Although statistically significant in some cases, these defects were less severe compared

to what we observed in the Pou3f4 or Epha4 mutants, probably owing to functional redundancy by other ephrins present or possibly a lack of complete ephrin-B2 elimination. Efnb2 cko cochleae showed a 6% increase in area consumed by axons and maximally a 4-fold increase in the number of axons that crossed between bundles ( Figures 7R and 7S). Because tamoxifen was given at a single dose at E9.5–E10.5, most defects were observed at the base and midmodiolar XAV-939 molecular weight regions of the cochlea, in accordance with previous studies with Ngn-CreERT2 ( Koundakjian et al., 2007). Importantly, the Efnb2 cko phenotype was similar to the fasciculation phenotypes observed in both the Pou3f4 PLX 4720 and Epha4 mutants lines, suggesting that ephrin-B2 may function as a ligand for EphA4 in this process. We next asked whether, in the developing otic mesenchyme, regulatory elements of Epha4 are a direct target of Pou3f4. Pou proteins are known to have a bipartite DNA binding system that includes a POU-specific domain,

as well as a homeobox domain ( Phillips and Luisi, 2000). Whereas the recognition sequences of several Pou proteins have been characterized, relatively little is known about mouse Pou3f4. However, one report has demonstrated that Pou3f4 preferentially recognizes the tandem homeobox sequence ATTATTA in the regulation of the dopamine receptor 1A gene D1A ( Okazawa et al., 1996). Therefore, we scanned the entire Epha4 genomic sequence and found four of these sites within introns in the first 70 kilobases (kb) and one approximately 8.5 kb upstream of exon 1 ( Figure 8A). We then performed chromatin immunoprecipitation (ChIP) using otic mesenchyme and a Pou3f4-specific IgY and assayed the resulting DNAs for these Epha4 regulatory regions using quantitative PCR ( Figure 8B). In these experiments β-actin, Neurogenin-1,

Idoxuridine and an Epha4 site that did not contain ATTATTA were used as negative controls; there was no significant association at these sites with the control or Pou3f4-specific IgY. However, all five of the putative Epha4 regulatory regions showed preferential association (with statistical significance) with the Pou3f4-specific IgY, with little to no association with the control IgY ( Figure 8B). These data suggest that Pou3f4 may directly regulate Epha4 in otic mesenchyme cells in order to initiate EphA4-mediated SGN fasciculation. The results presented in this study reveal a new and intriguing role for otic mesenchyme in the development of cochlear innervation. Moreover, the identification of additional auditory defects arising from the absence of Pou3f4 provides insights into the underlying basis for the deafness that occurs in both human and murine mutants.