Role Of The STAT3 Signaling Pathway During Mammalian Hair Cell Development

Hearing impairment incident is gradually increasing due to nosie,aminoglycosides and aging.Hearing loss,often caused by irreversible damage to hair cells or neuronal innervation defect,has been affecting millions of people in the world.Hair cells are mechanosensory cells that can directly convert sound into electrical signals.Therefore,understanding of mammalian hair cell differentiation mechanism and discovery of effective stimulators for hair cell generation are crucial for potential therapies of hearing loss.Hair cells,located adjacent to surrounding supporting in the organ of Corti,include one outer hair cell and three inner hair cells.Hair cell differentiation emerges at the mid-basal region of sensory epithelium at E14,and subsequently extends along the entire epithelium in basal-to-apical and medial-to-lateral fashions.Previous studies demonstrate that inner ear supporting cells,which retain stem cell features,have the capability to differentiate into hair cell in vitro.Over the past two decades,a number of genes and signaling pathways have been reported to regulate inner ear hair cell development.Of them,Notch signaling has been shown to be a major player in the specification of prosensory epithelium and regulation of hair cell differentiation.Activation of Notch signaling contributes to choosing the sensory progenitor fate and maintaining their undifferentiated status.Inactivation of Notch signaling in conditional knockout mouse models or by pharmacological inhibitors induces an increase in hair cell production.Activation of Wnt signaling pathway enhance sensory epithelial cells proliferantion and differentiation.Another important factor is Math1,a bHLH transcription factor.Math1 is not only sufficient to induce differentiation of supporting cells into hair cells,but also required for hair cell differentiation.More recently,Jin et al.reported that in the zebrafish the signal transducer and activator of transcription 3?STAT3?signaling,a classical pathway activated by extracellular factors,plays a role in regulation of zebrafish neuromast hair cell development.The zebrafish lateral line neuromasts are similar to the mammalian inner ear sensory epithelium in structure.STAT3 signaling is activated following hair cell damages and hair cell regeneration in the lateral line neuromasts.Knockdown of Stat3 decreases the number of hair cells by downregulating Math1 expression during hair cell development.However,the importance of STAT3 signaling for mammalian inner ear hair cell differentiation and the relationship between STAT3 and Notch signaling pathways during this process are still unknown.On the other hand,normal tissue homeostasis is maintained through symmetric and asymmetric cell divisions of stem/progenitor cells.Symmetric divisions are required for the expansion of progenitor numbers,while asymmetric divisions are operated to give rise to differentiated cells.For instance,in developing prostates,basal cells display symmetric division to produce daughter cells with self-renewal capacity,and undergo asymmetric division to generate daughter cells to achieve both self-renewal and differentiation potential.Up to now,the cell division modes that the inner ear supporting cells undergo have never been examined and whether STAT3 signaling influences these cell division modes during hair cell differentiation has not been reported.In this study,using conditional STAT3 and Notch1 knockout mouse models and pharmacological inhibitors in otosphere-adhesive differentiation or cochlear explant cultures,the present study provides evidences for a role of STAT3 signaling in supporting cell proliferation and hair cell differentiation in mammalian cochleae.In the first Chapter,we found that STAT3 was diffused expressed in the cochlear prosensory epithelium at E14,and gradually restricted in the hair cells.Meanwhile,STAT3 pS727 was expressed in the hair cells.RT-qPCR assay showed that,adhesive otosphere cells displayed an increased mRNA level of Stat3,concomitant with up-regulated expression of Math1,as hair cell differentiation proceeded.STAT3 and its activated form STAT3 pS727 and STAT3 pY705 were expressed in the newly hair cells in the process of hair cell differentiation in vitro.To assess the role of STAT3 in regulating hair cell development,we introduced Sox2CreER;STAT3^flox/flox mouse line in which Stat3 can be deleted following induction of Cre activity.Sox2CreER;STAT3^flox/floxlox/flox mice displayed sensory epithelium disorganization,a reduction in hair cell numbers,and down-regulation expression of hair cell-related genes.Importantly,hair cell loss detected appear to more severe in the apical turn than that in the basal turn.Similarly,using explant culture and otosphere-adhesive differentiation culture,we found the reduction of hair cell number when STAT3 signaling pathway is blocked.Moreover,S3I-201 treatment increased BrdU⁺/Sox2⁺and BrdU⁺/Prox1⁺cell numbers,but decreased BrdU⁺/Math1-GFP⁺cell number.By cell division mode analysis using double immunostaining for cell mitotic markers and supporting cell markers,or differentiated cell markers,we found that three cell division modes:symmetric self-renewal,symmetric commitment and asymmetric cell division.Inactivation of STAT3 signaling pathway induced a reduction in hair cell number,mediating by a shift from asymmetric division to symmetric division.In the second Chapter,we investigate the crosstalk between STAT3and Notch signaling pathway in regulation of hair cell development.We found that STAT3 and STAT3 pS727 were expressed in ectopic hair cells in the Sox2CreER;Notch1^flox/flox mouse.STAT3 activated form?STAT3pS727 and STAT3 pY705?were upregulated when Notch signaling pathway is inhibited,suggesting that perturbing Notch signaling may activate STAT3 siganling in the sensory epithelia.Inhibition of Notch signaling pathway led to the production of ectopic hair cells,an increase in hair cell number,a shift from symmetric division to asymmetrivc divisions of supporting cells.However,blocking STAT3 signaling attenuated the effects of the inhibition of Notch signaling,including a regression of disordered epithelium,a recovery from the reduction in the hair cell numbers,and a reversion of the cell division modes.In conclusion,STAT3 signaling is important for mammalian cochlear hair cell differentiation.It contributes to the down-stream of the Notch pathway in regulation of hair cell production via influencing the balance between symmetric and asymmetric division modes of supporting cells.