Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling

Journal name:
Nature
Volume:
545,
Pages:
234–237
Date published:
DOI:
doi:10.1038/nature22306
Received
Accepted
Published online

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

Figures

  1. Engineering of FZD-specific and cross-reactive LRP6–FZD heterodimerizers.
    Figure 1: Engineering of FZD-specific and cross-reactive LRP6–FZD heterodimerizers.

    a, Concept of Wnt surrogate-induced heterodimerization of FZD and LRP5 or LRP6. b, Binding affinities and specificities of B12 and scFv, determined by surface plasmon resonance (superscript 1), and yeast surface binding titration (superscript 2). nb, no binding. Kd, dissociation constant. c, Crystal structure of B12 (green) bound to FZD8 CRD (blue), with modelled binding of XWnt8 and Wnt lipid (purple). Zoomed-in view of the B12–FZD8 CRD binding interface, with side chains mediating critical contacts shown as sticks. Residues that substitute for Trp73 in different FZD proteins are listed. Crystallographic data and refinement statistics are summarized in Supplementary Table 1. d, e, Schematic representation (d) and typical single-molecule fluorescence images (e) for FZD8 (red) and LRP6 (blue) co-locomotion (magenta) mediated by scFv–DKK1c, B12–DKK1c and WNT3A in the plasma membrane of HeLa cells. Scale bars, 2 μm.

  2. FZD-specific activation of canonical Wnt signalling by Wnt surrogates.
    Figure 2: FZD-specific activation of canonical Wnt signalling by Wnt surrogates.

    a, b, Activation of the β-catenin-dependent STF luciferase reporter by Wnt surrogates (scFv–DKK1c, B12–DKK1c), XWnt8 or negative control proteins (B12, DKK1) (3–50 nM) in mouse L cells (endogenously expressing FZD7) without (a) and with (b) overexpression of FZD8. c, Activation of the STF reporter by B12–DKK1c (5, 25, 100 nM) in L cells overexpressing the 10 mouse FZD receptors. d, RSPO2 (5–75 nM) potentiates the activity of scFv–DKK1c (5–75 nM) and WNT3A conditioned media (CM) (25–45%), as demonstrated by enhanced expression of the STF reporter in HEK293 cells. The scFv–DKK1c–RSPO2 fusion replicates the enhanced activity of the individual proteins. e, Upregulation of early osteogenic marker Alpl mRNA in mouse C3H10T1/2 cells treated for 4 days 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. f, Upregulation of ALPL mRNA in human primary MSCs treated for 3 days with WNT3A conditioned media, 50 nM scFv–DKK1c and 50 nM scFv–DKK1c–RSPO2 in the presence and absence of 200 ng ml−1 BMP2. Error bars represent s.d. of n = 3 technical replicates from 1 of 3 (ac, e, f) and 5 (d) representative experiments.

  3. Activity of Wnt surrogates on human organoid cultures in vitro.
    Figure 3: Activity of Wnt surrogates on human organoid cultures in vitro.

    a, Representative bright-field images of organoids of human pancreas, colon, stomach (corpus) and liver, expanded for 12 days in basal media containing 3 μM IWP-2 and tissue-specific growth factors (Supplementary Table 2), and supplemented with 50% WNT3A and 2% RSPO3 conditioned media, 200 nM scFv–DKK1c plus 2% RSPO3 conditioned media, and 200 nM scFv–DKK1c–RSPO2. b, Quantification of cell proliferation and organoid expansion (live cells based on the presence of intracellular ATP in metabolically active cells) by luminescence. RLU, relative light units. Data are mean and s.d. from n = 2 (pancreas, liver and stomach) and 3 (liver) technical replicates, from 1 of 3 (colon), 2 (liver and pancreas) and 1 (stomach) representative experiments.

  4. Engineered Wnt surrogate is bioactive and upregulates Wnt signalling in vivo.
    Figure 4: Engineered Wnt surrogate is bioactive and upregulates Wnt signalling in vivo.

    a, Representative images of glutamine synthetase (GS; pericentral marker) and CK19 (bile duct/portal marker) immunofluorescence staining of 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). b, Quantification of the glutamine synthetase immunofluorescence staining area shown in a relative to liver tissue area, with vessel lumen area subtracted (n = 4 mice per group). c, d, Effects of scFv–DKK1c and scFv–DKK1c–RSPO2 on hepatocyte proliferation determined in surgically paired mice by parabiosis. The donor mice received adenoviruses intravenously 2 days before parabiosis surgery with recipient mice to establish cross-circulation and allow diffusion of the transgenes in both mice. Immunofluorescent staining (c) and quantification of proliferating hepatocytes (d) in recipient mice with PCNA and HNF4α 7 days after parabiosis surgery. CV, central vein; LP, liver parenchyma; PV, portal vein. n = 3 mice per group. Experiments demonstrating the regulation of liver zonation were performed at least twice for each condition (n = 4 mice per group); the parabiosis experiment was done once in the laboratory (n = 3 mice per group). Error bars represent s.e.m. from biological replicates.

