Yoon J et al. (2023), Wnt4 and ephrinB2 instruct apical constriction ...

XB-ART-60812

Nat Commun 2023 Jan 20;141:337. doi: 10.1038/s41467-023-35991-6.

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Wnt4 and ephrinB2 instruct apical constriction via Dishevelled and non-canonical signaling.

Yoon J , Sun J , Lee M , Hwang YS , Daar IO .

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Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube.

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Species referenced: Xenopus laevis
Genes referenced: dvl2 dvl3 efnb2 mlc1 rho ror2 shroom3 sia1 wnt4 wnt5a
???displayArticle.antibodies??? Efnb1 Ab1
???displayArticle.morpholinos??? dvl2 MO1 dvl3 MO1 efnb2 MO2 ror2 MO2 shroom3 MO2 wnt4 MO1
gRNAs referenced: efnb2 gRNA1 ror2 gRNA3 wnt4 gRNA1

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	Fig. 1: ephrinB2/Ror2 interaction regulates apical constriction. a Schematic of screening for ephrinB2-binding partners and selected candidates from ephrinB2 pulldown assay. Scale bar, 500 μm. See also Supplementary Data 1. b Schematic of the transverse section through neural plate and RNA in situ hybridization (HCR) assay. Neural plate (yellow), PM; pre-somatic mesoderm (magenta), NC; notochord (blue), AR; archenteron roof (green). Scale bar, 50 μm. c Co-IP assay with Ror2 (1 ng) and ephrin-B type ligands (500 pg) at Stage 13 Xenopus embryos. d Schematic of ephrinB2 serial deletion mutants in C-terminus. The PDZ-binding motif in red. e Schematic of Ror2 serial deletion mutants in the tail region. f Co-IP with Ror2 (1 ng) and ephrinB2 (500 pg) serial deletion mutants. g Co-IP with ephrinB2 (500 pg) and Ror2 serial deletion mutants (1 ng). h Representative embryo morphology by ephrinB2 knockdown (2 ng of MO). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side (50 pg). The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents the expression level of ephrinB2 wildtype (WT) and [delta]4 (25 pg). Data means SEM. Numbers of embryos/experiments: 85/3, 80/3, 78/3, and 79/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used (p-value <0.0001). n.s., not significant, **p < 0.0001. Scale bar, 500 μm. i Representative embryo morphology by Ror2 knockdown (2 ng of MO). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side. The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents expression levels of Ror2-WT, [delta]Fr, and [delta]PR (50 pg). Data means SEM. Numbers of embryos/experiments: 79/3, 79/3, 81/3, 82/3, and 81/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used (p-value <0.0001). n.s., not significant, **p < 0.0001. Scale bar, 500 μm.. Scale bar, 500 um.
	Fig. 2: ephrinB2/Ror2 interaction regulates contractile actin network formation. a Transverse section through the neural plate. Unilateral injection of MO (2ng) and RNA (ephrinB2s; 25pg, Ror2s; 50pg) as indicated. Membrane-RFP (100pg) labeled outline of the neural plate cells. GFP (50pg) indicates the MO-injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 50/5 for each column. One-way ANOVAs followed by Dunnetts multiple comparison tests were used (p-value<0.0001). n.s., not significant, ***p<0.0001. Scale bar, 500m. Schematic of measuring apical width. Scale bar, 50m. b Apical F-actin levels in neural plate cells by Phalloidin staining. Membrane-RFP indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 15/3 for each column. One-way ANOVAs followed by Dunnetts multiple comparison tests were used. n.s., not significant, p<0.005, and ****p<0.0001. Schematic of area where images taken. Scale bar, 50m. c Stills from time-lapse imaging of GFP-UtrCH (100pg) overexpressed neural plates from stage 14 to 15 embryos. See also Supplementary Movie 3 and 4. Scale bar, 10um.
