| Studies have consistently shown that there is extremely limited regenerative ability in the mammalian cochlea. It is possible, however, that genetic or pharmacological interventions may be capable of inducing a certain amount of cochlear repair. Analyses of the cochleae of transgenic mice have demonstrated the cyclin-dependent kinsae (CDK) inhibitor p27and the retinoblastoma (Rb) proteins are important regulators of cell cycle entry in mammalian supporting cells. Such molecules may be targets for temporary gene knockdown, in order to permit hair cell recovery. In addition, retrovirally-mediated introduction of the Atohl transcription factor is capable of causing some mature supporting cells to transdifferentiate into hair cells. However, whether the ectopic regenerated hair cells induced by overexpressing Atohl normally establish PCP during their development has been increasingly focused on.Depolymerization and severing of actin filaments produces new actin monomers and new free ends that facilitate dynamic changes in the actin cytoskeleton. These events are essential for several cellular processes including cell survival, shaping, cytokinesis, migration and chemotaxis. For example, during migration and chemotaxis, cell protrusions are formed as a result of localized actin polymerization in the leading edge of a motile cell. In dividing cells, actin depolymerization plays an important role in chromosome congression, cleavage plane orientation and furrow formation. It’s known that three genes encode actin depolymerization factors (ADFs) in mammals: Cofilinl (Cfll, non-muscle Cofilin, n-Cofilin), Cofilin2(Cfl2, muscle Cofilin) and Destrin (Dstn, also called ADF or Cornl). However, the developmental distribution of ADF/destrin in semicircular canal’s ampullar epithelia and ectopic regenerated hair cells are still unknown, and the role of PCP relevant proteins or cilia polarity formation in ectopic regenerated myo7(+) cells induced by Atohl has not been reported.In the present research, we compared dynamic distribution of ADF/destrin between normal developing inner ear sensory epithelia (cochlear basilar membrane, utricle, semicircular canal ampullar epithelia) by immunofluorescent staining and laser confocal microscope analysis, then investigate the dynamic location of ADF/destrin and other microtubule-associated proteins(α-tubulin、γ-tubulin) in both WT and regenerated hair cells after overexpressing Atohl. Our results indicate that:1)In the developing cochlear(E14.5-P6), it showed that ADF/destrin distributed in some cuticular plate’s border of both hair cells and supporting cells at E14.5, then expressed in the whole cuticular plate of hair cells and supporting cells at E18.5and P1, there would be no ADF/destrin in the hair cells after P4; in the developing utricle(E14.5-P6), it showed that ADF/destrin mainly distributed in supporting cells before birth and after birth, interestingly, it existed on the kinocilia of utricle for a short time at E18.5-P1; in the developing ampullar sensory epithelia of lateral semicircular canal, ADF/destrin distributed in the cuticular plate’s border of supporting cells at E14.5and supporting cells at E18.5, it still expressed mainly in supporting cells after birth but it began to exist in the hair cells and their kinocilia from the distal area of ampullar epithelia at PI.2) Some ectopic regenerated Myo7a(+) cells induced by overexpression of Atohl expressed ADF/destrin within one week and did not have ADF/destrin expression10days after transfecting to overexpress Atoh1.3) After ectopic regenerated Myo7a(+) cells developed their actin-rich stereocilia, their basal body(y-tubulin) moved from center of cuticular plate of myo(+) cells to the distal side, suggesting the underlying PCP establishment during the development of ectopic regenerated myo(+) cells.4) We also observed that level of BrdU(+) cells of cochlear LER(lesser epithelial ridge) of neonatal mouse increased in response to testosterone-BSA treatment, and level of ADF/destrin decreased after12-24h of this treatment, which could indicate that testosterone-BSA treatment was related to the increase of proliferation within cochlear LER cells in vitro.In summary, our results indicate that ADF/destrin participated in the development of sensory epithelia in mammalian inner ear and ectopic hair cells induced by overexpressing Atohl. PCP signaling existed in the development of ectopic hair cells and the movement of their basal body. Relatively high level of proliferation of neonatal cochlear LER cells after treatment of testosterone-BSA provide a new clue to further elucidate the pivotal roles of testosterone-BSA involved in cell division of cochlear LER cells. |
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