lgr6 knock-in mouse lines. E.S.-R., U.A., C.B., B.K., X.L., S.B., E.B.Y., C.L.C. and Z.O.U. performed whole-exome sequencing, chromosomal array analysis and sequencing analysis. E.S.-R., M.L., C.B.-L., M.K., H.T.T., T.N., R.N., A.H., N.S., T.T.T. and L.V. performed functional experiments. C.B.-L., M.K. and H.T.T. contributed equally to this work. E.S.-R., U.A., M.L., K.V., H.K. and B.R. wrote and revised the manuscript.

Corresponding authors

Correspondence to Kris Vleminckx, Nick Barker, Hülya Kayserili or Bruno Reversade.

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Extended data figures and tables

Extended Data Fig. 1 Pictures of affected fetuses, exome sequencing analysis in family 1 and genotyping analysis in family 3.

a, Pictures and radiographs of indicated fetal cases illustrating severe limb defects in fetuses with HFH-RTRD, and the complete absence of limbs in fetuses with TETAMS. b, Summary of exome sequencing analysis for family 1, revealing a single biallelic missense mutation in the RSPO2 gene. c, Summary of genotyping in family 3 by SNP-array and array-CGH analysis, revealing a homozygous deletion including exon 6 of RSPO2.

Extended Data Fig. 2 The RSPO2(R69C) and RSPO2(Q70X) mutants fail to bind ZNRF3.

a, The RSPO2 R69 residue (highlighted in purple) is highly conserved in vertebrates and within the human paralogues RSPO1–RSPO4. Conserved cysteine residues of the Furin-like 1 domain are highlighted in pink. Protein alignment performed with ClustalO. b, Western blotting of protein extracts and supernatants from HEK293T cells transfected with indicated constructs. Deletion of the RSPO2 C-terminal domain (RSPO2-ΔC) decreases its retention on the cell surface without affecting its receptor binding and WNT enhancement properties38. The RSPO2(R69C) mutant was almost undetectable in conditioned media but was greatly increased by the addition of heparin in the medium. c, Co-immunoprecipitation of wild-type and mutant forms of RSPO2-ΔC-AP with the ProteinG-Flag beads only. Asterisk indicates an unspecific band. d, Co-immunoprecipitation of wild-type and mutant forms of RSPO2-ΔC-AP with the ZNRF3-ECD-Flag E3 ligase. Asterisk indicates an unspecific band. e, Cell-surface binding assay of HEK293T cells transfected with empty vector, LGR5 or RNF43, using equivalent amounts of RSPO2-ΔC-AP conditioned media (western blot). Experiments in be were repeated three times. f, SUPERTOPFLASH assay in HEK293T-STF cells transfected with WNT3A in the presence of equivalent amounts of RSPO2-ΔC-AP conditioned media (western blot). n = 4 biological replicates. Data are mean ± s.e.m. NS, not significant. **P < 0.01, one-way ANOVA test with Bonferroni’s correction. For gel source data, see Supplementary Fig. 1

Source Data.

Extended Data Fig. 3 Mouse Lgr4/5/6 knock-in embryos do not recapitulate the Rspo2 and Rspo3 phenotypes.

a, Table indicating the proportion and numbers of analysed embryos. The different genotypes were obtained in Mendelian ratio (P > 0.05, χ2 test). b, Normal limbs of embryos with indicated genotypes including a Lgr4/5/6 triple-knockout embryo. All obtained embryos (see a) had a similiar phenotype. FL, forelimbs; HL, hindlimbs; L, left; R, right. Scale bar, 1 mm. c, Normal lungs of embryos with indicated genotypes including a Lgr4/5/6 triple-knockout embryo. Comparable lung length relative to body length is indicated. Scale bar, 1 mm. All obtained embryos (see a) had a similiar phenotype. d, H&E staining of coronal sections of the heads through the oral cavity. Scale bar, 1 mm. Close-ups of the palatal shelves illustrate the cleft palate present in the Lgr5/6 double-knockout (n = 2) and Lgr4/5/6 triple-knockout (n = 2) embryos (indicated by black arrow heads). e, Properly vascularized placenta of a Lgr4/5/6 triple-knockout embryo compared to a Lgr6-knockout embryo. All obtained embryos (see a) had a similiar phenotype. Scale bar, 1 mm.

Source Data.

