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5 Tie2cre;Snai1fl/+ compared to Snai1fl/+ embryos. Table?1 indicates all the genes that were differentially expressed (p?[http://www.selleckchem.com/products/ldn193189.html Selleck LDN 193189] immunoreactivity (red, Figs. 3B, C). To examine snai1 expression relative to mmp15 during valve development, qPCR was performed using cDNA from AVC regions at E10.5, E12.5, E14.5 and post natal stages ( Fig.?3D). Approximate transcript levels of snai1 and mmp15 showed similar temporal expression patterns throughout valve development with transcript levels being highest at E10.5 and gradually declining to post natal stages. Consistently, co-localization studies show that snai1 and mmp15 are both expressed in endothelial (arrowhead, Fig.?3F) and mesenchymal (arrow, Fig.?3F) cells of the valves at E13.5 ( Figs. 3E, F). Although due to the expected membrane localization of mmp15 and largely [http://en.wikipedia.org/wiki/Diglyceride diglyceride] nuclear distribution of snai1, complete overlap is not always observed. Together, these [http://www.selleckchem.com/products/a-1210477.html A-1210477 mouse] studies suggest that snai1 and mmp15 are similarly expressed in developing valve structures and Snai1 function is required for mmp15 expression during EMT stages. Understanding the mechanisms of EC EMT has been greatly enhanced by studies in the chick using collagen I gel explant systems (Bernanke and Markwald, 1982?and?Person et al., 2005). In the avian model, EC formation is initiated in the looped heart at HH St. 14, and by HH St.18, EMT is active in all cushion sets (Person et al., 2005). Worthy of mention, the EMT inductive signals in the chick appear conserved with the mouse system and include Bmp and Tgf�� signaling (Armstrong and Bischoff, 2004, Combs and Yutzey, 2009?and?Person et al., 2005). To investigate if Snai1 and MMP15 are sufficient to promote EMT in AVC endothelial cells, we employed a published in vitro collagen I gel explant assay (Bernanke and Markwald, 1982, Mjaatvedt et al., 1987?and?Runyan and Markwald, 1983). In this assay, HH St.14 AV canals are placed on a 3-dimensional (3D) rat-tail collagen I gel with the endothelial cell layer facing down, allowing cells to migrate onto the surface of the gel (Runyan and Markwald, 1983). With the myocardium intact, subsets of endothelial cells then invade the underlying gel as newly transformed mesenchymal cells (Inai et al., 2008, Mjaatvedt et al., 1987?and?Runyan and Markwald, 1983).