Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells

<div><p>Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.</p></div

Microarray analysis comparing gene expression of mESCs and iMOP cells.

<p>(A) Normalized mean intensity values of differentially expressed genes in untransfected mESC and iMOP cells (n = 3, linear fold change >2 and ANOVA p<0.05). Upregulated genes in iMOP versus mESCs are depicted in red and downregulated genes in green. (B) Hierarchal clustering of gene expression profiles for untransfected mESC and iMOP cells, including profiles for each cell type transfected with indicated expression vector. (C) Number of upregulated genes in transfected mESCs and iMOP cells relative to control pT transfected cells (n = 3, linear fold change ≥1.5 and ANOVA p<0.05). Indicated are upregulated HC-enriched genes (pink) and upregulated non-HC genes (red). (D) Number of downregulated genes in transfected mESCs and iMOP cells relative to control pT transfected cells (n = 3, linear fold change ≥1.5 and ANOVA p<0.05). Indicated are HC-enriched genes (bright green) and non-HC genes (dark green).</p

Unique combinational influence of Atoh1 and miR-183 family.

<p>(A) Linear fold change in gene expression for Atoh1-expressing mESCs (blue) and Atoh1/miR-183 family-expressing mESCs (red) compared to control. (B) Linear fold change in gene expression for Atoh1-expressing iMOP cells (blue) and Atoh1/miR-183 family-expressing iMOP cells (red) compared to control. Differentially expressed genes for each cell type were selected based on comparison of Atoh1/miR-183 family-expressing cells to Atoh1-expressing cells (n = 3, linear fold change ≥1.5 and ANOVA p<0.06).</p

Plasmid (p) and adenoviral (Ad) vector nomenclature and content.

<p>Plasmid (p) and adenoviral (Ad) vector nomenclature and content.</p

qRT-PCR validation of a subset of differentially expressed genes in mESC and iMOP cells.

<p>Graphs show linear fold change of differentially expressed genes by Taqman qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p<0.05).</p

miR-183 family impact on predicted target genes’ expression.

<p>(A) Heat maps depicting linear fold changes for 34 genes among miR-183 family predicted target genes in the transcriptomes of transfected mESC and iMOP cells compared to controls. The genes were selected by comparing miR-183 family-expressing cell to control for each cell type (n = 3, linear fold change <-1.25), and they represent the most downregulated genes in mESCs (left heat map) and iMOP cells (right heat map). Red boxes indicate upregulated genes, green boxes indicate downregulated genes, and black boxes indicate genes with no change in expression. Genes are ordered by greatest repression in miR-183 family-transfected cells (top) to least (bottom). Asterisks denote statistically significant differences (p<0.05) using a one-way ANOVA test. (B) Venn diagram depicting the commonality of differentially downregulated miR-183 family predicted target genes among miR-183 family-expressing mESC and iMOP cells. (C) Number of differentially downregulated miR-183 family predicted target genes in transfected mESC and iMOP cells. Bright green bars depict the subset of these genes with more than 50% repression, while dark green bars represent the subset exhibiting 25% to 50% repression.</p

Impact of Atoh1 on mESC versus iMOP cell transcriptomes.

<p>(A) Heat maps showing linear fold changes for 40 genes in the transcriptomes of transfected mESC and iMOP cells compared to controls. The genes were selected by comparing Atoh1-expressing cells to control for each cell type (n = 3, linear fold change ≥1.5 and ANOVA p<0.05), and they represent the most upregulated genes in mESCs (left heat map) and iMOP cells (right heat map). Red boxes indicate upregulated genes, green boxes indicate downregulated genes, and black boxes indicate genes with no change in expression. Genes are ordered by greatest fold change in Atoh1-transfected cells (top) to least (bottom). (B) Venn diagram depicting the commonality of differentially upregulated genes among Atoh1-expressing mESC and iMOP cells. (C, D) Graphs showing enriched gene ontological categories in Atoh1-expressing mESCs (C) and iMOP cells (D) with statistical significance (p<0.05). The number of genes within each category is indicated beside each bar.</p

Expression of repressed miR-183 family predicted target genes during inner ear development.

<p>(A) Venn diagram depicting the commonality of differentially downregulated miR-183 family predicted target genes among miR-183 family-expressing and the Atoh1/miR-183 family-expressing iMOP cells (n = 3, linear fold change <-1.25). (B) Enriched gene ontological categories among the 26 common repressed predicted target genes with statistical significance (p<0.05). The number of genes within each category is depicted beside each bar. (C) Heat map depicting relative expression levels (normalized read counts) for the 26 common repressed predicted target genes in late prenatal (E16) and early postnatal (P0, P4 and P7) mouse inner ear development of HCs (“p” designation) versus non-HCs (“n” designation) obtained from SHIELD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180855#pone.0180855.ref026" target="_blank">26</a>]. Red boxes indicate high expression levels, green boxes indicate low expression levels, and black boxes indicate moderate level of expression. Genes are ordered by greatest repression in miR-183 family-expressing iMOP cells (top) to the least (bottom).</p

Fluorescence visualization and flow cytometric analysis of mESC and iMOP transfected cells.

<p>(A) mESCs at 48 h post-transfection with each of four plasmid vectors. (B) iMOP cells at 48 h post-transfection with each of four plasmid vectors. Bright field and fluorescence images are shown for corresponding fields (bar = 400 μm). Graphs depict representative results from flow cytometric analysis, where the threshold for RFP-positive cells was defined based on analysis of background levels of fluorescence detection in untransfected control cells. The average percentage of RFP-positive cells is indicated (n = 3).</p