Background: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. Results: By embryonic day 13.5, all rescued Raldh2^−/− hearts exhibit severe, reproducible OFT septation defects, although wild‐type and Raldh2^+/− littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2^−/− mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2^−/− mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. Conclusions: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus. Developmental Dynamics 245:388–401, 2016.

DiGeorge syndrome (DGS) is a congenital disease causing cardiac outflow tract anomalies, craniofacial dysmorphogenesis, thymus hypoplasia, and mental disorders. It results from defective development of neural crest cells (NC) that colonize the pharyngeal arches and contribute to lower jaw, neck and heart tissues. Although TBX1 has been identified as the main gene accounting for the defects observed in human patients and mouse models, the molecular mechanisms underlying DGS etiology are poorly identified. The recent demonstrations that SDF1-CXCR4 axis is implicated in NC chemotactic guidance and impaired in cortical interneurons of mouse DGS models prompted us to search for genetic interactions between Tbx1, Sdf1/Cxcl12 and Cxcr4 in pharyngeal NC and to investigate the effect of altering CXCR4 signaling on the ontogeny of their derivatives affected in DGS. Here, we provide evidence that Cxcr4 and Sdf1/Cxcl12 are genetically downstream of Tbx1 during pharyngeal NC development and that reduction of CXCR4 signaling causes misrouting of pharyngeal NC in chick and dramatic morphological alterations in the mandibular skeleton, thymus and cranial sensory ganglia. Our results therefore underlines the possibility of a pivotal role for SDF1/CXCR4 axis in DGS etiology.

Aims Heart valve maturation is achieved by the organization of extracellular matrix (ECM) and the distribution of valvular interstitial cells. However, the factors that regulate matrix components required for valvular structure and function are unknown. Based on the discovery of its specific expression in cardiac valves, we aimed to uncover the role of Krox20 ( Egr-2 ) during valve development and disease. Methods and results Using series of mouse genetic tools, we demonstrated that loss of function of Krox20 caused significant hyperplasia of the semilunar valves, while atrioventricular valves appeared normal. This defect was associated with an increase in valvular interstitial cell number and ECM volume. Echo Doppler analysis revealed that adult mutant mice had aortic insufficiency. Defective aortic valves (AoVs) in Krox20−/− mice had features of human AoV disease, including excess of proteoglycan deposition and reduction of collagen fibres. Furthermore, examination of diseased human AoVs revealed decreased expression of KROX20 . To identify downstream targets of Krox20, we examined expression of fibrillar collagens in the AoV leaflets at different stages in the mouse. We found significant down-regulation of Col1a1 , Col1a2 , and Col3a1 in the semilunar valves of Krox20 mutant mice. Utilizing in vitro and in vivo experiments, we demonstrated that Col1a1 and Col3a1 are direct targets of Krox20 activation in interstitial cells of the AoV. Conclusion This study identifies a previously unknown function of Krox20 during heart valve development. These results indicate that Krox20-mediated activation of fibrillar Col1a1 and Col3a1 genes is crucial to avoid postnatal degeneration of the AoV leaflets.

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Gα_(q/11)-coupled-P2Y₂purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y₂receptors by extracellular UTP inhibits the IGF-I–induced p110α-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Gα_(q/11)—and not G_(i/o)—independently of phospholipase Cβ. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110α mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I–induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110α mutant, in a Gα(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Gα_(q/11)-coupled receptors, which mediate opposite effects on p110α-PI3K activity and keratinocyte migration.

α6β4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6β4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we report that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we establish that ERK1/2 activation by EGF leads to the mobilization of α6β4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6β4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we show that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

Among the aortic valve diseases, the bicuspid aortic valve (BAV) occurs when the aortic valve has two leaflets (cusps), rather than three, and represents the most common form of congenital cardiac malformation, affecting 1–2% of the population. Despite recent advances, the etiology of BAV is poorly understood. We have recently shown that Krox20 is expressed in endothelial and cardiac neural crest derivatives that normally contribute to aortic valve development and that lack of Krox20 in these cells leads to aortic valve defects including partially penetrant BAV formation. Dysregulated expression of endothelial nitric oxide synthase (Nos3) is associated with BAV. To investigate the relationship between Krox20 and Nos3 during aortic valve development, we performed inter-genetic cross. While single heterozygous mice had normal valve formation, the compound Krox20+/−;Nos3+/− mice had BAV malformations displaying an in vivo genetic interaction between these genes for normal valve morphogenesis. Moreover, in vivo and in vitro experiments demonstrate that Krox20 directly binds to Nos3 proximal promoter to activate its expression. Our data suggests that Krox20 is a regulator of nitric oxide in endothelial-derived cells in the development of the aortic valve and concludes on the interaction of Krox20 and Nos3 in BAV formation.