Semilunar valve (SLV) diseases, including bicuspid aortic valves (BAV), are remarkably common and yet their genetic and developmental origins are poorly understood. Likewise, it remains unclear how disrupted embryonic valve development progresses into overt valve disease. Our long-term goal is to understand how gene regulation drives sequential developmental processes that ultimately produce complex, patterned valves and how these processes go awry in SLV disease. These gene regulatory events require transcription factors to interface with a chromatinized genome, suggesting that chromatin regulators are key components of SLV developmental networks. One important event is an endocardial-to-mesenchymal transformation (EMT) that occurs early in valve development to populate endocardial cushions (ECs), including the proximal outflow tract (pOFT) cushions that contribute tissue to SLVs. Our objectives are to 1) understand how chromatin remodeling integrates with cell signaling during EMT, and 2) determine mechanisms by which disruptions of valve development progress into diseased SLVs. Our central hypothesis is that endocardial Brg1-associated factor (BAF) chromatin remodeling complexes interact with Wnt signaling effectors to promote pOFT EMT. As a result, when endocardial Brg1 is deleted a subtype of OFT mesenchyme is depleted. Without these cells, cusp overgrowth and fusion results in thickened and malpatterned SLVs, including BAV. The rationale for our efforts is that defining chromatin remodeling roles during EMT will shed light on how SLV disease originates. Further, our mouse models of SLV disease will enable an understanding of the cellular and molecular progression of valve disease.
Our specific aims are: 1) Determine the molecular networks that the BAF complex interfaces with to direct EMT and 2) Determine mechanisms of SLV disease progression in mice lacking endocardial- lineage Brg1. In pursuit of the first Aim, we will compare cellular and molecular pOFT defects seen in unpublished genetic models disrupting Brg1 and Wnt signaling. We will apply a transformative new TU-tagging technology to define dynamic, endocardial transcriptomes dependent on each pathway. Using new cell culture approaches, we will test biochemical interactions between BAF, Wnt effectors, and chromatin in EC cells. For the second Aim, we will use genetic lineage tracing to determine contributions of EMT-derived cells to distinct SLV regions, define interactions between SLV mesenchyme sub-populations, characterize misexpressed transcripts that may drive SLV disease progression, and describe a new mouse model of adult SLV disease of potential utility in preclinical trials. Our proposed research uses novel technological and paradigmatic approaches to pursue unresolved questions of SLV development and disease. These contributions will be significant as they will shed light on the human genetics of SLV disease and inform regenerative medicine approaches. Our newly identified transcripts associated with a BAV model may represent biomarkers for disease diagnostics or therapeutic targets to prevent congenitally abnormal valves from becoming diseased.
The project will identify genetic causes of congenital heart valve defects and generate mouse models of the most common defects underlying aortic valve disease. Research outcomes will impact the design of regenerative medicine approaches to repair diseased valves and identify potential therapeutic targets to prevent disease progression.
|Gay, Leslie; Karfilis, Kate V; Miller, Michael R et al. (2014) Applying thiouracil tagging to mouse transcriptome analysis. Nat Protoc 9:410-20|