Regenerating hair cells in vestibular sensory epithelia from humans
- University College London, United Kingdom
- Imperial College London, United Kingdom
- Boston Children's Hospital, United States
Figures
Figure 1
Undamaged utricular maculae.
(A–C) Fixed immediately after harvesting. (D) After 28 days in explant culture. (A) Hair cells labelled for myosin VIIa (red) are distributed over the entire utricle. Phalloidin labels actin (green) …
Figure 2
Hair cell loss in utricular maculae following incubation in gentamicin.
(A) Hair cells undergoing apoptosis (arrows), indicated by the condensed marginated chromatin in their nuclei, at 24 hr after incubation in gentamicin. Scale bar: 2 µm. (B) Apoptotic body inside a …
Figure 3
Sensory epithelia transduced with ATOH1 and GFP after incubation with gentamicin.
(A) GFP expression 2 days after transduction. Many supporting cells express GFP which delineates cell shape revealing several cells with thin, basally directed extensions (inset). Scale bar 20 µm. (B…
Figure 4
Numbers of myosin VIIa positive cells per unit area.
Number of labelled cells (25%–75% percentiles and maximum-minimum) in utricles following 48 hr incubation in gentamicin then maintained up to 28 days with no further treatment (Gentamicin only, …
Figure 5
Morphological characteristics of cells in utricles 18 days after transduction with ATOH1.
(A) Many cells across the epithelium bear elongated microvillar-like projections from their apical surface. Scale bar: 5 µm. (B, C) Cells with dense cluster of apical projections possess a single …
Figure 6
Average RQ values in Log2 fold-change for ATOH1 treated versus controls from Taqman (blue) compared to Log2 fold-change measured by RNA-seq (red).
https://doi.org/10.7554/eLife.34817.008Tables
Table 1
Transcription factors and chromatin modifiers that exhibit significant changes in gene expression upon Atoh1 transduction into human sensory epithelia.
The left column lists gene names followed by gene descriptions. AB T is the average transcript abundance in the Atoh1 treated samples (as FPKMs). AB C is the average abundance in the control …
| GENE | Description | Ab T | Ab C | LOG2 | Funct |
|---|---|---|---|---|---|
| ATOH1 | atonal bHLH transcription factor 1 | 37.8 | 0.3 | 6.9 | TF |
| RAX | retina and anterior neural fold homeobox | 0.7 | 0.1 | 2.5 | TF |
| ZNF296 | zinc finger protein 296 | 17.1 | 3.0 | 2.5 | TF |
| POU4F3 | POU class 4 homeobox 3 | 3.4 | 0.8 | 2.1 | TF |
| CITED4 | Cbp/p300 interacting transactivator | 56.6 | 13.7 | 2.0 | TF |
| GTF2IRD2 | GTF2I repeat domain containing 2 | 2.7 | 0.7 | 2.0 | TF |
| SNAI1 | snail family transcriptional repressor 1 | 30.7 | 8.9 | 1.8 | TF |
| ZNF775 | zinc finger protein 775 | 17.8 | 5.9 | 1.6 | TF |
| MEF2B | myocyte enhancer factor 2B | 2.1 | 0.7 | 1.6 | TF |
| SOX12 | SRY-box 12 | 10.1 | 3.6 | 1.5 | TF |
| ZNF784 | zinc finger protein 784 | 3.7 | 1.3 | 1.4 | TF |
| ZNF837 | zinc finger protein 837 | 1.8 | 0.7 | 1.4 | TF |
| IRF9 | interferon regulatory factor 9 | 137.3 | 53.5 | 1.4 | TF |
| ZNF692 | zinc finger protein 692 | 36.7 | 15.1 | 1.3 | TF |
| GTF2H3 | general transcription factor IIH subunit 3 | 9.3 | 21.5 | −1.2 | TF |
| ZNF382 | zinc finger protein 382 | 0.9 | 2.2 | −1.2 | TF |
| EP300 | E1A binding protein p300 | 10.9 | 26.3 | −1.3 | CM |
| ZFHX4 | zinc finger homeobox 4 | 1.0 | 2.4 | −1.3 | TF |
| ASH1L | ASH1 like histone lysine methyltransfer | 5.1 | 12.8 | −1.3 | CM |
