Study of abnormal inner ear development in Waardenburg-Shah syndrome using a Sox10-GEP mutant mouse model

Abstract

Sox10 is a high mobility group (HMG) domain transcription factor which is an important regulator for neural crest development. SOX10 mutations have been identified in Waardenburg-Shah syndrome type 4 (WS4) patients who suffer from sensorineural deafness. However, the mechanisms underlying the hearing defect of SOX10-mediated WS4 are unclear. The aim of this study is to elucidate the function of Sox10 during mouse inner ear development using a mutant mouse model, in order to reveal the underlying basis for SOX10 mutation associated sensorineural deafness in WS4 patients.

The mammalian inner ear originates from the otic placode epithelium as well as neural crest cells (NCCs). To understand the role of Sox10 in inner development, I investigated the contribution of cranial NCCs to the cochleovestibular ganglion (CVG) by lineage tracing analysis, using Wnt1-cre;ZEG mice in which all NCCs were marked by GFP. Co-expression of GFP-positive cells with the glial marker BFABP suggested that glial cells in the CVG were derived from NCCs. Furthermore, Sox10-expressing NCCs were found to invade the CVG at 30-somite stage. These results suggest a role of Sox10 in regulating cranial NCCs contribution to CVG glia.

In our laboratory we have generated a mouse mutant Sox10EGFP in which the Sox10 N-terminal domain was fused to the EGFP reporter. To investigate the function of Sox10 in NCCs invasion and gliogenesis of CVG, phenotypic analysis of Sox10NGFP mutant mouse were performed. EGFP expression in the CVG and inner ear epithelium of Sox10NGFP/+ embryos recapitulated the dynamic expression pattern of Sox10. Sox10NGFP/NGFP mutants displayed a reduced number of migrating NCCs and lacked NCCs or glia in their CVG. Moreover, loss of glial cell in the developing spiral ganglia of Sox10NGFP/NGFP mice led to disorganized fasciculation and degeneration of axonal filaments. These data suggest that Sox10 is required for maintaining the cranial NC stem cell pool, and is also essential for CVG gliogenesis and normal growth and innervation of spiral ganglion neurons.

To study the function of Sox10 in regulating cochlear morphogenesis, morphological and histological analysis of mutant cochlear were performed. As illustrated by paint-filling analysis, Sox10NGFP/NGFP mice developed a shortened cochlear duct, reduced cochlear turning and enlarged endolymph lumen. Sensory hair cell patterning in the organ of Corti was normal in the Sox10 mutant as shown by immunohistochemistry analysis, suggesting that cochlear lumen enlargement was not due to disrupted planar cell polarity (PCP) pathway. To explore the molecular basis of Sox10-mediated cochlear morphogenic defect, expression of genes related to cochlear development were examined by qRT-PCR. Candidate genes included those involved in fluid homeostasis, which are known to affect the size of cochlear lumen. Up-regulated expression of Aquaporin 3, a water channel protein in the cochlear epithelium that facilitates water transport across the cell membrane, was observed in Sox10NGFP/NGFP cochlear. These results suggest that Sox10 may regulate cochlear morphogenesis by controlling endolymph homeostasis.

In conclusion, Sox10 is required in multiple processes during inner ear development including NCC invasion, gliogenesis and cochlear morphogenesis, and their abnormal development can lead to sensorineural deafness in WS4 syndrome.