Restriction of Wnt signaling in the dorsal otocyst determines semicircular canal formation in the mouse embryo.

The mouse inner ear develops from a simple epithelial pouch, the otocyst, with the dorsal and ventral portions giving rise to the vestibule and cochlea, respectively. The otocyst undergoes a morphological change to generate flattened saclike structures, known as outpocketings, in the dorsal and lateral regions. The semicircular canals of the vestibule form from the periphery of the outpocketings, with the central region (the fusion plate) undergoing de-epithelialization and disappearing. However, little is known of the mechanism that orchestrates formation of the semicircular canals. We now show that the area of canonical Wnt signaling changes dynamically in the dorsal otocyst during its morphogenesis. The genes for several Wnt ligands were found to be expressed in the dorsal otocyst according to specific patterns, whereas those for secreted inhibitors of Wnt ligands were expressed exclusively in the ventral otocyst. With the use of whole-embryo culture in combination with potent modulators of canonical Wnt signaling, we found that forced persistence of such signaling resulted in impaired formation both of the lateral outpocketing and of the fusion plates of the dorsal outpocketing. Canonical Wnt signaling was found to suppress Netrin1 expression and to preserve the integrity of the outpocketing epithelium. In addition, inhibition of canonical Wnt signaling reduced the size of the otocyst, likely through suppression of cell proliferation and promotion of apoptosis. Our stage-specific functional analysis suggests that strict regulation of canonical Wnt signaling in the dorsal otocyst orchestrates the process of semicircular canal formation.