Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Abstract

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.

Ablation studies on the developing inner ear reveal a propensity for mirror duplications

“…Significant percentages resulted in duplicated inner ears, with two anterior or two posterior halves fused as if mirror images. These percentages were much higher than was ever seen with the amphibian rotation studies (Yntema, 1955) or even the zebrafish Hh signaling pathway mutants (Hammond et al, 2003). Labeled grafting experiments confirmed that the regenerated tissue was from the remaining placode and not surrounding tissues.…”

“…In zebrafish, Hh signaling is required for posterior identity; when it is inhibited, anterior structures are duplicated posteriorly (Hammond et al, 2003). Hip is a vertebrate-specific inhibitor of Hh signaling that was discovered in mice (Chuang and McMahon, 1999;Koebernick et al, 2003).…”

“…These patterning signals could be intrinsic and/or extrinsic to the inner ear. For example, the Hh signal for inner ear patterning in zebrafish, chicken, and mouse likely arises from adjacent tissues (Hammond et al, 2003;Bok et al, 2005;Riccomagno et al, 2005).…”

“…A zebrafish study identified that members of the Hedgehog (Hh) signaling pathway are involved in anteroposterior patterning of the developing inner ear (Hammond et al, 2003). This finding contrasts with the role of Hh signaling in mouse inner ear development, where Sonic hedgehog (Shh) plays a role in dorsoventral patterning by promoting ventral identity and restricting Wnt genes to the dorsal region (Riccomagno et al, 2002(Riccomagno et al, , 2005Ohyama et al, 2006).…”

“…Two zebrafish mutants known to eliminate Hh signaling were found to have partial duplications of both anterior inner ear structures and gene expression in the posterior half, as well as reduction of posterior gene expression. One of these mutations is in the Hh signal transducer Smoothened and is one of the strongest Hh signaling mutants so far isolated in zebrafish (Hammond et al, 2003). These effects were phenocopied by overexpression of the patched1 mRNA, which also reduces Hh signaling.…”