Role of BMP signaling in cardiac neural crest development - PROJECT SUMMARY/ABSTRACT Congenital heart defects are the most prevalent birth defects observed in humans, several of which result from abnormalities in an embryonic stem cell population called the cardiac neural crest (CdNC). These cells, which originate in the vertebrate hindbrain, are characterized by their extensive migratory ability and capacity to differentiate into numerous components of the heart, including cardiac ganglia and the smooth-muscle septum between the aortic and pulmonary vessels. The mechanisms that govern CdNC derivation and spatiotemporal differentiation remain largely a black box. However, observations from preliminary and historical work suggest that the Bone Morphogenetic Protein (BMP) signaling pathway and cell-cell interactions between the ectoderm and mesoderm play critical roles in modulating CdNC development and contributions to heart organogenesis. The present study will leverage the unique strengths of multiple in vivo (chick and mouse embryos) and in vitro (human embryonic stem cell) models to elucidate how BMP signaling is regulated intrinsically and extrinsically to govern the derivation of the CdNC and its contributions to the heart. Aim 1 will define the role of the BMP signaling antagonist, Noggin, which is specifically expressed in the CdNC, in establishing CdNC identity by assessing the specification and behavior of chick and mouse neural crest cells in vivo following genomic perturbation of Noggin, as well as by performing transcriptomic analysis of Noggin loss-of-function mutant cells to identify downstream BMP target genes with altered expression. Aim 2 will uncover the gene regulatory mechanism underlying the CdNC-specific expression of Noggin by using a two-pronged approach of knocking out CdNC-specific transcription factors co-expressed with Noggin by CRISPR-Cas9, and site-directed mutagenesis of a putative Noggin enhancer which perfectly mimics endogenous Noggin expression in the CdNC. Additionally, Aim 2 will also explore a potential BMP signaling feedback loop and dose-dependent autoregulation of Noggin expression within the CdNC. Aim 3 will unravel the molecular crosstalk between ectoderm-derived CdNC and the mesodermal cells of surrounding tissues, particularly the somitic mesoderm, and unmask the resulting impacts on CdNC cell fate, by employing novel in vitro cultures of human CdNC cells derived from human embryonic stem cells. Taken together, these experiments will resolve how intrinsic genetic circuitry and extrinsic molecular cues synergistically coordinate spatiotemporal patterning of CdNC cells within the hindbrain, and their contributions to cardiovascular development. The completion of this project and the associated career development plan will support Dr. Gandhi in his goal to become a principal investigator studying the etiology of cardiac-crest-derived congenital defects in a manner that maximizes relevance to future therapeutic applications.
