Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues1. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors2, 3, 4. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
At a glance
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: De novo design and engineering of B12. (584 KB)
a, Design strategy of a FZD8 CRD-specific binding domain. b, Designed binding of B12 (orange) to the FZD8 CRD (blue), with XWnt8 and Wnt lipid (purple) modelled onto the structure to highlight competitive binding modes. Residues outside of the ‘lipid groove helix’ were designed to make FZD8-specific contacts to promote specificity. c, Comparison between the designed (left) and observed (right) conformation. d, Affinity maturation of parental B12 by yeast cell surface display identified enriched point mutations that were assembled in a degenerate library and selected to yield the final, optimized B12.
- Extended Data Figure 2: FZD CRD binding characterization of B12 and scFv. (327 KB)
a, Binding specificity of the B12 and FZD CRD interaction, as determined by yeast cell surface titration. B12 was displayed on yeast and binding of monomeric FZD CRDs fluorescently labelled with streptavidin-Alexa647 was detected by flow cytometry. Error bars represent s.d. of n = 3 technical replicates from 1 of 2 representative experiments. b, c, Binding affinity of the B12–FZD5 CRD (b) and B12–FZD8 CRD (c) interaction, as determined by surface plasmon resonance. FZD5 and FZD8 CRDs were immobilized on a streptavidin chip, and B12 was flown through as analyte. d, Inhibition of XWnt8 induced signalling in A549 cells, as measured by BAR luciferase reporter assay. Error bars represent s.d. of n = 3 technical replicates from 1 of 2 representative experiments. e–h, Binding affinity of the scFv–DKK1c–FZD1 CRD (e). scFv–DKK1c–FZD5 CRD (f), scFv–DKK1c–FZD7 CRD (g) and scFv–DKK1c–FZD8 CRD (h) interaction, as determined by surface plasmon resonance. FZD CRDs were immobilized on a streptavidin chip, and scFv–DKK1c was flown through as analyte.
- Extended Data Figure 3: Co-locomotion analysis of LRP6 and FZD8 induced by Wnt protein and surrogate. (321 KB)
a, Dimer/oligo-merization of LRP6 and FZD8 quantified by co-locomotion of both receptors under different conditions. The negative control (black) is the dimerization of DY649-labelled FZD8 and a TMR-labelled model transmembrane protein, HaloTag with maltose-binding protein linked to an artificial transmembrane domain, co-expressed in HeLa cells. The addition of 2 μM IWP-2 for 20 h reduced the receptor dimerization to the background level of the negative control (inset). Receptor dimerization induced by XWnt8 was used as a positive control (blue). Box plot represents measurements of more than 18 individual cells for each condition. **P < 0.01, ***P < 0.001, t-test. b, Diffusion coefficients of LRP6 and FZD8 in the absence or presence of 100 nM Wnt surrogates and control Wnt proteins. Error bars represent s.d. from more than 25 individual cells. c, Co-locomoting LRP6 and FZD8 measured within 30 min of the addition of 100 nM scFv–DKK1c, B12–DKK1c, XWnt8 and WNT3A, or without treatment. Box plot represents measurements of individual cells for each condition (**P < 0.01, t-test). scFv–DKK1c (n = 40), B12–DKK1c (n = 32), XWnt8 (n = 27), WNT3A (n = 25), and untreated (n = 28). d–i, Dimer/oligo-merization of LRP6 and FZD8 as a function of time after the addition of 100 nM scFv–DKK1c (d), B12–DKK1c (e), XWnt8 (f) and WNT3A (g). More than 12 individual cells were evaluated for each condition. h, Time course control of untreated cell. i, Summarized representation of time course of LRP6–FZD8 complex formation. j, Kinetics of β-catenin accumulation in K562 cells after stimulation with 10 nM scFv–DKK1c, recombinant WNT3A, B12, or basal conditions only (complete growth medium). Error bars represent s.d. of n = 3 technical replicates from 1 of 2 representative experiments.
- Extended Data Figure 4: Single-molecule trajectories and step length analysis of LRP6 and FZD8 under different conditions. (603 KB)
a–e, Single-molecule trajectories obtained for LRP6 and FZD8 in the plasma membrane of representative HeLa cells under different conditions. Trajectories were obtained by single-molecule tracking of the dye-labelled LRP6 (blue) and FZD8 (red) in the dual-colour time-lapse single-molecule images within 150 frames. Fast diffusion results into spread-out trajectories (prominent in a), while slow diffusion leads to dot-like trajectories (prominent in e). f–j, Step-length histogram analyses for determining diffusion coefficients of LRP6 and FZD8, shown for representative individual cells under different conditions. Step lengths for a time lapse of three frames (96 ms) were calculated from trajectories shown in a–e. According to equation (1), a two-component model comprising a slow (blue) and a fast (green) fraction was used for fitting the histograms. Inset: diffusion coefficient and the corresponding fraction in percentage (in brackets).
