R-spondins are BMP receptor antagonists in early embryonic development

BMP signalling plays key roles in development, stem cells, adult tissue homeostasis, and disease. How BMP receptors are extracellularly modulated and in which physiological context, is therefore of prime importance. R-spondins (RSPOs) are a small family of secreted proteins that co-activate WNT signalling and function as potent stem cell effectors and oncogenes. Evidence is mounting that RSPOs act WNT-independently but how and in which physiological processes remains enigmatic. Here we show that RSPO2 and RSPO3 also act as BMP antagonists. RSPO2 is a high affinity ligand for the type I BMP receptor BMPR1A/ALK3, and it engages ZNRF3 to trigger internalization and degradation of BMPR1A. In early Xenopus embryos, Rspo2 is a negative feedback inhibitor in the BMP4 synexpression group and regulates dorsoventral axis formation. We conclude that R-Spondins are bifunctional ligands, which activate WNT- and inhibit BMP signalling via ZNRF3, with implications for development and cancer.


INTRODUCTION
coupled receptor 5 (LGR5), and two related proteins, LGR4 and LGR6, leading to the

Rspo2 is a negative feedback regulator in the Xenopus BMP4 synexpression group
In early vertebrate embryos, genes belonging to certain signaling networks form characteristic 112 synexpression groups, i.e. genetic modules composed of genes that show tight spatio-temporal 113 RNA coexpression and that function in the respective signaling pathway 40 . A well-characterized 114 example is the BMP4 synexpression group, members of which are expressed like this growth 115 factor-dorsally in the eye, heart and proctodeum of tailbud stage Xenopus embryos (Fig. 3a). 116 This group consists of at least eight members, which all encode positive or negative feedback 117 components of the BMP signaling cascade as studied in early development, including ligands, 118 receptors and downstream components of the pathway 41 . Interestingly, we found that rspo2 is 119 part of the BMP4 synexpression group, being coexpressed with bmp4 from gastrula to tadpole 120 stages (Fig. 3a), suggesting that its expression depends on BMP signaling as for other 121 synexpressed genes. To test this idea, we employed Xenopus animal cap explants, which express 122 low levels of rspo2 and bmp4 to monitor rspo2 induction upon bmp4 overexpression (Fig. 3b). 123 Indeed, bmp4 induced rspo2 expression by qRT-PCR (Fig. 3c) and in situ hybridization ( Fig. 3d-124 e), similar to bmp4 direct targets sizzled (Fig. 3c-e) and vent1 (Fig. 3c). To test whether rspo2 is 125 an immediate early target of BMP4, we blocked protein synthesis with cycloheximide (CHX) 41 . 126 Interestingly, while induction of the direct BMP4 targets sizzled and vent1 by bmp4 was 127 unaffected by CHX, rspo2 induction was inhibited (Fig. 3b-e). We conclude that rspo2 is a 128 negative feedback inhibitor within the BMP4 synexpression group and that it is an indirect BMP 129 target gene, whose expression may depend on transcription factors of the e.g. Vent or Msx 130 families 41,42 (Fig. 3f). 133 Given that RSPOs act by promoting receptor endocytosis 14,17 , we postulated that RSPO2 might 134 regulate BMP signaling through its receptors: ACVR1, BMPR1A and BMPR1B. To test this 135 hypothesis, we analyzed the effect of RSPO1-4 treatment on BMP signaling induced by 136 constitutively active ACVR1/BMPR1A/BMPR1B (ACVR1/BMPR1A/BMPR1B QD ). 137 Interestingly, RSPO2 and -3 treatment specifically inhibited BMPR1A QD but not ACVR1 QD or 138 BMPR1B QD , while RSPO1 and -4 had no effect to any of the constitutively active receptors (Fig.   139 4a-c). 140 Indeed, cell surface binding assay and in vitro binding assay revealed that RSPO2 and -3, but not  Fig. 5a). RSPO2 showed high affinity with BMPR1A ECD (Kd ≈ 4.8 nM) (Fig. 4f), comparable 143 to the RSPO-LGR interaction 24 . To further delineate the domains required for BMPR1A 144 binding, we analyzed deletion mutants of RSPO2 in cell surface binding assays with BMPR1A 145 ECD, and found BMPR1A binding required the TSP1-but not the FU domains of RSPO2, while, 146 conversely, LGR binding required the FU domains but not TSP1 ( Supplementary Fig. 5b- Supplementary Fig. 5f). However, unlike wild-type 157 RSPO1, R1-TSP R2 bound to BMPR1A (Supplementary Fig. 5f) and antagonized BMP signaling, 158 mimicking the effects of RSPO2 (Fig. 4k).

