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

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: major_revision

Abstract
Cardiac outflow tract morphogenesis requires coordinated interactions between multiple cell populations and is dependent on the contribution of cardiac progenitors from the second heart field. While neural crest cells. While neural crest cells have been proposed to impact second heart field development, how they regulate progenitor behaviour remains unclear. Here, we discover neural crest cells are a primary source of Dickkopf-1 (DKK1) which modulates Wnt signalling activity in the second heart field to influence the balance between cardiac progenitor maintenance and differentiation. We show that the ubiquitin ligase NEDD4 regulates DKK1, with disruption of Nedd4 leading to outflow tract defects. We further identify a new NEDD4 variant underlying human congenital heart disease. Our findings uncover an unexpected role for neural crest cells as a rheostat of Wnt signalling in cardiac progenitors, identifying a new molecular pathway promoting outflow tract morphogenesis, and a new causative factor of congenital heart disease.

Introduction
The cardiac outflow tract of the heart provides the pathway for blood to leave the ventricles and circulate to the body via the aorta, or to the lungs via the pulmonary artery. Outflow tract development is complex and requires the contribution of cell types from multiple embryological origins, including second heart field cardiac progenitor cells, cardiomyocytes, smooth muscle, endocardium, and neural crest cells1. During early development, the primitive heart tube expands through the progressive addition of cardiac progenitors from the anterior second heart field, which give rise to the myocardium of the outflow tract and right ventricle. As the outflow tract lengthens, it repositions to overlie the future interventricular septum. Inadequate addition of second heart field cells to the outflow tract leads to insufficient lengthening and underpins conotruncal defects2.

In the developing heart, neural crest cells form components of the outflow tract valves, arteries, and conotruncal septum. Classical defects associated with disrupted cardiac neural crest cell development include persistent truncus arteriosus, where the outflow tract fails to septate into the aorta and pulmonary arteries3. Prior to entering the outflow tract, neural crest cells migrate in close apposition to cardiac progenitors of the second heart field, and are suggested to play a role in modulating the growth dynamics of second heart field derivatives2. However, prior models in which neural crest cells have been surgically or genetically ablated4–7 have not been amenable to elucidate how neural crest cells normally interact with the second heart field to control its growth, differentiation, and morphogenesis.

NEDD4 is an E3 ubiquitin ligase that targets specific protein substrates for ubiquitination to modulate protein function and/or turnover8. Prior studies have determined a necessary role for Nedd4 in heart development, with full knockout mouse models exhibiting DORV and endocardial cushion defects9. However, the cell-type-specific mechanisms by which Nedd4 regulates cardiac development remained unexplored.

In this work, we determine an essential tissue-specific role for Nedd4 in neural crest cells for correct outflow tract development. Wnt1-Cre; Nedd4fl/fl embryos exhibit outflow tract defects typically associated with failed addition of second heart field cells to the outflow tract, suggesting a novel role for Nedd4 and cardiac neural crest cells in regulating second heart field development. Furthermore, we identify DKK1 as a previously unknown substrate of NEDD4-mediated ubiquitination, and find that this functionality is impaired in a human variant of NEDD4 associated with the outflow tract defect Tetralogy of Fallot. We propose NEDD4 controls DKK1 protein levels in neural crest cells, which in turn modulates Wnt signalling in the second heart field to balance cardiac progenitor maintenance versus myocardial differentiation. This provides new insight into the mechanisms underpinning normal outflow tract morphogenesis, and defines a potential pathogenic mechanism underlying congenital heart disease.

Results
Nedd4 is required in neural crest cells for outflow tract development
Prior studies have reported heart defects in Nedd4−/− embryos9. We specifically assessed outflow tract defects in Nedd4−/− embryos, revealing defects at full penetrance with variable phenotypes, most commonly persistent truncus arteriosus (PTA) and transposition of the great arteries (TGA) (Fig. 1A, E, I–L, Q). Nedd4 is broadly expressed throughout all embryonic tissues that contribute to early heart development (Supplementary Fig. 1). To determine the cell types in which Nedd4 activity is critically required, we conditionally removed Nedd4 using tissue-specific Cre-drivers for cell lineages important in outflow tract development. Ablation of Nedd4 in the second heart field (Mef2cAHF-Cre; Nedd4fl/fl) and endothelial/endocardial cells (Tie2-Cre; Nedd4fl/fl) had no discernible impact on the development of the outflow tract (Fig. 1C, D, G, H). In contrast, ablation of Nedd4 in the neural crest cells (Wnt1-Cre; Nedd4fl/fl) led to fully penetrant outflow tract defects such as double-outlet right ventricle (DORV) and overriding aorta (Fig. 1B, F, M–P, Q). Importantly, the outflow tract defects identified in Nedd4−/− and Wnt1-Cre; Nedd4fl/fl embryos are of the same class of outflow tract defect, that being artery-ventricle alignment defects, that present on a variable spectrum of severity. These findings point to a critical cell-type-specific role for Nedd4 in neural crest cells to orchestrate outflow tract development, consistent with previous demonstration of essential roles for Nedd4 in neural crest cells in craniofacial morphogenesis and peripheral nervous system development10–12.

Loss of Nedd4 in neural crest cells induces premature second heart field differentiation
The myocardial component of the outflow tract is derived from cardiac progenitors of the second heart field, with growth of the outflow tract dependent on the continued addition of cardiac progenitors to lengthen the outflow tract, allowing for heart looping and outflow tract rotation to establish correct artery-ventricle alignment1,2. Underpinning the artery-ventricle alignment defects in Wnt1-Cre; Nedd4fl/fl embryos, defective outflow tract lengthening at E10.5 (Fig. 2A and Supplementary Fig. 2) led to incomplete clockwise rotation of the outflow tract at E12.5, and mispositioning of the aorta over the right ventricle (Fig. 2B). Semilunar valve morphology was also altered (Fig. 2B). Such artery-ventricle alignment defects typically stem from failed addition of second heart field cells to the lengthening outflow tract. Since the removal of Nedd4 in second heart field derivatives (Mef2cAHF-Cre; Nedd4fl/fl) did not lead to such outflow tract defects (Fig. 1C, G), this suggests a novel role for Nedd4 and cardiac neural crest cells in regulating second heart field development.

Lineage tracing of neural crest cells using an EGFP reporter revealed that prior to entering the outflow tract, cardiac neural crest cells migrate in close proximity to the second heart field in E9.5 embryos (Fig. 3A, B). This spatial relationship presents an appropriate environment for neural crest cells to interact with the second heart field during development. It is noted that Wnt1-Cre; Nedd4fl/fl embryos did not exhibit any deficiency in cardiac neural crest cells (Fig. 3C), nor were changes in cell proliferation (Fig. 3D) or rate of cell death (Fig. 3E) observed in the tissue domain populated by the neural crest cells and second heart field tissue. Furthermore, expression of the second heart field marker Isl1 was unaffected (Fig. 3F and Supplementary Fig. 3C), as were additional markers of the second heart field and other cardiac components (Supplementary Fig. 3), suggesting the development of other cardiogenic tissues was not affected by loss of Nedd4 function in neural crest cells. Taken together, this further supports a role for disruption of Nedd4 function in neural crest cells, specifically impacting the morphogenesis of the outflow tract, and not due to loss or misspecification of either the neural crest or second heart field tissues.

Myocardial differentiation is initiated as second heart field cardiac progenitors enter the developing outflow tract, where they upregulate expression of various myocardial markers such as myosin heavy and light chains and other myogenic factors13. MF20, which recognises myosin heavy chain, is widely used to assess in vivo differentiation dynamics at the transition zone between the second heart field and outflow tract myocardium13–16. While in wild type embryos the second heart field maintains minimal expression of MF20, Wnt1-Cre; Nedd4fl/fl embryos exhibited precocious MF20 expression in the second heart field (Fig. 3G and Supplementary Fig. 4C, D). The enhanced MF20 expression was also observed in ex vivo second heart field explant cultures (Fig. 3H), supporting the inference that loss of Nedd4 in neural crest cells causes premature differentiation of second heart field cardiac progenitors. Precocious myocardial differentiation is reputed to be one mechanism underpinning defective outflow tract lengthening, due to reducing the pool of cardiac progenitors available for contribution to the growing outflow tract2. To assess the effects of premature differentiation on second heart field deployment into the outflow tract, we used EdU to label the highly proliferative second heart field (relative to the lowly-proliferative outflow tract myocardium) at E9.5, and tracked the extent of deployment of EdU+ve cells into the outflow tract 24h after labelling17,18. After a 1h pulse, EdU labelling of the second heart field was equivalent between genotypes (Fig. 3I). EdU incorporation was terminated 1h after the EdU pulse by injection of unlabelled thymidine, and EdU+ve cells were chased for 24h to assess their movement from the second heart field into the outflow tract. After a 24h chase at E10.5, EdU+ve cells had moved a shorter distance into the outflow tract of Wnt1-Cre; Nedd4fl/fl embryos (Fig. 3J and Supplementary Fig. 5), consistent with impeded deployment of second heart field cells into the outflow tract underpinning outflow tract lengthening defects.

Neural crest cells have been implicated in modulating BMP and FGF signalling in the second heart field to impact outflow tract development19,20. Expression of Bmp4 and Fgf8, as well as downstream signalling components phospho-SMAD1/5/9 and phospho-ERK1/2, was not affected in Wnt1-Cre; Nedd4fl/fl embryos (Supplementary Figs. 3E, F and 6). Hence, we hypothesised that neural crest cells may be regulating the differentiation dynamics of the second heart field via other molecular mechanisms or signalling pathways.

Neural crest-derived DKK1 modulates Wnt signalling in the second heart field
To investigate how loss of Nedd4 in neural crest cells causes premature second heart field differentiation, we used laser capture microdissection to isolate cells from a defined region of the anterior second heart field, as well as neural crest cells and pharyngeal endoderm in the immediate vicinity, from wild-type and Wnt1-Cre; Nedd4fl/fl embryos for transcriptome profiling (Fig. 4A and Supplementary Dataset 1). 897 differentially expressed genes (DEGs) with P<0.05 were identified (Fig. 4B). Gene ontology enrichment analysis of the top 30 DEGs (Fig. 4C) revealed Wnt signalling as the top signalling pathway with differential gene expression (Supplementary Fig. 7A, B). Dkk1, the gene encoding a secreted Wnt signalling antagonist, was the top DEG identified (Fig. 4C). We next performed immunostaining and in situ hybridisation to investigate cell-type specificity of gene expression. Immunostaining revealed DKK1 expression was localised exclusively to neural crest cells (that co-express the neural crest marker AP2α), with no expression in other cell lineages such as the second heart field or pharyngeal endoderm (Fig. 4D). Importantly, DKK1 was only expressed in cardiac neural crest cells that had reached the peri-second heart field region in their migration trajectory, and not in earlier post-delaminating or migrating neural crest cells (Supplementary Fig. 8). In situ hybridisation, in parallel with immunostaining, validated the enhanced Dkk1 expression and enriched DKK1 abundance in cardiac neural crest cells in Wnt1-Cre; Nedd4fl/fl embryos (Fig. 4E–G). Given the function of DKK1 as a canonical Wnt signalling inhibitor, and the proximity of cardiac neural crest cells to the anterior second heart field, we hypothesised that neural crest-derived DKK1 may antagonise Wnt signalling in the adjacent second heart field. Employing β-catenin protein stabilisation and nuclear translocation as a readout of canonical Wnt signalling activity21, immunostaining for active forms of β-catenin (non-pS45, ABC (non-pS37/T41) and pY48922,23) revealed a specific reduction in nuclear localised staining intensity in the anterior second heart field of Wnt1-Cre; Nedd4fl/fl embryos (Fig. 4H and Supplementary Fig. 9A–E). Furthermore, we observed a reduction in Pitx2 expression, a canonical Wnt signalling target gene24 in the second heart field of Wnt1-Cre; Nedd4fl/fl embryos (Supplementary Fig. 9G, H). As well as signalling functions, β-catenin is also involved in regulating cell-adhesion via interactions with cadherins and the actin cytoskeleton25. Recent work has demonstrated requisite roles for cell-adhesion, polarity, and epithelial-like properties of the second heart field in outflow tract development13,26. Immunostaining for markers of cell-adhesion, polarity, and ECM components did not reveal any changes of these cellular attributes in the second heart field region of Wnt1-Cre; Nedd4fl/fl embryos (Supplementary Fig. 10). Taken together, our data support a role for neural crest-derived DKK1 in modulating canonical Wnt signalling activity in the second heart field.

