Abstract 1568: Mef2c Downstream Targets Direct Cardiac Morphogenesis
Mef 2c is an essential MADS-box transcriptional regulator that directs cardiac morphogenesis. Embryos lacking Mef2c are nonviable and have perturbed cardiac morphogenesis. While gene disruption strategies emphasize the importance of this transcription factor, its downstream targets remain ill-defined. To identify Mef2c downstream targets, we utilized transgenic and molecular biological technologies. We have previously established that the 6 kb Nkx2–5 EYFP transgenic model directs reporter expression to the cardiac progenitors early during cardio-genesis. We utilized this transgenic model and combinatorially mated it into the Mef2c wildtype and null backgrounds. Nkx2–5-EYFP labeled cardiac progenitors were isolated using FACS analysis. RNA was isolated and transcriptome analysis was undertaken to identify dysregulated transcripts in stage match Mef2c null cardiac progenitors compared to wildtype controls. Using dCHIP software analysis to evaluate significant changes in transcript expression, we identified distinct and 34 common downregulated transcripts in E8.25 and E9.5 Mef2c null cardiac progenitors. Transcripts downregulated in Mef2c null cardiac progenitors at both developmental time points include Anf, Actn2, Smyd1, Nppa, Myl3, and Cryab. These results were confirmed using qRT-PCR and immunohistochemical techniques to examine the spatial and temporal expression pattern for these putative downstream targets in stage matched wildtype and Mef2c null embryos (E8.25 and E9.5). Additionally, we utilized an adenoviral overexpres-sion strategy to examine transcripts that were induced by Mef2c in cultured neonatal cardiomyocytes and C2C12 myoblasts. This strategy further confirmed the induction of these candidate targets by Mef2c. These studies support the notion that Mef2c is an upstream regulator for transcriptional regulators and structural genes that are essential for cardiac morphogenesis.