- Extended Data Figure 5: Single-molecule intensity analysis of LRP6 and FZD8 under different conditions. (1,055 KB)
Single-molecule intensity analysis for quantifying LRP6 (a–f) and FZD8 (g–l) oligomerization in representative cells under different conditions. Raw images after treatment with scFv–DKK1c (b, h), B12–DKK1c (c, i), XWnt8 (d, j), WNT3A (e, k) and without treatment (a, g). On the basis of their intensities, individual diffraction-limited spots in the raw images were classified as monomers (blue circle), dimers (green circle), trimers (yellow circle) and higher oligomers (red circle), respectively. f, l, m, Different oligomer fractions summarized as the ratio of the classified species number to the total number of detected diffraction-limited spots. More than 7,200 single complex intensities were examined for each condition.
- Extended Data Figure 6: FZD-specific activation of canonical Wnt signalling by Wnt surrogates. (357 KB)
a–d, Activation of Wnt pathway by decreasing concentration of scFv–DKK1c, B12–DKK1c, XWnt8 or negative control proteins B12, DKK1, IL-2, IL-4 and EPO, as assayed by the BAR and STF reporters in L cells (50–3 nM) (a), A375 cells (250–15 nM) (b), SH-SY5Y cells (250–15 nM) (c) and A549 cells (100–1 nM) (d). Error bars represent s.d. of n = 3 technical replicates from 1 of 2 representative experiments. The relative quantities of human FZD mRNA in the relative cell lines determined by qRT–PCR are shown as insets, error bars represent s.d. of n = 3 technical replicates. e, f, Selective inhibition of B12–DKK1c and scFv–DKK1c activity in A549 cells by B12, DKK1, FZD1 CRD–Fc and FZD8 CRD–Fc, as assayed by the BAR reporter, correlates with binding specificity. Error bars represent s.d. of n = 3 technical replicates. g, Immunoblot analysis of LRP6 phosphorylation and β-catenin accumulation in A375 BAR cells treated with scFv–DKK1c and human SIRPα (0.1, 10, 50 nM), WNT3A conditioned media and mock conditioned media (30% and 50%). Data shown from 1 of 2 representative experiments. h, AXIN2 transcription relative to GAPDH in SH-SY5Y BAR and A375 BAR cells treated with 50 nM scFv–DKK1c, B12 (negative control), and 30% WNT3A conditioned media, as analysed by qRT–PCR. Error bars represent the s.d. of biological triplicates performed in technical triplicates (n = 9). i, AXIN2 transcription relative to GAPDH in A549 BAR cells treated with 50 nM B12–DKK1c variants and XWnt8, as analysed by qRT–PCR. Error bars represent s.d. of biological triplicates performed in technical triplicates (n = 9). j, Activation of Wnt signalling with distinct amplitudes by XWnt8 and B12–DKK1c variants, as assayed by the BAR reporter in A549 cells. Error bars represent s.d. of n = 3 technical replicates from 1 of 3 representative experiments.
- Extended Data Figure 7: Wnt surrogates enhance upregulation of alkaline phosphatase in MSCs. (327 KB)
Upregulation of alkaline phosphatase (ALP) assessed by the cell surface enzymatic activity with the ALP substrate NBT/BCIP (a), and qRT–PCR of mRNA levels (b) in C3H10T1/2 cells treated for 4 days with increasing concentration of WNT3A conditioned media, scFv–DKK1c (0.5, 5, 50 nM) and scFv–DKK1c–RSPO2 (0.5, 5, 50 nM) in osteogenic media. c, Induction of ALP in C3H10T1/2 cells treated with WNT3A conditioned media, 50 nM scFv–DKK1c and 50 nM scFv–DKK1c–RSPO2 in the presence of 200 ng ml−1 BMP2 in osteogenic media for 4 days, assayed by cell-surface ALP enzymatic activity with the ALP substrate NBT/BCIP. d, Relative changes of Col2a1 mRNA levels, an early marker of chondrogenesis, in C3H10T1/2 cells treated with WNT3A conditioned media, scFv–DKK1c and scFv–DKK1c–RSPO2 in the presence of BMP2 in osteogenic media for 4 days. e, Upregulation of ALP cell surface enzymatic activity assessed with the ALP substrate NBT/BCIP in C3H10T1/2, mouse primary MSCs, and human primary MSCs treated for 3 days with WNT3A conditioned media, 50 nM scFv–DKK1c, and 50 nM scFv–DKK1c–RSPO2 in osteogenic media in the presence and absence of 200 ng ml−1 BMP2. f–h, Pixel quantification of images in e using ImageJ. Error bars represent s.d. of n = 3 technical replicates from 1 of 3 (C3H10T1/2 and human MSCs) and 1 (mouse MSCs) representative experiments.