To assess canonical Wnt signalling activity in Wnt1-Cre; Nedd4fl/fl embryos, we intersected our laser-capture tissue mRNAseq dataset with a β-catenin ChIPseq dataset27 to analyse expression of canonical Wnt target genes. We identified 179 differentially expressed Wnt target genes, with 49 upregulated and 130 downregulated (Fig. 4I). To validate and determine cell-type specificity of expression, we performed in situ hybridisation for ten candidate genes on tissue sections of the outflow tract region from E9.5 wildtype and Wnt1-Cre; Nedd4fl/fl embryos. Downregulated Wnt target genes, e.g., Axin2, SP5, Pitx2c, Fam49a, Mical2, were localised to the second heart field (Fig. 4K and Supplementary Fig. 11), consistent with the reduction in nuclear β-catenin in the second heart field. Of note, upregulated expression of Wnt target genes was observed in the cardiac neural crest cells (Fig. 4J), suggesting there may be a cell-autonomous role for Nedd4 in regulating Wnt activity in neural crest cells.

Disruption of Wnt signalling activity underpins outflow defects
To ascertain that disruption of Wnt signalling activity causes outflow tract defects in Wnt1-Cre; Nedd4fl/fl embryos, we manipulated canonical Wnt signalling in vivo by in utero treatment with small molecule activators (CHIR99021) and inhibitors (iCRT3) of Wnt signalling over the developmental time-period when neural crest cells interact with the second heart field (Fig. 5A). Treatment with Wnt agonist CHIR99021, which may enhance Wnt signalling activity, did not rescue the outflow tract defects in Wnt1-Cre; Nedd4fl/fl embryos by E13.5 (Fig. 5B, C and Supplementary Fig. 12B), suggesting that escalating Wnt signalling activity could not counteract the effect of elevated DKK1 activity. However, when Wnt signalling was blocked with the chemical inhibitor iCRT3, Wnt1-Cre; Nedd4fl/+ heterozygous embryos, which do not normally exhibit structural heart defects, now exhibited outflow tract rotation defects (Fig. 5B, C and Supplementary Fig. 12C). Thus, loss of one copy of Nedd4 in neural crest cells sensitises the phenotypic response to disruption of Wnt signalling, and points to genetic interaction between Nedd4 and the Wnt signalling pathway.

To determine if the outflow tract rotation deficiency in iCRT3-treated Wnt1-Cre; Nedd4fl/+ heterozygous embryos is caused by premature second heart field differentiation, we examined the differentiation of second heart field tissue in E9.5+6h iCRT3-treated embryos by MF20 immunostaining (Fig. 5D). iCRT3-treated Wnt1-Cre; Nedd4fl/+ heterozygous embryos exhibited precocious second heart field differentiation, like that in Wnt1-Cre; Nedd4fl/fl knockout embryos (Fig. 5E, F and Supplementary Fig. 4E, F). Furthermore, examination of Wnt signalling activity using β-catenin pY489 and non-pS45 immunostaining revealed reduced nuclear-localised β-catenin in the second heart field of iCRT3-treated Wnt1-Cre; Nedd4fl/+ heterozygous embryos, indistinguishable from that of Wnt1-Cre; Nedd4fl/fl knockout embryos (Supplementary Fig. 13). These findings suggest that a reduced level of canonical Wnt signalling underpins premature second heart field differentiation, and is causative of outflow tract rotation deficiency.

To determine if ectopic Dkk1 overexpression is a contributing factor to outflow tract defects, we generated compound Wnt1-Cre; Nedd4fl/fl; Dkk1+/− embryos in which Dkk1 expression was reduced in the Wnt1-Cre; Nedd4fl/fl background, to test for genetic rescue of outflow tract defects. Compared to Wnt1-Cre; Nedd4fl/fl embryos, Wnt1-Cre; Nedd4fl/fl; Dkk1+/− embryos exhibited improvement in outflow tract development at E13.5 (Fig. 5G, H and Supplementary Fig. 12D), including restoration of aortic valve diameter (Fig. 5I and Supplementary Fig. 14A) partially rescued pulmonary valve diameter (Fig. 5J and Supplementary Fig. 14B) and partial rescue of outflow tract tissue mass (Fig. 5K and Supplementary Fig. 14C). While artery-ventricle alignment was not completely rescued in Wnt1-Cre; Nedd4fl/fl; Dkk1+/- embryos, there was a greater extent of outflow tract rotation (Fig. 5L and Supplementary Fig. 12D). Taken together, the enhanced outflow tract development and rotation suggest that ectopic Dkk1 overexpression is one major factor underpinning defective outflow tract development in Wnt1-Cre; Nedd4fl/fl embryos.

DKK1 is a substrate for NEDD4-mediated ubiquitination
Expression of DKK1, at both transcript and protein levels, was elevated in neural crest cells with loss of Nedd4 function (Fig. 4). Given NEDD4 is an E3 ubiquitin ligase with important roles in regulating protein abundance, we investigated if DKK1 may be a ubiquitinated substrate of NEDD4. Computational analysis on the UbiBrowser bioinformatic platform28 predicts DKK1 can be ubiquitinated, with NEDD4 predicted to be the targeting E3 ligase. Using in vitro ubiquitination assays, we demonstrated that wild type NEDD4 indeed ubiquitinates DKK1, while an inactive version of NEDD4 in which the catalytic cysteine is mutated (NEDD4CS) is unable to ubiquitinate DKK1 (Fig. 6A and Supplementary Fig. 15A). Moreover, NEDD4 specifically ubiquitinates DKK1, but not other DKK family members such as DKK2 (Fig. 6B). This ubiquitination likely targets DKK1 for protein degradation, as overexpression of NEDD4WT in HEK293T cells reduces the steady-state levels of DKK1-Myc, whereas overexpression of NEDD4CS did not affect DKK1-Myc levels (Fig. 6C). Overexpression of DKK1-GFP and FLAG-NEDD4 in HeLa cells revealed an inverse correlation of fluorescence intensity that measured protein abundance between DKK1 and NEDD4 protein, but correlation was observed between DKK1 and NEDD4CS (Fig. 6D and Supplementary Fig. 16A). Expression of a GFP-only construct with NEDD4WT and NEDD4CS revealed no inverse correlation (Fig. 6E), suggesting that DKK1 is the substrate targeted by NEDD4. Furthermore, high-magnification imaging of cardiac neural crest cells in vivo revealed co-localisation of NEDD4 and DKK1 in wild-type neural crest cells, with greatly increased DKK1 fluorescent intensity and abundance in Wnt1-Cre; Nedd4fl/fl cardiac neural crest cells (Fig. 6F), consistent with the inverse correlation of fluorescence intensity that was observed in the in vitro HeLa cell assay. Interestingly, NEDD4 exhibited a particularly enhanced expression and punctate subcellular localisation specifically in cardiac neural crest cells (Supplementary Fig. 1E and Fig. 6F), suggesting a potentially unique function for NEDD4 in this cell type. Taken together, the increased levels of DKK1 in the neural crest cells of Wnt1-Cre; Nedd4fl/fl embryos can be accounted for in part by the absence of NEDD4-mediated ubiquitination and consequent degradation of DKK1.

NEDD4 is clinically implicated in congenital heart defects
We identified a homozygous missense variant in NEDD4 in a child with Tetralogy of Fallot. The child and parents were recruited and assessed as part of an Australian congenital heart disease genome sequencing study29 (now extended to n=363 trios and singletons). Analysis of genome sequencing data from the trio revealed the recessive inheritance of a NEDD4 variant by the proband from parents carrying one allele each (Fig. 7A). The variant NEDD4:c.2297A>G:p.K766R (GenBank: NM_006154.4) changes the reference lysine to an arginine, which is predicted to be damaging by in silico metrics CADD and PolyPhen (CADD: 25, PolyPhen 0.995). No predicted damaging variants were identified in other genes presently known to cause congenital heart disease, making the NEDD4 variant a compelling candidate for causing disease in this family. The parents were not consanguineous. While the family also reported congenital heart disease in distant relatives on the maternal side of the family, no clinical data were available. Furthermore, an additional NEDD4 missense variant was recently documented in another individual with Tetralogy of Fallot in an independent study30, strengthening disease links to this gene.

The K766R amino acid change is located in the HECT domain of NEDD4, which is responsible for ubiquitin ligase activity (Fig. 7B). Given that NEDD4 can ubiquitinate DKK1, in vitro assays to assess the effect of K766R amino acid substitution on NEDD4 function revealed NEDD4 K766R had reduced capacity to ubiquitinate DKK1 (Fig. 7C and Supplementary Fig. 15B). Furthermore, analysis of fluorescence intensity revealed no correlation between DKK1 and NEDD4 K766R protein levels (Fig. 7D and Supplementary Fig. 16B), confirming NEDD4 K766R is a loss-of-function variant for ubiquitination of DKK1.

To investigate the in vivo consequences of NEDD4 K766R substitution, we generated a CRISPR-engineered Nedd4K766R/K766R mouse model. Homozygous embryos of this mutant mouse strain were present at expected mendelian ratios at all developmental stages and phenotypically normal (Fig. 7E). Examination of hearts in E15.5 to E17.5 Nedd4K766R/K766R homozygous embryos revealed a range of developmental cardiac defects (Fig. 7F). While artery-ventricle alignment defects such as overriding aorta, which is a clinical feature of Tetralogy of Fallot, were not present, there were defects consistent with deficiency of right ventricular development. This included a thin ventricular wall which was restricted to the right ventricle, often with a rugged appearance (Fig. 7G, H) suggestive of defective right ventricular compaction, a disorganised interventricular septum, and ventricular septal defects (Fig. 7G). Hence, Nedd4K766R/K766R embryos exhibit heart defects, including some features of Tetralogy of Fallot, such as right ventricular wall and interventricular septal defects, implicating the NEDD4 K766R variant as a cause of congenital heart disease.

Taken together, NEDD4 regulation of DKK1 in neural crest cells unveils a novel mechanistic paradigm in heart development, which when disrupted leads to heart defects in mice and humans.