Extended Data Fig. 4 Mouse Lgr4, Lgr5 and Lgr6 knock-in embryos recapitulate the knockout phenotypes.

a, Illustration of the GFP knock-in in exon 1 of the Lgr4, Lgr5 and Lgr6 genes, which cause loss-of-function mutations. b, Liver weight in single Lgr4−/− (n = 2) and triple Lgr4/5/6−/− (n = 2) compared to wild-type (n = 3) E14.5 embryos. Data are mean ± s.e.m. NS, not significant. *P < 0.05, one-way ANOVA test with Bonferroni’s correction. c, Lgr4−/− embryos (n = 4) have a smaller liver. Liver weight is indicated. Scale bar, 1 mm. d, Lgr4−/− embryos (n = 2) show female-to-male sex reversal. Blue arrowheads point to male-specific coelomic vessels. Genetic genders (XY or XX) are indicated. Scale bar, 0.1 mm. e, H&E staining of coronal sections of the heads through the oral cavity. Scale bar, 1 mm. Close-ups of the tongue illustrate the ankyloglossia phenotype (tongue attached to the mouth floor, black arrow heads) in Lgr5−/− embryos (n = 4), whereas the tongue is detached for other genotypes (white arrowheads).

Source Data.

Extended Data Fig. 5 Mouse Lgr4, Lgr5 and Lgr6 knock-in cause loss-of-function mutations.

a, b, qPCR analyses for Lgr4, Lgr5 and Lgr6 expression in E14.5 limbs (a) and lungs (b) of wild-type, heterozygous and homozygous mutant embryos for the respective genes. c, d, qPCR analyses for Lgr4 and Lgr5 in embryonic intestine (c) and embryonic liver (d) of embryos with indicated genotype. e, qPCR analyses for Lgr6 in embryonic and adult skin of wild-type versus homozygous animals. n indicates number of embryos. Data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test with Bonferroni’s correction or two-tailed unpaired t-test with Welch’s correction when less than three groups were compared (for Lgr6 qPCR analysis). f, Duplex RNAscope for the indicated gene (blue) and Rspo2 (pink) in transverse sections of wild-type E14.5 lungs. Strongly expressed genes are denoted in bold (summary on the right). Scale bars, 0.2 mm. Experiment repeated with three different wild-type embryos.

Source Data.

Extended Data Fig. 6 Expression analyses in cells derived from mutant embryos.

a, qPCR analyses for Lgr4, Lgr5 and Lgr6 in NPCs derived from embryos of indicated genotypes. n = 4 biological replicates. b, qPCR analyses for Lgr4 and Lgr6 in iPS cells derived from NPCs of indicated genotypes. n = 3 biological replicates. c, qPCR analyses for Lgr4 and Lgr5 in SV40-immortalized dermal fibroblasts derived from embryos of indicated genotypes. n = 3 biological replicates. d, qPCR analyses for Rspo2, Rspo3 and Znrf3 in Lgr4/5/6 triple-knockout SV40-immortalized fibroblasts, transfected with indicated siRNAs. n = 3 biological replicates. Data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test with Bonferroni’s correction.

Extended Data Fig. 7 Extreme phenotypes of Xenopus tropicalis mutant tadpoles.

a, Control and rspo2 CRISPR–Cas9-injected tadpole showing a complete tetra-amelia phenotype probably due to incomplete cleavage at the time of injection and leakage of the CRISPR–Cas9 between the two blastomeres (n = 3 froglets). Scale bar, 1 cm. b, Alizarin red and alcian blue staining of a double-mutant rnf43/znrf3 TALEN-injected tadpole showing complete mirror-image diplopodia of both hindlimbs with 10 digits each (n = 4 froglets). Scale bar, 0.2 cm.

Extended Data Table 1 Clinical characteristics of affected individuals with biallelic RSPO2 mutations
Extended Data Table 2 List of primers

Supplementary information

Supplementary Figure 1

This file contains gel source data for immunoblots presented in the main and extended data figures including molecular weight markers (kDa).

Reporting Summary

Supplementary Table 1 (Related to Fig. 4)

This file contains an overview of the cutting efficiencies of the CRISPR gRNAs and TALENs as determined by PCR amplification and sequencing (MiSeq) of the targeted regions

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Szenker-Ravi, E., Altunoglu, U., Leushacke, M. et al. RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6. Nature 557, 564–569 (2018). https://doi.org/10.1038/s41586-018-0118-y

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