EFNB encodes EPHRIN B a member of the Eph

EFNB1 encodes EPHRIN-B1, a member of the Eph/ephrin family of membrane-linked signaling molecules, and abnormal signaling between bcr-abl inhibitor expressing wild-type EPHRIN-B1 and cells that are functionally EPHRIN-B1-null may occur in the mosaic state (Compagni et al., 2003; Wieacker and Wieland, 2005). During development, Eph/ephrin signaling plays an important role in boundary formation, an essential process that requires signaling between adjacent cells and often involves segregation between different cell types (Batlle and Wilkinson, 2012; Cayuso et al., 2015; Fagotto, 2014; Fagotto et al., 2014). Differential expression of Eph receptors and ephrins in vivo can restrict cell intermingling in the vertebrate hindbrain (Xu et al., 1999), limb bud (Compagni et al., 2003; Davy et al., 2004), eye (Cavodeassi et al., 2013), somites (Barrios et al., 2003; Durbin et al., 1998), cranial sutures (Merrill et al., 2006; Ting et al., 2009), and intestinal crypts (Holmberg et al., 2006), as well as in the Drosophila wing disc (Umetsu et al., 2014). In culture, expressing an Eph receptor in one population of cells and an ephrin in another restricts intermingling of cells from the two populations (Jorgensen et al., 2009; Mellitzer et al., 1999; Poliakov et al., 2008). Further, cell segregation occurs in developing Efnb1 mouse limb (Compagni et al., 2003) and secondary palate (Bush and Soriano, 2010), supporting the idea that XCI-induced mosaicism leads to segregation of Ephrin-B1 expressing and non-expressing cells. The role of Eph/ephrin signaling in boundary formation and supporting data from mouse models suggest that mosaicism for EPHRIN-B1 expression may lead to aberrant cell segregation in human CFNS patients (Compagni et al., 2003; Twigg et al., 2004, 2006, 2013; Wieacker and Wieland, 2005; Wieland et al., 2004). However, it has proven difficult to determine the mechanism of cellular interference, and EPHRIN-B1-mediated cell segregation has not been demonstrated in CFNS patients. Here, we report the generation of an hiPSC model to study defects in morphogenesis in a congenital craniofacial disorder. We demonstrate that cell segregation is a consequence of EPHRIN-B1 mosaicism in CFNS, providing evidence that this cell behavior is relevant to CFNS pathogenesis in humans. The CFNS hiPSC model provides proof of principle that hiPSC-derived cell types can be used both to model structural anomalies and to gain valuable insights into fundamental cellular mechanisms of morphogenesis in patient cells.
Results
Discussion Here, we have generated an hiPSC model of a human craniofacial condition and have used it to address an outstanding question: does mosaicism for EFNB1 expression result in cell segregation in human CFNS? The c.712delG mutation found in this CFNS family occurred 5′ to the transmembrane domain-encoding region of EFNB1, and we found that EFNB1 mutant hNE cells did not express EPHRIN-B1, indicating that this mutation results in an unstable EPHRIN-B1 protein and most likely null loss of function. To enable us to model CFNS, it was essential that loss of EFNB1 function not prevent CFNS patient-derived HDFs from undergoing reprogramming to hiPSCs. We did not observe differences in reprogramming ability between EFNB1 mutant and control HDFs, leading us to conclude that EPHRIN-B1 expression is not necessary for reprogramming. Further, we found that both control and CFNS hiPSCs possessed differentiation potential to all three germ layers; loss of EPHRIN-B1 expression does not apparently prevent differentiation. This is consistent with our qRT-PCR data demonstrating that transcripts of EFNB1 and several other Eph/ephrin signaling family members are expressed at very low levels in hiPSCs relative to hNE cells, suggesting that these signaling molecules may not play critical roles in hiPSCs. Previous human genetic studies have indicated that mosaicism for EFNB1 mutation is central to CFNS pathology, a phenomenon termed cellular interference suggested to result in cell segregation based on evidence from model organisms (Compagni et al., 2003; Twigg et al., 2004, 2006, 2013; Wieacker and Wieland, 2005; Wieland et al., 2004). Whether cell segregation occurs in CFNS, however, and in what cell types, was not known. Based on evidence of cell segregation in the neural plate NE in Efnb1 mice (O’Neill et al., 2016), we differentiated hiPSCs to hNE cells to address this question.