Methods
Mice
All experiments were carried out in accordance with the ethical guidelines of the University of South Australia Animal Ethics Committee. Mice are housed with a 12h dark/light cycle, with temperature maintained between 19-23°C and 40-70% relative humidity. Mice used for timed matings were between 6-12 weeks of age. All mouse lines were maintained on a mixed C57BL/6 background. Nedd4-/- mice and Nedd4fl/fl have been described previously49,50. To remove Nedd4 in specific tissues, we crossed Nedd4fl/fl females to Wnt1-Cre;Nedd4fl/+51, Mef2cAHF-Cre;Nedd4fl/+52 or Tie2-Cre;Nedd4fl/+53 males. Wnt1-Cre;Nedd4fl/fl mice were intercrossed with Dkk1+/−47 mice (Wnt1-Cre;Nedd4fl/+ males crossed to Nedd4fl/fl;Dkk1+/− females) to generate Wnt1-Cre;Nedd4fl/fl;Dkk1+/- embryos (as well as littermate controls) for assessing the rescue effect of DKK1 dosage compensation. The following reporter lines were also intercrossed with Wnt1-Cre;Nedd4fl/+ mice; Mef2cAHF-LacZ54 and Z/EG55. Nedd4K766R mice were generated using CRISPR targeting. As embryos that contained the variant on both chromosomes (homozygotes) or in combination with a frameshifting indel (compound heterozygotes) were likely to have compromised cardiac function, we used a dual template competition strategy. SaCas9 KKH mRNA was microinjected with two single-stranded DNA oligonucleotide donor templates. The first oligonucleotide contained silent mutations at residues K766, I765 & L764 and retained WT function when integrated (sequence: TTCCGACGCGTTTCCTTTCCTTCTAGGGATTTTTTGAACTGATACCACAGGATttgATtAAaATATTTGATGAAAATGAGCTAGAGGTAAGAACTATTTCTGCATGTGCTTGGGAATGTAG). The second oligonucleotide encoded the K766R variant and a silent CTC (L764) > TTG (L764) mutation to abolish the PAM site and prevent re-cutting of the targeted DNA by the nuclease (sequence: TTCCGACGCGTTTCCTTTCCTTCTAGGGATTTTTTGAACTGATACCACAGGATTTGATCAGGTATTTGATGAAAATGAGCTAGAGGTAAGAACTATTTCTGCATGTGCTTGGGAATGTAG). The K766R oligonucleotide also introduced a BclI recognition site for subsequent genotyping. Both oligonucleotides had 60 bp homology arms on either side of the targeted mutations.

To obtain mouse embryos of defined gestational ages, mice were mated in the evening, and the morning of vaginal plug formation was counted as E0.5. In all experiments, stage-matched embryo littermates were selected for comparison using stage-appropriate parameters such as somite number, crown-rump length, and forelimb/digit maturity. For embryonic analyses, we did not genotype for sex.

Drug treatments of pregnant dams
Pregnant dams were intraperitoneally injected with 16.6 mg/kg CHIR99021 (Selleck Chem) or 10 mg/kg iCRT3 (Selleck Chem) at the indicated time points in 15% DMSO, 30% PEG 300, PBS vehicle. Embryos were harvested at E13.5 or E9.5, and processed for immunostaining.

Histology and immunostaining
Embryos were fixed in 4% paraformaldehyde in PBS. Cryosections were stained with Hematoxylin and Eosin to classify heart defects at E15.5-E17.5. For immunolabelling, cryosections, whole embryos, or fixed cells were blocked in 10% DAKO block, 0.2% BSA, 0.2% Triton X-100 in PBS, and stained with the indicated primary antibodies. Antibodies used were rabbit anti-NEDD4 (Abcam 14592) 1:300; rabbit anti-NEDD4 (purified serum, gift from S. Kumar) 1:300; mouse anti-alpha smooth muscle actin (Sigma A2547) 1:2000; rat anti-CD31 (Biolegend 102502) 1:150; goat anti-SOX10 (R&D Systems AF2864) 1:200; chicken anti-GFP (Abcam ab13970) 1:1000; mouse anti-Isl1 (DSHB 39.4D5 or 40.3A4) 1:50; mouse anti-MF20 (DSHB) 1:100; goat anti-DKK1 (R&D Systems AF1765) 1:200; mouse anti-AP2a (DSHB 3B5) 1:20; rabbit anti-beta-catenin non-pS45 (Cell Signaling Technology 19807) 1:200; mouse anti-beta-catenin ABC (8E7) (Millipore 05-665) 1:100; mouse anti-beta-catenin pY489 (DSHB) 1:50; mouse anti-FLAG (Sigma F3165) 1:1000; mouse anti-GM130 (BD 610822) 1:100; rabbit anti-phospho-Histone H3 (Millipore 06-570) 1:500; rabbit anti-cleaved-Caspase-3 (Cell Signaling Technology 9661) 1:500; rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology 13820) 1:200; rabbit anti-phospho-ERK1/2 (Cell Signaling Technology 4370) 1:100; goat anti-Scribble (Santa Cruz Biotechnology sc-11048) 1:50; rabbit anti-Laminin (Sigma L9393) 1:1000; rabbit anti-Fibronectin (DakoCytomation A0245) 1:1000; mouse anti-N-cadherin (Cell Signaling Technology 14215) 1:100; Alexa Fluor 647 conjugated Phalloidin (Invitrogen) 1:200. EdU staining was performed following manufacturers recommendations (Invitrogen Click-iT EdU Alexa Fluor 555 Cell Proliferation Kit). Slides were mounted in Fluoro-mount G with DAPI (ProSciTech). Histology and whole embryo/heart images were captured using Olympus DP2-AOU1.1 software or Improvision OpenLab 5 software. Confocal images were acquired with a Zeiss LSM800 microscope and ZEN 2.6 (Blue edition) software.

Fluorescence intensity and image quantitation
Mean fluorescence intensity (MFI) was measured using ZEN 3.4 (Blue edition) software (Zeiss) and represents the total fluorescence intensity of the region of interest, normalised to the area of the region of interest. For MF20 measurements in the second heart field at E9.5, a line of 100 μm was drawn downwards perpendicular to the outflow tract, and the single layer of cells of the second heart field (pericardial wall) was traced around only to the end of the 100 μm line. Hence, an equivalent area of MF20 fluorescent intensity was measured across different embryo samples. A minimum of 2 and up to 4 sagittal sections per embryo (at the medial position relative to the left and right side of the second heart field) were used to calculate MFI as described above, and averaged. Individual points on graphs represent the mean value calculated from 1 embryo. A minimum of 5 and up to 12 embryos per genotype were used for MF20 quantitation. In addition, MF20 MFI in the second heart field was normalised to MFI measurements taken of the adjacent unaffected atria within the same acquired microscopy image, and presented in the Supplementary Figs. MFI was measured from a 100 μm length of MF20+ve atria tissue, with the normalised value representing MFI in the second heart field / MFI in atria.

For MF20 MFI measurements of SHF explants, the entire DAPI+ve region was selected, and MFI was measured in this region of interest. Individual values represent MFI for independent explants (i.e., biological replicates) of the indicated genotype.

For DKK1 MFI measurements, a minimum of three and up to six sagittal sections per embryo were measured and averaged. An area encompassing the cardiac neural crest cells was drawn using the boundaries of the basal sides of the pharyngeal endoderm and dorsal pericardial wall for width, and ~100 μm for height (area ~2000–3000 μm2). Given DKK1 is only expressed in cardiac neural crest cells in the pericardiac region, it was not possible to assess any other DKK1+ve areas of immunostaining for the purposes of normalisation to an unaffected structure.

For β-catenin immunostaining, high-magnification images were assessed, and co-localisation of β-catenin immunostaining with DAPI fluorescence was counted as nuclear β-catenin+ve. A 100 μm length of second heart field nuclei were counted, which ~20–25 nuclei per section. β-catenin+ve nuclei were represented as a percentage of total nuclei counted. A minimum of 3 and up to 8 sagittal sections were quantified per embryo and averaged. Individual points on graphs represent the mean value calculated from 1 embryo. A minimum of 4 and up to 9 embryos per genotype were used for β-catenin quantitation.

For EdU quantitation in SHF after 1h pulse, a 100 μm length of second heart field nuclei were counted, which ~20–25 nuclei per section. EdU+ve nuclei were represented as a percentage of total nuclei counted. Five sagittal sections were quantified per embryo and averaged. Individual points on graphs represent the mean value calculated from 1 embryo, with 6 embryos assessed per genotype. For EdU migration front distance at E10.5 after 24h chase, the EdU+ve migration front in the OFT was measured as the distance (in μm) of the last EdU+ve cell in the MF20+ve OFT from the pericardial wall. Three to four sagittal sections were quantified per embryo and averaged. Individual points on graphs represent the mean value calculated from 1 embryo. A minimum of 5 and up to 6 embryos per genotype were used for quantitation. EdU+ve migration front distance was also normalised to atrio-ventricular valve primordium thickness (an unaffected structure present in the same microscopy images) as a means to account for any variation in embryo size, and is presented in the Supplementary Figs.

For HeLa cell assays, the entire cell outline was traced to define the region of interest, and MFI was measured for NEDD4 and DKK1 or GFP-only within this region of interest. Approximately eight images were acquired at random per transfection condition, from n=3 experiments. Data points on graphs represent individual cells, with a minimum of 40 cells measured per transfection condition in total.

Semi-quantitative optical density measurements of whole-mount in situ hybridisation were performed in ImageJ. Images were subjected to colour deconvolution to generate 8-bit grayscale images of BCIP/NBT substrate staining. Mean grey values were calculated from a region of interest corresponding to the second heart field/dorsal pericardial wall. Optical density was calculated using the formula OD=Log10(255/mean grey value). A minimum of three and up to seven whole-mount embryos per genotype were assessed.

In situ hybridisation
Whole embryos or cryosections were hybridised with digoxigenin-labelled antisense probes for the following genes Dkk1, Dlx2, Prrx1, Msx1, Msx2, Col1a1, Axin2, SP5, Pitx2c, Fam49a, Mical2, Tbx1, Tbx20, Isl1, HoxB1, Fgf8, Bmp4. Signal was detected using anti-DIG-AP conjugated Fab fragments (Roche), and staining with NBT/BCIP (Roche). Whole embryos were imaged with Olympus CZX10, and tissue sections were imaged with Olympus IX73.

β-galactosidase staining
Fixed embryos were incubated in staining solution: 19 mM sodium dihydrogen phosphate, 81 mM Disodium hydrogen phosphate, 2 mM MgCl2, 5 mM EGTA, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal substrate, at 37°C until blue staining was sufficient.

SHF explant
The second heart field region was dissected from E9.5 embryos by removing the head above pharyngeal arch 2, cutting off the outflow tract and heart, cutting down the lateral sides of the pharynx to create a tissue flap, and then separating this second heart field tissue flap at the posterior end from the rest of the embryo. This was transferred to tissue culture dishes coated with 50 μg/ml Collagen and 50 μg/ml Fibronectin, and cultured for 5 days in 5% FCS + DMEM. Explants were fixed in 4% PFA.

SHF deployment assay
In methods similar to refs. 17,18, pregnant dams were intraperitoneally injected with 100 mg/kg EdU (5-ethynyl-2’-deoxyuridine) at E9.5 to pulse-label proliferating cells. Embryos were harvested after 1h to assess the extent of EdU labelling. To chase EdU-labelled proliferating cells, pregnant dams were injected with 500 mg/kg of thymidine 1h after the EdU pulsing period to compete for remaining EdU label, and were harvested 24h later at E10.5. Embryos or tissue sections were stained with Click-iT EdU Kit Alexa Fluor-555 conjugated (Life Technologies) following staining with primary antibodies.

Laser capture and RNA preparation
Tissue preparation and laser capture were based on ref. 56. Briefly, mouse embryos were fresh-frozen in OCT, cryosectioned, and collected on PEN membrane slides. Slides were stained with 1% Cresyl Violet to visualise tissue sections, air-dried, and then laser dissection was performed promptly (Leica LMD) to specifically capture the central anterior second heart field region (with associated neural crest and pharyngeal endoderm tissue). RNA was prepared from tissue by lysing in 4 M guanidine isothiocyanate solution (Invitrogen) and precipitating with sodium acetate and ethanol. After 75% ethanol wash, the RNA pellet was resuspended in 12 μl H2O, with 1 μl reserved for Bioanalyser quality control of RNA, and 9.5 μl used for subsequent cDNA synthesis.

