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EGFR is required for Wnt9a–Fzd9b signalling specificity in haematopoietic stem cells

Abstract

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand–receptor (Wnt–Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit β-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a–Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a–Fzd9b–LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt–Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.

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Data availability

MS data have been deposited in ProteomeXchange with the primary accession code PXD010649 through MassIVE (MSV000082677). Summary data are seen in Supplementary Figs. 6e,f and 7d,e. Source data for the main and supplementary figures have been provided as Supplementary Table 3. Previously published sequencing data that were re-analysed here are available from the European Nucleotide Archive under the accession number PRJEB4197. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank C. Fine and J. Olvera for FACS assistance; R. Espin-Palazon, J. Nussbacher and I. J. Huggins for manuscript reading; I. J. Huggins for providing cetuximab; N. Gohad for microscopy assistance; and M. Boutros for cell lines. S.G. was supported by awards from the American Heart Association (14POST18340021) and the Leukemia and Lymphoma Society (5431-15). Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the US National Institutes of Health (NIH) under award number K99HL133458 (awarded to S.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. J.R. was supported in part by the UCSD Interdisciplinary Stem Cell Training Program (CIRM TG2-01154) and by the American Heart Association Predoctoral Fellowship (16PRE27340012). This work was supported in part by funding to K.W. from the UCSD Stem Cell Program and was made possible in part by the CIRM Major Facilities grant (FA1-00607) to the Sanford Consortium for Regenerative Medicine. D.T. was supported by a Scholar Award (1657-13) from The Leukemia and Lymphoma Society and CIRM (RB4-06158). This project was also supported by the NIH/National Heart, Lung, and Blood Institute grant R01HL135205 (awarded to D.T. and K.W.) and by the NIH/National Institute of General Medical Sciences grant R01GM110304 (awarded to K.W.). J.M.W. was supported by the Graduate Training in Cellular and Molecular Pharmacology Training Grant NIH T32 GM007752. I.A.D. was supported by the Harvard Stem Cell Institute and NIDDK grant UH2/3DK107372.

Author information

S.G. conceived, designed and conducted the experiments and analysis, and wrote the manuscript. N.N. and J.R. designed and conducted the experiments and analysis. J.S., B.L., R.B., C.H.O. and J.H. conducted the experiments and analysis. J.M.W., M.C.-T. and D.G. performed MS and conducted the analysis. C.N.K. and I.A.D. provided the zebrafish lines. D.T. and K.W. supervised the experiments and edited the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Karl Willert or David Traver.

Integrated supplementary information

  1. Supplementary Fig. 1 Fzd screening.

    a, Schematic of strategy to screen for fzd expression in endothelial cells as marked by fli1a-GFP at 16.5 hpf. b, Expression of all 14 fzd transcripts in Fli1a+ cells at 16.5 hpf; n = 1 experiment shown as representative data from qPCR on pooled sorted cells; trend reproduced in 3 independent experiments. Bars represent the mean of three technical replicates. c, In the Wnt-off state, the STF reporter is silent and luciferase is not expressed. d, In the presence of a Wnt signal, β-catenin is translocated to the nucleus, where it activates the STF reporter and luciferase is expressed. e, Quantification of the HEK293T cell STF assay with Wnt9a and Fzd8a or Fzd9b in the context of STF or FOP:FLASH reporters; n = 3 biological replicates each. f, Quantification of the HEK293T (FZD1, FZD2 and FZD7 KO) cell STF assay with zebrafish Wnt3a and zebrafish Fzd8a, Fzd9a, Fzd9b or human FZD7; n = 3 biological replicates each. In all STF assays, the bars represent the mean, the error bars represent the standard deviation and the dots represent individual biological replicates; one-way ANOVA with Dunnett’s test. All STF assays were repeated independently with a similar trend.