- Extended Data Figure 8: Activity of Wnt surrogates on human organoid cultures in vitro. (428 KB)
a, b, Representative bright-field images of organoids of human pancreas, stomach (corpus), and liver expanded for 12 days in the absence (a) or presence (b) of 3 μM IWP-2 in basal media (as detailed in Supplementary Table 2), supplemented with 50% WNT3A conditioned media, 2% RSPO3 conditioned media, 200 nM scFv–DKK1c and 200 nM scFv–DKK1c–RSPO2, or combinations thereof as indicated. c–e, Quantification of cell proliferation by luminescence. Bars represent mean of n = 2 technical replicates from 1 of 3 (colon), 2 (liver and pancreas) and 1 (stomach) representative experiments. f, Representative bright-field images of colon organoids from a patient with cystic fibrosis with characteristic budded morphology owing to mutations in the ion channel CFTR, grown in expansion media with 50% WNT3A conditioned media, 2% RSPO3 conditioned media, 100 and 1,000 nM scFv–DKK1c and 100 and 1,000 nM scFv–DKK1c–RSPO2, or combinations thereof as indicated.
- Extended Data Figure 9: Wnt surrogate activate Wnt signalling in vivo and alter Wnt-driven hepatic zonation. (836 KB)
a–d, qRT–PCR validation of adenoviral transgene expression in mouse livers 7 days after intravenous injection with Ad-Wnt3a, Ad-scFv–DKK1c, Ad-RSPO2–Fc or Ad-scFv–DKK1c–RSPO2 from the same experiment as in g. n = 4 mice per condition. e, Schematic representation of Ad-scFv–DKK1c and Ad-scFv-DKK1c-RSPO2 constructs for adenoviral transgene expression. f, Detection of scFv–DKK1c and scFv–DKK1c–RSPO2 in mice sera by western blot at the indicated days after adenovirus injection. g, Images from biological replicate mice depicting effects of adenoviral expression of various Wnt agonists on liver zonation from Fig. 4a, b. Mice (n = 4 per condition) received single intravenous injection of adenovirus expressing Fc (2.5 × 108 p.f.u.), scFv–DKK1c (1.2 × 107 p.f.u.), WNT3A (1.2 × 107 p.f.u.), RSPO2–Fc (2.5 × 108 p.f.u.), scFv–DKK1c–RSPO2 (1.2 × 107 p.f.u.), or combinations as indicated. In all cases, the total virus dose was increased to 2.5 × 108 p.f.u. by Ad-Fc as filler. After 7 days, liver was analysed for glutamine synthetase expression by immunofluorescence. Anti-CK19 immunofluorescence was performed to mark portal areas. Each panel is a representative image from a different mouse. h, i, qRT–PCR analysis of periportal marker Cyp2f2 (h) and Wnt target gene Axin2 (i) in livers from mice that received adenoviruses expressing the mouse IgG2α Fc fragment, mouse WNT3A, scFv–DKK1c, human RSPO2–Fc, WNT3A plus RSPO2–Fc, scFv–DKK1c plus RSPO2–Fc or scFv–DKK1c–RSPO2, 7 days after adenovirus injection (n = 4 mice per group). Error bars indicate s.e.m. of biological replicates.
- Extended Data Figure 10: Wnt surrogate enhance hepatocyte proliferation. (507 KB)
a, Scheme depicting parabiosis experiment design. Age-matched and gender-matched mice were selected and housed together. The donors received intravenous adenovirus injection of (1) Ad-Fc, (2) Ad-scFv–DKK1c-His plus Ad-Fc or (3) Ad-scFv–DKK1c–RSPO2 plus Ad-Fc with liver infection of the donors. Two days later, parabiosis surgery was performed to surgically pair the adenovirus injected donor mice with the recipient mice that did not receive adenovirus injection. Ad-Fc was deliberately added to all treatment groups as a tracer to monitor the successful establishment of cross-circulation (see b). b–d, Detection of scFv–DKK1c (b), scFv–DKK1c–RSPO2 (c) or the tracer Fc (d) in the serum of recipients and donor mice 5 days after parabiosis by western blotting. e, f, Liver qRT–PCR validation of adenoviral transgene expression from donor and recipient mice at 7 days after surgery (9 days after adenoviral injection) for scFv–DKK1c or scFv–DKK1c–RSPO2. Note the absence of transgene expression in the recipient livers. g–i, Analysis of livers by qRT–PCR for Glul, Cyp2f2 and Axin2 mRNA with induction of Glul and Axin2 and repression of Cyp2f2 in both donors and recipients. j, Liver weight of recipients 7 days after parabiosis surgery. n = 3 mice per condition. Error bars indicate s.e.m. of biological replicates.
- Video 1: Co-locomoting Lrp6/Fzd8 measured without treatment (666 KB, Download)
- Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-colour single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell without treatment. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.
- Video 2: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of scFv-DKK1c (654 KB, Download)
- Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of scFV-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of scFV-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.
- Video 3: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of B12-DKK1c (664 KB, Download)
- Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of B12-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.
- Video 4: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of XWnt8 (612 KB, Download)
- Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of XWnt8. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.
- Video 5: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of Wnt3a (659 KB, Download)
- Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of Wnt3a. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.
- Supplementary Information (414 KB)
This file contains Supplementary Tables 1-2 and Supplementary Figure 1, the uncropped western blots.