	Fig. 3: Wnt4 interacts with ephrinB2 via Ror2 to regulate apical constriction. a Co-IP assay with ephrinB2 (500 pg) and Wnt ligands (100 pg). b Co-IP assay with Wnt4 (100 pg) and ephrinB2 (100 pg) in Ror2 morphants. c Representative embryo morphology by Wnt4 knockdown (2 ng). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side. The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents expression levels of Wnt4 and Wnt5a (50 pg). Data means SEM. Numbers of embryos/experiments: 55/3, 64/3, 57/3, and 57/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 500 μm. d Transverse section through the neural plate. Unilateral injection of MO and RNA as indicated. Membrane-RFP labeled outline of the neural plate cells. GFP (Cyan) indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 40/4 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, *p < 0.0001. Scale bar, 50 um. e Apical actin levels in the neural plate by Phalloidin staining. Membrane-RFP indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 20/4 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.005, and ****p < 0.0001. Scale bar, 50 μm. f Stills from time-lapse imaging of GFP-UtrCH (100 pg) overexpressed in neural plates from stage 14 to 15 embryos. See also Supplementary Movie 6. Scale bar, 10 um. g Spatial expression pattern of Wnt4. WISH of stage 12, 12.5, and 14 embryos with anti-Wnt4 probes. Scale bar, 500 um.
	Fig. 4: Wnt4, ephrinB2, Ror2, Dsh2, and Shroom3 form a signaling complex. a Co-IP assay with ephrinB2 (100 pg) and Ror2 (100 pg) in Dsh2/3 morphants. b Schematic of Dsh2 serial deletion in the tail region (Ctail). c Co-IP assay with ephrinB2 (500 pg) and Dsh2 (1 ng) deletion mutants. d Co-IP assay with Ror2 (1 ng) and Dsh2 (1 ng) deletion mutants. e Co-IP assay with ephrinB2 (500 pg) and conserved domain deletion mutants of Shroom3 (1 ng). f Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in the presence of exogenously expressed ephrinB2 (25 pg) and Wnt4 (50 pg). g Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in neural plates of ephrinB2 or Wnt4 morphants. h Co-IP assay with Dsh2 (200 pg) and Shroom3 (100 pg) in the presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg). i Co-IP assay with Shroom3 and Rock in presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg).
	Fig. 5: EphrinB2 and Ror2 regulate Shroom3 accumulation at tricellular junctions. a Co-IP assay with ephrinB2 (100 pg) and Ror2 (100 pg) in Dsh2/3 morphants. b Schematic of Dsh2 serial deletion in the tail region (Ctail). c Co-IP assay with ephrinB2 (500 pg) and Dsh2 (1 ng) deletion mutants. d Co-IP assay with Ror2 (1 ng) and Dsh2 (1 ng) deletion mutants. e Co-IP assay with ephrinB2 (500 pg) and conserved domain deletion mutants of Shroom3 (1 ng). f Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in the presence of exogenously expressed ephrinB2 (25 pg) and Wnt4 (50 pg). g Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in neural plates of ephrinB2 or Wnt4 morphants. h Co-IP assay with Dsh2 (200 pg) and Shroom3 (100 pg) in the presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg). i Co-IP assay with Shroom3 and Rock in presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg).
	Fig. 6: WERDS signaling complex controls apical constriction. a Ectopic apical constriction analysis with WERDS components. Representative images of ectopic apical constriction induced by exogenously expressed Shroom3 (200 pg). The red arrowheads indicate ectopic apical constriction in the ectoderm. Data means SEM. Numbers of embryos/experiments: 82/3, 89/3, 80/3, 77/3, 85/3, 102/3, 97/3, 91/3, 103/3, and 82/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 500 μm. See also Supplementary Fig. 6c. b Ectopic apical constriction analysis with the WERDS complex and ephrinB2-Δ4. Data means SEM. Numbers of embryos/experiments: 66/3, 68/3, 69/3, and 75/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6d. c Ectopic apical constriction analysis with the WERDS complex and Ror2-ΔPR. Data means SEM. Numbers of embryos/experiments: 73/3, 70/3, 79/3, and 79/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6f. d Ectopic apical constriction analysis with the WERDS complex and Wnt5a. Data means SEM. Numbers of embryos/experiments: 75/3, 77/3, 78/3, and 78/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6g. e F-actin (Phalloidin) and phospho-MLC levels in ectodermal explants. Data means SEM. The number of ectodermal explants/experiments: 9/3 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 50 μm. f A model for WERDS signal complex.

