Figure 5

E2F1 expression during HC regeneration in the chick BP. (A) Schematic representation of the experimental timeline of organotypic cultures of E18 chick BPs cultured in DMEM and 1% FBS for 2 days with 78 µM streptomycin (Strep) followed an additional 3 or 6 days in vitro (DIV) in the same media without streptomycin. The arrows indicate when the tissue was harvested. (B) Immunofluorescence of BPs showing the expression of E2F1 and ATOH1 in HCs (labelled with Phalloidin) and SCs (labelled with SOX2). Cultures maintained for 2 days in media with Strep show ATOH1 re-activation in some SCs whereas a downregulation of nuclear E2F1 expression is observed in comparison to control cultures maintained in DMEM (hollow arrowheads in zoom in areas in row (I) and (IV); scale bar: 10 µm. In BPs cultured for 3 additional days in streptomycin-free media (2d Strep + 3 DIV), nuclear E2F1 expression is observed in SCs (hollow arrowheads in zoom in areas in row II); scale bar: 10 µm). New HCs labelled with Phalloidin are formed in cultures maintained for 6 days in streptomycin-free media after drug treatment (2d Strep + 6 days DIV). In these cultures, ATOH1 is downregulated in SCs labelled with SOX2 whereas E2F1 is expressed in SCs and HCs (arrowheads in row III in E2F1 panel). Control experiments were conducted in parallel in DMEM and at the same timings plus additional days of in vitro (DIV). Technical replicates were three for each experiment with at least three biological samples.