  2. Supplementary Fig. 2 Fzd9b is required for HSPC development.

    a, Endothelial cell emerging from the aorta is labelled by fzd9b:Gal4; UAS:Cre; loxP-BFP-loxP-dsRed (pseudo-coloured green) at 3 dpf, during HSPC emergence (left). Scale bar, 10 µm. Cells in the thymus are labelled by fzd9b:Gal4; UAS:Cre; loxP-BFP-loxP-dsRed at 7 dpf (right). Scale bar, 25 µm. Images are representative of ten embryos from three experiments. b, Expression of Fzd9b-mKate fusion protein in fzd9b MO injected samples and no MO control at 3 dpf. Scale bar, 200 µm. Representative from n = 10 biological samples. c, Quantification of Runx1+ cells in the aorta of uninjected (n = 15) and fzd9b MO injected (n = 12) fish at 30 hpf. d, Quantification of runx1 transcripts by qPCR in uninjected and fzd9b MO injected fish at 30 hpf; n = 3 zebrafish each. e, Nascent HSPCs were labelled by gata2b and kdrl fluorescent transgenes at 46 hpf in uninjected and fzd9b MO injected fish. Scale bar, 10 µm. f, Quantification of Gata2b+ cells per millimetre of the aortic floor from e; n = 5 biological replicates each. g, WISH for the T cell marker rag1 at 5 dpf in uninjected and fzd9b MO injected fish. Scale bar, 200 µm. Representative from 30 biological samples as indicated. h, WISH for aortic (dlc, dll4 and notch1b), vascular (kdrl) and pronephros (cdh17) markers in uninjected and fzd9b MO-injected fish at 24 hpf. Scale bar, 500 µm. The numbers in the upper right corners represent the number of animals with the shown phenotype, over the total examined. i, WISH for cmyb in uninjected, fzd9b MO injected and fzd9b MO + fzd9b mRNA injected fish at 40 hpf. Scale bar, 30 µm. j, Quantification of i; n = 10, 7 and 10 biological replicates from left to right. k, Quantification of Cmyb+ cells in the aorta of fzd9b mutants and siblings injected with fzd9b MO or uninjected controls; n = 16, 6, 16 and 9 biological replicates from left to right. l, WISH for cmyb at 40 hpf in endothelial-specific fzd9b mutants. Scale bar, 30 µm. m, Quantification of l; n = 13 and 8 from left to right. In all graphs, each dot represents a biological replicate, the bars represent the mean and the error bars represent the standard deviation. Statistical analyses were done by ANOVA compared to controls as indicated. All STF assays were repeated independently with a similar trend.

  3. Supplementary Fig. 3 WNT9A and FZD9 are required for HSPC development.

    Expression of WNT9A (n = 4 biological replicates each) (a), FZD9 (n = 3 biological replicates each) (b), CD34 (n = 8 biological replicates each) (c), CD31 (n = 8 biological replicates each) (d), CD45 (n = 7, 8 and 8 biological replicates from left to right) (e) and CMYB (n = 7, 4 and 8 biological replicates from left to right) (f), after 14 d of haematopoietic-directed differentiation in shControl, shWNT9A and shFZD9 cells as measured by qPCR. The bars represent the mean and the error bars represent the standard deviation of biological replicates from three experiments, which are represented by individual dots. Statistical analyses were done by an unpaired, two-tailed t-test in a and b, and by ANOVA in cf, compared to control shRNA. Representative FACS lots of TRA1-81 and SSEA4 staining in undifferentiated control (g), WNT9A (h) and FZD9 (i) shRNA-expressing hES cells; representative plots from two independent experiments. All STF assays were repeated independently with a similar trend.

  4. Supplementary Fig. 4 LRP6-KO HEK293T STF cell line validation.

    a, WISH for cmyb at 40 hpf in zebrafish injected with suboptimal MO dosages (0.1 ng wnt9a and 0.5 ng fzd9b). For quantification, see Fig. 2g. Scale bar, 30 µm. b, Quantification of the HEK293T cell STF assay with zebrafish Wnt9a, Fzd9b and Lrp6; n = 3 biological replicates each. Statistical analyses are compared to Wnt9a alone. c, Quantification of the HEK293T cell STF assay screen with mouse Wnt3a in WT and LRP6-KO cells; n = 3 biological replicates each. Statistical analyses were done by ANOVA as indicated. d, Quantification of the HEK293T cell STF assay with 500 nM CHIR in WT and LRP6-KO cells; n = 3 biological replicates each. In all graphs, the dots represent biological replicates from one experiment, the bars represent the mean and the error bars represent the standard deviation. Statistical analyses were done by ANOVA compared to controls as indicated. All STF assays were repeated independently with a similar trend.

  5. Supplementary Fig. 5 Fzd9b intracellular domains are required for signalling.

    a,b, Immunoblots of Fzd9b-V5 chimaeric proteins. Representative of two independent experiments. See also Supplementary Fig. 7. c, Quantification of the HEK293T cell STF assay with zebrafish Wnt9a, Fzd9b and Fzd4 chimaeras; n = 3 biological replicates each. d, Quantification of the HEK293T cell STF assay with human WNT9A, FZD9 and FZD4 chimaeras; n = 3 biological replicates each. e, Schematic of Fzd9b indicating the immunogen region used for antibody generation. f, Non-permeabilized immunofluorescence for Fzd9b chimaeras. Scale bar, 30 µm. Data are representative of ten fields of view from two independent experiments. g, Histograms of flow cytometry for Fzd9b chimaeras. Data are representative of two independent experiments using 10,000 cells for each condition. All STF assays were repeated independently with a similar trend. In c,d, the dots represent biological replicates from a single experiment, the bars represent the mean and the error bars represent the standard deviation; statistical analyses were by ANOVA compared to controls as indicated.