  5. De novo design and engineering of B12.
    Extended Data Fig. 1: De novo design and engineering of B12.

    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.

  6. FZD CRD binding characterization of B12 and scFv.
    Extended Data Fig. 2: FZD CRD binding characterization of B12 and scFv.

    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. eh, 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.

  7. Co-locomotion analysis of LRP6 and FZD8 induced by Wnt protein and surrogate.
    Extended Data Fig. 3: Co-locomotion analysis of LRP6 and FZD8 induced by Wnt protein and surrogate.

    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). di, 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.

  8. Single-molecule trajectories and step length analysis of LRP6 and FZD8 under different conditions.
    Extended Data Fig. 4: Single-molecule trajectories and step length analysis of LRP6 and FZD8 under different conditions.

    ae, 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). fj, 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 ae. 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).

  9. Single-molecule intensity analysis of LRP6 and FZD8 under different conditions.
    Extended Data Fig. 5: Single-molecule intensity analysis of LRP6 and FZD8 under different conditions.

    Single-molecule intensity analysis for quantifying LRP6 (af) and FZD8 (gl) 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.

  10. FZD-specific activation of canonical Wnt signalling by Wnt surrogates.
    Extended Data Fig. 6: FZD-specific activation of canonical Wnt signalling by Wnt surrogates.

    ad, 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.

  11. Wnt surrogates enhance upregulation of alkaline phosphatase in MSCs.
    Extended Data Fig. 7: Wnt surrogates enhance upregulation of alkaline phosphatase in MSCs.

    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. fh, 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.

  12. Activity of Wnt surrogates on human organoid cultures in vitro.
    Extended Data Fig. 8: Activity of Wnt surrogates on human organoid cultures in vitro.

    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. ce, 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.

  13. Wnt surrogate activate Wnt signalling in vivo and alter Wnt-driven hepatic zonation.
    Extended Data Fig. 9: Wnt surrogate activate Wnt signalling in vivo and alter Wnt-driven hepatic zonation.

    ad, 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.

  14. Wnt surrogate enhance hepatocyte proliferation.
    Extended Data Fig. 10: Wnt surrogate enhance hepatocyte proliferation.

    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). bd, 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. gi, 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.

Videos

  1. Co-locomoting Lrp6/Fzd8 measured without treatment
    Video 1: Co-locomoting Lrp6/Fzd8 measured without treatment
    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.
  2. Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of scFv-DKK1c
    Video 2: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of scFv-DKK1c
    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.
  3. Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of B12-DKK1c
    Video 3: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of B12-DKK1c
    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.
  4. Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of XWnt8
    Video 4: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of XWnt8
    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.
  5. Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of Wnt3a
    Video 5: Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of Wnt3a
    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.

