EPHRIN-B1 and EPHRIN-B2 are transmembrane proteins that can activate both forward signaling to EPH receptors as well as reverse signaling through their cytoplasmic domain. We have investigated the function of both EPHRINS during mouse development. Complete ablation of the X-linked Efnb1 resulted in peri-natal lethality associated with a range of phenotypes, including defects in craniofacial development leading to a cleft palate and other neural crest cell (NCC)-derived phenotypes, as well as abnormal skeletal patterning. Conditional deletion of Efnb1 demonstrated that EPHRIN-B1 acts autonomously in NCCs, and controls their migration (Davy et al., 2004). Mutation in X-linked Efnb1 lead to similar phenotypes as in humans, where it causes Craniofrontonasal Syndrome (CFNS), a disease which affects female patients more severely than males. In Efnb1 heterozygous mutants, X-inactivation generates Efnb1 expressing and non-expressing cells that sort out, resulting in mosaic Efnb1 expression. We found that Efnb1^+/- mutants exhibit impaired differentiation of osteogenic precursors and calvarial defects that correlate with sorting of Efnb1 positive and negative cells following X-inactivation. Gap junction communication (GJC) is inhibited at ectopic EPHRIN boundaries and EPHRIN-B1 interacts directly with CONNEXIN43 and regulates its distribution. In turn, regulation of GJC influences cell sorting. These results uncover a novel role for EPH/EPHRINS in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues (Davy et al., 2006).

We have generated mice harboring a series of targeted point mutations in the Efnb1 gene which independently ablate specific reverse signaling pathways, while maintaining forward signaling capacity. We found that both PDZ and phosphorylation-dependent reverse signaling by EPHRIN-B1 are dispensable for craniofacial and skeletal development, whereas PDZ-dependent reverse signaling by EPHRIN-B1 is critical for the formation of a major commissural axon tract, the corpus callosum. Efnb1 is strongly expressed within axons of the corpus callosum, and reverse signaling acts autonomously in cortical axons to mediate an avoidance response to its signaling partner EPHB2 (Bush and Soriano, 2009). By integrating phospho-proteomic and transcriptomic approaches, we found that EPHRIN-B1 controls proliferation in the palate by regulating the ERK/MAPK signal transduction pathway. The alteration in proliferation rates resulting from ectopic Eph-ephrin expression boundaries correlates with the more severe dysmorphogenesis of Efnb1^+/- heterozygotes that is a hallmark of CFNS (Bush and Soriano, 2010).

Genetic studies have previously implicated EPHRIN-B2 in blood vessel formation, cardiac development and remodeling of the lymphatic vasculature. We have found that loss of Efnb2 also leads to defective somite development and to defects in populations of cranial and trunk NCCs. Expression of one copy of a mutant version of Efnb2 that lacks tyrosine phosphorylation sites was sufficient however to rescue the embryonic phenotypes associated with loss of Efnb2. These results suggest that EPHRIN-B2 exerts many of its embryonic function via activation of forward signaling (Davy and Soriano, 2007). However, conditional rescue experiments indicate that the neural crest defects observed in Efnb2 mutants are non autonomous and originate from defects in the vasculature (Lewis et al., 2015).