Abstract
Malformations of the cardiovascular system are the most common type of birth defect in humans, frequently affecting the formation of valves and septa. During heart valve and septa formation, cells from the atrio-ventricular canal (AVC) and outflow tract (OFT) regions of the heart undergo an epithelial-to-mesenchymal transformation (EMT) and invade the underlying extracellular matrix to give rise to endocardial cushions. Subsequent maturation of newly formed mesenchyme cells leads to thin stress-resistant leaflets. TWIST1 is a basic helix-loop-helix transcription factor expressed in newly formed mesenchyme cells of the AVC and OFT that has been shown to play roles in cell survival, cell proliferation and differentiation. However, the downstream targets of TWIST1 during heart valve formation remain unclear. To identify genes important for heart valve development downstream of TWIST1, we performed global gene expression profiling of AVC, OFT, atria and ventricles of the embryonic day 10.5 mouse heart by tag-sequencing (Tag-seq). Using this resource we identified a novel set of 939 genes, including 123 regulators of transcription, enriched in the valve forming regions of the heart. We compared these genes to a Tag-seq library from the Twist1 null developing valves revealing significant gene expression changes. These changes were consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. To study the role of TWIST1 at the DNA level we performed chromatin immunoprecipitation and identified novel direct targets of TWIST1 in the developing heart valves. Our findings support a role for TWIST1 in the differentiation of AVC mesenchyme post-EMT in the mouse, and suggest that TWIST1 can exert its function by direct DNA binding to activate valve specific gene expression.
Highlights
The transformation of the heart tube into a four-chambered organ divided by valves and septa is a critical event during mammalian heart development and is required for proper function
atrioventricular canal (AVC) and outflow tract (OFT) Share Significant Gene Expression Since expression of Twist1 is spatially restricted to the endocardial cushions in both the AVC and OFT of the developing heart, we first assessed the diversity of genes that are normally enriched in the AVC and OFT
We examined 16 genes previously characterized as enriched in the AVC and involved during its development to establish whether our Tag-seq libraries captured known patterns of gene expression (Table S3)
Summary
The transformation of the heart tube into a four-chambered organ divided by valves and septa is a critical event during mammalian heart development and is required for proper function. The embryonic heart is a linear tube composed of endocardial and myocardial cell layers separated by an acellular extracellular matrix (ECM) termed the cardiac jelly. During formation of the heart valves and septa, the cardiac jelly, which gives rise to the endocardial cushions [1], begins to accumulate in the region between the atria and ventricles, known as the atrioventricular canal (AVC), and the junction between the ventricles and the major arteries, known as the outflow tract (OFT). During EMT, groups of endocardial cells destined to undergo transformation are first specified and become hypertrophic. These cells lose polarity markers and intercellular cadherins and adherens junctions. Removal of excess mesenchyme cells by apoptosis starts at E12.5 in mice, and patterning of ECM molecules relative to direction of blood flow turns the endocardial cushions into thin stress-resistant AV valve leaflets and semilunar valve cusps by the first week after birth [10,11,12]
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