References [+] :

Aigouy, Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila. 2010, Pubmed

	Fig. 1: ephrinB2/Ror2 interaction regulates apical constriction. a Schematic of screening for ephrinB2-binding partners and selected candidates from ephrinB2 pulldown assay. Scale bar, 500 μm. See also Supplementary Data 1. b Schematic of the transverse section through neural plate and RNA in situ hybridization (HCR) assay. Neural plate (yellow), PM; pre-somatic mesoderm (magenta), NC; notochord (blue), AR; archenteron roof (green). Scale bar, 50 μm. c Co-IP assay with Ror2 (1 ng) and ephrin-B type ligands (500 pg) at Stage 13 Xenopus embryos. d Schematic of ephrinB2 serial deletion mutants in C-terminus. The PDZ-binding motif in red. e Schematic of Ror2 serial deletion mutants in the tail region. f Co-IP with Ror2 (1 ng) and ephrinB2 (500 pg) serial deletion mutants. g Co-IP with ephrinB2 (500 pg) and Ror2 serial deletion mutants (1 ng). h Representative embryo morphology by ephrinB2 knockdown (2 ng of MO). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side (50 pg). The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents the expression level of ephrinB2 wildtype (WT) and [delta]4 (25 pg). Data means SEM. Numbers of embryos/experiments: 85/3, 80/3, 78/3, and 79/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used (p-value <0.0001). n.s., not significant, **p < 0.0001. Scale bar, 500 μm. i Representative embryo morphology by Ror2 knockdown (2 ng of MO). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side. The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents expression levels of Ror2-WT, [delta]Fr, and [delta]PR (50 pg). Data means SEM. Numbers of embryos/experiments: 79/3, 79/3, 81/3, 82/3, and 81/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used (p-value <0.0001). n.s., not significant, **p < 0.0001. Scale bar, 500 μm.. Scale bar, 500 um.
	Fig. 2: ephrinB2/Ror2 interaction regulates contractile actin network formation. a Transverse section through the neural plate. Unilateral injection of MO (2ng) and RNA (ephrinB2s; 25pg, Ror2s; 50pg) as indicated. Membrane-RFP (100pg) labeled outline of the neural plate cells. GFP (50pg) indicates the MO-injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 50/5 for each column. One-way ANOVAs followed by Dunnetts multiple comparison tests were used (p-value<0.0001). n.s., not significant, ***p<0.0001. Scale bar, 500m. Schematic of measuring apical width. Scale bar, 50m. b Apical F-actin levels in neural plate cells by Phalloidin staining. Membrane-RFP indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 15/3 for each column. One-way ANOVAs followed by Dunnetts multiple comparison tests were used. n.s., not significant, p<0.005, and ****p<0.0001. Schematic of area where images taken. Scale bar, 50m. c Stills from time-lapse imaging of GFP-UtrCH (100pg) overexpressed neural plates from stage 14 to 15 embryos. See also Supplementary Movie 3 and 4. Scale bar, 10um.
	Fig. 3: Wnt4 interacts with ephrinB2 via Ror2 to regulate apical constriction. a Co-IP assay with ephrinB2 (500 pg) and Wnt ligands (100 pg). b Co-IP assay with Wnt4 (100 pg) and ephrinB2 (100 pg) in Ror2 morphants. c Representative embryo morphology by Wnt4 knockdown (2 ng). See also Supplementary Movie 1. Schematic of D1.1 microinjection in the 16-cell stage embryo. Membrane-GFP indicates injected side. The lateral boundaries of the neural plate are marked by dotted lines (green; uninjected side and magenta; injected side). Immunoblotting represents expression levels of Wnt4 and Wnt5a (50 pg). Data means SEM. Numbers of embryos/experiments: 55/3, 64/3, 57/3, and 57/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 500 μm. d Transverse section through the neural plate. Unilateral injection of MO and RNA as indicated. Membrane-RFP labeled outline of the neural plate cells. GFP (Cyan) indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 40/4 