HES-1 and COUP-TFI shRNA Knocking Down Give Rises to New Hair Cells and Supporting Cells in Organ of Corti Organotypic Culture

Notch pathway proteins, including Hes-1, play a role in keeping supporting cells phenotype and prevent them from becoming hair cells by lateral inhibition mechanism. COUP-TFI is expressed during early otic vesicle development and is correlated with the differentiation of hair and support cells in the organ of Corti. The aim of this study was to compare the expression of hair and supporting cells and quantify the mRNA levels after knocking down Hes-1 and COUP-TFI transcripts in organ of Corti organotypic cultures of postnatal day 3 mouse. Forty-eight hours after lentiviral transfection, RNA from the organ of Corti was extracted from six conditions: control, scrambled, shRNA for Hes1 and shRNA for COUP-TF1, two different sequences for each target. Real-time PCR assess the amount of Hes-1 and COUP-TFI silencing, as well as Myo7a, Pax2, Sox2 and p27. Among the conditions willing to silence Hes1 gene, the one with the lowest level of silencing (Hes1.2 10%) showed interesting results such as increased levels of expression of Myo7a (200%), Sox2 (150%), and p27(10%). These findings suggest minor silencing of Hes1 gene leads to cell proliferation of both hair and supporting cells. On the contrary, among the conditions willing to silence Coup-Tf1 gene, the one with highest level of silencing (COUP10 30%) revealed increased levels of expression of Myo7a (70%), Sox2 (60%), Pax2 (130%), and p27 (40%). These findings suggest major silencing of Coup-Tf1 gene leads to cell proliferation, of both hair and supporting cells, and was supported by immunofluorescence analysis of the organ of Corti cryostat sections.