Poster Sessions

DEV-13

Yon Ju Ji

Y. J. Ji, H. Lee, I. Daar

Flotillin Regulates Neural Tube Formation in the Wnt/Planar Cell Polarity (PCP) Pathway

Eph receptors are the largest family of receptor tyrosine kinases and their ligands are membrane bound proteins known as ephrins. Unlike other signaling mechanisms, Eph/ephrin signaling is bi-directional, where both the receptor and ligand expressing cells transduce signals. We focus on ephrinB1, a transmembrane Eph ligand, and how it is able to accomplish reverse signaling through its intracellular domain. We have discovered that ephrinB1 functions in retinal progenitor cell movement and cell-cell adhesion. Since there is strong evidence that de-regulation of the Eph/ephrin signaling pathway is involved in several metastatic cancers, studying the precise mechanism of how ephrinB1 regulates cell movement and cell adhesion becomes more urgent. To determine relevant in vivo proteins that may complex with ephrinB1, we utilized mass spectrometric analysis of ephrinB1 immune-complexes from frog embryos over-expressing ephrinB1. Among the identified candidates, Flotillin-2 was selected due to its strong avidity to ephrinB1. Flotillins are known as lipid raft proteins which function in the formation of membrane domains. After confirming the ephrinB1/Flotillin2 interaction by co-immunoprecipitation (Co-IP) analysis, an additional form of Flotillin (Flotillin1) was assessed and determined to have a robust interaction with ephrinB1. To assess the possible role of Flotillin during embryogenesis, antisense-morpholino oligonucleotides (MOs) were introduced into frog embryos to knockdown expression. Loss of Flotillin expression caused a dramatic defect, failure of neural tube closure. Neural tube formation is regulated by the Wnt/PCP pathway and Flotillin is involved in Wnt signaling in fly. Since the Wnt/PCP pathway regulates convergent extension (CE) movements in vertebrates, an ectodermal explant elongation assay that mimics CE movements was performed. Activin treatment of explants caused 90% of the explants to elongate, however, only 5% of explants elongated where Flotillin1 expression was blocked by MOs, suggesting that Flotillin1 function is required for CE movements. Co-IP analysis demonstrated that Flotillin also formed a complex with Dsh (a critical signal transducer in the Wnt signaling pathway), supporting the role for Flotillin in the Wnt/PCP pathway. Taken together, Flotillin functions in the dynamic process of neural tube formation in the context of the Wnt/PCP pathway.