Neural crest cell-derived DKK1 and NEDD4 modulate Wnt signalling in the second heart field to orchestrate outflow tract development

Figures

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  Fig. 1. Nedd4 activity in neural crest cells is required for cardiac outflow tract development. A–H Whole E17.5 hearts from Nedd4 full- or conditional-knockout mouse strains. Hearts were injected with Evans Blue solution in the right and left ventricles to aid visualisation of the outflow tract and branching arteries. A aorta, P pulmonary artery, RS right subclavian artery, RC right common carotid artery, LC left common carotid artery, LS left subclavian artery, PTA persistent truncus arteriosus, DORV double outlet right ventricle. Representative images from n = 3 embryos per genotype. I–P Transverse sections through the heart and outflow tract of E15.5 embryos at the level of the pulmonary or aortic valve region from wildtype and Nedd4−/− strains (I–L), and from wildtype and Wnt1-Cre; Nedd4fl/fl strains (M–P), stained with H&E. Dashed arrow indicates ventricle patency with the pulmonary artery, and solid arrow indicates ventricle patency with the aorta. RV right ventricle, LV left ventricle. Q Frequency of outflow tract defects in indicated mouse strains, classified by histological staining of tissue sections as in (I–P).

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  Fig. 2. Hearts of Wnt1-Cre; Nedd4fl/fl embryos exhibit shortened outflow tract and incomplete outflow tract rotation. A Hearts of wildtype and Wnt1-Cre; Nedd4fl/fl E10.5 embryos showing expression of Mef2cAHF-LacZ reporter gene in the right ventricle (RV) and outflow tract (OFT). Right panel: OFT + RV length was measured as the distance along the dashed lines as indicated. LV left ventricle. Data points represent biological replicates from n = 7 wildtype and n = 5 Wnt1-Cre; Nedd4fl/fl embryos from three independent experiments. Graph represents mean +/− SEM, ****P = 0.000011 (unpaired t test, two-tailed). B Coronal sections through the outflow tract region of wildtype and Wnt1-Cre; Nedd4fl/fl E12.5 embryos immunostained for smooth muscle actin (SMA) and CD31. Representative images from n = 6 embryos per genotype from six independent experiments. Incomplete clockwise outflow tract rotation is observed in Wnt1-Cre; Nedd4fl/fl embryos, evidenced by misalignment of the aortic valve over the right ventricle. Valve leaflet defects are also observed. L left, R right, A aortic artery/valve, P pulmonary artery/valve. Source data are provided as a Source Data file.

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  Fig. 3. Wnt1-Cre; Nedd4fl/fl embryos display precocious differentiation and defective deployment of anterior second heart field derivatives. A Whole E9.5 Wnt1-Cre; Z/EG and Wnt1-Cre; Z/EG; Nedd4fl/fl embryos immunostained for EGFP, which labels neural crest cells and derivatives. The inset image shows the migratory cardiac neural crest cells in the 3rd, 4th, and 6th pharyngeal arches. Representative images from n = 3 embryos per genotype from three independent experiments. B Sagittal sections through the outflow tract of E9.5 Wnt1-Cre; Z/EG and Wnt1-Cre; Z/EG; Nedd4fl/fl embryos immunostained for EGFP, CD31, and Isl1, revealing localisation of neural crest cells (NCCs) in close proximity to the anterior second heart field (SHF), and outflow tract (OFT). RV right ventricle, PE pharyngeal endoderm. Representative images from n = 8 embryos per genotype from eight independent experiments. C Transverse sections of the post-otic region of E9.5 wildtype and Wnt1-Cre; Nedd4fl/fl embryos immunostained for NCC markers AP2α and SOX10. AP2α is also expressed in the embryonic epithelium; these cells were not included in NCC quantitation. NT neural tube. The graph represents the total number of…

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  Fig. 4. Laser capture mRNA sequencing reveals disrupted Wnt signalling in Wnt1-Cre; Nedd4fl/fl embryos. A Diagram indicating tissue region (dashed box) that was excised by laser capture for mRNA sequencing, from n = 3 wildtype and Wnt1-Cre; Nedd4fl/fl embryos. Created in BioRender. Schwarz, Q. (2026) https://BioRender.com/g0bv7cx. B Volcano plot representing differentially expressed genes (DEGs) in Wnt1-Cre; Nedd4fl/fl tissue samples relative to wildtype. C List of top 30 DEGs identified from laser capture mRNA sequencing. D Sagittal section through the outflow tract of an E9.5 wildtype embryo immunostained for DKK1 and AP2α, which marks the nucleus of cardiac neural crest cells (cNCCs). Inset: higher magnification demonstrates DKK1 is expressed in neural crest cells, but not in anterior second heart field (aSHF) or pharyngeal endoderm (PE). Representative images from n = 3 independent experiments. E Wholemount in situ hybridisation of E9.5 wildtype and Wnt1-Cre; Nedd4fl/fl embryos, showing Dkk1 expression in cardiac NCCs. Representative images from n = 5 embryos per genotype from 5 independent experiments. F In situ hybridisation of E9.5 wildtype and Wnt1-Cre; Nedd4fl/fl embryo sagittal…