RNA sequencing and bioinformatics analyses
Isolated RNA samples were submitted to the ACRF Genomics Facility for library preparation and sequencing. Extracted RNA was quantified using the Bioanalyser Picochip, and 9.5 μl (containing 5–17 ng of RNA) was input into SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio) for generation and amplification of double-stranded full-length cDNA (using nine cycles of LD-PCR). Prior to library preparation, cDNA was sheared to 200–500 bp fragments using the Covaris system. NGS libraries were prepared using the ThruPLEX DNA-seq Kit (Takara Bio) together with the DNA Unique Dual Index Kit (Takara Bio), using 11 cycles of PCR for library amplification. Indexed libraries from three biological replicates for each wild type and Wnt1-Cre; Nedd4fl/fl samples were pooled equally for sequencing using 2×75 bp reads on an Illumina NextSeq High Output kit. Raw data, averaging 47 million reads per sample, were analysed and quality checked using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were mapped against the mouse reference genome (mm10) using the STAR spliced alignment algorithm57 (version 2.6.1d with default parameters and --chimSegmentMin 20, --quantMode GeneCounts), returning an average unique alignment rate of 81%. The resulting mapped reads were found to possess a high rate of duplicates (≈64%). These were marked and subsequently removed using Picard Tools (version 2.16.0)58 leaving, on average 18 million deduplicated reads per sample. Differential expression analysis was evaluated from TMM normalised gene counts using R (version 3.2.3), edgeR (version 3.3)59 and Degust60 following protocols as described61. Alignments were visualised and interrogated using the Integrative Genomics Viewer v2.3.8062. Graphical representations of differentially expressed genes were generated using Glimma63, Degust60, and MaGIC Volcano Plot Tool64. Gene ontology analysis was performed using DAVID65,66.

Ubiquitination assays
For cell-based ubiquitination assays, HEK293T cells originally obtained from ATCC (CRL-3216) were grown in DMEM supplemented with 10% foetal calf serum. Plasmid transfections were carried out with Lipofectamine 2000 (Thermo Fisher). HEK293T cells were co-transfected with an equal amount of pcDNA3-FLAG-tagged NEDD4 WT, NEDD4 CS, or NEDD4 K766R, pCDNA3-HA-tagged ubiquitin (gift from Sharad Kumar), and GFP-tagged DKK1 or DKK2. The DKK1-GFP and DKK2-GFP constructs were generated by cloning a PCR fragment of the open reading frames from pCS2-DKK1-FLAG (Addgene plasmid #16690) or pCS2-DKK2-FLAG (Addgene plasmid #15495) in frame to EGFP N3 using EcoRI and BamHI restriction sites. Cells were grown for 24–48h at 37°C with 5% CO2 in a humidified atmosphere. Transfected cells were treated with 10 μM MG132 and 100 μM of Chloroquine for 3h prior to protein extraction.

Immunoprecipitation and western blot
Cells were lysed in NP-40 buffer (137 mM NaCl, 10 mM Tris pH 7.4 (108319 Merck), 10% glycerol, 1% NP-40, 2 mM sodium fluoride, and 2 mM sodium vanadate) containing Complete Protease Inhibitors. For purification of GFP-fusion proteins, GFP-Trap beads were used following the manufacturer’s protocols (gta-10 Chromotek). Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (88585 Thermo Fisher). Membranes were blocked in 5% skim milk powder in tris-buffered saline (TBS, 50 mM Tris pH 7.5, 150 mM NaCl) with 0.1% Tween 20 (P1379 Merck). The following primary antibodies were used: mouse anti-HA (6E2, Cell Signaling Technologies 2367) 1:1000; mouse anti-Myc (9B11, Cell Signaling Technologies 2276) 1:1000; rabbit anti-GFP (ClonTech 632592) 1:1000; Goat anti-DKK1 (R&D Systems AF1765) 1:500; mouse anti-beta-Actin (Sigma A5441) 1:1000; rabbit anti-14-3-3 zeta C-16 (sc1019 Santa Cruz) 1:1000. Western blot images were captured using BioRad Image Lab Touch Software or FUJIFILM ImageQuant LAS4000 software. Densitometry of all western blot data was performed with ImageJ.

HeLa protein abundance correlation assays
HeLa cells (kindly provided by the laboratory of Prof. Stuart Pitson (Centre for Cancer Biology, Adelaide) were grown in DMEM supplemented with 10% foetal calf serum. Plasmid transfections were carried out with X-tremeGENE HP (Roche) in eight-well glass-bottom imaging slides (Eppendorf). Cells were co-transfected with an equal amount of FLAG-tagged NEDD4 WT, NEDD4 CS, or NEDD4 K766R, and DKK1-GFP. Cells were grown for 24–48h at 37°C with 5% CO2 in a humidified atmosphere. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, and immunostained following the protocol described above. Mean fluorescent intensity for each cell was calculated using ZEN 3.4 software (Zeiss). Approximately eight images were acquired at random per transfection condition, from n=3 experiments. Data points on graphs represent individual cells, with a minimum of 40 cells measured per transfection condition in total.

Human patient recruitment
Ethics approval was obtained from the Sydney Children’s Hospital Network Human Research Ethics Committee (approval number HREC/16/SCHN/73). The family was recruited through the Kids Heart Research DNA Bank at the Children’s Hospital at Westmead, Sydney, Australia. Informed, written consent was obtained from the family. Heart defects in the proband were confirmed by echocardiography.

Patient sequencing and genome data analysis
Genomic DNA was extracted, as described previously67. DNA sample libraries were prepared using the Illumina TruSeq Nano DNA HT Library Prep Kit and genome sequenced (Illumina HiSeq X Ten, 150 base paired-end reads) at Genome.One, Garvan Institute of Medical Research, Sydney, Australia. Calling and annotation of sequencing data were performed as per ref. 29. In short, sequence reads were mapped to the human reference genome hg38 using Burrows-Wheeler Aligner (BWA-mem v0.7.12)68. Single-nucleotide variants (SNVs) and small insertions or deletions were called using Platypus v0.8.169. The variant call files (VCFs) were annotated using ANNOVAR v2016 Feb 0170. Variants were prioritised as described in ref. 29 by screening for a list of curated CHD genes, followed by an unbiased comprehensive analysis for all genes. Variant frequencies were in reference to the gnomAD database (v2.1.1)71. A variant of interest identified in the family, NEDD4:c.2297A>G:p.K766R (GenBank: NM_006154.4), had an allele frequency of 0.004438.

