Promotion of Hair Cell Fate by Atoh1 and MicroRNA-183 Family

Hair cells are the mechanoreceptors responsible for hearing and balance in the inner ear. Multiple factors cause hair cell loss including aging, noise exposure and genetic predisposition. A barrier to hearing restoration after hair cell loss is the inability of mammalian auditory hair cells to spontaneously regenerate. Multiple factors are shown to be crucial for hair cell development and represent good candidates for regenerative studies. Atoh1 expression is widely accepted to be necessary and contextually sufficient for driving hair cell fate. Furthermore, the miRNA-183 family is known to be expressed at the time of hair cell differentiation, and mutations in miR-96 (one member of the family) can cause deafness both in mouse and human. I hypothesize that combining Atoh1 with miR-183 family is efficient in promoting hair cell fate. Our goal is to identify and compare the impact of expressing Atoh1 and miR-183 family separately and in combination on the transcriptome of two different developmental models; mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells. Our lab has previously developed an array of plasmid vectors expressing miR-183 family alone or in combination with Atoh1 , along with red fluorescent protein (RFP) from a single transcript. HEK293 cells were used to validate protein expression by western blot analysis, RFP expression by flow cytometry, and miRNA expression by quantitative real time PCR. mESCs and iMOP cells were transfected with different plasmid vectors, and RFP-positive cells were sorted 48 hours post-transfection using fluorescence activated cell sorting. Gene expression profiling was assessed using Affymetrix Mouse Gene ST Arrays. Transcriptome analysis of the two cell lines expressing Atoh1 alone, miR-183 family alone, or in combination revealed a set of differentially expressed genes that are relevant to hair cell development. Results demonstrate that the role of Atoh1 in development is contextually dependent with more hair cell-specific effects at a later developmental stage. Furthermore, miR-183 family expression fine tunes gene expression and potentiates the effect of Atoh1 in hair cell programming. The combination of Atoh1 and miR-183 family is necessary to upregulate expression of some hair cell-specific genes. To complement the gain-of-function study, the effects of miR-183 family member loss-of-function were investigated utilizing a miR-183/96 knockout mouse model. Gene expression profiling of knockout cochlea revealed a set of downregulated genes relevant to hair cell function including intracellular calcium buffer protiens. A subset of these genes was validated using quantitative real time PCR and immunohistochemistry. Our analyses reveal new roles for Atoh1 and iR-183 family in hair cell development that can be utilized in future regenerative studies.