The vertebrate inner ear mediates hearing and balance through a complex arrangement of mechanosensory hair cells, nonsensory

INTRODUCTION The vertebrate inner ear mediates hearing and balance through a complex arrangement of mechanosensory hair cells, nonsensory supporting cells and sensory neurons. All of these cell types derive from the otic placode, a transient ectodermal thickening adjacent to the developing hindbrain (Barald and Kelley, 2004; Fritzsch et al., 2006). Otic placode formation is a multistep process initiated by the establishment of the preplacodal region, which surrounds the anterior neural plate and contains precursors for all sensory placodes (Streit, 2007). Subsequently, the otic-epibranchial progenitor domain (OEPD), a common territory for otic and epibranchial precursors, is specified (Ladher et al., 2010; Chen and Streit, 2012). In zebrafish, the OEPD also appears to contain the progenitors of the anterior lateral line ganglion (McCarroll et al., 2012). Studies in various species have shown that OEPD formation is triggered by Fibroblast growth factor (Fgf) ligands secreted by the hindbrain and subjacent mesendoderm (Phillips et al., 2001; Léger and Brand, 2002; Maroon et al., 2002; Alvarez et al., 2003; Wright and Mansour, 2003; Ladher et al., 2005). In response to these signals, cells express Pax8 and Pax2, two members of the Pax2/5/8 transcription factor family, which are crucial regulators of OEPD formation and proper inner ear development (Brand et al., 1996; Pfeffer et al., 1998; Hans et al., 2004; Mackereth et al., 2005; Bouchard et al., 2010; Freter et al., 2012). The subsequent segregation of otic and epibranchial progenitors is mediated by Wnt signaling in a Pax2-dependent manner (Freter et al., 2008; McCarroll et al., 2012). With respect to otic fate, a two-phase model has been proposed to summarize genetic interactions during otic induction in zebrafish (Hans et al., 2004; Solomon et al., 2004). According to this model, the forkhead transcription factor Foxi1 enables expression of Pax8 during the early phase, and the homeodomain transcription factors Dlx3b and Dlx4b (Dlx3b/4b) provide competence to activate Pax2a during the second phase (Hans et al., 2004; Solomon et al., 2004). Previous work has shown that dlx3b, dlx4b and foxi1 are regulated initially independently in a BMP-dependent manner in the same region at late gastrula stages and whereas foxi1 is progressively restricted to the presumptive OEPD, dlx3b and dlx4b are maintained in a stripe corresponding to cells of the preplacodal region (Akimenko et al., 1994; Ellies et al., 1997; Nissen et al., 2003; Solomon et al., 2003; Solomon et al., 2004; Hans et al., 2007). Subsequently, downregulation of foxi1 in a Pax2aand Pax8-dependent manner is required for proper otic development, whereas dlx3b and dlx4b are maintained in the cells of the future otic placode (Akimenko et al., 1994; Padanad and Riley, 2011). Loss of Foxi1 or Dlx3b/4b results in compromised otic induction and development of smaller otic vesicles (Solomon and Fritz, 2002; Liu et al., 2003; Nissen et al., 2003; Solomon et al., 2003), and combined loss of both factors eliminates all indications of otic specification (Hans et al., 2004; Solomon et al., 2004). However, the functional differences of Foxi1 and Dlx3b/4b during otic induction have not been addressed thus far and remain elusive. After placode formation, otic tissue develops into the otic vesicle where sensory neurons and mechanosensory hair cells are born. The neuronal precursors delaminate as neuroblasts from an anteriorventral position in the otic vesicle and give rise to the eighth cranial or statoacoustic ganglion, whereas hair cells are generated in the sensory epithelia of the otic vesicle (Haddon and Lewis, 1996; Technische Universität Dresden, Biotechnology Center and DFG-Center for Regenerative Therapies Dresden Cluster of Excellence, Tatzberg 47-49, 01307 Dresden, Germany.