The Effect Of Sonic Hedgehog On The Differentiation Of Cochlear Neural Progenitors Of Mice In Vitro

Objective:To study the influence of sonic hedgehog(Shh) on the differentiation of cochlear neural progenitors(CNPs) of mice.Methods: Cochlear neural progenitors were isolated from the postnatal day 1 organ of Corti of mice. Cells were divided into three groups: Shh treatment,phosphate-buffered saline (PBS) control, and Shh treatment plus LY294002(a specific inhibitor for the Akt pathway). Mammalian atonal homolog1(Math1 or Atoh1), a transcription factor, is used as a marker for the initial differentiation of auditory hair cells. The expression of Math1 mRNA was evaluated by RT-PCR and luciferase assays at 24 hours. The expression of Math1 protein was evaluated by immunohistochemistry and flow cytometry at 48 hours. The effect of Shh on the proliferation of CNPs were evaluated with comprehensive methods: i) cell cycle progression (flow cytometry), ii) 5-bromo-2-deoxyuridine incorporation, and iii) cell apoptosis.Results: Shh increased the expression of Math1 mRNA transcripts by RT-PCR,(p<0.05) the promoter activity of Math1 by luciferase assays,(p<0.05) the expression of Math1 protein by immunohistochemisty and flow cytometry(p<0.05). LY294002 significantly attenuated the Shh-induced promoter activity of Math1 by luciferase assays and the Shh-induced expression of Math1 protein by immunohistochemistry(p<0.05).It is demonstrated that shh is not a mitogen for the CNPs through the proliferation assays and the apoptosis assays from the postnatal day 1 organ of Corti of mice.Conclusions: Shh increases the differentiation of CNPs derived from the postnatal day 1 organ of Corti of mice,but has little influence on the proliferation of the cells. This study shows that Shh affects the differentiation of CNPs under the cell culture condition.