Abstract
SUMMARYEndothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.
Highlights
Epithelial mesenchymal transition (EMT) is a cellular process that mediates transition from two-dimensional cellular sheets to a threedimensional structure (Hay, 1995)
OLFM1 protein and antibody treatments did not result in the expected regulation of several EMT transcription factors, and did not reduce the expression of adhesion molecules that are normally lost upon EMT
OLFM1 regulated the expression of several secreted mesenchymal cell markers and the expression of proteases that are consistent with cell invasion
Summary
Epithelial mesenchymal transition (EMT) is a cellular process that mediates transition from two-dimensional cellular sheets to a threedimensional structure (Hay, 1995). A common view is that induction or stabilization of one or more transcriptional repressors such as SNAI1 or ZEB1 leads to loss of cell-cell adhesion and initiates a cascade of processes leading to EMT (Nieto, 2011). The details of this cascade are still being explored but its interruption could lead to epithelial plasticity or a metastable state rather than a complete transition (Klymkowsky and Savagner, 2009). In avian heart valve formation, EMT is mediated by activities of transforming growth
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