INTRODUCTION The inner ears of vertebrates are responsible for the sensations of hearing and of balance. Although the two ears are symmetric about the midline of the organism, each individual organ in most vertebrate species is asymmetric about all three axes [anteroposterior/rostrocaudal (AP), dorsoventral (DV) and mediolateral (ML)]. The zebrafish otic field (defined by the expression of pax8) is induced at the lateral edges of the neural plate, adjacent to several presumptive rhombomeres (r) of the developing hindbrain. By about 14 hours post fertilisation (hpf), condensation of cells gives rise to distinct thickenings (the otic placodes) immediately opposite r5 (Kimmel et al., 1995; Pfeffer et al., 1998; Phillips et al., 2001). The expression of several genes, for example pax2a (formerly pax2.1), dlx3b (formerly dlx3) and eya1, is now detected throughout these placodes (Krauss et al., 1991; Akimenko et al., 1994; Sahly et al., 1999). The only known genes with restricted patterns of expression in the otic placode at this stage are the Delta genes. These are expressed in anterior and posterior domains, symmetrical about the AP and DV axes, but restricted to the medial side of the placode (Haddon et al., 1998). It is therefore likely that at 14 hpf only the ML axis of the otic placode has been specified. Asymmetric gene expression patterns about both the AP and the DV axes are obvious by 18 hpf, when the placode begins to cavitate to form an otic vesicle. nkx5.1 (hmx3 – Zebrafish Information Network), which is currently the earliest known marker of an asymmetry about the AP axis, is expressed in an anterior domain from around 16 hpf; pax5 is detectable in the anterior epithelium from 17.5 hpf and dacha is detected in the dorsal otic epithelium by 17-18 hpf, suggesting that all axes of the ear have been specified by this time (Pfeffer et al., 1998; Adamska et al., 2000; Hammond et al., 2002). By 24 hpf, several further otic genes are expressed asymmetrically, and this presumably both reflects and reinforces axis specification. pax5, nkx5.1 and fgf8 are expressed anteriorly, bmp7 and follistatin posteriorly, dlx3b and dacha dorsally, eya1 ventrally, and pax2a and dacha medially (Krauss et al., 1991; Akimenko et al., 1994; Pfeffer et al., 1998; Reifers et al., 1998; Sahly et al., 1999; Adamska et al., 2000; Mowbray et al., 2001; Hammond et al., 2002). Sensory epithelium now thickens and stratifies, and fingers of non-sensory epithelium protrude into the otic lumen and fuse to form the semicircular canal system (reviewed by Whitfield et al., 2002). Fekete and colleagues have proposed a model in which tissues surrounding the ear provide inductive signals for both axis specification and further otic differentiation (Fekete, 1996; Brigande et al., 2000a; Brigande et al., 2000b). They propose that signals from the hindbrain have dorsalising activity, and may also be important in providing AP information and medialising signals to the otic vesicle. Several lines of 1403 Development 130, 1403-1417 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00360 Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (con tf18b ) and slow muscle omitted (smu b641 ) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle. Key words: Hedgehog, Sonic hedgehog, Tiggy-winkle hedgehog, Inner ear, Zebrafish, Mirror image duplication, slow muscle omitted, chameleon, Otic vesicle, Axis formation SUMMARY Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle Katherine L. Hammond, Helen E. Loynes, Amos A. Folarin, Joanne Smith and Tanya T. Whitfield* Centre for Developmental Genetics, University of Sheffield School of Medicine and Biomedical Science, Western Bank, Sheffield S10 2TN, UK *Author for correspondence (e-mail: t.whitfield@sheffield.ac.uk) Accepted 18 December 2002