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, *p < 0.0001. Scale bar, 50 um. e Apical actin levels in the neural plate by Phalloidin staining. Membrane-RFP indicates the injected side. The box plot shows Min to Max with all data points. Numbers of embryos/experiments: 20/4 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.005, and ****p < 0.0001. Scale bar, 50 μm. f Stills from time-lapse imaging of GFP-UtrCH (100 pg) overexpressed in neural plates from stage 14 to 15 embryos. See also Supplementary Movie 6. Scale bar, 10 um. g Spatial expression pattern of Wnt4. WISH of stage 12, 12.5, and 14 embryos with anti-Wnt4 probes. Scale bar, 500 um.
	Fig. 4: Wnt4, ephrinB2, Ror2, Dsh2, and Shroom3 form a signaling complex. a Co-IP assay with ephrinB2 (100 pg) and Ror2 (100 pg) in Dsh2/3 morphants. b Schematic of Dsh2 serial deletion in the tail region (Ctail). c Co-IP assay with ephrinB2 (500 pg) and Dsh2 (1 ng) deletion mutants. d Co-IP assay with Ror2 (1 ng) and Dsh2 (1 ng) deletion mutants. e Co-IP assay with ephrinB2 (500 pg) and conserved domain deletion mutants of Shroom3 (1 ng). f Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in the presence of exogenously expressed ephrinB2 (25 pg) and Wnt4 (50 pg). g Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in neural plates of ephrinB2 or Wnt4 morphants. h Co-IP assay with Dsh2 (200 pg) and Shroom3 (100 pg) in the presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg). i Co-IP assay with Shroom3 and Rock in presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg).
	Fig. 5: EphrinB2 and Ror2 regulate Shroom3 accumulation at tricellular junctions. a Co-IP assay with ephrinB2 (100 pg) and Ror2 (100 pg) in Dsh2/3 morphants. b Schematic of Dsh2 serial deletion in the tail region (Ctail). c Co-IP assay with ephrinB2 (500 pg) and Dsh2 (1 ng) deletion mutants. d Co-IP assay with Ror2 (1 ng) and Dsh2 (1 ng) deletion mutants. e Co-IP assay with ephrinB2 (500 pg) and conserved domain deletion mutants of Shroom3 (1 ng). f Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in the presence of exogenously expressed ephrinB2 (25 pg) and Wnt4 (50 pg). g Co-IP assay with Ror2 (100 pg) and Dsh2 (200 pg) in neural plates of ephrinB2 or Wnt4 morphants. h Co-IP assay with Dsh2 (200 pg) and Shroom3 (100 pg) in the presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg). i Co-IP assay with Shroom3 and Rock in presence of other WERDS components, Wnt4 (50 pg), Ror2 (100 pg), and ephrinB2 (25 pg).
	Fig. 6: WERDS signaling complex controls apical constriction. a Ectopic apical constriction analysis with WERDS components. Representative images of ectopic apical constriction induced by exogenously expressed Shroom3 (200 pg). The red arrowheads indicate ectopic apical constriction in the ectoderm. Data means SEM. Numbers of embryos/experiments: 82/3, 89/3, 80/3, 77/3, 85/3, 102/3, 97/3, 91/3, 103/3, and 82/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 500 μm. See also Supplementary Fig. 6c. b Ectopic apical constriction analysis with the WERDS complex and ephrinB2-Δ4. Data means SEM. Numbers of embryos/experiments: 66/3, 68/3, 69/3, and 75/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6d. c Ectopic apical constriction analysis with the WERDS complex and Ror2-ΔPR. Data means SEM. Numbers of embryos/experiments: 73/3, 70/3, 79/3, and 79/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6f. d Ectopic apical constriction analysis with the WERDS complex and Wnt5a. Data means SEM. Numbers of embryos/experiments: 75/3, 77/3, 78/3, and 78/3 from left to right for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, p < 0.0001. See also Supplementary Fig. 6g. e F-actin (Phalloidin) and phospho-MLC levels in ectodermal explants. Data means SEM. The number of ectodermal explants/experiments: 9/3 for each column. One-way ANOVAs followed by Dunnett’s multiple comparison tests were used. n.s., not significant, **p < 0.0001. Scale bar, 50 μm. f A model for WERDS signal complex.