7. PROJECT ABSTRACT Congenital heart disease (CHD) is the most common human birth defect, with approximately 40,000 newborns affected annually in the United States. CHD is characterized by any defect in the formation of the heart and is often most severe when involving defects of the aortic arch artery (AAA) vasculature. The AAAs route oxygenated blood from the heart throughout the body and are formed from the remodeling of three pairs of pharyngeal arch arteries (PAAs), namely the 3rd, 4th and 6th. PAA-to-AAA remodeling depends upon a population of stem-like cells known as cardiac neural crest cells (cNCCs), and their differentiation to vascular smooth muscle cells (vSMCs) in the PAAs. Defective cNCC-to-vSMC differentiation results in AAA abnormalities including interrupted aortic arch, a severe defect of the 4th PAA which necessitates surgery upon birth. The extreme nature of arch artery defects resulting from defective cNCC-to-vSMC differentiation presents the need to investigate normal processes of cNCC-to-vSMC differentiation with a particular focus on the sensitivity of the 4th PAA to defects. Our lab previously found that the extracellular matrix protein, Fibronectin (Fn1), is essential for the process of cNCC-to-vSMC differentiation. Fn1 is expressed in cNCCs along a spatiotemporal gradient in murine pharyngeal arches. At E9.5, its expression is detected in migrating cNCCs; but by E10.5, when cNCCs are filling the PAAs, its expression is attenuated. By E11.5, just prior to cNCC-to-vSMC differentiation, its expression is restored in such a manner that cNCCs closest to the PAA lumen exhibit the highest expression while those farther away exhibit less, phenocopying the pattern of cNCC-to-vSMC differentiation. Ablation of Fn1, specifically in cNCCs, results in attenuated vSMC differentiation and defective PAA-to-AAA remodeling. This phenotype is not rescued by exogenous Fn1 or the presence of other matrix proteins, indicating the cell-autonomous function of Fn1 in cNCCs. As critical as Fn1 is in cNCC-to-vSMC differentiation and downstream PAA-to-AAA remodeling, how its expression is regulated in cNCCs is unknown. We hypothesize that the induction of Fn1 within cNCCs is initiated by blood-flow induced TGF? signaling in cNCCs or the endothelium. We propose to investigate the role of the TGF? superfamily (via Smad4) in regulating Fn1 in cNCCs for various reasons: (1) The role that the TGF? family plays in regulating Fn1 during embryogenesis is not well understood (2) TGF? is a mechanoresponsive pathway which may regulate the spatiotemporal gradient of Fn1 in response to blood flow (3) there is controversy over the role of TGF? in cNCC development and (4) there are improved genetic tools which will allow us to investigate TGF? signaling in our system. Our preliminary data suggests that Smad4 is important for cNCC-specific Fn1 production in the pharyngeal arches, yet we do not know the cell type in which Smad4 must function for this response. We propose to address the role of Smad4 in a cell-type specific manner with the experiments outlined in this F31 application (see Research Strategy). Completion of the experiments outlined in this proposal will help to elucidate factors which may contribute to the etiology of arch-artery CHDs.
The differentiation of cardiac neural crest cells (cNCCs) to vascular smooth muscle cells (vSMCs) is critical for remodeling of the pharyngeal arch arteries (PAAs) to the aortic arch artery vasculature (AAA). Defective cNCC- to-vSMC differentiation can result in various arch artery defects, which constitute some of the most severe forms of congenital heart disease- the most common human birth defect. We have found that Fibronectin is essential for cNCC-to-vSMC differentiation and PAA-to-AAA remodeling; thus, we propose to investigate how Fn1 is regulated in cNCCs to clarify factors which may contribute to severe CHD etiologies.
