Multi-Lineage Modulation of cardiac neural crest cells during cardiac development

Project Abstract Congenital heart disease (CHD) is the most common birth defect with complex CHD causing significant morbidity/

mortality. Therapies targeting adult heart failure have been ineffective in treating heart failure in patients with CHD, which implies different pathomechanisms of congestive heart failure in patients with CHD compared with those with

structurally normal hearts. Therefore, there is an urgent need to understand the genetic basis of CHDs. Genetic analysis in CHD patients is confounded by the genetic heterogeneity of the human population. Hence, we used large-scale chemical/N-ethyl-N-nitrosourea (ENU) mutagenesis in inbred mice to recover mutations that cause CHD. We recovered

mutations in ten vesicular/endocytic trafficking proteins, including a missense mutation (C4232R) in the highly conserved epidermal growth factor (EGF) repeat domain encoding low-density lipoprotein receptor-related protein 1 (LRP1). All Lrp1m/m mutant mice exhibited CHD comprising outflow tract (OFT) malalignment defects and/or

atrioventricular (AV) septal defects. Conditional knockout of Lrp1 in different cell lineages that are crucial in cardiac development demonstrated ablation of cardiac neural crest cells (CNCC) using Wnt1-Cre driver recapitulate the phenotypes of ENU-induced mutants. Interestingly, these Cre deletion experiments also showed Lrp1 deficiency in

other cell lineages along the CNCC migratory path generated with Nkx2-5+/cre, Tie2+/cre also yielded either double outlet

right ventricle (DORV), atrioventricular septal defect (AVSD), or ventricular septal defect (VSD) phenotypes. As these are observed with variable penetrance, they suggest CNCC migration to the heart is supported by redundant signaling to

ensure high fidelity of CNCC deployment to the heart. In addition, the combined double knockout of Nkx2-5+/cre Tie2+/cre: Lrp1f/f mutants demonstrated synergistic effects which reflect the complex genetics in human CHD. Based on findings from a large body of preliminary data, we hypothesize that LRP1 is required in the cardiac neural crest lineage and its

migratory path for proper OFT alignment and development of the atrioventricular cushions (AVC). We will investigate this with a combination of in vivo and in vitro experiments conducted using Wnt1+/cre: Lrp1f/f mutant mice with Lrp1

deletion targeted to the CNCCs and double knockout of Nkx2-5+/cre Tie2+/cre: Lrp1f/f mutants. First, we will investigate the deployment of CNCCs with in vitro migration studies in combination with in vivo cell lineage fate mapping analysis (Aim

1). We will further investigate the cellular and molecular basis for how Lrp1 deficiency in CNCCs can lead to OFT and AV valve defects by conducting single-cell RNA sequencing analysis, as well as in vivo and in vitro analysis of endocytic

trafficking (Aim 2). This will include the examination of endocytic trafficking related to cell signaling pathways identified in the CNCC and its migratory path. We will also investigate the clinical relevance of LRP1 mutations in human CHD by generating CRISPR gene-edited mice bearing LRP1 mutations recovered from CHD patients (Aim 3). This will include an

analysis of heterozygous mutations to assess a dominant genetic model of disease, as well as compound heterozygous mutations to investigate a recessive genetic model of disease. Together, these studies will provide novel mechanistic insights into the role of endocytic trafficking in the pathogenesis of human CHD.