Statistics
In all graphical representations, individual values represent independent biological replicates. Unless otherwise specified, P values were calculated using unpaired two-tailed t test. For grouped analysis, significance was assessed using 1 or two-way ANOVA multiple comparisons. All graphs and statistical analyses were performed using GraphPad Prism 10.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Wiszniak et al. describe a striking phenotype resulting from the ablation of Nedd4 specifically in neural crest cells. The authors report that this genetic alteration leads to premature differentiation of second heart field (SHF) progenitors, resulting in outflow tract (OFT) defects, including shortening and mispositioning. Mechanistically, these defects are attributed to upregulated expression of DKK1 in neural crest cells, which under normal conditions express this gene at lower levels. This, in turn, leads to reduced Wnt signaling within the SHF and consequently, premature differentiation of its progenitors.\nWhile the research is well conducted and supported by a strong genetic approach, a more direct elucidation of neural crest cell function during normal OFT development would require in vivo evidence that NEDD4-mediated ubiquitination and degradation of DKK1 occurs specifically within neural crest cells.\nI have a few additional comments:\n1. Could the authors provide detailed data on Nedd4 expression? Is it expressed in branchial arches, the heart, and neural crest cells?\n2. In Fig 1, Could the authors comment on differences in distributions of OFT defects observed in Nedd4−/− vs Wnt1-Cre; Nedd4fl/fl embryos?\n3. Fig 3C, why is there no apparent loss of Isl1+ SHF tissue, given the precocious differentiation into myocardium?\n4. Why is enhanced MF20 expression interpreted as a marker of premature SHF differentiation? Please clarify for the readers.\n5. In Figure 4H, β-catenin appears to localise primarily at cell-cell junctions rather than in the nucleus. In the mutants, the reduction is most apparent at the junctional level. The reviewer does not observe a reduction in nuclear β-catenin. Could the authors clarify this, possibly by providing higher magnification images? The evidence for reduced Wnt signaling in the SHF, based only on β-catenin staining, is not fully convincing. Could the authors validate this using additional Wnt reporters such as TCF/Lef:H2B-GFP, Axin2-lacZ, etc..?\n6. Could the authors discuss why enhancing Wnt signaling with CHIR99021, a well-known Wnt pathway activator, failed to rescue the OFT defects in Wnt1-Cre; Nedd4fl/fl embryos? Could the author analyse Wnt signalling activity under CHIR99021 and iCRT3 conditions?\n7. In Figure 4G, there appears to be no upregulation of MF20 in Wnt1-Cre; Nedd4fl/fl embryos. Could the authors clarify this inconsistency?\n8. In Figure 4G, the concept of an \"EdU migration front\" is unclear. How is this defined, and how does it relate to the extent of SHF incorporation into the myocardium?\n9. Lastly, could the authors discuss the phenotypic differences between Nedd4K766R embryos and Nedd4fl/fl embryos, and explain why the K766R mutation, which replicates a human variant, does not phenocopy neural crest-specific loss of Nedd4?", "reviewer_2": "In the manuscript “Neural crest cell-derived DKK1 modulates Wnt signalling in the second heart field to orchestrate cardiac outflow tract development, “Wiszniak et al examine the role of NEDD4 expressed in neural crest cells during cardiac development. The authors demonstrate that the deletion of NEDD4 results in defects in the development of the cardiac outflow tract and the positioning of the great arteries relative to the right and left ventricles. The authors examine the evidence that this effect is due to increased levels of Dkk1 in their mutants. While the idea is very interesting, the data does not support the authors’ conclusions. In many experiments, the experimental setup is not appropriate for what the authors intended to study.\nMajor Concerns:\n1) A major concern is that for multiple types of experiments, normalization is not provided.\na) For example, the length of the outflow tract is not normalized to an unaffected embryonic structure (e.g., otic vesicle) to control for embryo size in Figure 2A. In addition, it is not clear how authors consistently found a boundary between the proximal OFT and the right ventricle. This needs to be described. Alternatively, the RV needs to be included in the measurements because it is difficult to find such a boundary, and customarily, the RV is included in the measurements of OFT length at E10.5.\nb) Another example is the measurement of valve diameter in Figure 5G. This measurement also needs to be normalized to an unaffected embryonic structure to control for embryo size. These normalizations are essential since no single embryo develops at the same rate, and differences in embryo stages and sizes are common even within the same litter.\nc) It is not proper to report fluorescence intensity as a measurement without normalization, since fluorescence intensity will vary based on many factors beyond the experimenter’s control, such as fluctuations in laser power, differences in cover slip thickness, and the exact plane of focus. Check the Bioimaging guide for best practices (https://www.bioimagingguide.org/03_Image_analysis/Intensity.html)\n2) Lines through the ventricles seem to be drawn in random orientation in the mutants in Figure 5. While in controls, the line is drawn through the lumens of LV and RV, in the mutants, the line does not appear to go through the lumen of LV. Other section levels or coronal sections may be more appropriate. Multiple levels like those shown in Fig. 2B may also work well to demonstrate altered alignment.\n3) On line 14, page 6, the authors state that NC numbers are not affected in the mutants. Since this is very important for the rest of the study, the authors need to provide quantification to support this statement.\n4) On line 19, page 6, the authors state that the expression of additional SHF markers were not affected in the mutants. However, Sup. Fig 2 shows that Hoxb1 and BMP4 are downregulated and Fgf8 is upregulated in the mutants. Please state how many times each experiment was conducted; how many controls and mutants were analyzed for each staining, and provide quantification.\n5) For the experiment with the explant in Fig. 3E, FACS is more appropriate to determine the proportion of MF20+ cells and intensity. Western blot is also appropriate, but the measurements shown are not up to the accepted standards for quantification.\n6) The EdU experiment in Fig. 3G is not appropriate to show cell migration. For this experiment, EdU pulse-chase needs to be implemented: after one hour of EdU, thymidine needs to be injected to compete EdU off. Otherwise, the experiment in 5G can be interpreted as a decrease in proliferation rather than migration. See doi: 10.1016/j.ydbio.2016.02.017 as an example of such an assay in vivo. Also see papers by Elaine Fuchs, as additional examples for how this is done.\n7) Figure 4H. There is no nuclear β-catenin in controls or mutants. It is not clear what is being measured and how.\n8) Figure 4K: Expression of Axin2 can be used as a readout for Wnt signaling. However, the authors point to Axin2 staining in the atrial chamber in a control embryo (arrowhead) and not the SHF in the dorsal pericardial wall contiguous with the OFT. The SHF appears not to express Axin2 in the control. In the mutant shown, the Axin2 staining did not seem to work because there is no staining in the atrial chamber, a tissue unaffected by the neural crest. This is an important experiment, and it needs to be done properly to determine whether Wnt signaling is indeed downregulated in the SHF in the mutants.\n9) Figure 5E: MF20 IF intensity is reported without normalization and thus is unreliable. In addition to properly measuring and normalizing MF20 intensity, the authors should measure the length of SHF occupied by the ectopic MF20+ cells.\n10) Figure 6: How do authors explain the mechanism by which NEDD4 regulates the ubiquitination of Dkk1? Dkk1 is a secreted protein (i.e., it is present inside the ER, Golgi, and secretory vesicles), while NEDD4 is a cytoplasmic protein that is always outside the ER, Golgi, etc. These two proteins are never in the same cellular compartment.\n11) Figure 7G. The hearts look smaller in the mutants, suggesting that VSD and other “defects” may be due to delayed embryo development at E15.5 in the mutants. E18.5 is a more appropriate time point for the evaluation of structural heart defects.\na. Are Nedd4-K766R homozygous mutants viable?\nMinor:\n1) on line 14 page 4, the authors state that “neural crest cells form structural components of the outflow tract valves.” This is incorrect. Neural crest cells die at late gestation and do not contribute to mature outflow tract valves. See papers from Jonathan Epstein lab.\n2) On line 19-21 page 4, the authors state “However, prior models in which neural crest cells have been surgically or genetically ablated4-7 have not been amenable to elucidate how neural crest cells interact with the second heart field to control its growth, differentiation and morphogenesis.” This is inaccurate. The authors should mention the work from Margaret Kirby’s lab showing the importance of NC in regulating BMP4-FGF8 cross talk between NCCs and SHF in preventing precocious differentiation of cardiac progenitors in the SHF.", "reviewer_3": "The MS by Wiszniak et al reports a novel regulatory mechanism whereby the ubiquitin ligase NEDD4 affects the protein levels of DKK1in neural crest cells that modulates Wnt signaling thereby affecting the timing of second heart field differentiation and outflow tract morphogenesis. A combination of in vivo genetic studies in mice and in vitro examination of NEDD4 function provide convincing evidence for the major conclusions of the study. Notably, a DDK1 deficient mouse is provides evidence for a rescue of the NEDD4 deficiency in neural crest cells which is important support for the proposed mechanism. In addition, variants in NEDD4 were identified in individuals with congenital heart malformations and loss of function demonstrated experimentally.\nThis is a strong study with convincing data to support a new regulatory contribution of Wnt signaling in cardiac outflow tract development with human clinical implications.\nMinor comments\n1. The title does not mention NEDD4 which is a major focus of the study.\n2. Were abnormalities in outflow tract endocardial cushions or semilunar valve primordia observed?\n3. The Discussion is long and includes information on BMP and FGF signaling that seem peripheral to the current study."}, "author_response": "Manuscript # NCOMMS-25-39806-T (Wiszniak et. al.)\nWe thank the reviewers for their time and expertise in reviewing our manuscript. Please find our detailed responses to the reviewers concerns and Reviewer Figures 1-3 below.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Wiszniak et al. describe a striking phenotype resulting from the ablation of Nedd4 specifically in neural crest cells. The authors report that this genetic alteration leads to premature differentiation of second heart field (SHF) progenitors, resulting in outflow tract (OFT) defects, including shortening and mispositioning. Mechanistically, these defects are attributed to upregulated expression of DKK1 in neural crest cells, which under normal conditions express this gene at lower levels. This, in turn, leads to reduced Wnt signaling within the SHF and consequently, premature differentiation of its progenitors.\nWhile the research is well conducted and supported by a strong genetic approach, a more direct elucidation of neural crest cell function during normal OFT development would require in vivo evidence that NEDD4-mediated ubiquitination and degradation of DKK1 occurs specifically within neural crest cells.\nAssays to investigate protein ubiquitination typically require overexpression of constructs in cell lines to yield very high levels of expression, combined with MG132/Chloroquine treatment to inhibit protein degradation, with 1-2mg of total cell lysate often used as input into immunoprecipitations for in vitro ubiquitination assays. While we demonstrate that NEDD4 ubiquitinates and degrades DKK1 in several in vitro assays (eg. overexpression experiments in HEK293 and HeLa cells, Figure 6A,D) demonstrating ubiquitination in vivo remains challenging. It is not possible to yield enough protein lysate from E9.5 neural crest cells in vivo to perform any such ubiquitin assay/western blot/TUBE assay. We have developed a protocol to generate neural crest cells from human iPSCs, with good success after a standardised 10 day differentiation protocol. scRNAseq of these iPSC-derived NCCs reveals broad expression of markers of NCCs including TFAP2A (AP2a) and SOX10 (Reviewer Fig. 1A). DKK1 is only expressed in a subset of neural crest cells in vivo at E9.5 (specifically the cardiac neural crest cells (Figure 4D,E, Supp. Fig. 10, and Reviewer Fig. 1C E9.5 cluster 14), and indeed we find that a small proportion of iPSC-derived NCCs express DKK1 (Reviewer Fig. 1A cluster 12) suggesting that these likely correspond to cardiac NCCs. Furthermore, these cells share expression profiles of DKK1+ve cardiac NCCs in vivo (eg. SOX10 -ve, HAND1+ve, HAND2+ve, GATA3+ve). While we have attempted to perform some in vitro assays using these iPSC-derived NCCs, the proportion of cells expressing DKK1 is too low to yield enough experimental material to proceed with any successful assays (eg. Reviewer Fig. 1B). We are continuing with development of our iPSC-derived NCC differentiation protocol, and hope in future to enrich for DKK1+ve cardiac neural crest cell populations, for which there is no currently published protocol and is a missing tool in the cardiac neural crest cell research field.\nTowards providing “in vivo evidence that NEDD4-mediated ubiquitination and degradation of DKK1 occurs specifically within neural crest cells”, we now present additional figures to clarify the cardiac neural crest cell-specific expression pattern of DKK1. In Supplementary Fig. 10 we show that DKK1 is only expressed in neural crest cells via co-localisation with the neural crest cell marker AP2a; hence if NEDD4 is regulating DKK1, this must be only occurring in neural crest cells. Furthermore, DKK1 is not expressed in all neural crest cells, but rather only becomes expressed once neural crest cells have reached the second heart field region in their migration trajectory (Supp. Fig. 10), suggesting specificity of DKK1 expression to the cardiac neural crest. We have now also performed immunostaining for NEDD4 and DKK1 in cardiac neural crest cells in vivo (Figure 6F), and demonstrate subcellular co-localisation, suggesting that it is feasible that NEDD4 could be interacting with and ubiquitinating DKK1 in neural crest cells in vivo. Interestingly, NEDD4 demonstrates a particularly heightened and punctate expression pattern, specifically in cardiac neural crest cells (Supp. Fig, 1E), suggesting NEDD4 may play unique functional roles in this cell type.