Opposing activity of SOX9 and Wnt in the developing otocyst ensures SOX10 control of endolymphatic development

Proper development of the inner ear, including specification and differentiation of progenitors for the endolymphatic sac, is crucial for hearing and balance. In a mouse model of the SOX9^Y440X/+ mutation that causes campomelic dysplasia and deafness, impaired development of the endolymphatic system at mid-gestation was associated with SOX10 downregulation. The underlying molecular causes and significance are unclear. Here, we found in Sox9^Y440X/+ mice, Sox10 expression was downregulated, prior to overt differentiation, in the dorsomedial aspect of the E10.5 otic vesicle that normally becomes the endolymphatic duct and sac. Single-cell transcriptomic profiling of E10.5 otic vesicles showed increased expression of Wnt pathway genes that was associated with the domain-specific decrease in Sox10 expression. Forced elevation of Wnt signaling by expression of stabilized β-catenin in the early otocyst and more developed inner ears, suppressed Sox10 expression. Wnt exerted an inhibitory effect on SOX10 expression in otocyst-like organoids derived from human pluripotent stem cells. In vitro transactivation assays reveal antagonistic action of SOX9 and Wnt signaling on Sox10 transactivation mediated by conserved enhancers with neighboring/overlapping SOX9- and TCF/LEF-binding (Wnt effectors) motifs. We propose a conserved SOX9-Wnt antagonism acting via conserved enhancers, regulates SOX10 to modulate endolymphatic cell fate. The SOX9^Y440X/+ mutation disrupts this regulatory circuit through both dominant-negative and haploinsufficient effects culminating in impaired establishment of progenitors in the otocyst that are essential for the development of the endolymphatic system for hearing and balance.