Olfactomedin-1 activity identifies a cell invasion checkpoint during epithelial-mesenchymal transition in the embryonic heart

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.

Olfactomedin proteins: central players in development and disease

“…OLFM1 and OLFM3 are neuronal olfactomedin proteins, with OLFM1 corresponding to the rat neuronal olfactomedin found throughout the brain and OLFM3, also known as optimedin (Torrado et al, 2002 ), expressed in cerebellum (Kulkarni et al, 2000 ) and in the eye (Torrado et al, 2002 ). In addition to its expression in brain, OLFM1 is also expressed in embryonic heart and has been implicated in heart development (Lencinas et al, 2013 ). OLFM2 is expressed in pancreas and prostate, and OLFM4 in colon, small intestine, and prostate (Kulkarni et al, 2000 ; Figure 5 ).…”

Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies

“…The emerging functional picture is that this truncated species trimerizes via the collagen domains, binds to the first three fibronectin-III-like domains of neurofascin 186 to recruit axonal sodium channels and thus facilitates the formation of, and helps maintain, the Nodes of Ranvier [ 10 , 19 – 21 ]. Olfactomedin-1 has been linked to neurogenesis and neural crest formation [ 22 , 23 ], cortex development [ 24 ], valve formation in the developing embryo heart [ 25 ], spheroid attachment onto endometrial cells [ 26 ], and formation of actin stress fibers in podocytes [ 27 ]. Olfactomedin-1 has been shown to interact with select binding partners [ 8 , 28 ], of which just two involve binding to the olfactomedin-1 OLF domain [ 24 , 29 ].…”

Deletion in the N-terminal half of olfactomedin 1 modifies its interaction with synaptic proteins and causes brain dystrophy and abnormal behavior in mice

“…The olfactomedin domain is encoded by the last two 3′-exons found in the AMZ (or pancortin-1) and BMZ (or pancortin-2) forms. Available data suggest that Olfm1 plays a role in promoting neuronal cell death, cortical cell migration and axon growth in mice (Cheng et al, 2007; Nakaya et al, 2012; Rice et al, 2012), neural crest production and cell invasion during epithelial-mesenchymal transition in the embryonic heart in chicken (Barembaum et al, 2000; Lencinas et al, 2012), maintenance of neuronal precursor cells in Xenopus (Moreno and Bronner-Fraser, 2005), eye size regulation and optic nerve arborization in the optic tectum in zebrafish (Nakaya et al, 2008). …”