Background: Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells. One of the key signaling pathways regulating cardiac neural crest cell (CNCC) development involves the endothelin-A receptor (Ednra). However, the exact function of Ednra signaling in CNCC is unknown. Results: The fate mapping of CNCC in Ednra embryos indicated that the migration of these cells is aberrant in the cardiac outflow tract (OFT), but not in the pharyngeal arches. This premature arrest of CNCC migration occurs independently of CNCC proliferation and apoptosis changes and major gene expression changes. Analysis of the Rho family of small GTPases in the mutant embryos revealed that Cdc42 failed to localize normally in the CNCC migrating in the OFT. The inhibition of Cdc42 activity in cultured embryos recapitulated the migratory phenotype observed in Ednra mice. Further analyses revealed that Cdc42 is part of the signaling pathway activated by endothelin specifically in OFT CNCC to control their migration. Conclusions: These results indicated that the activation of Cdc42 by endothelin signaling is important for CNCC migration in the OFT but this pathway is not involved in mandibular or pharyngeal arch artery patterning.K E Y W O R D S cardiovascular malformation, Cdc42, endothelin, fate mapping, migration, neural crest cell

Congenital cardiovascular malformations are the most common birth defects affecting children. Several of these defects occur in structures developing from neural crest cells (NCC) that migrate ventrally to populate the pharyngeal arches. Cardiac NCCs (CNCC) participate in the asymmetric remodeling of the pharyngeal arch arteries (PAA) into the great vessels and the septation of the cardiac outflow tract (COFT) into the pulmonary and aortic outflows. One of the key signaling pathways regulating CNCC development involves the Endothelin‐A receptor (Ednra). The absence of Ednra signaling in the mouse causes severe cardiovascular defects, including patent ductus arteriosus, coarctation of the aorta, persistent truncus arteriosus and ventricular septal defects. However, the exact function of Ednra signaling in CNCCs is unknown.CNCC fate mapping in the Ednra mouse revealed that the migration of these cells is aberrant in the COFT of the mutant embryos, but not in the pharyngeal arches. This premature arrest of CNCC migration appeared to be independent of CNCC proliferation and apoptosis changes. These results suggest that Ednra has a patterning function in the pharyngeal arches and controls CNCC migration in the COFT. Based on these results, we tested whether CNCC migration was arrested prematurely because CNCCs fail to activate specific cell migratory mechanisms by looking at migratory markers of the Rho family of GTPases. In the mutant embryos, Cdc42 fails to localize at the migratory front of the CNCCs migrating in the COFT. Cultured E9.25 whole embryos treated with the Cdc42 inhibitor ML141 recapitulated the phenotype. To confirm that Ednra and Cdc42 were acting in the same pathway, primary NCC from the cardiac area and mandibular pharyngeal arch were isolated for culture and treated with the Ednra ligand Edn1, ML141 and Ednra antagonist TBC3214. Western blot analysis revealed that Edn1 stimulated the phosphorylation of the Cdc42 downstream target ACK only in CNCCs and the addition of either inhibitor prevented ACK phosphorylation. Erk1/2 phosphorylation was observed in the mandibular NCCs upon Edn1 treatment but this was not prevented by the addition of the Cdc42 inhibitor. Wound healing scratch assay showed that the addition of Edn1 stimulated CNCC migration, which was repressed by the addition of either the Ednra or Cdc42 inhibitor. However, no such stimulation or inhibition was observed with the mandibular NCCs.In conclusion, these results demonstrate that endothelin signaling controls the migration of NCCs in the COFT via the specific activation of Cdc42 and this pathway is not necessary for the migration of NCC in the pharyngeal arches and the rearrangement of the pharyngeal arch arteries.Support or Funding InformationAmerican Heart Association 11BGIA 5670010NIH T32 DE018380Baylor Oral Health FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.