Mouse functional analysis of genes for congenital heart disease

ABSTRACT ? PROJECT 3 This P01 renewal application is in response to RFA, HD-16-009 from the NICHD Institute of NIH. The scientific goal for Project 3 is to determine the developmental mechanisms of congenital heart disease using the mouse as a model system. The class of congenital heart disease that is the focus of the P01 is termed, conotruncal and related aortic arch defects, referred to as CTRDs. As part of the P01, we will identify genes and noncoding regions of interest from patients with 22q11.2 deletion syndrome (22q11.2DS; aka DiGeorge syndrome velo-cardio-facial syndrome; Project 1) and non-syndromic disease. Among the 40 genes deleted in patients with 22q11.2DS, two of them, TBX1, encoding a T-box transcription factor and CRKL, encoding a cytoplasmic adaptor for intracellular signaling, are considered the strongest candidate genes. During the current P01, we created and utilized conditional loss of function mutant mouse models for Tbx1 and Crkl to understand their function. The cardiac outflow tract and aortic arch develop from progenitor anterior heart field and neural crest cells that lie within the embryonic pharyngeal apparatus. The Tbx1 gene is expressed in the epithelia (endoderm and ectoderm) as well as anterior heart field mesoderm of the pharyngeal apparatus, while Crkl is ubiquitously expressed, with highest expression in adjacent neural crest cells. Tbx1 is not expressed in the developing heart, implicating its function truly in the migrating cells entering the heart. Previous studies demonstrated that Tbx1 and Crkl genetically interact, but the mechanistic basis of this has only been partially explored. In Aim 1 of this renewal program, we will understand the tissue specific mechanism by which Tbx1 and Crkl interact in forming and remodeling the pharyngeal arch arteries. In Aim 2, we will perform RNA-seq on the microdissected distal pharyngeal apparatus in wildtype and mutant mouse embryos (Tbx1, Crkl and Lgdel/+) and create an interactive gene network termed the PA-INet to provide new insights to cardiovascular development and to send to Projects 1 and 2. In Aim 3, we will uncover regions of open chromatin from the pharyngeal tissue, so as to prioritize loci harboring noncoding variants from SNP genotyping arrays and whole genome sequencing. In addition, Aim 3 will utilize a pipeline to perform functional analysis of genes and loci found in Projects 1 and 2, starting with computational in silico analysis, in vitro or cell culture assays and finally testing in mouse models. Overall, Project 3 is designed to advance the science by determining developmental mechanisms and to enhance discoveries made in Projects 1 and 2. !