Embryonic morphogenesis requires coordinated communications among diverse cell types. Dysregulation of these interactions results in birth defects. Signaling among cells in vivo occurs in the context of the extracellular matrix (ECM), and both the chemical and mechanical properties of the ECM regulate cell signaling and progenitor cell fates. Mechanisms whereby components of the ECM regulate embryonic morphogenesis are not well understood. In the course of the studies supported by our first R01, we made the intriguing discovery that synthesis of the ECM glycoprotein fibronectin (Fn1) is dynamically regulated during mouse embryonic development, and that the expression of Fn1 is highly enriched in distinct embryonic progenitors important for cardiovascular morphogenesis. Fn1 is a highly conserved vertebrate glycoprotein encoded by a single gene in mice and humans, and previous studies showed that Fn1 is required for cardiovascular development. Therefore, we hypothesized that different cellular sources of Fn1 played distinct, biologically significant roles in the development of the cardiovascular system. Preliminary data presented in this grant demonstrate that Fn1 synthesized by the neural crest (NC) is essential for the development of the aortic arch arteries (AAAs), a system of blood vessels that routs oxygenated blood from the heart to the rest of the body. Fn1 mRNA and protein become highly induced in NC cells surrounding the pharyngeal arch arteries as these blood vessels mature into the AAAs, and we show that Fn1 synthesized by NC cells around the pharyngeal arch arteries regulates the differentiation of NC cells into vascular smooth muscle cells (VSMCs). We present evidence suggesting that Fn1 synthesized by the NC regulates NC-to-VSMC differentiation in a cell- autonomous manner by signaling through integrin ?5?1 and regulating actin polymerization, which in turn, regulates the translocation of the myocardin-related transcription factor B (MRTFB) from the cytoplasm into the nucleus. In the nucleus, MRTFB can directly activate the transcription of smooth muscle genes, and we show that a deletion mutant of MRTFB that constitutively localizes to the nucleus rescues smooth muscle differentiation in Fn1-deficient NC cells. In this renewal application we propose to determine the mechanisms by which Fn1 regulates NC-to-VSMC differentiation and the role of tissue biomechanics in this process by addressing the following Specific Aims: 1) To test the hypothesis that Fn1 regulates the differentiation of NC cells into VSMCs by signaling through integrin ?5?1; 2) To test the hypothesis that Fn1 and tissue biomechanics regulate NC-to-VSMC differentiation by modulating actin cytoskeletal dynamics and nuclear localization of MRTFB; and 3) To test the hypotheses that EIIIA+ and EIIIB+ forms of Fn1 regulate NC-to-VSMC differentiation and that the induction of Fn1 in NC cells surrounding the endothelium of the pharyngeal arch arteries is mediated by SMAD2-dependent signaling.
Frequency of congenital heart disease (CHD) has been estimated to be at about 1% among live births. Defects in the development of the aortic arch arteries give rise to some of the most severe forms of CHD, which remain life threatening even following corrective surgery. Elucidation of molecular and genetic pathways regulating normal aortic arch artery patterning is required to understand what goes awry in CHD.
