Formation of the pharyngeal arch arteries (PAAs) is defective in patients with DiGeorge syndrome, the most common chromosomal abnormality in humans. However, the cellular and molecular mechanisms regulating PAA formation are not well understood. For many years, it was believed that PAAs developed by the process of angiogenesis, whereby PAAs were thought to form as branches stemming from the dorsal aorta that invade the avascular pharyngeal arches. However, recent studies in mutant mice with defective PAA formation suggested that the mutant phenotypes and the presence of a primitive vascular plexus within the pharyngeal arches were incompatible with the idea that PAAs formed via angiogenesis. Instead, these studies suggested that PAAs develop by the process of vasculogenesis, whereby PAAs arise de-novo from pharyngeal vascular endothelial progenitors. We employed transient labeling and cell lineage tracking to resolve this issue and demonstrated that endothelial cells of PAAs arise from the second heart field. Furthermore, our time-resolved, 3D confocal imaging showed that PAAs form by reorganization of the primitive vascular plexus into the PAA. In the course of our studies, we discovered that the extracellular matrix glycoprotein fibronectin (Fn1) is required for the formation of the 4th pair of PAAs, and that conditional deletion of Fn1 in the Isl1 lineage caused severe congenital aortic arch defects, similar to those observed in DiGeorge syndrome. We found that the number and density of endothelial cells in Fn1flox/-;Isl1Cre animals is much lower than in controls, and hypothesize that Fn1 mediates PAA formation by regulating the differentiation of SHF-derived endothelial progenitors into endothelial cells or by regulating the migration of endothelial progenitors from the SHF into the pharyngeal arches. In this grant application, we propose to test these hypotheses and to refine the spatio-temporal mechanisms regulating the development of PAA progenitors. We then propose to apply our methodology to systematically elucidate the basis for the PAA formation defects in Tbx1+/- mutants that model the DiGeorge syndrome. We plan to achieve these goals by addressing the following specific aims: 1) To determine the mechanisms, by which Fn1 regulates PAA morphogenesis; 2) To determine the role of VEGFR2positive cells within the SHF in PAA development; and 3) To determine the stage(s) of PAA formation regulated by Tbx1. We will use conditional mutagenesis, time-resolved confocal imaging and 3D reconstruction to test our hypotheses, and investigate the role of Fn1 in growth factor signaling during PAA formation. Furthermore, we will employ an innovative live multi-photon microscopy to investigate the dynamics of endothelial cells during PAA formation in living embryos, and the roles of Fn1 and Tbx1 in this process. Completion of these studies will provide important insights into the mechanisms of normal PAA formation and into alterations that cause defective PAA development in patients with congenital heart disease.

Public Health Relevance

Defects in the development of the aortic arch arteries (AAAs) are common in patients with congenital heart disease. Mouse models indicated that abnormal formation of the pharyngeal arch arteries, the precursors of the AAAs, underlies AAA pathologies associated with DiGeorge syndrome, the most common chromosomal abnormality in humans. Completion of the studies outlined in this proposal will provide important insights into the physiological regulation of AAA formation and the alterations causing disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL134935-03
Application #
9829751
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Schramm, Charlene A
Project Start
2017-08-05
Project End
2021-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Rutgers University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
078795851
City
Newark
State
NJ
Country
United States
Zip Code
07103