The Role Of Rb In The Proliferation Of Cochlear Supporting Cells

Cochlear hair cells (HCs) are mechanosensory receptors that transduce sound into electrical signals. But HCs are very sensitive and can be easily damaged by age, adverse reaction of certain medications and noise. Once HCs are damaged, it will cause permanent hearing loss, because mammalian HCs cannot regenerate. HC damage in non-mammalian vertebrates induces surrounding supporting cells (SCs) to divide, transdifferentiate and replace lost HCs, suggesting that SCs are the origin of HC regeneration. Isolated SCs from the postnatal mouse cochlea can proliferate and transdifferentiate into HCs in vitro, and the down-regulation of the cell cycle inhibitor, p27Kip1, was observed in the process. Thus, postmitotic mammalian cochlear SCs may be good candidates for HC regeneration and the down-regulation of cell cycle inhibitors may play important roles in initiating cell cycle reentry. Rb is a key cell cycle inhibitor which suppresses genes required for entering and progressing through the cell cycle. Deletion of Rb in cochlear HC and SC progenitors produces supernumerary progenitors, which subsequently acquire features of differentiated HCs and SCs and later undergo cell death. The acute elimination of Rb in postmitotic HCs causes cell cycle reentry and mitosis; however, HCs die at different stages of the cell cycle before division is complete. The effect of inactivating Rb in postmitotic SCs remains unknown. Here, we induce the acute elimination of Rb in postnatal SCs and examine their capability to proliferate and transdifferentiate in vivo, in order to understand the potential use of SCs in the regeneration of mammalian HCs.(1) Creating mouse line that can visualize cells with Cre recombinase activity.①Cross Prox1-CreERT2 mice with Rosa-LacZ reporter mice, to create Prox1-CreERT2/ROSA-LacZ mice that expressβ-galactosidase (β-gal) in the cells with Cre recominase activity.②Use X-gal staining to detect cells with Cre activity in the Prox1-CreERT2 cochlea. (2) Inducing Cre activity in Prox1-CreERT2 mice. Administrate intraperitoneal (IP) injection of tamoxifen once daily at P0 and P1 to Prox1-CreERT2/ROSA-LacZ mice at 3mg/40g body weight, to induce Cre activity.(3) Detecting induced Cre activity in the Prox1-CreERT2 cochlea.①Administrate IP injection of tamoxifen to neonatal Prox1-CreERT2/ROSA-LacZ mice, following the dosage above.②Collect cochlea at P6 from Prox1-CreERT2/ROSA-LacZ mice that has received tamoxifen injection at P0 and P1, and use X-gal staining in combination with co-staining of a HC marker, Myo7a, to observe the localization of cells with Cre activity in the Prox1-CreERT2 cochlea.(4) Study in detail the subtype of Cre positive SCs.①Cross Prox1-CreERT2 mice with Rosa-EYFP reporter mice, to create Prox1-CreERT2/ROSA-EYFP mice that express enhanced yellow fluorescent protein (EYFP) in the cells with Cre recominase activity.②Administrate IP injection of tamoxifen to neonatal Prox1-CreERT2/ROSA-EYFP mice, following the dosage above.③Collect cochlea at P4 from Prox1-CreERT2/ROSA-EYFP mice that has received tamoxifen injection at P0 and P1, and then observe the relative localization of EYFP-positive cells to the HCs and SCs by immunofluerecent staining under the confocal microscopy.(1) Creating mouse line that can ablate Rb gene specifically in the neonatal cochlear SCs.①Cross Prox1-CreERT2 mice with RbloxP/loxP mice, to create Prox1-CreERT2/RbloxP/loxP mice.②Administrate tamoxifen injection to Prox1-CreERT2/RbloxP/loxP mice following the description above, to induce Rb gene knock-out specifically in the neonatal SCs mediated by Cre recombinase activity.(2) Investigating cell cycle reentry of cochlear SCs after Rb ablation.①Administrate IP injections of BrdU or EdU at P4 or P6 to the Prox1-Rb-/- mice that has received P0-P1 tamoxifen injection.②Co-stain BrdU or EdU with HC or SC markers, to investigate whether postmititic SCs reentered the cell cycle.③Count the number of BrdU-positive cells in the basil, middle and apical turn of Prox1-Rb-/- cochleae.