Pax3 function is required specifically for inner ear structures with melanogenic fates

• Inner ear morphology is affected indirectly by neural tube defects in Pax3 mutants.
• Pax3-expressing cells migrate from the neural tube to various inner ear structures.
• Pax3 lineages with melanogenic fates are specifically absent in Pax3 null inner ears.
• Pax3 is required for melanogenesis, but not for migration of the Pax3 lineage.

Pax3 mutations result in malformed inner ears in Splotch mutant mice and hearing loss in humans with Waardenburg’s syndrome type I. In the inner ear, Pax3 is thought to be involved mainly in the development of neural crest. However, recent studies have shown that Pax3-expressing cells contribute extensively to multiple inner ear structures, some of which were considered to be derived from the otic epithelium. To examine the specific functions of Pax3 during inner ear development, fate mapping of Pax3 lineage was performed in the presence or absence of functional Pax3 proteins using Pax3Cre knock-in mice bred to Rosa26 reporter (R26R) line. β-gal-positive cells were widely distributed in Pax3Cre/+; R26R inner ears at embryonic day (E) 15.5, including the endolymphatic duct, common crus, cristae, maculae, cochleovestibular ganglion, and stria vascularis. In the absence of Pax3 in Pax3Cre/Cre; R26R inner ears, β-gal-positive cells disappeared from regions with melanocytes such as the stria vascularis of the cochlea and dark cells in the vestibule. Consistently, the expression of Dct, a melanoblast marker, was also absent in the mutant inner ears. However, when examined at E11.5, β-gal positive cells were present in Pax3Cre/Cre mutant otocysts, whereas Dct expression was absent, suggesting that Pax3 lineage with a melanogenic fate migrated to the inner ear, yet failed to differentiate and survive without Pax3 function. Gross inner ear morphology was generally normal in Pax3Cre/Cre mutants, unless neural tube defects extended to the cranial region. Taken together, these results suggest that despite the extensive contribution of Pax3-expressing cells to multiple inner ear tissues, Pax3 function is required specifically for inner ear components with melanogenic fates.