Stemness of the Organ of Corti Relates to the Epigenetic Status of Sox2 Enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti

Quantitative High-Resolution Cellular Map of the Organ of Corti

The organ of Corti harbors highly specialized sensory hair cells and surrounding supporting cells that are essential for the sense of hearing. Here, we report a single cell gene expression data analysis and visualization strategy that allows for the construction of a quantitative spatial map of the neonatal organ of Corti along its major anatomical axes. The map displays gene expression levels of 192 genes for all organ of Corti cell types ordered along the apex-to-base axis of the cochlea. Statistical interrogation of cell-type-specific gene expression patterns along the longitudinal gradient revealed features of apical supporting cells indicative of a propensity for proliferative hair cell regeneration. This includes reduced expression of Notch effectors, receptivity for canonical Wnt signaling, and prominent expression of early cell-cycle genes. Cochlear hair cells displayed expression gradients of genes indicative of cellular differentiation and the establishment of the tonotopic axis

Comparative functional characterization of novel non-syndromic GJB2 gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu

We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein

EGF interferes with the epigenetic regulation of Sox2 expression and affects the self-renewal potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) P4 OC-derived otospheres after 5 DIV; labeling for Sox2 combined with EdU and DAPI (<b>A</b>) Otospheres grown under FGF/IGF-only conditions. (<b>B</b>) Otospheres supplemented with EGF as an additional growth factor (Scale Bars: A,B, 100 µm). (<b>C</b>) Absolute numbers of primary spheres isolated per OC with (n = 7) and without EGF (n = 8) supplementation. Data were analyzed by student's t-test and are presented as means ±SDs. (<b>D</b>) Mean diameter of the primary sphere population measured in a range from 25 to 60 µm with (n = 7) and without EGF (n = 8) supplementation. Data are presented as means ±SDs. (<b>E</b>) Methylation profiles of the otic Sox2 enhancers NOP1/2 in P21 OC, proliferating OCSCs and OCSCs supplemented with EGF (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>F</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin VIIa, p27Kip and Prox1) in standard OCSCs and in OCSCs supplemented with EGF. Relative expression levels of standard OCSCs were compared to those of OCSCs supplemented with EGF. Transcript levels were normalized to HPRT1/TbP levels. Averages of three independent experiments are shown with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2E</a>).</p

Epigenetic and transcriptional characterization of Sox2 during OCSC isolation.

<p>(<b>A</b>,<b>B</b>,<b>C</b>) Methylation profile of the Sox2 enhancers (<b>A</b>) SRR1/2, (<b>B</b>) NOP1 and (<b>C</b>) NOP2 in OCSCs as compared to the OC at P4 and E13.5 (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>D</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) in OCSCs and the OC at E13.5 and P4. Relative expression levels of OCSCs and E13.5 OC were compared with those of P4 OC. Transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of three independent experiments with SDs are depicted (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2C</a>).</p

Differentiation potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) Methylation profiles of the otic Sox2 enhancers (<b>A</b>) NOP1 and (<b>B</b>) NOP2 in the mature OC (P21), proliferating OCSC spheres and epithelial patches differentiated from OCSC spheres (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>C</b>) Relative expression levels of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) after 14 and 28 days of differentiation (n = 3) were compared with those of the proliferating OCSC spheres by qPCR. Transcript levels were normalized to TbP/Ubiquitin C levels. Shown are averages of three independent experiments (and two independent experiments for 28 days for the differentiation group) with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2D</a>). (<b>D</b>–<b>G</b>) <i>In situ</i> cell type-specific marker expression of the maturing OC (P4): Sox2 antibody (<b>F</b>) labels all supporting cells of the sensory domain (<b>G</b>), whereas S100-antibody (<b>D</b>) detects pillar and Deiters' cells only (<b>G</b>). Myosin VIIa (<b>E</b>) expression is associated with inner and outer hair cells (<b>G</b>). (<b>H</b>–<b>K</b>) OCSC-derived progeny differentiated under <i>in vitro</i> culture conditions. OCSC progeny were labeled by an EdU pulse (during the last day of 5 DIV) under proliferative culture conditions and a pulse chase after 14 DIV under differentiation-inducing culture conditions. EdU-labeling in supporting cell (Sox2, S100) (<b>H</b>) and hair cell-like (myosin VIIa) (<b>I</b>) cells. Hair cell-like cells were additionally characterized based on membrane-localized prestin (<b>J</b>) and F-actin-stained (<b>K</b>) membrane protrusions (Scale Bars: D,E,F,H,I,J,K, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s006" target="_blank">Figure S6</a>).</p

Characterization of Sox2 translation during OCSC isolation.

<p>(<b>A</b>,<b>B</b>) Double-labeling of Sox2 with PCNA, Bmi1, Jag1 and Hes1 in the OC at E13.5 and P4 compared to OCSCs. (<b>A</b>) Representative immunostaining images of longitudinal cryosections of the prosensory domain in the proximal cochlea duct at E13.5 (basilar membrane on top, luminal surface on the bottom). (<b>B</b>) Immature (P4) OC in mid-modiolar sections of the basal cochlea turn (medial to the left). (<b>C</b>) P4 OC-derived otic spheres after 5 DIV. Due to the requirements for the different tissue types investigated, the fixation, staining protocols and image acquisition settings were not identical (Scale Bars: A,B,C, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s005" target="_blank">Figure S5</a>).</p

Epigenetic, transcriptional and translational characterization of Sox2 expression during OC development.

<p>(<b>A</b>–<b>C</b>) OC during development. (<b>A</b>) Upper panel: Schematic of the sensory domain, which contains the proximal cochlea duct, showing interkinetic nuclear migration at E13.5. Sox2 expression is indicated by red nuclei. Remaining panels: marker expression at E13.5. All proliferating Ki-67-positive cells are co-labeled for Sox2. (<b>B</b>) Upper panel: schematic of the different cell types found in the maturating OC at P4. Inner hair cell (ihc, arrowhead), three outer hair cells (ohc, arrowheads) and different supporting cells: inner sulcus cells (is); interphalangeal cells (i); pillar cells (p); Deiters' cells (d); Hensen's cells (h); and Claudius cells (c). Remaining panels: marker expression at P4. The quiescence of Sox2-positive supporting cells is indicated by co-labeling with p27Kip1. (<b>C</b>) Upper panel: schematic of the different cell types found in the functional OC at P21. Remaining panels: marker expression at P21. Senescence of Sox2-positive cells is indicated by p16Ink4a expression. (<b>D</b>) RT-PCR of pluripotency marker, hair cell marker and supporting cell marker expression in the OC (E13.5, P4, P21). HPRT1 was used as the loading control. (<b>E</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, Myosin VIIa, p27Kip1 and Prox1) during OC development (E13.5, P4 and P21). The relative expression levels of P4 and P21 were compared with those at E13.5. The transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of the three independent experiments with SDs are shown (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2B</a>). Depending on the temporal expression pattern, genes were assigned to early, transition or differentiation groups (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s003" target="_blank">Figure S3</a>). (<b>F</b>,<b>G</b>) Bisulfite methylation of the Sox2 enhancers (f) (NOP1/2) and (g) (SRR1/2) during OC development (E13.5, P4, P21) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (Scale Bars: A,B,C, 10 µm).</p

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells

Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution

The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification