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 ...
A transgenic mouse line expressing the CreER TM fusion protein under the control of the Math1 enhancer was generated. Expression of the transgene in the postnatal mouse was restricted to hair cells of the inner ear and granule neurons in the external granule layer of the cerebellum in a temporally regulated manner. Cre activity was virtually nonexistent in uninduced mice; however, treatment of newborn pups with tamoxifen, leading to nuclear translocation of the fusion protein, resulted in efficient recombination at LoxP sites in the appropriate cell types. Up to two thirds of cerebellar granule neurons and 80 -90% of cochlear hair cells underwent Cre-specific recombination. This mouse line provides a powerful tool to dissect gene function at early and late stages in development of the cerebellum and inner ear.
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