Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells

Abstract

Unlike lower vertebrates, mammals are unable to replace damaged mechanosensory hair cells (HCs) in the cochlea. Recently, ablation of the retinoblastoma protein (Rb) in undifferentiated mouse HC precursors was shown to cause cochlear HC proliferation and the generation of new HCs, raising the hope that inactivation of Rb in postmitotic HCs could trigger cell division and regenerate functional HCs postnatally. Here, we acutely inactivated Rb in nearly all cochlear HCs of newborn mice, using a newly developed HCspecific inducible Cre mouse line. Beginning 48 h after Rb deletion, Ϸ40% of HCs were in the S and M phases of the cell cycle, demonstrating an overriding role for Rb in maintaining the quiescent state of postnatal HCs. Unlike Rb-null HC precursors, such HCs failed to undergo cell division and died rapidly. HC clusters were restricted to the less differentiated cochlear regions, consistent with differentiation-dependent roles of Rb. Moreover, outer HCs expressed the maturation marker prestin, suggesting an embryonic time window for Rb-dependent HC specification. We conclude that Rb plays essential and age-dependent roles during HC proliferation and differentiation, and, in contrast to previous hypotheses, cell death after forced cell-cycle reentry presents a major challenge for mammalian HC regeneration from residual postnatal HCs. Genetic and environmental factors, such as noise and ototoxic drugs, can damage HCs, leading to their death and to permanent hearing loss. Mammalian HCs are not regenerated, whereas nonmammalian vertebrates are able to replenish lost sensory HCs by proliferation and differentiation of nearby SCs within the sensory epithelium (5-7). The critical difference in the regenerative ability of mammalian cochleae and nonmammalian hearing organs appears to be their proliferative responsiveness after trauma.A key inhibitor of proliferation is the retinoblastoma protein (Rb), a member of the family of pocket domain proteins. These proteins bind to E2F transcription factors and can repress genes required during the S phase, thereby maintaining cells in a quiescent state (reviewed in ref. 8). Rb is expressed in embryonic and postnatal HCs; its absence in HC precursors of mice led to the production of supernumerary HCs that appeared functional, demonstrating that mammalian HC precursors can indeed be manipulated to proliferate (9, 10). However, HC injury typically occurs after birth; our aim was therefore to determine whether the inactivation of Rb could trigger cell-cycle reentry in postnatal HCs. The consequences of Rb loss in vivo have been studied in many tissues, using mouse models (11); in this study, we inactivated Rb in postnatal HCs, using a newly developed tamoxifen-inducible Cre mouse line. We observed that HCs rapidly reentered the cell cycle after the loss of Rb but died without generating supernumerary HCs. Thus, forced cell-cycle reentry by Rb inactivation had an entirely different outcome in postnatal HCs than in embryonic precursor cells for HCs and SCs, having limited use for ...

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

“…For instance, during sensory hair cell regeneration in the chick, the majority of hair cells arise by proliferation of neighboring support cells (19). Given that mammalian support cells do not respond to hair cell death by proliferating, molecular studies in chick have focused on identifying the mitogenic signals as well as the target genes that induce re-entering of the cell cycle in support cells (11,(20)(21)(22). For example, the transcription factors Atoh1 and Sox2 and the Wnt/β-catenin and Notch pathways have been shown to play crucial roles (23), and gene-expression analyses using microarrays have identified hundreds of genes involved in chick hair cell regeneration (18,24).…”

Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

“…After injection, it takes time for tamoxifen to enter the blood stream, be hydrolyzed to its active form, reach cells in the inner ear, and activate the CreER molecule for translocation to the nucleus. Cre-mediated recombination has been detected at the level of the genome 24-30 h after tamoxifen injection (Weber et al 2008); however, detection using different reporter lines may vary molecule (Madisen et al 2010). For this reason, most labs wait 5-10 days after tamoxifen injection to analyze reporter samples.…”

“…These data also suggest that, as in the cochlea, Atoh1 is expressed in vestibular progenitor cells before HC and SC fates are specified (Yang et al 2010a , deletion of Rb in neonatal HCs using Atoh1-CreER TM produced S phase reentry followed by cell death. Thus, Rb plays an age-dependent role in HC proliferation (Weber et al 2008). Atoh1-CreER TMmediated deletion of Pkd1 ruled out the involvement of this protein as the major component in the mechanoelectrical transduction channel complex or in planar cell polarity mechanisms, but demonstrated its requirement for normal stereocilia number and structure (Steigelman et al 2011).…”

The Cyclin Dependent Kinase Inhibitor p27Kip1 Maintains Terminal Differentiation in the Mouse Organ of Corti

“…In contrast to Rb-/-hair cells [27,28] or p27-/-, p19 -/-neurons in the central nervous [29], cell cycle reentry in the terminally differentiated supporting cells of the p27-/-organ of Corti is not coupled to apoptosis. In the retina of p27-/-mice, cytotoxic injury induces proliferation of Mueller glia without immediate apoptosis.…”

“…This suggests a general role for the Rb pathway in the maintenance of the G o state in terminally differentiated cells. However, Rb knockout mice possess hair cells that can re-enter the cell cycle [27], and for the most part rapidly enter apoptosis [28]. In contrast, deletion of p27 does not induce hair cells to re-enter the cell cycle and proliferate.…”