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Primary accessions

Protein Data Bank

References

  1. Clevers, H. & Nusse, R. Wnt/β-catenin signaling and disease. Cell 149, 11921205 (2012)
  2. Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448452 (2003)
  3. Takada, R. et al. Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev. Cell 11, 791801 (2006)
  4. Janda, C. Y., Waghray, D., Levin, A. M., Thomas, C. & Garcia, K. C. Structural basis of Wnt recognition by Frizzled. Science 337, 5964 (2012)
  5. Cong, F., Schweizer, L. & Varmus, H. Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP. Development 131, 51035115 (2004)
  6. Holmen, S. L., Robertson, S. A., Zylstra, C. R. & Williams, B. O. Wnt-independent activation of beta-catenin mediated by a Dkk1–Fz5 fusion protein. Biochem. Biophys. Res. Commun. 328, 533539 (2005)
  7. Liu, G., Bafico, A. & Aaronson, S. A. The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts. Mol. Cell. Biol. 25, 34753482 (2005)
  8. Mulligan, K. A. et al. Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility. Proc. Natl Acad. Sci. USA 109, 370377 (2012)
  9. Gurney, A. et al. Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors. Proc. Natl Acad. Sci. USA 109, 1171711722 (2012)
  10. Ahn, V. E. et al. Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6. Dev. Cell 21, 862873 (2011)
  11. Bourhis, E. et al. Reconstitution of a Frizzled8·Wnt3a·LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6. J. Biol. Chem. 285, 91729179. (2010)
  12. Bilic, J. et al. Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science 316, 16191622 (2007)
  13. Wilmes, S. et al. Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling. J. Cell Biol. 209, 579593 (2015)
  14. Hao, H. X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195200 (2012)
  15. Zhong, Z., Ethen, N. J. & Williams, B. O. WNT signaling in bone development and homeostasis. Wiley Interdiscip. Rev. Dev. Biol. 3, 489500 (2014)
  16. Salazar, V. S., Ohte, S., Capelo, L. P., Gamer, L. & Rosen, V. Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2. Development 143, 43524367 (2016)
  17. Kretzschmar, K. & Clevers, H. Organoids: modeling development and the stem cell niche in a dish. Dev. Cell 38, 590600 (2016)
  18. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262265 (2009)
  19. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 2536 (2010)
  20. Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247250 (2013)
  21. Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 19, 939945 (2013)
  22. Wei, K., Kuhnert, F. & Kuo, C. J. Recombinant adenovirus as a methodology for exploration of physiologic functions of growth factor pathways. J. Mol. Med. (Berl.) 86, 161169 (2008)
  23. Wang, B., Zhao, L., Fish, M., Logan, C. Y. & Nusse, R. Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver. Nature 524, 180185 (2015)
  24. Benhamouche, S. et al. Apc tumor suppressor gene is the “zonation-keeper” of mouse liver. Dev. Cell 10, 759770 (2006)
  25. Planas-Paz, L. et al. The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size. Nat. Cell Biol. 18, 467479 (2016)
  26. Yan, K. S. et al. Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal. Nature http://dx.doi.org/10.1038/nature22313 (this issue)
  27. Lemmon, M. A. & Schlessinger, J. Transmembrane signaling by receptor oligomerization. Methods Mol. Biol. 84, 4971 (1998)
  28. Spangler, J. B., Moraga, I., Mendoza, J. L. & Garcia, K. C. Insights into cytokine-receptor interactions from cytokine engineering. Annu. Rev. Immunol. 33, 139167 (2015)
  29. Xu, Q. et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883895 (2004)
  30. Ke, J. et al. Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex. Genes Dev. 27, 23052319 (2013)
  31. Chang, T. H. et al. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan. eLife 4, (2015)
  32. Veeman, M. T., Slusarski, D. C., Kaykas, A., Louie, S. H. & Moon, R. T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13, 680685 (2003)
  33. Biechele, T. L. & Moon, R. T. Assaying β-catenin/TCF transcription with β-catenin/TCF transcription-based reporter constructs. Methods Mol. Biol. 468, 99110 (2008)
  34. Cooper, S. et al. Predicting protein structures with a multiplayer online game. Nature 466, 756760 (2010)
  35. Azoitei, M. L. et al. Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334, 373376 (2011)
  36. Fleishman, S. J. et al. RosettaScripts: a scripting language interface to the Rosetta macromolecular modeling suite. PLoS One 6, e20161 (2011)
  37. Whitehead, T. A. et al. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing. Nat. Biotechnol. 30, 543548 (2012)
  38. Kunkel, T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA 82, 488492 (1985)
  39. Chao, G., Cochran, J. R. & Wittrup, K. D. Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display. J. Mol. Biol. 342, 539550 (2004)
  40. Kabsch, W. Xds. Acta Crystallogr. D 66, 125132 (2010)
  41. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007)
  42. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 21262132 (2004)
  43. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213221 (2010)
  44. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 1221 (2010)
  45. Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protocols 1, 755768 (2006)
  46. Boder, E. T. & Wittrup, K. D. Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol. 328, 430444 (2000)
  47. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373382 (2008)
  48. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128136 (2008)
  49. Muster, B. et al. Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells. PLoS One 5, e11910 (2010)
  50. Löchte, S., Waichman, S., Beutel, O., You, C. & Piehler, J. Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane. J. Cell Biol. 207, 407418 (2014)
  51. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100107 (2009)
  52. VandeVondele, S., Vörös, J. & Hubbell, J. A. RGD-grafted poly-l-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion. Biotechnol. Bioeng. 82, 784790 (2003)
  53. Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Ed. Engl. 47, 54655469 (2008)
  54. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 27752783 (2002)
  55. Gould, T. J., Verkhusha, V. V. & Hess, S. T. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat. Protocols 4, 291308 (2009)
  56. Sergé, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods 5, 687694 (2008)
  57. Schütz, G. J., Schindler, H. & Schmidt, T. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys. J. 73, 10731080 (1997)
  58. You, C. et al. Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells. ACS Chem. Biol. 8, 320326 (2013)
  59. Wedeking, T. et al. Single cell GFP-trap reveals stoichiometry and dynamics of cytosolic protein complexes. Nano Lett. 15, 36103615 (2015)
  60. Shah, S. M., Kang, Y. J., Christensen, B. L., Feng, A. S. & Kollmar, R. Expression of Wnt receptors in adult spiral ganglion neurons: frizzled 9 localization at growth cones of regenerating neurites. Neuroscience 164, 478487 (2009)
  61. Zhong, Z. A., Ethen, N. J. & Williams, B. O. Use of primary calvarial osteoblasts to evaluate the function of Wnt signaling in osteogenesis. Methods Mol. Biol. 1481, 119125 (2016)
  62. van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933945 (2015)
  63. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 17621772 (2011)
  64. Bartfeld, S. et al. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology 148, 126136 (2015)
  65. Stange, D. E. et al. Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155, 357368 (2013)
  66. Boj, S. F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324338 (2015)
  67. Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 27082721 (2013)
  68. Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299312 (2015)
  69. Wei, K. et al. A liver Hif-2α–Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nat. Med. 19, 13311337 (2013)
  70. Rocha, A. S. et al. The angiocrine factor Rspondin3 is a key determinant of liver zonation. Cell Reports 13, 17571764 (2015)
  71. Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297, 22562259 (2002)

