At the earliest stages of development, the atrioventricular ECs contain cells which are predominantly GFP-positive in a flk1::GFP transgenic line (Beis et al.,2005). However, as seen in Figure 1D, by 16 dpf a significant number of cells in the leaflet are flk1::GFP-negative, suggesting that some AV cells are no longer endothelial as flk1 expression is a hallmark of endothelial cells (Liao et al.,1997; Sumoy et al.,1997; Kamei et al.,2004; Jin et al.,2005). To determine the identity of these cells, immunohistochemistry was performed using antibodies that are specific for either epithelial or mesenchymal cells and which identify cells transitioning between these identities. Epithelial cells are typically identified by an organized structure, the presence of tight junctions which contain ZO-1, and cytokeratins (Lee et al.,2006). Mesenchymal cells are expected to stain for vimentin, be surrounded by ECM components such as Versican, and have increased focal adhesions and focal adhesion kinase. An intermediate cell type also exists which shows markers of both epithelial and mesenchymal cells along with a loss of tight junctions (Lee et al.,2006). Our data show that at 12 dpf (Fig. 3A–C), the developing AV valves are positive for ZO-1–containing junctions (Fig. 3A), punctate focal adhesion kinase staining (Fig. 3B), are pancytokeratin positive (Fig. 3G), and lack significant Vimentin (Fig. 3H) indicating the epithelial identity of these cells. However, by 16 dpf, cells are found in the developing leaflet which have lost their ZO-1–containing tight junctions (Fig. 3D), stain for both pancytokeratin (Fig. 3J) and vimentin (Fig. 3K), and which have large depositions of focal adhesion kinase (Fig. 3E), which suggest that AV valve cells are transitioning between an epithelial and mesenchymal phenotype, which is indicative of a intermediate cell type. Taken together, these data suggest that the flk1:GFP-negative cells at 16 dpf are intermediate—demonstrating both epithelial and mesenchymal traits. These data also suggest that the developing valve leaflets are initially composed primarily of sheets of epithelial endocardium, but that expansion and organization of the valves takes place as endocardial cells enter the valve mesenchyme.
Figure 3. Valve leaflet cells become intermediate or mesenchymal between 12 and 16 days postfertilization (dpf). A–L: Five-micrometer paraffin sections of flk1::GFP (GFL, green fluorescent protein) transgene-carrying zebrafish embryos/larvae were immunostained to identify epithelial and mesenchymal cell phenotypes in the developing leaflets. Atria (At) and ventricles (Ve) are labeled (yellow letters), and yellow lines outline the extent of the atrioventricular (AV) valves. A–F: Staining identifies ZO-1 (A, D; green in C and F), focal adhesion kinase (B, E; red in C and F), and cell nuclei (DAPI: blue in C and F). The presence of ZO-1 reveals tight junctions that form to make cell to cell connections, which is indicative of an epithelial phenotype. FAK reveals whether there are transient (punctate) or long lived (large) cell:matrix adhesions occurring between these cells. A–C: At day 12, AV valve cells are organized, have ZO-1–containing tight junctions, and punctate FAK staining. D–F: By day 16, cells are less organized and some lack ZO-1–containing junctions. G–L: Staining identifies pancytokeratin (G, J; red in I and L), Vimentin (H, K; green in I and L), and cell nuclei (DAPI, 4′,6-diamidine-2-phenylidole-dihydrochloride; blue in I and L). G–I: At 12 dpf, the valves have a high level of pancytokeratin staining indicating that surrounding cells have a predominantly epithelial phenotype. J–L: By 16 dpf, a large increase in Vimentin is seen, indicating a shift toward mesenchymal cell phenotypes. Scale bar = 20 μm in A.
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