\nI have a few additional comments:\n1. Could the authors provide detailed data on Nedd4 expression? Is it expressed in branchial arches, the heart, and neural crest cells?\nWe have previously documented expression of Nedd4 in the branchial arches and neural crest in our publication (Wiszniak et. al. 2013, Dev Biol). Additionally, we have now performed detailed immunostaining for NEDD4 at E9.5, which we present in Supplementary Figure 1. This demonstrates broad expression of NEDD4 throughout all tissues including neural crest cells, pharyngeal arches, heart and second heart field. We also show that expression of NEDD4 is specifically absent in neural crest cells of Wnt1-Cre; Nedd4fl/fl embryos. Notably, this new data identifies heightened expression and altered localisation of NEDD4 in cardiac neural crest cells, which was previously unknown.\n2. In Fig 1, Could the authors comment on differences in distributions of OFT defects observed in Nedd4−/− vs Wnt1-Cre; Nedd4fl/fl embryos?\nWhile the distribution of OFT defects differs between Nedd4-/- and Wnt1-Cre; Nedd4fl/fl embryos, the broader class of OFT defect observed is the same, ie. artery-ventricle alignment defects. We have now modified a sentence in the manuscript to emphasise this more clearly (“Importantly, the outflow tract defects identified in Nedd4-/- and Wnt1-Cre; Nedd4fl/fl embryos are of the same class of outflow tract defect, that being artery-ventricle alignment defects, that present on a variable spectrum of severity.”). Heart defects often present on a scale of variable severity. In terms of artery-ventricle alignment defects, severity can range due to the degree of defective outflow tract rotation. A small reduction in OFT rotation may lead to overriding aorta (where the aorta is positioned between right and left ventricles, but pulmonary is normal), further reduction in OFT rotation may lead to DORV (where the aorta and pulmonary are both aligned with the right ventricle), with greatly reduced OFT rotation leading to TGA (where the aorta and pulmonary arteries are completely switched ie. aorta aligns with right ventricle and pulmonary with left ventricle). PTA is the most severe defect, with no OFT rotation and no aorticopulmonary septum, leading to a single OFT vessel connecting to the heart, and straddling both ventricles. The distribution of OFT defects observed in Nedd4-/- and Wnt1-Cre; Nedd4fl/fl embryos is consistent with Nedd4-/- embryos exhibiting defects at the very severe end of this spectrum, while Wnt1-Cre; Nedd4fl/fl embryos exhibit defects at the moderate-severe end.\nNedd4-/- embryos have severe growth retardation, and from E12.5 onwards are approximately half the size of their wildtype littermates (Cao et. al. 2010, Science Signaling 1:ra5, and Reviewer Fig. 2). We hypothesise that these severe systemic defects in whole embryo development likely impact on heart development, and as such leads to OFT rotation defects that are more pronounced. There may also be important roles for NEDD4 in other tissue lineages that contribute to heart development that were not tested in our conditional knockout analyses, such as the first heart field or pharyngeal mesoderm.\n3. Fig 3C, why is there no apparent loss of Isl1+ SHF tissue, given the precocious differentiation into myocardium?\nIsl1 is expressed in the SHF, but is also expressed in MF20+ve myocardium of the outflow tract (See wildtype examples in Figure 3B and 3C). Isl1 immunostaining only diminishes once myocardial cells have reached the right ventricle. Hence we do not expect to see any reduction in Isl1 in the SHF given the precocious MF20+ve differentiation in Wnt1-Cre; Nedd4fl/fl embryos.\n4. Why is enhanced MF20 expression interpreted as a marker of premature SHF differentiation? Please clarify for the readers.\nWe now add a sentence to our manuscript to clarify for the readers why enhanced MF20 expression is considered a marker of premature SHF differentiation, and reference other publications that have used MF20 immunostaining for this analysis.\n“Myocardial diTerentiation is initiated as second heart field cardiac progenitors enter the developing outflow tract, and upregulate expression of various myocardial markers such as myosin heavy and light chains and other myogenic factors 13. MF20, which recognises myosin heavy chain, is widely used to assess in vivo diTerentiation dynamics at the transition zone between the second heart field and outflow tract myocardium 13-16.”\n5. In Figure 4H, β-catenin appears to localise primarily at cell-cell junctions rather than in the nucleus. In the mutants, the reduction is most apparent at the junctional level. The reviewer does not observe a reduction in nuclear β-catenin. Could the authors clarify this, possibly by providing higher magnification images? The evidence for reduced Wnt signaling in the SHF, based only on β-catenin staining, is not fully convincing. Could the authors validate this using additional Wnt reporters such as TCF/Lef:H2B-GFP, Axin2-lacZ, etc..?\nWe now present higher magnification images of the β-catenin (non-phospho S45) immunostaining shown in Figure 4H in Supplementary Figure 11A. Furthermore, we have now highlighted examples of nuclear localised β-catenin (solid arrowheads) vs examples of no nuclear β-catenin (outlined arrowheads) to make our quantitation methods clearer for the reader.\nIn addition to the previous immunostaining shown for β-catenin non-phospho S45 (which is considered an active form of β-catenin), we have now performed additional immunostaining using another two antibodies that react to active forms of β-catenin. These include Active Beta Catenin (ABC) (clone 8E7), and β-catenin phospho Y489, and are presented and quantified in Supplementary Figure 11C-F. We have also examined the canonical Wnt signalling target gene Pitx2 by immunostaining for Pitx2 protein, which we present and quantify in Supplementary Figure 11G,H. All three additional antibodies examined revealed reduced nuclear staining in the second heart field of Wnt1-Cre; Nedd4fl/fl embryos, consistent with our initial finding in Figure 4H.\nWe agree that a Wnt signalling reporter would be an ideal way to demonstrate changes in Wnt signalling activity. However, we are limited by availability of crossing such a reporter to our mouse line, given strict import and quarantine requirements in Australia, which based on our previous experience would require a minimum ~12 months. In lieu of this lengthy timeframe, we now provide additional immunostaining, as well as our prior analysis in Figure 4K that shows downregulation of Wnt-regulated genes specifically in the SHF by in situ hybridisation. Taken together, our data provide multiple lines of evidence to support reduced Wnt signalling in the SHF of Wnt1-Cre; Nedd4fl/fl embryos.\n6. Could the authors discuss why enhancing Wnt signaling with CHIR99021, a well-known Wnt pathway activator, failed to rescue the OFT defects in Wnt1-Cre; Nedd4fl/fl embryos? Could the author analyse Wnt signalling activity under CHIR99021 and iCRT3 conditions?\nWe hypothesise several possible reasons that CHIR99021 treatment failed to rescue OFT defects in Wnt1-Cre; Nedd4fl/fl embryos. One reason may be that the effect of precocious DKK1 overexpression in neural crest cells is too strong to be overcome by increasing canonical Wnt signalling via CHIR99021. Another reason may be that systemic CHIR99021 treatment may cause pleiotropic deleterious elects due to precocious Wnt signalling activation in the whole embryo. Given this, we chose to use a genetic approach (Dkk1+/- cross) to try to assess rescue of embryonic phenotypes.\nWe have now also analysed Wnt signalling activity in iCRT3 treated embryos by assessing β-catenin non-phospho S45 and β-catenin phospho Y489 immunostaining at E9.5 (Supp. Fig. 14). This revealed a reduction in Wnt signalling in the second heart field of iCRT3 treated embryos, and importantly caused a significant reduction in Wnt signalling in Wnt1-Cre; Nedd4fl/+ (Het) embryos such that these were indistinguishable from Wnt1-Cre; Nedd4fl/fl (mutant) embryos. This is consistent with the notion that premature second heart field differentiation can be induced by inhibition of Wnt signalling in genetically susceptible embryos (eg. Wnt1-Cre; Nedd4fl/+ (Het)), and strengthens our conclusion that part of the phenotype arises from altered Wnt signalling.\n7. In Figure 4G, there appears to be no upregulation of MF20 in Wnt1-Cre; Nedd4fl/fl embryos. Could the authors clarify this inconsistency?\nWe now show an adjacent tissue section for the Wnt1-Cre; Nedd4fl/fl embryo from the same experiment, which demonstrates upregulation of MF20, and is a truer representative image of the MF20 staining observed in embryos of this genotype. Upon close examination we found the original image in Fig 4G had a small DAPI-negative area in this region, indicating a small piece of second heart field tissue was torn as a sectioning artefact, explaining the loss of MF20 staining.\n8. In Figure 4G, the concept of an \"EdU migration front\" is unclear. How is this defined, and how does it relate to the extent of SHF incorporation into the myocardium?\nWe have added a paragraph in the methods to describe how the EdU migration front is defined and measured:\n“For EdU migration front distance at E10.5 after 24h chase, the EdU +ve migration front in the OFT was measured as the distance (in µm) of the last EdU +ve cell in the MF20+ve OFT from the pericardial wall. 3-4 sagittal sections were quantified per embryo, and averaged. Individual points on graphs represent the mean value calculated from 1 embryo. A minimum of 5 and up to 6 embryos per genotype were used for quantitation. EdU +ve migration front distance was also normalised to atrio-ventricular valve primordium thickness (an unaffected structure present in the same microscopy images) as a means to account for any variation in embryo size, and is presented in the Supplementary figures.”\nEdU labelling is able to be utilised as a form of lineage tracing to assess incorporation of SHF cells into the OFT myocardium (eg. see references 17 and 18 cited in text). This is possible owing to the SHF being highly proliferative which readily incorporates EdU, while the OFT is essentially quiescent, and does not take up EdU label. Hence, after a 1hr pulse of EdU which only labels the SHF, any EdU+ve cells in the OFT after a 24h chase are derived from the earlier labelled cells in the SHF. Given that SHF cells move progressively from the SHF into the OFT, the extent of EdU ‘migration’ distance into the OFT can be used as a measure to assess the extent of SHF cell deployment.\n9. Lastly, could the authors discuss the phenotypic differences between Nedd4K766R embryos and Nedd4fl/fl embryos, and explain why the K766R mutation, which replicates a human variant, does not phenocopy neural crest-specific loss of Nedd4?\nThere are many possible complex reasons why the Nedd4K766R mutation in mice does not phenocopy the neural crest-specific loss of Nedd4, or additionally the phenotype of Nedd4-/- mice. The Nedd4K766R variant represents a single amino acid change. Given Nedd4K766R mice at E15.5 and E17.5 are grossly phenotypically normal compared to their wildtype littermates (Figure 7E), while Nedd4-/- mice are severely growth restricted with notable cranial defects (Cao et. al. 2010, Science Signaling 1:ra5; Wiszniak et. al. 2013, Developmental Biology 383:186-200), this indicates Nedd4K766R is not a complete loss-of-function mutation. While Nedd4K766R has lost ability to ubiquitinate DKK1, it is likely that this variant maintains other functional abilities, and has perhaps lost or even altered function in respect to a subset of ubiquitinated targets. Hence, it is likely this variant still maintains some functional capacity in neural crest cells, and hence why this does not phenocopy full loss of Nedd4 function in neural crest cells in Wnt1-Cre; Nedd4fl/fl mice.\nWe now include an additional sentence in our discussion to highlight phenotypic differences between Nedd4K766R and Nedd4fl/fl mice:\n“While the heart defects exhibited in Nedd4K766R/K766R mice do not fully phenocopy the defects observed in Wnt1-Cre; Nedd4fl/fl mice, this is likely due to functional diTerences between hypomorphic NEDD4 K766R protein expression in the whole embryo versus complete removal of NEDD4 protein in neural crest cells of Wnt1-Cre; Nedd4fl/fl mice.”\nReviewer #2 (Remarks to the Author):\nIn the manuscript “Neural crest cell-derived DKK1 modulates Wnt signalling in the second heart field to orchestrate cardiac outflow tract development, “Wiszniak et al examine the role of NEDD4 expressed in neural crest cells during cardiac development. The authors demonstrate that the deletion of NEDD4 results in defects in the development of the cardiac outflow tract and the positioning of the great arteries relative to the right and left ventricles. The authors examine the evidence that this effect is due to increased levels of Dkk1 in their mutants. While the idea is very interesting, the data does not support the authors’ conclusions. In many experiments, the experimental setup is not appropriate for what the authors intended to study.\nMajor Concerns:\n1) A major concern is that for multiple types of experiments, normalization is not provided.\nWe have now added a sentence in the methods under ‘Mice’ to clarify that normalisation was an important consideration for all analyses performed in our manuscript, and that multiple methods were used to ensure stage-matched littermates were examined. “In all experiments, stage-matched embryo littermates were selected for comparison using stage-appropriate parameters such as somite number, crown-rump length, and forelimb/digit maturity.” Furthermore, to clarify for the reviewer, we do not observe any developmental delay with Wnt1-Cre; Nedd4fl/fl embryos relative to their wildtype littermates, with embryos indistinguishable from each other in terms of size and somite numbers. This can be observed in examples throughout the manuscript such as in Fig. 3A, Fig. 4E, Supp. Fig. 2 and Supp. Fig. 5.