Mechanisms underlying abnormal inner ear development caused by the Sox9Y440X campomelic dysplasia mutation

Abstract

Campomelic dysplasia is a severe congenital disease caused by SOX9 mutations characterized by skeletal malformation. Hearing impairment is a common associated phenotype, but its mechanisms are unknown. Among the mutations, the SOX9Y440X mutation is one of the most common.

To understand the etiology of Campomelic dysplasia, we established a mouse model that expresses the equivalent SOX9Y440X upon Cre induction. These mice exhibited sensorineural deafness. One hypothesis for the underlying cause of this phenotype is the disruption of endolymph homeostasis, as a loss of endocochlear potential was found. However, the mechanism leading to the imbalance of endolymph remains to be determined.

The endolymphatic sac, an inner ear compartment essential for endolymph regulation, was examined at E14.5 with single-cell transcriptomic profiling. In Sox9Y440X/+, maturation of progenitors was impaired and the expression profile was altered, with downregulation of a predicted Sox10 regulon and ion regulators. Further investigation revealed that the loss of SOX10 plays a significant role in the maturation defect of the endolymphatic sac.

Interestingly, the downregulation of SOX10 was only observed in the endolymphatic sac and duct, but not in other inner ear compartments. To better understand the cause of this differential reduction, SOX10 expression was examined during early otic development. Domain-specific SOX10 reduction began at E10.5 in the medial otic vesicle, and later in the dorsomedial derivatives. Further analysis revealed that SOX9Y440X was not exclusively expressed in the affected domain, indicating the presence of additional factor(s) that act in combination with SOX9Y440X to cause the differential downregulation.

To gain further insights, analyses were conducted using compound mutants to enable otic-conditional and temporal induction of the Sox9Y440X mutation. The findings suggested that the differential reduction of SOX10 was cell-autonomous, as the otic-conditional mutant recapitulated the observed SOX10 reduction. Additionally, a time-series induction of Sox9Y440X/+ in mice carrying Sox9CreERT2 revealed stage-dependency regarding the molecular impact. Further comparison with Sox9 heterozygous null mutants revealed a dominant negative mechanism.

To identify the factor(s) that acts collectively with Sox9Y440X/+ to cause the SOX10 downregulation, single-cell transcriptomic profiling was performed on E10.5 otic vesicles. Canonical WNT signalling was identified as a potential factor that exhibited differential elevation in the dorsomedial domain associated with the specific reduction of SOX10. Further investigation with stabilization of β-catenin revealed that WNT antagonizes SOX9 in the transactivation of Sox10 and inhibits the expression of Sox10.

The WNT-SOX9-SOX10 regulatory relationship was subsequently shown to be conserved in the human inner ear system with the use of inner ear organoids as the experiential platform. To further investigate the molecular interactions involving SOX9Y440X, the SOX9Y440X mutation was introduced into human pluripotent stem cells, enabling molecular investigations.

The investigation of the disease mechanism revealed a regulatory relationship between WNT, SOX9, and SOX10 in the otic system. This finding sheds light on the molecular interactions involved in the development and function of the inner ear. Additionally, the study provided evidence supporting the dominant negative nature of the SOX9Y440X mutation.