Sox2 dosage-dependent role in prosensory versus nonsensory lineage decision during otic development

Abstract

The mammalian inner ear is a complex organ housing different types of specialized cells which descend from common progenitors in a structure named the otic vesicle (OV) or otocyst. Early in otic development, the OV is already patterned into different compartment to give rise to different lineages and the compartments are characterized by a combination of gene expression. The common progenitors are specified to become three major lineages, neuronal lineage forming the otic neurons, prosensory lineage forming the six sensory organs, and finally the nonsensory lineage forming the architectural components. All the specialized cell types forming the inner ear arise from these progenitors must be in precise locations and proportions to function co-ordinately to sense balance and detect soundwaves. In particular, Sox2 is both necessary and sufficient to specify the prosensory lineage.

Two Sox2 allelic enhancer mouse mutants, Sox2Ysb and Sox2Lcc contain disruption in the regulatory region of Sox2 and present recessive circling behaviour and deafness. Sox2Lcc, losing a longer 3’ region, is completely deaf. Sox2Ysb, losing a shorter 3’ region, on the other hand responds only to loud noise. The degree of disruption in the Sox2 regulatory region correlates with severity of inner ear phenotypes and effects on prosensory/neurosensory development. To understand the relationship between prosensory/neurosensory development and SOX2 dosage on protein level, here, the SOX2 expression levels in a Sox2 hypomorphic mutant series compromising Sox2Ysb/Ysb, Sox2Lcc/Lcc, Sox2EGFP/Lcc and Sox2EGFP/cKO were characterized. Interestingly, the level of SOX2 protein in Sox2 heterozygous null otocysts (Sox2EGFP/+) was found to be unchanged compared to wildtype in E9.5 OV. In addition, Sox2Ysb/Ysb was found to express a 96% SOX2 level compared to 43% in Sox2Lcc/Lcc, indicating multiple different temporal-specific Sox2 enhancers are disrupted in Sox2Ysb/Ysb and Sox2Lcc/Lcc.

To further dissect the impact of the dosage reduction of Sox2, the fates of the Sox2 descendant cells at later otic developmental stages were studied using an inducible SOX2CREERT2 to tag and trace the Sox2 lineages. The expression of nonsensory markers such as GATA2 in Sox2 loss-of-function mutant descendants suggested a possible fate change that depends on the expression level of SOX2 protein. In addition, excessive apoptosis was detected in the cells adjoining the Sox2 mutant cells. To further investigate the impact on molecular level, single-cell transcriptomes of Sox2EGFP/+ and Sox2EGFP/Lcc EGFP+ otocyst cells were compared at E9.5. In Sox2EGFP/+ control, two major cell populations with gene expression characteristic of neuronal and otic epithelium (OE) progenitors were identified. In mutant Sox2EGFP/Lcc, in addition to a smaller proportion of neuronal and OE progenitor populations, an additional ectopic cell population was identified, which is enriched in cells expressing dorsal otic vesicle markers (e.g. Lmx1a, Wnt6). These cells express a lower level of neurosensory markers (Neurog1, NeuroD1, Pax2) and genes novel to the otic vesicles (e.g. Col3a1, Mme). Interestingly these cells also co-expressed genes characteristic of multiple endolymphatic epithelium early progenitors (e.g. Gbx2) and differentiated cells (e.g. Oc90). The data suggest a dysregulation of the otic cell fate decision at early stages with reduced Sox2 level. The mutant cells lose neurosensory identity and acquire a hybrid state that resembles a nonsensory-like identity that is partially endolymphatic progenitor cell-like combined with a differentiated state. It was also revealed that Sox2Lcc cells possibly gain ectopic Mme expression via rearrangement of enhancers.

In humans, enlarged vestibular aqueduct (EVA) caused by defects in the endolymphatic system contributes 5 – 15% of sensorineural hearing loss in children. These findings have important significance to recognizing the critical value of maintaining appropriate levels of SOX2 in regulating inner ear development but also has implications for the development of therapeutic approaches for sensorineural hearing loss.