  6. Supplementary Fig. 6 Fzd9b-5GS-APEX2 cell line validation and EGFR mediates Wnt9a–Fzd9b signalling.

    a, Immunoblot for streptavidin after treatment of cells with vehicle, biotin-phenol, H2O2 or both, representative of five independent experiments. See also Supplementary Fig. 7. b, Immunoblots of cells for Fzd9b, FLAG (APEX2) and actin; representative of three independent experiments. See also Supplementary Fig. 7. c, Immunofluorescence for FLAG showing APEX2 localization on the membrane; representative of three independent experiments. Scale bar, 30 µm. d, Quantification of the HEK293T cell STF assay with zebrafish Wnt9a conditioned medium, WT Fzd9b and Fzd9b-5GS-APEX2; n = 3 biological replicates each; statistical analysis was done by ANOVA compared to Wnt9a conditioned medium alone. e, Fold enrichment of GO terms for biological processes identified from the top 5% of changed proteins in Fzd9b-APEX2 HEK293T cells treated with Wnt9a. The P values are listed next to each bar; n = 3 biological replicates; see the Methods for analysis and statistics. f, Normalized intensity of the top three transmembrane proteins detected in the APEX assay; n = 3 biological replicates. g, Immunoblot for EGFR in siControl-treated or siEGFR-treated HEK293T cells; representative of three independent experiments. See also Supplementary Fig. 7. h, Quantification of the HEK293T cell STF assay with human WNT9A and FZD9 with siEGFR or siControl; n = 3 biological replicates. i, WISH for cmyb at 40 hpf; quantified in j; n = 10 biological replicates each. Scale bar, 30 µm. k,l, STF assay with zebrafish Wnt9a and Fzd9b (k; n = 8 biological replicates each) or human WNT9A and FZD9 (l; n = 3 biological replicates each), treated with the selective EGFR inhibitor AG1478. m, Expression of runx1 and gata2b in vehicle and EGFR inhibitor zebrafish treated from 15–36 hpf; n = 4 biological replicates each. n, Expression of known EGFR ligands as transcripts per million (TPM) in HEK293T cells as reported from RNA-sequencing data sets42. The red dashed line indicates the commonly accepted cut-off for expression levels by TPM; n = 6 biological replicates from 2 experiments. o, Quantification of the HEK293T cell STF assay in Fzd9b-expressing cells cultured in serum-free conditions with neutralizing antibody for HBEGF as indicated; n = 3 biological replicates each. In all graphs, the dots represent biological replicates from a single experiment (except for panel n, which is pooled from two experiments), the bars represent the mean and the error bars represent the standard deviation. Statistical analyses were done by ANOVA compared to controls as indicated. All STF assays were repeated independently with a similar trend.

  7. Supplementary Fig. 7 Immunoblots.

    The original immunoblot images used in this study. Boxes indicate the cropped region shown in respective figures as indicated.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7, Supplementary Table titles and legends

  2. Reporting Summary

  3. Supplementary Table 1

    Primers used

  4. Supplementary Table 2

    Source reagents

  5. Supplementary Table 3

    Statistics source data

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Fig. 1: Fzd9b is required for zebrafish HSC development.
Fig. 2: Fzd9b interacts genetically with Wnt9a.
Fig. 3: Wnt9a–Fzd9b specificity is mediated intracellularly.
Fig. 4: EGFR mediates Wnt9a–Fzd9b signalling.
Fig. 5: EGFR is required to phosphorylate the Fzd9b CTT in response to Wnt9a.
Fig. 6: Clathrin-mediated endocytosis is required for the Wnt9a–Fzd9b signal.
Supplementary Fig. 1: Fzd screening.
Supplementary Fig. 2: Fzd9b is required for HSPC development.
Supplementary Fig. 3: WNT9A and FZD9 are required for HSPC development.
Supplementary Fig. 4: LRP6-KO HEK293T STF cell line validation.
Supplementary Fig. 5: Fzd9b intracellular domains are required for signalling.
Supplementary Fig. 6: Fzd9b-5GS-APEX2 cell line validation and EGFR mediates Wnt9a–Fzd9b signalling.
Supplementary Fig. 7: Immunoblots.