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The importance of the TSP1 domain in BMP inhibition was further corroborated in Xenopus, 160 where we took advantage of the fact that the TSP1-domain is encoded by a distinct exon in the 161 3'-end of the rspo2 gene. We generated a rspo2 Mo (rspo2 ∆TSP Mo), which specifically abolished 162 TSP1-domain splicing, yielding 3' truncated rspo2 mRNA lacking the TSP1 domain but 163 retaining the FU domains (Fig. 5a). Microinjection of rspo2 ∆TSP Mo resulted in ventralized 164 tadpoles with shorter axis and reduced heads compared to control tadpoles, which was partially 165 rescued by introducing a non-targeted rspo2 mRNA (Supplementary Fig. 6a-b). rspo2 ∆TSP

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RSPO2 ΔFU1 lost ZNRF3 binding ( Supplementary Fig. 8b), yet it bound LGR4 (Supplementary 214 Fig. 8c), but did not inhibit BMP4 signaling (Fig. 7f). Conversely, RSPO2 ΔFU2 bound ZNRF3 but 215 not to LGR4 ( Supplementary Fig. 8b-c), yet it still antagonized BMP4 signaling (Fig. 7g)    Taken together, our results support a model ( Supplementary Fig. 10d) wherein RSPO2 bridges 252 ZNRF3 and BMPR1A and routes the ternary complex towards clathrin-mediated endocytosis for 253 lysosomal degradation, thereby antagonizing BMP signaling. We suggest that a similar 254 mechanism applies to RSPO3 but not RSPO1 and -4. TGFβ family members, whose unproductive binding to type II receptors prevents signal 275 transmission. Relatedly, the BMP antagonist BAMBI is a BMP pseudoreceptor lacking kinase 276 activity, which also leads to formation of a dead-end complex with BMP receptors 44 . In contrast, 277 RSPO2 and -3 share no sequence homology with TGFβ family members, they inhibit type I 278 instead of type II BMP receptors, and they do so by a novel mechanism, which engages the 279 ZNRF3 E3 transmembrane ubiquitin ligase to internalize BMPR1A. RSPO2 thereby routes 280 BMPR1A to clathrin-mediated endocytosis for lysosomal degradation. This mode of action resembles the function of the Spastic Paraplegia related gene NIPA1, a transmembrane 282 antagonist, which promotes BMP receptor type II endocytosis and lysosomal degradation 45 .

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However, we found that RSPO2 specifically binds to BMPR1A but not to ACVR1 or BMPR1B 285 (data not shown), which explains why ACVR1 and BMPR1B signaling were not antagonized by 286 RSPO2 in human cells (Fig. 4a-c) Spemann organizer genes in Xenopus embryos, which is characteristic not only for WNT but 296 also BMP inhibition 43 . In WNT signalling, the role of RSPO2 is to protect WNT receptors from 297 ubiquitination and internalisation by ZNRF3, by forming a ternary complex with LGR4-6 and 298 triggering endocytosis. In contrast, during BMP signalling, RSPO2 directly forms a ternary 299 complex with ZNRF3-BMPR1A to internalize and degrade the type I receptor. Our data also 300 imply a possible function of RNF43 in antagonizing BMP signalling, inviting a closer inspection 301 of its loss-of-function phenotypes 25,26 .
A number of studies emphasized the importance of the Furin domains in RSPOs, which are 303 necessary and sufficient for activation of WNT signaling 17,20,26,28 , however, the role of the TSP1 304 domain has received less attention. We found that the specificity of RSPOs for BMP signaling is 305 dictated by the TSP1 domain, which binds directly to BMPR1A. Unlike RSPO2 and -3, RSPO1   to Renilla. TOPFlash luciferase assays were carried out as previously described 62 . Data are 412 displayed as average of biological replicates with SD. Statistical analyses were made with the 413 PRISM7 software using unpaired t-test or one-way ANOVA test. Not significant (ns) P > 0.05, 414 *P < 0.05 **P < 0.01, ***P < 0.001, and ****P < 0.0001.   Table 4) 64 , or used as previously described 27   Xenopus tropicalis T7 Endonuclease I assay 505 To validate CRISPR/Cas9-mediated genome editing, three embryos of each injection set were 506 lysed at stage 30 for genotyping PCR reactions as described 64 (Supplementary Table 4). All (1:500) were applied for 2 hours at room temperature. Tyramide Signal Amplification for 545 detecting RSPO-HRP was carried out as previously described 24,39 . Quantification was executed 546 using ImageJ. Dot plots show average and SD from every cells analyzed with unpaired t-test.

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For X.laevis embryos, bmpr1a-EYFP and membrane-RFP mRNAs were coinjected with the 548 indicated mRNAs or Mos. Embryos were dissected for animal or ventrolateral explants at stage 549 9 or stage 11.5, respectively. Explants were immediately fixed with 4% PFA for 2 hours and 550 mounted with Fluoromount-G (ThermoFisher 00495802). Images were obtained using LSM 700 551 (Zeiss). Data are representative images from two independent experiments. For quantification, 552 Pearson's correlation coefficient for EYFP and RFP was analyzed using 16-30 random areas 553 harboring 10 cells chosen from 6-10 embryos per each set. Dot plots show an average and SD 554 from every plane analyzed with unpaired t-test.

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Cell surface binding assay 556 Cell surface binding assays were carried out as previously described 39 with few modifications.

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In brief, human BMPR1A-HA and Xenopus tropicalis LGR4 DNA were transfected in HEK293T cells, and incubated with 1.5 U ml -1 conditioned media for 3 hours on ice. After 559 several washes and crosslinking, cells were treated with 2 mM Levamisole for 20 min to 560 inactivate endogenous AP activities and developed with BM-Purple (Sigma 11442074001). Cells 561 were mounted with Fluoromount G. Images were obtained using LEICA DMIL 562 microscope/Canon DS126311 camera.

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Reverse transcription and PCR amplification were performed as described before 62 . For 567 Xenopus laevis, animal cap explants were harvested at stage 10 and qRT-PCR was executed as 568 previously described 43 . Primers used in this study are listed in Supplementary Table 1

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The Authors declare no competing interests.