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Author information

  1. These authors contributed equally to this work.

    • Claudia Y. Janda &
    • Luke T. Dang

Affiliations

  1. Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Claudia Y. Janda,
    • Dirk Siepe &
    • K. Christopher Garcia
  2. Department of Biochemistry, Howard Hughes Medical Institute, and the Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA

    • Luke T. Dang,
    • James D. Moody &
    • David Baker
  3. Division of Biophysics, Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany

    • Changjiang You &
    • Jacob Piehler
  4. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Junlei Chang,
    • Kelley S. Yan,
    • Xingnan Li &
    • Calvin J. Kuo
  5. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

    • Wim de Lau &
    • Hans Clevers
  6. Program for Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, Michigan 49503, USA

    • Zhendong A. Zhong &
    • Bart O. Williams
  7. Hagey Laboratory for Pediatric Regenerative Medicine and Department of Surgery, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    • Owen Marecic

Contributions

C.Y.J. designed experiments, performed biophysical measurements, determined crystal structures, performed in vitro functional assays and prepared the manuscript. D.S. analysed data. D.B., L.T.D. and J.D.M. designed the B12 binding module, and performed affinity maturation. J.P. and C.Y. performed TIRF microscopy, analysed data and contributed to manuscript preparation. Z.A.Z. and B.O.W. performed osteogenesis assays, and analysed data. W.d.L. and H.C. performed organoid culture assays, analysed data and contributed to manuscript preparation. J.C., K.S.Y. and X.L. carried out in vivo experiments in mice and analysed data. O.M. performed parabiosis surgery. C.J.K. designed and supervised in vivo experiments, analysed data and contributed to manuscript preparation. K.C.G. conceived of the project, analysed data, supervised execution of the project, and prepared the manuscript.

Competing financial interests

K.C.G., C.J.K. and C.Y.J. are founders of Surrozen, Inc.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks W. DeGrado, Y. Jones and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. 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.

  2. 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. eh, 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.

  3. 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). di, 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.

  4. Extended Data Figure 4: Single-molecule trajectories and step length analysis of LRP6 and FZD8 under different conditions. (603 KB)

    ae, 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). fj, 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 ae. 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).

  5. 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 (af) and FZD8 (gl) 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.

  6. Extended Data Figure 6: FZD-specific activation of canonical Wnt signalling by Wnt surrogates. (357 KB)

    ad, 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.

  7. 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. fh, 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.

  8. 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. ce, 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.

  9. Extended Data Figure 9: Wnt surrogate activate Wnt signalling in vivo and alter Wnt-driven hepatic zonation. (836 KB)

    ad, 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.

  10. 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). bd, 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. gi, 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.

Supplementary information

Video

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.

PDF files

  1. Supplementary Information (414 KB)

    This file contains Supplementary Tables 1-2 and Supplementary Figure 1, the uncropped western blots.

Additional data