The number of mesenchyme cells in Epha4−/− cochleae appeared unchanged compared to wild-type,

suggesting that the fasciculation defects in the SGNs are not due to a loss of surrounding mesenchyme ( Figures 5G and 5H). Similar to SGNs from Pou3f4y/− mice, SGNs from Epha4−/− mice showed an approximately 4-fold increase in the number of axons that crossed between bundles ( Figure 5J). Finally, we examined Epha4−/− cochleae at P7 to determine Pomalidomide mouse whether these animals (like Pou3f4y/− mice) had defects in hair cell innervation and ribbon synapse formation ( Figure S2). As a result of early postnatal lethality of this line, we were only able to examine four mutant ears, and thus our conclusions are based on a limited sampling. Compared to controls ( Figures S3A, S3C, and S3D), Epha4−/− mice showed a nearly 2-fold reduction in the number of ribbon synapses ( Figures S2B, S2E, and S2F), further supporting that SGN fasciculation is important for target innervation. Overall, the fasciculation and synapse defects in Epha4 and Pou3f4 mutants are very similar, consistent Imatinib molecular weight with their participation in a common developmental

process. To investigate whether EphA4 plays a non-cell-autonomous role during SGN fasciculation, we asked whether exogenous EphA4 extracellular domains (serving as a substitute for mesenchyme) could promote SGN fasciculation in vitro. E12.5 spiral ganglia were cultured for 24 hr (without otic mesenchyme) on a layer of Matrigel infused with either control Fc or EphA4-Fc (Figures 5K–5M) to determine the effects on fasciculation. We also examined the effects of preclustering EphA4-Fc to determine whether “activating” (preclustered form) or “blocking” (unclustered

form) ephrin ligands on the SGNs had different effects on fasciculation (Davis et al., 1994). In the presence of control Fc (preclustered), SGN explants projected mostly single neurites and some rudimentary fascicles (Figure 5K, see arrowheads), suggesting that SGNs may have some intrinsic axoaxonal binding capacity. However, explants unless cultured with preclustered EphA4-Fc showed dramatically enhanced fasciculation, with thick bundles projecting away from the soma (Figure 5L) and very few single neurites. Interestingly, EphA4-Fc treatments did not reduce the length of axons (growth cone collapse) compared to controls (data not shown). When we stained these cultures with anti-integrin-α6 antibodies to mark the auditory glia, we found their growth pattern to be almost identical to that of the SGNs, raising the possibility that the glia may also respond to EphA4. However, when the SGNs were eliminated in the presence of high pH culture medium (Mukai et al., 2011) but grown in preclustered EphA4-Fc, the glia persisted but did not bundle (Figure 5M), suggesting that SGN fasciculation is not normally a secondary response to aggregating glia.