Epithelial mesenchymal transition (EMT) is a cellular process that mediates transition from two-dimensional cellular sheets to a three- dimensional structure (Hay, 1995). EMT is widely utilized by the embryo for gastrulation, neural crest cell formation, heart valve

632 INTRODUCTION Epithelial mesenchymal transition (EMT) is a cellular process that mediates transition from two-dimensional cellular sheets to a threedimensional structure (Hay, 1995). EMT is widely utilized by the embryo for gastrulation, neural crest cell formation, heart valve formation and other developmental processes (Hay, 1995; Yang and Weinberg, 2008). In the adult, EMTs occur in the pathologies of both metastasis and organ fibrosis (Thiery et al., 2009). EMT is initiated through a variety of signals including growth factors, inflammatory signals and extracellular proteases (Nieto, 2011; Thiery et al., 2009). 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 factor (TGF)β2 and TGFβ3, but sequential regulation has not been identified in any other EMT (Boyer et al., 1999). We identify here a novel regulator of cell invasion during EMT in the avian heart, Olfactomedin-1 (OLFM1). These findings extend the observation that EMT is mediated by sequential signals and that the ability of a cell to invade a matrix is a regulated element of EMT. OLFM1 (variant 1) (also known as Noelin-1 and Pancortin-1) is an extracellular matrix protein expressed in the developing heart (reported here), the central nervous system and in a variety of tumor tissues (Kim et al., 2010; Moreno and Bronner-Fraser, 2005; Sun et al., 2006). It is a member of the olfactomedin family of proteins and contains a conserved 250 amino acid olfactomedin domain. Olfactomedin domains are binding sites for protein-protein interaction between olfactomedin family members such as Myocilin and Olfactomedin-3 (Tomarev and Nakaya, 2009). There are four numbered variants of OLFM 1, with variant 1 being the longest isoform (Barembaum et al., 2000). Though called Noelin-1 in the avian species, we use OLFM1 here as the conserved gene name. Avian OLFM1 is conserved across species with 92% amino acid sequence similarity with Xenopus and 85% similarity to murine OLFM1. Mouse and chick transcripts have numbered isoforms, 14, generated through alternative splicing. The nomenclature in the human is different as OLFM1 is a homolog of Noelin-1 and Pancortin-1 but human Olfactomedins 2-4 are unique gene products. Alternately spliced forms of OLFM1 in humans are referred to as variants 1-4. OLFM1 was found to induce additional neural crest cells in the embryo (Barembaum et al., 2000), a process that includes EMT as well as cell differentiation and migration. Because its role in neural Disease Models & Mechanisms 6, 632-642 (2013) doi:10.1242/dmm.010595