The long-term objectives of this proposal are to understand the genetic control of a specific subpopulation of endocardium, the pro-valve endocardial cells (PVECs), and determine their role in valve formation. During embryogenesis, NFATd expression is restricted to the endocardium and its expression is accentuated in the PVECs (endocardial cells that do NOT undergo mesenchymal transformation during the formation of cardiac valves). These observations suggest that a focus on NFATd regulation may provide unique insights into the transcriptional program orchestrating pro-valve endocardial development and valve formation. Having recently identified a 243 bp sequence within the first intron of NFATd that serves as a PVEC specific transcriptional enhancer required for accentuation of NFATd gene expression and having determined that a conserved 58 bp sequence within this enhancer region functions as a critical cis-regulatory element, controlling both the intensity and specificity of NFATd expression in the PVEC population, we hypothesized that this NFATd enhancer region defines an enhanceosome that plays a critical role in the regulation of NFATd expression essential for normal PVEC development and valve formation. Therefore, we propose to 1) Determine the molecular components and interactions required for activation of the PVEC-specific NFATd transcriptional enhancing complex using a novel enodardial cell line for gel shift, DNA affinity purification and mass psectrometry, 2) Define the in vivo role of the PVEC enhanceosome in PVEC development and valve formation utilizing a cre-loxp based dual-genetic mouse model for sequential analysis of enhanceosome suppression and activation of NFATd expression, and 3) Delineate the in vivo role of the PVEC endocardial subpopulation by spatiotemporal ablation of these cells using bigenic cre-/loxp, puAtk conditional genetic ablator mouse line. This work will define critical regulatory mechanisms required for heart valve development and will inform novel tissue-regeneration remedies for valve degeneration.

R01HL078881

2006-07-01

2012-05-31