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

Amino acid sequence comparisons of the three structural features of the gerbil, lizard and frog prestins.

<p>Id = identities; Pos = Positives.</p

Transport activity measured using radioisotope technique with [¹⁴C] formate as the substrate.

<p>Human pendrin was used as a positive control and EGFP plasmid was used as a negative control. Inhibition of formate uptakes by DIDS was also presented. All the data were acquired from 3 wells in each plate and repeated 3 times.</p

Heterogenic expression and NLC measured from prestin orthologs on transfected HEK cells.

<p><i>(A)</i> Examples of confocal microscopy images of HEK cells transfected by lPres in the attached (left panel) and detached conditions (middle panel), and by fPres (right panel). Bar: 10 µm. <i>(B)</i> Means (heavy lines) and standard deviations (light color lines) of NLC obtained from HEK cells transfected by lPres and fPres, respectively. The mean capacitance-voltage responses were fitted with Boltzmann function. Linear capacitance (C_lin) was used to normalize NLC.</p

Examples of motile responses measured from HEK cells transfected by gPres, lPres, fPres, and EGFP vector, respectively.

<p>The cell was approximately 50% inserted into the microchamber and length change of the extruded segment was measured by a photodiode-based displacement-measurement system. The electrical stimulus (bottom panel) was a 100-Hz sinusoidal voltage burst with duration of 100 ms. No time registered motile response was seen from lPres-, fPres- and EGFP-transfected HEK cells. Motility responses measured were observed from gPres-transfected HEK cells. The responses were the results of 200 averages.</p

Voltage-dependent NLC of prestin and its orthologs.

<p>(<i>A</i>) Means of NLC responses measured from gPres (n = 11), pPres (n = 9), cPres (n = 12), lPres (n = 10) and fPres (n = 8). The mean capacitance-voltage responses were fitted with Boltzmann function. (<i>B</i>) Four parameters obtained from curving fitting using Boltzmann function.</p

Alignment of consensus amino acid sequences of SLC26A5 of gerbil (Meriones unguiculatus, Mungu), frog (Xenopus tropicalis, Xtrop) and anole lizard (Anolis carolinensis, Acaro).

<p>Color of each residue represents identity at the residue among three different species. Blue: Full identity at a residue; Black: Partial identity (2/3 sequences) at a residue; Red: complete disparity at a residue. Gaps in the aligned sequences are indicated by the dashed line. The red box marks the motif that is remarkably conserved among mammalian species but highly variable among non-mammalian orthologs. This area may reflect a structural adaptation that facilitates gain of motor function in mammalian prestin.</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

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