Septation of the cardiac outflow tract (OFT) from a single channel into two tubes of similar size represents a distinctive morphogenetic event during embryonic development. Neural crest cells (NCCs), originating from the dorsal neural tube, form two opposing streams in the OFT to ensure an even division. Many conotruncal defects originate from the disproportional division of the OFT, but the regulatory mechanisms governing the positioning of NCC streams within the OFT remain unclear. Mechanistic insights have emerged from analysis of a mouse line recovered from a mutagenesis screen exhibiting conotruncal defects including persistent truncus arteriosus (PTA). Although the entry of NCCs into the lower part of the OFT was normal in these mutants, the two NCC streams remained in close proximity instead of diverging to assume opposite positions along the OFT, thereby leading to abnormal OFT division. Genetic analysis identified a L16P missense mutation in Pdia3, encoding a disulfide isomerase located in the endoplasmic reticulum (ER) that facilitates protein folding. However, we did not detect enhanced ER stress in mutant OFT. Using a substrate trapping strategy, we identified Pdgfra, which is essential for OFT development, as a substrate of Pdia3. Co-immunoprecipitation confirmed Pdia3 interaction with Pdgfra in the OFT. The expression of Pdgfra and its downstream target was significantly reduced in cardiac NCCs of Pdia3 mutants. NCC-specific deletion of Pdia3 recapitulated the NCC malpositioning defect observed in the Pdia3 missense mutant. Loss of Pdgfra signaling led to premature differentiation of NCCs into smooth muscle cells and failed migration in response to polarized Pdfga expression in the OFT wall. Clinical relevance is indicated by the enrichment of rare PDIA3 variants in congenital heart disease patients. These findings indicate that Pdia3 regulates PDGF crosstalk to guide the diametric positioning of two NCC streams in the OFT, which is a prerequisite for an even division of the OFT.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Cardiac outflow tract formation requires the contribution of cell types from multiple embryological origins, and critically depends on the continued addition of cardiac progenitor cells from the anterior second heart field to ensure sufficient outflow tract growth. Neural crest cells are known to migrate into the outflow tract and later form structural components, such as the septum. Prior to entering the outflow tract, neural crest cells migrate in close apposition to the second heart field, and have historically been suggested to play important roles in regulating second heart field growth dynamics. However, the molecular mechanisms by which neural crest cells signal to the second heart field to control its growth have remained a critical unanswered question in the field. We have discovered that removal of the ubiquitin ligase Nedd4 specifically in neural crest cells (Wnt1-Cre; Nedd4fl/fl) results in outflow tract defects reminiscent of those observed clinically (DORV, TGA). Lineage tracing reveals no deficiency of neural crest cells, however the second heart field exhibits premature cardiomyocyte differentiation, causing insufficient outflow tract lengthening. Laser capture microdissection and mRNAseq of the second heart field region revealed potential modulation of the Wnt signalling pathway, as well as an upregulation of the secreted Wnt signalling inhibitor Dkk1. Spatial expression analysis revealed Dkk1 is localised to the cardiac neural crest cells, and acts to inhibit canonical Wnt signalling in the adjacent second heart field. Modulation of Wnt signalling in vivo by injection of agonists/inhibitors into Wnt1-Cre; Nedd4fl/fl pregnant dams revealed loss of Nedd4 in neural crest cells sensitises embryos to Wnt modulation. Furthermore, we discover Dkk1 is a ubiquitinated target of Nedd4, and that a human congenital heart disease variant of Nedd4 has lost ability to ubiquitinate Dkk1. We propose a new and unexpected role for neural crest cells as a rheostat of Wnt signalling in cardiac progenitors to influence the balance of progenitor maintence vs differentiation, identifying a new molecular pathway underpinning correct outflow tract morphogenesis, and new origins of congenital heart disease.

The mammalian heart is divided into four chambers by septa that arose during vertebrate evolution to isolate the systemic and pulmonary circulation. Cardiac septa are hotspots for congenital heart defects (CHD), and ventricular and atrial septal defects account for 35% and 15% of CHD, respectively. Despite this, septal morphogenesis is poorly understood. Cardiac septa arise at the interface between cells derived from the first (FHF) and second (SHF) heart fields. Previous work has shown that retinoic acid (RA) signaling is required to activate the FHF and venous pole progenitor cell regulator TBX5 in the posterior SHF for subsequent atrial septation. In order to further investigate the role of RA signaling at the heart field interface we expressed a conditional dominant negative RA receptor in the SHF. Mef2c-AHF-Cre;RARaDN hearts are characterized by a common arterial trunk and a failure of muscular interventricular septum morphogenesis resulting in a deep interventricular cleft and a bifid ventricular phenotype that emerges by E12.5. The septal phenotype resembles the bifid hearts of marine mammals of the order Sirenia, implicating RA signaling in the evolution of mammalian ventricular morphology. Genetic and pharmacological experiments have defined the spatiotemporal requirements for RA signal reception in ventricular septum morphogenesis, revealing a late role during septum formation in cardiomyocytes derived from the heart field interface, distinct from requirements for outflow tract development. Moreover bifid hearts have impaired cardiomyocyte maturation and increased extracellular matrix gene expression. Our results support a model by which ventricular septal morphogenesis involves infolding of the compact myocardial wall during chamber ballooning associated with convergence of the right and left ventricular walls through an RA-dependent zipper-like mechanism.

Amongst the mechanisms involved in tissue development, cell volume regulation emerges as an important modulator of morphogenesis. In zebrafish, endocardial cell (EdC) volume decrease is pivotal for atrioventricular valve formation. However, the cellular and molecular processes underlying EdC volume changes remain unknown. Here, we demonstrate that the interplay of Piezo1, Notch1b, and an aquaporin (Aqp) water channel controls EdC volume reduction. We show that Aqp8a.1 is required for EdC volume regulation, and aqp8a.1 mutant larvae display heart valve defects, altered cell polarity, and absence of F-actin remodelling. Furthermore, we demonstrate that aqp8a.1 expression is modulated by Notch signalling. Mechanistically, overexpression of Piezo1 using the genetically encoded Piezo1 biosensor GenEPi leads to downregulation of notch1b and subsequently aqp8a.1. At the protein level, the Ca2+-binding calmodulin directly binds to Aqp8a.1, driving its localisation in response to mechanical stimuli. In parallel, F-actin remodelling through the actin crosslinking protein Marcksl1 arises as an essential event for atrioventricular EdC volume reduction. Altogether, this study identifies new effectors of EdC volume regulation and heart morphogenesis, thereby advancing our understanding of early organogenesis.