\nWe agree that the reviewers suggestions for additional normalisation for specific experiments is warranted, and now provide these below.\na) For example, the length of the outflow tract is not normalized to an unaffected embryonic structure (e.g., otic vesicle) to control for embryo size in Figure 2A. In addition, it is not clear how authors consistently found a boundary between the proximal OFT and the right ventricle. This needs to be described. Alternatively, the RV needs to be included in the measurements because it is difficult to find such a boundary, and customarily, the RV is included in the measurements of OFT length at E10.5.\nAs suggested, we have now included the RV in the measurements of OFT length, and present a new quantified figure for OFT+RV length in Figure 2A. Furthermore, we have also normalised the OFT+RV length to an unaffected structure (eye diameter) which we had previously acquired in whole embryo images of the same samples, now presented in Supp. Fig. 2. Both the ‘absolute’ OFT+RV length and the ‘normalised’ OFT+RV length demonstrate the same outcome, with reduced OFT+RV length in Wnt1-Cre; Nedd4fl/fl embryos, with a p-value of <0.0001. We prefer to present the ‘absolute’ OFT+RV length in the main figure, as we believe the length in µm is more informative to the reader than the arbitrary ratio of OFT+RV / eye.\nb) Another example is the measurement of valve diameter in Figure 5G. This measurement also needs to be normalized to an unaffected embryonic structure to control for embryo size. These normalizations are essential since no single embryo develops at the same rate, and differences in embryo stages and sizes are common even within the same litter.\nWe have now normalised all of the valve and OFT measurements in Figure 5G to an unaffected structure, that being the neural tube (Supp. Fig. 15D,E). The neural tube was present in all tissue sections for which the valves and OFT were measured. We have now measured the medial neural tube diameter (averaged from 3x sections) for each embryo examined. We present all normalised measurements in Supp. Fig. 15A,B,C, which all demonstrate the same outcomes as the ‘absolute’ measurements presented in Figure 5 I-K. We prefer to present the ‘absolute’ values in the main figure, as we believe the diameter in µm or area in µm2 is more informative to the reader than the arbitrary ratio of diameter / neural tube.\nc) It is not proper to report fluorescence intensity as a measurement without normalization, since fluorescence intensity will vary based on many factors beyond the experimenter’s control, such as fluctuations in laser power, differences in cover slip thickness, and the exact plane of focus. Check the Bioimaging guide for best practices (https://www.bioimagingguide.org/03_Image_analysis/Intensity.html)\nWe have now added a section to the methods ‘Fluorescence intensity and image quantitation’, in which we clarify our methods for Mean Fluorescence Intensity (MFI) measurements, and other quantitation methods. We thank the reviewer for providing the suggested link for best practices in image analysis, of which we address in this comment and in the further comments below.\nAll images presented throughout the manuscript, and from which quantitation was performed, represent maximum intensity projections of 3x acquired z-stacks per image to account for differences in the exact focal plane. Images were always acquired in the same microscopy session on the same day with the same settings for paired analyses to minimise any variances in laser power or PMT sensitivity/acquisition. Images were carefully acquired such that no saturated pixels were present, and the raw (.czi) files were always used for quantitative analysis. The fluorescence intensity measurements presented are the ‘mean fluorescence intensity’ measurements; ie. the absolute fluorescence in the region of interest is normalised to the area of the region of interest. Equivalent areas of ‘region of interest’ were selected (as now described in greater detail in our methods, as well as indicated in our figures, eg. Supp. Fig. 6).\nImportantly, we have carefully selected our representative images in all figures to demonstrate what we observe many times over for individual analyses. We use MFI as a way to quantify this for the purpose of statistical analysis to confirm and add power to our visual observations.\nWhere possible, we have now incorporated additional normalisations into our MFI measurements, as documented below.\n2) Lines through the ventricles seem to be drawn in random orientation in the mutants in Figure 5. While in controls, the line is drawn through the lumens of LV and RV, in the mutants, the line does not appear to go through the lumen of LV. Other section levels or coronal sections may be more appropriate. Multiple levels like those shown in Fig. 2B may also work well to demonstrate altered alignment.\nWe now show multiple levels of sections (like for Fig. 2B) to better demonstrate differences in OFT rotation, and present these in Supp. Fig. 13. All additional sections shown are from the original embryos shown in Fig. 5B and 5H, except for the vehicle treated Wnt1-Cre; Nedd4fl/fl embryo in 5B as the section through the valve was damaged. We have picked another littermate imaged in the same experiment that had complete and undamaged sections for the artery/valve/ventricle, and now present these in both Fig. 5B and Supp. Fig. 13A.\n3) On line 14, page 6, the authors state that NC numbers are not affected in the mutants. Since this is very important for the rest of the study, the authors need to provide quantification to support this statement.\nWe now provide data to support this statement and present neural crest cell number quantitation in Supp. Fig. 3, with no significant differences between genotypes.\n4) On line 19, page 6, the authors state that the expression of additional SHF markers were not affected in the mutants. However, Sup. Fig 2 shows that Hoxb1 and BMP4 are downregulated and Fgf8 is upregulated in the mutants. Please state how many times each experiment was conducted; how many controls and mutants were analyzed for each staining, and provide quantification.\nWe have now included higher magnification images of the second heart field region of the whole-mount embryos, to more clearly demonstrate the region of interest that was assessed when comparing wildtype versus Wnt1-Cre; Nedd4fl/fl embryos, and show this in Supplementary Figure 5. Furthermore, we have performed semi-quantitative analysis of BCIP/NBT substrate staining in the SHF region from the multiple experiments that were performed. At least 3 (and up to 7) embryos were analysed per probe per genotype. We did not observe any significant differences in gene expression in the SHF region between wildtype and Wnt1-Cre; Nedd4fl/fl embryos. We have now added the following paragraph to our methods section:\n“Semi-quantitative optical density measurements of whole-mount in situ hybridisation were performed in ImageJ. Images were subjected to colour deconvolution to generate 8-bit grayscale images of BCIP/NBT substrate staining. Mean grey values were calculated from a region of interest corresponding to the second heart field/dorsal pericardial wall. Optical density was calculated using the formula OD = Log (255/mean 10 grey value). A minimum of 3 and up to 7 whole mount embryos per genotype were assessed.”\n5) For the experiment with the explant in Fig. 3E, FACS is more appropriate to determine the proportion of MF20+ cells and intensity. Western blot is also appropriate, but the measurements shown are not up to the accepted standards for quantification.\nWe agree that flow cytometry would provide more quantitative measures of MF20+ve cell proportions and staining intensity. However, based on other current work in our laboratory (work up of E9.5 SHF cell dissociation for scRNAseq, yielding around 2000 cells per SHF tissue) it is not possible to dissociate a single SHF in individual tubes, and have sufficient cells for performing subsequent analyses (in this case, fixing, permeabilising and staining cells for MF20, then flow cytometry analysis).\nWe politely disagree that western blotting for MF20 is more appropriate than immnostaining. Western blotting would require scraping the SHF tissue explant from the growth substrate, potentially leaving behind or damaging cells such there will be inherent differences between samples. Sample integrity and the consistency between samples is much better maintained by simply fixing the tissue in place with PFA. Staining for the MF20 antigen via western blot with primary and secondary antibody is equivalent to staining the MF20 antigen via immunofluorescence with primary and secondary antibody. Then the principles for quantitation of both methods are similar (densitometry of a grayscale image). We have also now included further information on our method of quantitation: “For MF20 MFI measurements of SHF explants, the entire DAPI+ve region was selected, and MFI measured in this region of interest. Individual values represent MFI for independent explants (ie. biological replicates) of the indicated genotype.”\nGiven we already show MF20 is upregulated in vivo, the SHF explant experiments are intended to be confirmatory, and do not change the conclusions of our paper. If the reviewer does not agree our quantitation methods for this experiment are valid, we are satisfied to remove the graph in Figure 3E and simply show the representative immunofluorescence image of each genotype.\n6) The EdU experiment in Fig. 3G is not appropriate to show cell migration. For this experiment, EdU pulse-chase needs to be implemented: after one hour of EdU, thymidine needs to be injected to compete EdU off. Otherwise, the experiment in 5G can be interpreted as a decrease in proliferation rather than migration. See doi: 10.1016/j.ydbio.2016.02.017 as an example of such an assay in vivo. Also see papers by Elaine Fuchs, as additional examples for how this is done.\nWe have now performed this EdU pulse-chase experiment as per the reviewers suggestion, and present results in an updated Figure 3G and Supp. Fig. 7. We have also updated our methods to reflect the 1hr EdU pulse, followed by thymidine injection, and then a 24h chase.\nImportantly, the pulse-chase approach confirmed our original findings, with reduced migration of EdU labelled cells into the outflow tract in Wnt1-Cre; Nedd4fl/fl embryos. We also normalised the EdU migration distance to an unaffected structure (A-V valve thickness) to account for any variability in embryo size.\n7) Figure 4H. There is no nuclear β-catenin in controls or mutants. It is not clear what is being measured and how.\nWe now present higher magnification images of β-catenin immunostaining in Supplementary Figure 11. Furthermore, we have now highlighted examples of nuclear localised β-catenin (solid arrowheads) vs examples of no nuclear β-catenin (outlined arrowheads) to make our quantitation methods clearer for the reader.\n8) Figure 4K: Expression of Axin2 can be used as a readout for Wnt signaling. However, the authors point to Axin2 staining in the atrial chamber in a control embryo (arrowhead) and not the SHF in the dorsal pericardial wall contiguous with the OFT. The SHF appears not to express Axin2 in the control. In the mutant shown, the Axin2 staining did not seem to work because there is no staining in the atrial chamber, a tissue unaffected by the neural crest. This is an important experiment, and it needs to be done properly to determine whether Wnt signaling is indeed downregulated in the SHF in the mutants.\nWe now provide uncropped images of the Axin2 in situ hybridisation presented in Fig. 4K in Reviewer Figure 3. This demonstrates that the in situ procedure worked well, as Axin2 expression is evident in unaffected structures such as pharyngeal arch 1 and the posterior pharyngeal endoderm. We also use dashed lines to indicate the boundaries of the dorsal pericardial wall, outflow tract, and atrial chamber, which highlight expression of Axin2 in the dorsal pericardial wall in the wildtype section, but not in the Wnt1-Cre; Nedd4fl/fl section.\n9) Figure 5E: MF20 IF intensity is reported without normalization and thus is unreliable. In addition to properly measuring and normalizing MF20 intensity, the authors should measure the length of SHF occupied by the ectopic MF20+ cells.\nWhile our prior analysis was carefully performed with bioimaging best practices considered (as described in response to point 1c above), we have now additionally performed further normalisation as per the reviewers suggestion. The atrial chamber, which is also positive for MF20 immunostaining, is considered an unaffected structure in our analyses, given neural crest cells do not make contact with or influence it’s development at the stage assessed (E9.5). The atrial chamber is also adjacent to the second heart field in sagittal sections, and as such was acquired in all images used previously to quantify MF20 mean fluorescence intensity in the second heart field. We have now measured fluorescence intensity in a region of the atrial wall (as described in our methods and shown in Supp. Fig. 6A), and have used this to normalise our previously measured MFI measurements in the SHF. We have performed normalisation for all MF20 MFI measurements in the SHF shown in Fig. 3D and 5E, and present these in Supp. Fig. 6C and Supp. Fig. 6E. Furthermore, we have also measured the length of SHF occupied by the ectopic MF20-positive cells, and present this in Supp. Fig. 6D and Supp. Fig. 6F. Importantly, all analysis methods revealed statistically significant differences in MF20 immunostaining in the SHF.\n10) Figure 6: How do authors explain the mechanism by which NEDD4 regulates the ubiquitination of Dkk1? Dkk1 is a secreted protein (i.e., it is present inside the ER, Golgi, and secretory vesicles), while NEDD4 is a cytoplasmic protein that is always outside the ER, Golgi, etc. These two proteins are never in the same cellular compartment.\nWe now show 63x high magnification images of cardiac neural crest cells in vivo immunostained for NEDD4, DKK1 and GM130 (a cis-Golgi marker) in Figure 6F. This demonstrates co-localisation of NEDD4 and DKK1, suggesting it is feasible that NEDD4 could make contact with DKK1 in the same cellular compartment.\nPrior publications also provide evidence for roles for NEDD4 in other cellular compartments such as Golgi and endosomes, including some from the current authorship team of this manuscript.