The functions of suppressor of fused (SUFU) and Hedgehog signaling in mammalian inner ear development

Abstract

Development of mammalian auditory epithelium, the organ of Corti (oC), requires precise control of both cell cycle withdrawal and differentiation. Sonic Hedgehog (Shh) signaling has been suggested to regulate the coordinated spatiotemporal control of differentiation and cell cycle exit of sensory hair cell (HC) progenitors, but the underlying mechanisms remain unclear. Moreover, the regulation of non-sensory cell development by Shh signaling has not been explored.

In this study, the roles of key Shh signaling components, including Suppressor of Fused (Sufu), Spop, Ptch1 and Gli, in cochlear development were investigated by mouse mutant analyses. Sufu negatively regulates Shh signaling by retaining Gli transcriptional activator in the cytoplasm and promoting the formation of Gli repressor. Analysis of E16.5 Emx2Cre;Sufuf/f mutants showed that outer HCs were completely absent and Atoh1 protein expression was restricted to inner HCs only. In addition, Gli2 was ectopically expressed in the oC. In E16.5 Emx2Cre;Sufuf/f;Gli2f/f compound mutants, the HC differentiation defect was largely restored. Intriguingly, outer HCs could be detected and ectopic Gli2 expression in the oC became absent in Emx2Cre;Sufuf/f mutants at E18.5, suggesting that HC differentiation was delayed in Sufu mutants and down-regulation of Gli2 level is required for HC differentiation. Spop promotes ubiquitination and degradation of Gli2 activator. Emx2Cre;Sufuf/f;Spopf/f mutants exhibited robust Gli2 activation and severely disrupted HC differentiation at both E16.5 and E18.5. Ptch1 also functions as a negative regulator of Shh signaling. Consistently, activation of Gli2 and severe HC differentiation defect at E16.5 and E18.5 were observed in Emx2Cre;Ptch1f/f mutants. In summary, the mutant analyses demonstrated that Gli2 inhibits HC differentiation.

In Emx2Cre;Sufuf/f mutants, prolonged expression of sensory progenitor marker Sox2 was detected. It has been demonstrated that high level of Sox2 would inhibit hair cell differentiation. At E16.5, the undifferentiated progenitors in the cochlear apical region expressed higher level of Sox2 compared with differentiating cells in the basal region as visualized by the Sox2-EGFP reporter. Combining the Sox2Ysb hypomorphic mutant allele and the Sox2EGFP knock-in mutant allele, the level of Sox2 protein expression was significantly reduced and detectable only in the apical region. In Emx2Cre;Sufuf/f;Sox2EGFP/Ysb and Emx2Cre;Ptch1f/f;Sox2EGFP/Ysb mutants, Sox2 protein expression was largely restored. These results showed that Shh signaling regulates differential expression level of Sox2 along the basal-apical axis of the cochlea.

The control of non-sensory cell differentiation, including the Stria Vascularis (SV) in the dorsal cochlea, is largely unknown. In this study, mutant analysis showed that Shh signaling controls the formation of SV. Emx2Cre;Ptch1f/f and Emx2Cre;Sufuf/f;Spopf/f exhibited significant reduction of marginal, intermediate and basal cells of the SV and prolonged maintenance of progenitor marker Otx2. Moreover, SV differentiation defect is largely restored in Emx2Cre;Ptch1f/f;Gli2f/f, indicating Gli2 inhibits SV differentiation. Intriguingly, Ptch1 is only transiently expressed in the ventral prosensory region, suggesting an indirect non-cell-autonomous regulation of Shh in SV development.

Taken together, this study provides novel molecular insights on Shh mediated sensory HC differentiation and discovers new functions of Shh signaling in regulating development of non-sensory cells in the cochlea.