④Compare the distribution of proliferating cells with that of Cre activity in the Prox1-CreERT2 mice.(3) Investigating the completion of cell division and proliferation of Prox1-Rb-/- SCs.①Immunostaining of pH3, a marker of M phase nuclei, to determine whether Prox1-Rb-/ SCs can enter mitosis (M) phase.②Stain BrdU in combination with FISH, which allows discrimination between cells with unduplicated or duplicated chromosomes, to test whether Prox1-Rb-/- SCs could complete the cell cycle.③Double label BrdU and EdU in Prox1-Rb-/- cochleae, to confirm that Prox1-Rb-/- SCs progressed through more than one round of S phase.④Quantify SC number, to confirm that Prox1-Rb-/- SCs proliferated.(1) Cell death in Prox-Rb-/- cochleae.①Use TUNEL staining to observe cell death in Prox1-Rb-/- cochlea at different ages.②Analyze the TUNEL-positive SC number in the P9 Prax1-Rb-/- cochleaer wholemount.③Stain Myo7a or Myo6 on Prox1-Rb-/- cochlear wholemounts at different ages, to detect the change of HC number.(2) The cell fate decision of Prox1-Rb-/- SCs during proliferation.①Administrate IP injection of EdU once daily into Prox1-Rb-/- mice at P4-P5 or P8-P14, and then co-stained with HC or SC markers, like Myo6, Prox1 and Sox2, to determine the cell fate of Prox1-Rb-/- SCs during proliferation.②Investigate the influence of HC death to the cell fate decision of the proliferating SCs, and discuss a best way to manipulate SCs to regenerate HCs.(1) Inducible Cre activity in DCs and PCs of postnatal Prox1-CreERT2 cochlea. After injection of tamoxifen at P0 and P1, Prox1-CreERT2;Rosa-LacZ cochleae showed restricted Cre activity in DCs and PCs, and the number of cells with Cre activity increased from basal to apical turn. In the apical turn of Prox1-CreERT2;Rosa-EYFP cochlea,72.77%±1.12% of YFP positive SCs are DCs while 27.23%±1.12% are PCs, i.e. the majority of Cre-positive SCs are DCs.(2) Inactivation of Rb resulted in cell cycle reentry and proliferation of cochlear SCs. BrdU-positive DCs and PCs were frequently detected in the Prox1-Rb-/- cochlea, but never in the cochlea of control mice. Although BrdU-positive nuclei were also observed in the HC layer, no Myo7a-positive cells were labeled by BrdU and all EdU-positive SCs were labeled by Proxl. The number of BrdU-positive cells displayed a gradient from basal to apical turn, consistent with the gradient of Cre-positive cells in Prox1-CreERT2 cochlea. More PCs than DCs were labeled with BrdU in the apical turn at P4. Prox1-Rb-/-cochleae showed a significant increase in SC number at P6. PCs and DCs with strong pH3 labeling, or with BrdU-positive condensed chromosome, were abserved in Prox1-Rb-/- cochlear whole mounts at various ages. BrdU-positive PCs showed G1 or early S phase features (i.e., two FISH signals) at P6. BrdU/EdU double positive PCs were also observed in Prox1-Rb-/- cochleae.(3) Cell death of Prox1-Rb-/- SCs. In Prox1-Rb-/- cochleae, no TUNEL-positive SCs were observed by cross section before P5. At P7, we first detected dying cells in the organ of Corti using TUNEL staining. These dying cells are likely DCs whose nuclei have migrated into the HC layer. HC numbers were not significantly changed at P9. At P12 scattered HC loss was clearly evident in the Rb-/- cochlea and was more severe at 7 weeks of age. Moreover, HC loss was more prominent in the apical turn than in middle and basal turns at P12 and P21. Furthermore, ABR thresholds of Rb-/- mice at 5 weeks of age were significantly increased from 6-32 kHz compared to controls.(4) Proliferating Prox1-Rb-/- DCs and PCs still maintain SC fate. All EdU-positive cells were also labeled with Proxl and Sox2 at P4, demonstrating that proliferating cells still maintained their SC fate during cell cycle reentry. In the area where HCs were lost, EdU-positive SCs were also found co-labeled with the SC marker, Sox2. No EdU/Myo6 double positive cells were observed, suggesting that Prox1-Rb-/-SCs did not transdifferentiate into HCs even after the loss of neighboring HCs in one week.