\nEg:\nhttps://www.nature.com/articles/s12276-025-01396-2 (Shows NEDD4 ubiquitinates key golgi protein GM130).\nhttps://www.nature.com/articles/s41467-025-57944-x (Shows Nedd4(NE) in endosomes. Our own unpublished work has also discovered this NE version of Nedd4 is present in mouse too, not just ‘primate’ specific as per the paper title.)\nhttps://www.sciencedirect.com/science/article/pii/S0006291X21005623?via%3Dihub (Shows Nedd4 interaction helps traffic a protein from Golgi to late endosomes).\nhttps://www.sciencedirect.com/science/article/pii/S0021925819363045?via%3Dihub (Paper from the authorship research team, showing ubiquitination of a Golgi protein (Ndfip2) by Nedd4).\n11) Figure 7G. The hearts look smaller in the mutants, suggesting that VSD and other “defects” may be due to delayed embryo development at E15.5 in the mutants. E18.5 is a more appropriate time point for the evaluation of structural heart defects.\na. Are Nedd4-K766R homozygous mutants viable?\nYes, indeed the hearts are smaller in Nedd4K766R/K766R mutants. This is observed at both E15.5 and E17.5 (Figure 7G,H), and we have now added example images for E17.5 to the main figures of our manuscript to show these defects persist at older stages of development. Our data is consistent with this being a specific defect and not likely due to developmental delay, given Nedd4K766R/K766R embryos are equivalent in size to their wildtype littermates and gross embryo development appears normal (Figure 7E). We evaluated structural heart defects over a range of stages at late gestation from E15.5 – E17.5, with the defects listed in Figure 7F present across all ages examined. We chose E15.5 onwards as several other studies have reported this to be an appropriate time to phenotype cardiac defects as all major structures of the heart have formed by this stage (eg. Schneider et. al. 2004 BMC Dev Biol 4:16; Liu et. al 2013 Circulation: Cardiov. Im. 7:31; Kim et. al. 2013 Circulation: Cardiov. Im. 6:551.) E15.5 onwards is also considered appropriate to assess VSDs without concern for developmental delay, as the interventricular septum has normally completed development by E13.5 (eg. Anderson et. al. 2014 Anat. Rec. 297:1414; Webb et. al. 1998 Circulation 82:645; Krishnan et. al. 2014 Pediatric Res. 76:500).\nLimitations in terms of compliance with University of South Australia Animal Ethics Committee guidelines have precluded pups being born from Nedd4K766R/+ x Nedd4K766R/+ crosses, hence we have not yet been able to assess if Nedd4K766R/K766R mutants are viable post-birth.\nMinor:\n1) on line 14 page 4, the authors state that “neural crest cells form structural components of the outflow tract valves.” This is incorrect. Neural crest cells die at late gestation and do not contribute to mature outflow tract valves. See papers from Jonathan Epstein lab.\nWe have now revised this sentence to reflect that neural crest cells form components of the heart during the early development stages, even though they undergo apoptosis in some structures (such as the valves) later in developmental maturation.\n“In the developing heart, neural crest cells form structural components of the outflow tract valves, arteries, and conotruncal septum.”\n2) On line 19-21 page 4, the authors state “However, prior models in which neural crest cells have been surgically or genetically ablated4-7 have not been amenable to elucidate how neural crest cells interact with the second heart field to control its growth, differentiation and morphogenesis.” This is inaccurate. The authors should mention the work from Margaret Kirby’s lab showing the importance of NC in regulating BMP4-FGF8 cross talk between NCCs and SHF in preventing precocious differentiation of cardiac progenitors in the SHF.\nOur intention with this sentence was to emphasise that these prior models have completely ablated the neural crest cells, hence it is not possible to assess how these neural crest cells would have normally signalled to or interacted with the second heart field (as they are now not even present to assess). We wholeheartedly agree that these ablation models were critical to discover that neural crest cells play important roles in heart development, and do indeed cite how these models have uncovered roles for BMP-FGF signalling and cross-talk in our discussion section.\nWe have modified our statement: “However, prior models in which neural crest cells have been surgically or genetically ablated4-7 have not been amenable to elucidate how neural crest cells normally interact with the second heart field to control its growth, differentiation and morphogenesis.”\nReviewer #3 (Remarks to the Author):\nThe MS by Wiszniak et al reports a novel regulatory mechanism whereby the ubiquitin ligase NEDD4 affects the protein levels of DKK1in neural crest cells that modulates Wnt signaling thereby affecting the timing of second heart field differentiation and outflow tract morphogenesis. A combination of in vivo genetic studies in mice and in vitro examination of NEDD4 function provide convincing evidence for the major conclusions of the study. Notably, a DDK1 deficient mouse is provides evidence for a rescue of the NEDD4 deficiency in neural crest cells which is important support for the proposed mechanism. In addition, variants in NEDD4 were identified in individuals with congenital heart malformations and loss of function demonstrated experimentally.\nThis is a strong study with convincing data to support a new regulatory contribution of Wnt signaling in cardiac outflow tract development with human clinical implications.\nMinor comments\n1. The title does not mention NEDD4 which is a major focus of the study.\nWe have now included NEDD4 in the title.\n2. Were abnormalities in outflow tract endocardial cushions or semilunar valve primordia observed?\nYes, we do indeed observe abnormalities in the morphology of the endocardial cushions of the semilunar valves, as can be seen in Figure 2B. While we eluded to this in the Figure Legend “Valve leaflet defects are also observed”, we did not include this in the main manuscript text. We have now briefly mentioned this in the main text:\n“Semilunar valve morphology was also altered (Fig. 2B).”\nWe believe this is a very interesting phenotype that is independent of the second heart field differentiation mechanism, and is currently being investigated in a separate project in our laboratory, and will be the focus of a new manuscript in the near future.\n3. The Discussion is long and includes information on BMP and FGF signaling that seem peripheral to the current study.\nWe agree that the discussion regarding additional signalling pathways is somewhat peripheral to the current study that is focused on Wnt signalling. However, we have decided to maintain the information regarding BMP and FGF signalling as this was a specific request for inclusion from Reviewer 2.\nReviewer Figure 1\nA Human iPSC-derived NCCs\nday10 differentiation\ncluster 12 TFAP2A SOX10 DKK1\nDKK1+ve\nNEDD4 HAND2 HAND1 GATA3\nB\nNEDD4 iPSC d10 diff\nDKK1\nDAPI Reviewer Figure 1 - Building an in vitro model of cardiac neural crest cells\nA: Human iPSCs were directed to differentiate into neural crest cells in our established unpublished protocol (briefly, SMAD inhibition + Wnt induction, followed by FGF2, BMP4, RA treatment for 10 days). scRNAseq reveals broad induction of NCC markers such as TFAP2A and SOX10. Cluster 12 is SOX10-ve and DKK1+ve, consistent with expression profile of cardiac neural crest cells at E9.5. This population is also +ve for other cardiac NCC markers such as HAND2, HAND1 and GATA3. B: Immunostaining reveals a small proportion of these cells express DKK1 (arrowheads). C: scRNAseq of E9.5 Wnt1-Cre;EGFP FACS-sorted neural crest cells. Dkk1 is specifically expressed in cluster 14, along with other cardiac neural crest cell markers. Importantly, this highlights Dkk1 as a novel cardiac neural crest cell marker.\n20μm\nC Mouse E9.5 Wnt1-Cre;EGFP +ve NCCs\nFrom: De Bono et. al (2023) Single-cell transcriptomics uncovers a non-autonomous Tbx1-dependent genetic program controlling cardiac neural crest cell development. Nature Communications 14:1551\nTfap2a Sox10 Dkk1\ncluster 14\nDkk1+ve\nNedd4 Hand2 Hand1 Gata3\nReviewer Figure 2\nReviewer Figure 2 - Nedd4-/- embryos have severe growth restriction and vascular defects\nExample images of wildtype and Nedd4-/- embryos at E11.5 and E12.5. Nedd4-/- embryos exhibit growth restriction from around E11.5 onwards, and have severe cardiovascular defects.\nReviewer Figure 3\nwildtype Wnt1-Cre;Nedd4fl/fl\nAxin2\npa1 pa1\npe OFT pe OFT\nV\nV\nA dpcw\ndpcw A\nReviewer Figure 3 - Axin2 in situ hybridisation\nUncropped images of in situ hybridisation for Axin2 as shown in Figure 4K. Expression is evident in other areas such as the pharyngeal arch and posterior pharyngeal endoderm.\nDashed lines in lower panels show overlay of tissue boundaries.\npa1, pharyngeal arch 1; pe, pharyngeal endoderm; OFT, outflow tract; dpcw, dorsal pericardial wall; A, atrium; V, ventricle."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am satisfied with the authors’ response to my previous comment. Overall, this is an insightful and compelling study with strong genetic evidence. For clarity, I would still recommend moving Supplementary Figure 4A and 4B into the main figures.", "reviewer_2": "The revisions of the manuscript entitled “Neural crest cell derived DKK1 and NEDD4 modulate Wnt signalling in the second heart field to orchestrate cardiac outflow tract development,” has a resulted in a really beautiful manuscript with high-quality data reporting novel and interesting findings that are also relevant to human congenital heart disease. The revised manuscript has addressed all previous critiques. The following are minor concerns remaining:\n1) Reviewer Figure 3 - Axin2 in situ hybridization should be included in the manuscript.\n2) Please mention which Wnt1-Cre strain was used? Wnt1-Cre1 or Wnt1-Cre2? Wnt1-Cre1 ectopically expresses large amounts of Wnt1 protein (Lewis AE, Vasudevan HN, O’Neill AK, Soriano P, & Bush JO (2013). The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Dev Biol, 379(2), 229–234. 10.1016/j.ydbio.2013.04.026).\n3) In the methods, please add the genetic background of each parental strain used. In general, it would be useful to know the parental genotypes of the crosses that were used to generate various embryo genotypes. This information should be added to the methods as well.\nPlease add to the methods whether Wnt1-Cre; Nedd4fl/fl; Dkk1+/- embryos used for quantifications in Fig. 5G are littermates of Wnt1-Cre; Nedd4fl/fl; Dkk1+/+. If they were not littermates, please note whether or not differences in phenotypes between Wnt1-Cre; Nedd4fl/fl; Dkk1+/- and Wnt1-Cre; Nedd4fl/fl; Dkk1+/+ may be due to potential differences in genetic background.\n4) Please add a reference to your EdU pulse-chase method\n5) Sup. Fig. 8: pErk1/2 in staining in Sup. Fig. 8 is too dim and is hard to see."}, "author_response": "Response to reviewers’ comments:\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nI am satisfied with the authors’ response to my previous comment. Overall, this is an insightful and compelling study with strong genetic evidence. For clarity, I would still recommend moving Supplementary Figure 4A and 4B into the main figures.\nWe have now moved both Supplementary Figure 3 and 4 into the main figures, as new panels in Figure 3 (now Fig. 3C-E).\nReviewer #2 (Remarks to the Author):\nThe revisions of the manuscript entitled “Neural crest cell derived DKK1 and NEDD4 modulate Wnt signalling in the second heart field to orchestrate cardiac outflow tract development,” has a resulted in a really beautiful manuscript with high-quality data reporting novel and interesting findings that are also relevant to human congenital heart disease. The revised manuscript has addressed all previous critiques. The following are minor concerns remaining:\n1) Reviewer Figure 3 - Axin2 in situ hybridization should be included in the manuscript.\nWe now include this as Supp. Fig. 11.\n2) Please mention which Wnt1-Cre strain was used? Wnt1-Cre1 or Wnt1-Cre2? Wnt1-Cre1 ectopically expresses large amounts of Wnt1 protein (Lewis AE, Vasudevan HN, O’Neill AK, Soriano P, & Bush JO (2013). The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Dev Biol, 379(2), 229–234. 10.1016/j.ydbio.2013.04.026).\nThe Wnt1-Cre strain used (Wnt1-Cre1) is listed in our methods (ref. 51). This is the gold-standard line for the field. As per the reference included above, and from our own and other researchers’ observations in the neural crest field, the Wnt1-Cre1 line only causes significant adverse effects when maintained in a Wnt1-Cretg/tg homozygous state. We only ever use this line in a hemizygous Wnt1-Cretg/+ state.\n3) In the methods, please add the genetic background of each parental strain used. In general, it would be useful to know the parental genotypes of the crosses that were used to generate various embryo genotypes. This information should be added to the methods as well.\nPlease add to the methods whether Wnt1-Cre; Nedd4fl/fl; Dkk1+/- embryos used for quantifications in Fig. 5G are littermates of Wnt1-Cre; Nedd4fl/fl; Dkk1+/+. If they were not littermates, please note whether or not differences in phenotypes between Wnt1-Cre; Nedd4fl/fl; Dkk1+/- and Wnt1-Cre; Nedd4fl/fl; Dkk1+/+ may be due to potential differences in genetic background.\nAll embryos used for analyses in Fig. 5G-L were littermates. Our experiment was carefully designed this way to eliminate any potential effects due to genetic background. Embryos were derived from litters of Wnt1-Cre; Nedd4fl/+ males crossed to Nedd4fl/fl; Dkk1+/- females, yielding embryos of all listed genotypes.\nWe now add this to the methods section:\n“Wnt1-Cre; Nedd4fl/fl mice were intercrossed with Dkk1+/- 47 mice (Wnt1-Cre; Nedd4fl/+ males crossed to Nedd4fl/fl; Dkk1+/- females) to generate Wnt1-Cre; Nedd4fl/fl; Dkk1+/- embryos (as well as littermate controls) for assessing the rescue effect of DKK1 dosage compensation.”\n4) Please add a reference to your EdU pulse-chase method.\nWe now add references to the methods section:\n“In methods similar to 17,18, pregnant dams were intraperitoneally injected with 100 mg/kg EdU (5-ethynyl-2’-deoxyuridine) at E9.5 to pulse-label proliferating cells.”\n5) Sup. Fig. 8: pErk1/2 in staining in Sup. Fig. 8 is too dim and is hard to see.\nWe have increased the brightness of all panels in pERK1/2 figure (now Supp. Fig. 6B)."}]

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