Supplementary Materials Supplemental Textiles (PDF) JCB_201604097_sm. Our data demonstrate that dramatic ephrin-B1Cmediated cell segregation occurs in the early neuroepithelium. In contrast to the paradigm that repulsive bidirectional signaling drives cell segregation, unidirectional EphB kinase signaling leads to cell sorting by the Rho kinaseCdependent generation of a cortical actin differential between ephrin-B1C and EphB-expressing cells. These results define mechanisms of Eph/ephrin-mediated cell segregation, implicating unidirectional regulation of cortical actomyosin contractility as a key effector of this fundamental process. Introduction Normal development requires the self-organization of cells by sorting or segregation to establish and maintain boundaries and ultimately form distinct tissues in the adult organism (Fagotto, 2014). Cellular segregation can be achieved by three general cellular mechanisms, namely differential adhesion, cellCcell repulsion, and differential interfacial tension (Batlle and Wilkinson, 2012; Fagotto et al., Rabbit Polyclonal to BCAR3 2014; Cayuso et al., 2015). The differential adhesion hypothesis proposes that differences in adhesion between cell populations driven by qualitative or quantitative differences in the expression of cell adhesion molecules will drive the more adhesive populace to segregate to the inside of the less adhesive populace (Steinberg, 1963, 1970; Batlle and Wilkinson, 2012). Models of segregation involving cellCcell repulsion predict that segregating cells are repelled by and migrate directionally away from one another, eventually resulting in segregation. In contrast, the differential interfacial tension hypothesis (DITH) proposes that segregation is usually caused by differences in cortical tension between cells leading to a change in the pressure of the cellCcell contact, or interfacial tension. The actomyosin cytoskeleton is usually thought to be critical for interfacial tensionCdriven segregation, with prominent F-actin cables often forming at heterotypic boundaries, and cells with differences in Rho kinase (ROCK) activity segregate in vitro (Krieg et al., 2008; Monier et al., 2010). The Eph/ephrin family of signaling molecules mediates boundary formation during many developmental processes across organisms, including the development of rhombomeres, the eye, limb buds, somites, cranial sutures, and intestinal crypts in vertebrates, as well as in wing discs (Durbin et al., 1998; Xu et al., 1999; Santiago and Erickson, 2002; Barrios et al., 2003; Wada et al., 2003; Holmberg et al., 2006; Merrill et al., 2006; Ting et al., 2009; Cavodeassi et al., 2013; Umetsu et al., 2014). B-type ephrins are transmembrane proteins that bind to EphB receptors on neighboring cells and can signal bidirectionally. They stimulate forward signaling by activation of EphB receptor signaling and reverse signaling through SH2 and PDZ (named for PSD95, Dlg1, and ZO-1) adaptor proteins that bind to conserved phosphorylated tyrosines and PDZ ligands, respectively, around the intracellular domain name c-Fms-IN-9 of B-type ephrins. Forward signaling has been proposed to occur via both kinase-dependent and kinase-independent mechanisms (Birgbauer et al., 2000; Holmberg et al., 2006; Dravis and Henkemeyer, 2011). Numerous cell culture and explant studies have investigated Eph/ephrin downstream signaling pathways, revealing important functions for Rho family GTPase signaling, though no genetic research in unchanged embryos have however been reported (Tanaka et al., 2003; Rohani et al., 2011; Nievergall et al., 2012). The existing paradigm, produced from overexpression research mainly, proposes that bidirectional Eph/ephrin signaling mediates adjustments in adhesion and repulsive migration to operate a vehicle cell segregation; whether differential interfacial stress plays a part in c-Fms-IN-9 Eph/ephrin cell segregation is certainly unidentified (Xu et al., 1999; Poliakov et al., 2008; J?rgensen et al., 2009; Rohani et al., 2011; Prospri et al., 2015). To review the mechanisms root Eph/ephrin-mediated cell segregation, we work with a hereditary mouse model regarding mosaicism for ephrin-B1. This model arose out appealing in the individual disease craniofrontonasal symptoms (CFNS; MIM304110). CFNS outcomes from mutation from c-Fms-IN-9 the X-linked gene display serious manifestations of CFNS (Twigg et al., 2013). This.