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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
Glinka, A. et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 12, 1055–1061 (2011).
Carmon, K. S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl Acad. Sci. USA 108, 11452–11457 (2011).
Niswander, L. Pattern formation: old models out on a limb. Nat. Rev. Genet. 4, 133–143 (2003).
Niemann, S. et al. Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family. Am. J. Hum. Genet. 74, 558–563 (2004).
Kazanskaya, O. et al. R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534 (2004).
Kim, K. A. et al. R-Spondin proteins: a novel link to β-catenin activation. Cell Cycle 5, 23–26 (2006).
Nam, J. S., Turcotte, T. J., Smith, P. F., Choi, S. & Yoon, J. K. Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257 (2006).
Ohkawara, B., Glinka, A. & Niehrs, C. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis. Dev. Cell 20, 303–314 (2011).
Zebisch, M. et al. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat. Commun. 4, 2787 (2013).
Bell, S. M. et al. R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis. Development 135, 1049–1058 (2008).
Aoki, M., Kiyonari, H., Nakamura, H. & Okamoto, H. R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development. Dev. Growth Differ. 50, 85–95 (2008).
Nam, J. S. et al. Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb. Dev. Biol. 311, 124–135 (2007).
Yamada, W. et al. Craniofacial malformation in R-spondin2 knockout mice. Biochem. Biophys. Res. Commun. 381, 453–458 (2009).
Brüchle, N. O. et al. RSPO4 is the major gene in autosomal-recessive anonychia and mutations cluster in the furin-like cysteine-rich domains of the Wnt signaling ligand R-spondin 4. J. Invest. Dermatol. 128, 791–796 (2008).
Başaran, S. et al. Tetra-amelia, lung hypo-/aplasia, cleft lip-palate, and heart defect: a new syndrome? Am. J. Med. Genet. 51, 77–80 (1994).
Sousa, S. B. et al. Tetra-amelia and lung hypo/aplasia syndrome: new case report and review. Am. J. Med. Genet. A. 146A, 2799–2803 (2008).
Xie, Y. et al. Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin. EMBO Rep. 14, 1120–1126 (2013).
Proffitt, K. D. et al. Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer. Cancer Res. 73, 502–507 (2013).
Mazerbourg, S. et al. Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality. Mol. Endocrinol. 18, 2241–2254 (2004).
Morita, H. et al. Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol. Cell. Biol. 24, 9736–9743 (2004).
Snippert, H. J. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010).
Styrkarsdottir, U. et al. Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits. Nature 497, 517–520 (2013).
Sulem, P. et al. Identification of a large set of rare complete human knockouts. Nat. Genet. 47, 448–452 (2015).
Koizumi, M. et al. Lgr4 controls specialization of female gonads in mice. Biol. Reprod. 93, 90 (2015).
Jin, Y. R., Turcotte, T. J., Crocker, A. L., Han, X. H. & Yoon, J. K. The canonical Wnt signaling activator, R-spondin2, regulates craniofacial patterning and morphogenesis within the branchial arch through ectodermal-mesenchymal interaction. Dev. Biol. 352, 1–13 (2011).
Aoki, M. et al. R-spondin3 is required for mouse placental development. Dev. Biol. 301, 218–226 (2007).
van de Glind, G. C. et al. RNA-seq analysis of Lgr6+ stem cells and identification of an Lgr6 isoform. Exp. Dermatol. https://doi.org/10.1111/exd.13453 (2017).
Lebensohn, A. M. & Rohatgi, R. R-spondins can potentiate WNT signaling without LGRs. eLife 7, e33126 (2018).
Simon, A. & Tanaka, E. M. Limb regeneration. Wiley Interdiscip. Rev. Dev. Biol. 2, 291–300 (2013).
Hao, H. X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195–200 (2012).
Koo, B. K. et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665–669 (2012).
Seshagiri, S. et al. Recurrent R-spondin fusions in colon cancer. Nature 488, 660–664 (2012).
Bond, C. E. et al. RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis. Oncotarget 7, 70589–70600 (2016).
Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608–611 (2009).
Wang, D. et al. Structural basis for R-spondin recognition by LGR4/5/6 receptors. Genes Dev. 27, 1339–1344 (2013).
Peng, W. C. et al. Structure of stem cell growth factor R-spondin 1 in complex with the ectodomain of its receptor LGR5. Cell Reports 3, 1885–1892 (2013).
Li, S. J. et al. Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function. Cell. Signal. 21, 916–925 (2009).
Krug, U., Patzschke, R., Zebisch, M., Balbach, J. & Sträter, N. Contribution of the two domains of E. coli 5′-nucleotidase to substrate specificity and catalysis. FEBS Lett. 587, 460–466 (2013).
Monsoro-Burq, A. H. A rapid protocol for whole-mount in situ hybridization on Xenopus embryos. CSH Protoc. 2007, pdb.prot4809 (2007).
Van Nieuwenhuysen, T. et al. TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis. Oncoscience 2, 555–566 (2015).
Naert, T., Van Nieuwenhuysen, T. & Vleminckx, K. TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models. Genesis 55, https://doi.org/10.1002/dvg.23005 (2017).
Moreno-Mateos, M. A. et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods 12, 982–988 (2015).
Boel, A. et al. BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment. Sci. Rep. 6, 30330 (2016).
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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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 b–e 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.
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.
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.
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.
This file contains gel source data for immunoblots presented in the main and extended data figures including molecular weight markers (kDa).
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
About this article
Cite this article
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
LGR4, Not LGR5, Enhances hPSC Hematopoiesis by Facilitating Mesoderm Induction via TGF-Beta Signaling Activation
Cell Reports (2020)
Frontiers in Endocrinology (2020)
R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth
Cell Death & Differentiation (2020)
R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors