Table 1.
Induction of endogenous Atoh1 expression and hair cell differentiation by Eya1, Six1 and Sox2
| Constructs | GFP+/Explants 6 DIV |
Atoh1+ Myo7a 6 DIV |
Atoh1+ Myo7a− 6 DIV |
Atoh1− Myo7a 6 DIV |
%/% (Atoh1+:GFP+/Myo7a+:GFP+) | %/% (Atoh1+:Myo7a+/Atoh1−:Myo7a+) |
%(Atoh1+:GFP+) (Explants 2, 3 or 4 DIV |
%(Pou4f3+: GFP+) (Explants 2, 3, 4 or 6 DIV |
|---|---|---|---|---|---|---|---|---|
| Eya1 | 325/3 | 22 | 2* | 1 | 7.4±0.6/7.1±0.5 | 95.7±0.9/4.3±0.3 | 7.3±0.8(9:124)(3), 2 DIV | |
| Six1 | 271/3 | 15 | 2* | 3 | 6.3±0.7/6.6±0.6 | 83.3±1.0/16.7±0.9 | 6.2±0.2(11:178)(3), 2 DIV | |
| Eya1/Six1** | 441/6 | 152 | 0 | 240 | 34.5±1.2/88.9±0.5 | 38.8±0.7/61.2±0.5 | 33.6±1.0(80:238)(3), 2 DIV | 88.2±1.1(284:322) (2), 6 DIV |
| Eya1/Six4 | 266/4 | 15 | 1* | 23 | 6.0±0.2/14.3±0.3 | 39.5±1.0/60.5±0.7 | 6.2±0.9(28:449)(5), 2 DIV | |
| Eya4/Six1 | 438/5 | 51 | 32* | 206 | 19.0±0.9/58.7±1.1 | 19.8±0.7/80.2±0.3 | 19.0±0.9(98:517)(5), 2 DIV | |
| Eya1phosΔ/Six1 | 351/4 | 11 | 5* | 72 | 4.6±1.1/23.7±0.8 | 13.3±0.7/86.7±0.5 | 4.9± 1.1(22:451)(5), 2 DIV | |
| Sox2 | 498/5 | 0 | 6* | 0 | 1.2±0.3/0 | 0/0 | 3.1±0.3(4:129)(3), 2 DIV | |
| Sox2/Eya1 | 520/7 | 1 | 2* | 3 | 0.5±0.8/0.7±0.8 | 25.0±0.8/75.0±0.8 | 9.3±0.9(26:280)(3), 2 DIV | |
| Sox2/Six1 | 336/4 | 0 | 0 | 1 | 0/0.3±0.7 | 0/100 | 7.5±1.2(20:268)(3), 2 DIV | |
| Sox2/Eya1/Six1 | 389/4 | 15 | 6* | 23 | 5.4±0.7/9.8±0.8 | 39.5±1.6/60.5±2.1 | 39.2±0.9(133:339)(4), 2 DIV | |
| 29.9±0.7(88:290)(4), 3 DIV | 2.0±0.8(7:290)(4), 3 DIV | |||||||
| 30.0±0.4(78:259)(4), 4 DIV | 2.3±0.6(6:259)(4), 4 DIV | |||||||
| Sox2/Atoh1 | 234/3 | 0 | 1 | 0 | 0/0*** | 0/0*** | 95.6±4.5(214:220)(3), 2 DIV | 16.7±2.4(38:220)(3), 2 DIV |
| 66±1.0(40:167)(2), 3 DIV | 7.2±0.1(12:167)(2), 3 DIV | |||||||
| 29.2±0.9(31:181)(2), 4 DIV | 2.7±0.5(5:181)(2), 4 DIV | |||||||
| Atoh1 | 311/4 | 309 | 0 | 0 | 99.4±0.6/99.4±0.6 | 100/0 | 99.1± 0.9(205:207)(3), 2 DIV | 17.0±2.5(36:207)(3), 2 DIV |
| 100(150:150)(2), 3 DIV | 48.7±0.1(73:150)(2), 3 DIV | |||||||
| 100(148:148)(2), 4 DIV | 78.9±1.1(117:148)(2), 4 DIV | |||||||
| Control | 288/3 | 0 | 0 | 0 | 0 | 0 | 0(0:221)(3), 3 DIV | 0(0:221)(3), 3 DIV |
Transfected cells in the GER of E13.5–14.0 explants were identified as GFP+ cells. Explants after 2–6 DIV were processed for in situ hybridization for detecting Atoh1 expression and immunohistochemistry for detecting Pou4f3 or Myo7a expression.
Note that some of the Atoh1+ cells were negative for Myo7a, indicating that they failed to differentiate into hair cells.
Coexpression of Eya1/Six1 promoted Atoh1 expression and hair cell induction. Among the ectopic Myo7a+ cells, ~38.8% were Atoh1+, but ~61.2% were Atoh1−.
When Sox2 was coexpressed with Atoh1, it antagonized the effect of Atoh1 to induce hair cells. Although Pou4f3 expression was initiated normally at 2 DIV when compared with those induced by Atoh1 alone, its expression was decreased after 2 DIV, in contrast to that induced by Atoh1 alone. In the absence of hair cell differentiation, Atoh1 expression eventually disappeared. Atoh1+, Pou4f3+ or Myo7a+ or GFP+ cells were counted from each explant and the ratios of Atoh1+ vs. GFP+, Myo7a+ vs. GFP+ or Pou4f3+ vs. GFP+ from each explant were quantified and values are expressed as means±standard deviations. Significance was determined by comparing each of the sample groups using StatView’s t-test. P-values were between P<0.0001 and P=0.0379.