| ATRX | ATRX, chromatin remodeler | 7.0 | 22.4 | −1.7 | CM |
| SATB2 | SATB homeobox 2 | 0.8 | 3.0 | −1.9 | TF/CM |
| HDAC9 | histone deacetylase 9 | 5.7 | 20.9 | −1.9 | CM |
| TCF19 | transcription factor 19 | 0.7 | 0.2 | 1.8 | TF |
| HES1 | hes family bHLH transcription factor 1 | 78.1 | 24.1 | 1.7 | TF |
| ZNF467 | zinc finger protein 467 | 4.3 | 1.5 | 1.5 | TF |
| GSC | goosecoid homeobox | 0.8 | 0.3 | 1.5 | TF |
| SALL1 | spalt like transcription factor 1 | 9.7 | 3.9 | 1.3 | TF |
| KCNIP3 | potassium voltage-gated channel protein 3 | 10.1 | 4.3 | 1.3 | TF |
| THAP3 | THAP domain containing 3 | 30.6 | 13.2 | 1.2 | TF |
| MSX1 | msh homeobox 1 | 8.4 | 3.7 | 1.2 | TF |
| SCAND1 | SCAN domain containing 1 | 58.9 | 26.8 | 1.1 | TF |
| ING2 | inhibitor of growth family member 2 | 13.2 | 6.1 | 1.1 | CM |
| MLXIP | MLX interacting protein | 8.8 | 4.2 | 1.1 | TF |
| ZNF652 | zinc finger protein 652 | 11.4 | 5.7 | 1.0 | TF |
| PATZ1 | POZ/BTB and AT hook containing zincfinger | 18.1 | 9.3 | 1.0 | TF/CM |
| RARG | retinoic acid receptor gamma | 18.0 | 9.2 | 1.0 | TF |
| GLI4 | GLI family zinc finger 4 | 13.1 | 6.8 | 1.0 | TF |
| THAP8 | THAP domain containing 8 | 7.5 | 4.3 | 0.8 | TF |
| ING5 | inhibitor of growth family member 5 | 17.5 | 10.4 | 0.8 | CM |
| TAF6 | TATA-box binding protein associated factor | 42.6 | 26.1 | 0.7 | TF |
| ZNF768 | zinc finger protein 768 | 47.2 | 29.3 | 0.7 | TF |
| CREBBP | CREB binding protein | 11.6 | 21.8 | −0.9 | TF |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample | Ad2 -CMV ATOH1:CMV-GFP viral vector | Kesser et al., Laryngoscope 118:821–831 | ||
| Antibody | anti-myosin VIIa | Developmental Studies Hybridoma Bank | DSHB myo7a138-1 | (1:100) |
| Antibody | anti-espin | J. Bartles (Gift) | (1:50) | |
| Antibody | anti sox 2 | Abcam | ab97959 | (1:100) |
| Antibody | anti-ATOH1 | Aviva Bio Systems | ARP 32365_P050 | (1:100) |
| Antibody | FITC, TRITC secondaries | Sigma | (1:200) | |
| Other | Vectorshield with DAPI | Vector Laboratories | H-1200 |
Additional files
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Supplementary file 1
Known and putative hair cell markers that exhibit upregulation in gene expression upon Atoh1 transduction into human sensory epithelia.
The left column lists gene names followed by gene descriptions. AB T is the average transcript abundance in the Atoh1 treated samples (as FPKMs). AB C is the average abundance in the control samples. LOG2 is the log base2 fold change of Atoh1 transfected compared to the controls. REFERENCE indicates the published results showing supportive evidence that the given gene is a putative hair cell marker (1) Cai et al. (2015); (2) Scheffer et al., 2015; (3) Shin et al. (2013). Names in bold are genes that show a > 2 fold change in expression (and p<0.05) across all three comparisons (they are part of the 441 differentially expressed genes, see materials and methods and results). Genes with an asterisk (*) are putative Atoh1 downstream targets based on Groves et al publication. Genes in plain text and above the thick bottom line show statistically significant upregulation in all comparisons, but only pass > 2 fold in at least one comparison. Genes below the thick bottom line do not pass any of the above significance thresholds and show at least upregulation in all comparisons.
- https://doi.org/10.7554/eLife.34817.010
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Transparent reporting form
- https://doi.org/10.7554/eLife.34817.011