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  Fig. 5. Reduced canonical Wnt signalling underpins precocious second heart field differentiation. A Protocol for drug administration at E8.5, E9.5, and E10.5, and sample collection at E13.5. B Coronal sections through the outflow tract region of E13.5 wildtype, Wnt1-Cre; Nedd4fl/+ and Wnt1-Cre; Nedd4fl/fl embryos immunostained for smooth muscle actin (SMA) and CD31. RV and LV indicate the positions of the right ventricle and left ventricle, respectively. Outflow tract rotation was measured as the degree of clockwise rotation, indicated from the curved arrow from a perpendicular reference plane (the transverse plane of the embryo). C Quantitation of outflow tract rotation. Wnt1-Cre; Nedd4fl/fl embryos exhibit deficient rotation in all treatment conditions, while Wnt1-Cre; Nedd4fl/+ embryos also exhibit deficient rotation only when treated with the canonical Wnt signalling inhibitor iCRT3, and are indistinguishable from Wnt1-Cre; Nedd4fl/fl embryos. Data points represent biological replicates n = 14 wildtype, n = 13 Wnt1-Cre; Nedd4fl/+ and n = 14 Wnt1-Cre; Nedd4fl/fl embryos from 9 independent experiments. Graph represents mean +/− SEM. **1 P = 0.0024; **2 P = 0.0063; ***P = 0.0006; ns,…

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  Fig. 6. NEDD4 ubiquitinates DKK1 to regulate protein levels. A Western blot of an in vitro ubiquitination assay. HEK293T cells were transfected with DKK1-GFP, Ub-HA, and either no NEDD4, FLAG-NEDD4 CS (cysteine mutant) or FLAG-NEDD4 WT (wild-type). Immunoprecipitation for GFP demonstrates HA positivity with NEDD4 WT, indicating ubiquitination of DKK1, but minimal ubiquitination with NEDD4 CS or no NEDD4. Representative image from n = 3 independent experiments. B Ubiquitination assay as in (A), but with DKK2-GFP construct. Minimal ubiquitination is observed in all conditions. Representative image from n = 3 independent experiments. C Western blot of HEK293T cells transfected with DKK1-Myc and FLAG-NEDD4 as indicated. Representative image from n = 5 biological experimental replicates. Quantitation of integrated density normalised to Actin shows that steady-state DKK1 protein levels are reduced with NEDD4 WT compared to either no NEDD4 or NEDD4 CS. ns not significant; **1 P = 0.0012; **2 P = 0.0061 (unpaired t test, two-tailed). D HeLa cells transfected with DKK1-GFP and FLAG-NEDD4 WT or CS, immunostained for FLAG and DKK1. Representative images from n = 3 independent experiments. Mean…

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  Fig. 7. A NEDD4 variant identified in an individual with congenital heart disease has impaired ubiquitination of DKK1. A Pedigree of family with a NEDD4 variant. Genomic sequencing of the trio (parents and proband) revealed a homozygous NEDD4 missense variant c.2297 A > G (p.K766R) in the proband with Tetralogy of Fallot. Hole in the heart was the term used by the family to describe distant relatives, with no clinical data available. B Protein domain structure of NEDD4, indicating the position of the K766R amino acid substitution. C Western blot of in vitro ubiquitination assay. Representative image from n = 3 independent experiments. HEK293T cells were co-transfected with DKK1-GFP, Ub-HA, and either NEDD4 WT, FLAG-NEDD4 CS (cysteine mutant), or FLAG-NEDD4 K766R. Immunoprecipitation for GFP demonstrates HA positivity with NEDD4 WT, indicating ubiquitination of DKK1, but minimal ubiquitination with NEDD4 CS or NEDD4 K766R. D HeLa cells transfected with DKK1-GFP and FLAG-NEDD4 WT, CS, or K766R, immunostained for FLAG and DKK1. Representative images from n = 3 independent experiments. Mean fluorescence intensity of DKK1 and NEDD4 was quantified for individual cells and plotted graphically.…