Although this hypothesis cannot be tested without a mouse model in which endocochlear potential is preserved, it would need check details to be considered in terms of developing any potential therapeutic interventions. The results presented here identify a molecular signaling pathway in which Pou3f4 expression in otic mesenchyme cells directly activates Epha4, leading to the expression of EphA4 on the surface of these cells ( Figure 8C). The presence of EphA4 provides a cue that acts, along with the spatial distribution of otic mesenchyme, to promote fasciculation of SGNs via

binding to ephrin-B2 on their surfaces. Furthermore, these data predict that EphA4 activates ephrin-B2 to generate a reverse signaling response to segregate the SGNs and mesenchyme in a manner classically documented in zebrafish animal cap assays ( Mellitzer et al., 1999). However, the mechanism(s) by which ephrin-B2 promotes fasciculation among the SGN axons remains unclear. Ephrin-B2

reverse signaling may induce filopodial collapse ( Cowan and Henkemeyer, 2001) by the SGN growth cones, which, by default, may lead them to preferentially associate with neighboring axons. Or ephrin-B2 reverse signaling may promote SGN interaxonal adhesion by signaling to other cell-surface factors known to regulate fasciculation, such as IgCAMs ( Lin et al., 1994) and/or integrins ( Baum and Garriga, 1997). These results reveal the Ulixertinib mouse molecular basis for the organizing effects of otic mesenchyme and show a paracrine mode of action for Pou3f4 in axon guidance. Interestingly, Pou4F2 (Brn3b) is known to regulate axon pathfinding and fasciculation in retinal ganglion cells through an autocrine signaling pathway (Pan et al., 2008), and in Drosophila,

deletion of the Pou3f4 ortholog ventral veins lacking (vvl) leads to defects in fasciculation and steering of axons in the fly brain, although much of these defects may be secondary to effects on neuronal specification ( Meier et al., 2006). In addition, although this demonstrates a role for Eph-ephrin signaling in the initial development of the peripheral auditory system, elegant work by Huffman and Cramer (2007) has already demonstrated a role for EphA4 in hearing and central auditory plasticity ( Hsieh et al., 2007 and Huffman Farnesyltransferase and Cramer, 2007). These authors showed that, after surgically removing the cochlea (and peripheral input to the brain), the expression of Epha4 was critical for target selection during remodeling ( Hsieh et al., 2007 and Miko et al., 2008). The functions of ephrin-Eph receptor interactions probably go well beyond those presented here and in previous publications. As shown in Figure S4 and in previous reports, several other ephrins and Ephs are expressed in the cochlea ( Bianchi and Gale, 1998 and Zhou et al., 2011) and may serve a variety of additional functions.