Abstract 1479: Cardiac Neural Crest Specific Deletion of Notch2 Causes Defective Development of the Outflow Tract.
Notch signaling is an evolutionarily conserved pathway that influences cell fate decisions required for the development of most organ systems including the heart. To study the functional requirements for Notch2 signaling during cardiovascular development, we used Pax3-Cre transgenic mice to inactivate Notch2 specifically in the cardiac neural crest derived cells that contribute to the smooth muscle tissue of the aorta and pulmonary arteries. Whole mount and histological analysis showed that the Pax3-Cre; Notch2flox mice, mice that have Notch2 inactivated, had abnormally narrow aortas and pulmonary arteries. Consequently, about half of the Pax3-Cre; Notch2flox mice die shortly after birth apparently due to a decrease in cardiovascular function. To determine if Notch2 function is required for the migration of cardiac neural crest derived cells, Pax3Cre; Notch2flox mice were analyzed using the GtROSA26 lacZ Cre reporter strain to track the fate of cells where Cre is active, and Notch2 is inactivated. At E11.5 all Pax3-Cre;Notch2flox;GtROSA26 embryos showed no change in the number or location of lacZ+ cells present in the outflow tract relative to littermates that were Pax3-Cre;GtROSA26. In contrast, the number of lacZ+ cells in the outflow tract of embryos at E16.5, E18.5 and neonatal mice (P3) were significantly reduced in mice that have Notch2 inactivated. These results show that loss of Notch2 expression in cardiac neural crest cells did not affect their migration but instead either reduced proliferation or induced apoptosis in post-migratory neural crest cells. Since cardiac neural crest cells form the vascular smooth muscle cells of the outflow tract, we performed fluorescent immunohistochemistry with anti BrdU and α-smooth-muscle-actin antibodies. At E16.5 cell proliferation of smooth muscle positive cells was greater in the wild type mice 36 ± 0.9 %, than in mutant mice 10.75 ± 1.0 %. No increase in apoptotic cells was observed when mutants were compared to the wild type using TUNEL assay. Our findings demonstrate that Notch2 is required cell autonomously for proper formation of the heart outflow tract and provides insights into the role of Notch2 in vascular smooth muscle development and cardiovascular defects associated with Alagille syndrome.