The transcription factor Pou4f3 is essential for the survival of postnatal and adult mouse cochlear hair cells and normal hearing.

Introduction: Hair cells (HCs) of the cochlea are responsible for sound transduction and hearing perception in mammals. Genetic mutations in the transcription factor Pou4f3 cause non-syndromic autosomal dominant hearing loss in humans (DFNA15) which varies in the age of onset depending on the individual mutation. Mouse models with germline deletion or mutations in Pou4f3 have previously demonstrated its critical role in the maturation and survival of cochlear HCs during embryonic development. However, the role of Pou4f3 in auditory function and in the survival or maintenance of cochlear HCs after birth and during adulthood has not been studied. Methods: Therefore, using the inducible CreER-loxP system, we deleted Pou4f3 from mouse cochlear HCs at different postnatal ages, relevant to specific stages of HC maturation and hearing function. Results and discussion: Elevated auditory brainstem response thresholds and significant HC loss were detected in mice with Pou4f3 deletion compared to their control littermates, regardless of the age when Pou4f3 was deleted. However, HC loss occurred more rapidly when Pou4f3 was deleted from immature HCs. Additionally, HC loss caused by Pou4f3 deletion did not affect the number of cochlear supporting cells, but caused a delayed loss of spiral ganglion neurons at 4 months after the deletion. In conclusion, Pou4f3 is necessary for the survival of cochlear HCs and normal hearing at all postnatal ages regardless of their maturation state. Our data also suggest that Pou4f3 indirectly regulates the survival of spiral ganglion neurons.

Multiple supporting cell subtypes are capable of spontaneous hair cell regeneration in the neonatal mouse cochlea.

Supporting cells (SCs) are known to spontaneously regenerate hair cells (HCs) in the neonatal mouse cochlea, yet little is known about the relative contribution of distinct SC subtypes which differ in morphology and function. We have previously shown that HC regeneration is linked to Notch signaling, and some SC subtypes, but not others, lose expression of the Notch effector Hes5 Other work has demonstrated that Lgr5-positive SCs have an increased capacity to regenerate HCs; however, several SC subtypes express Lgr5. To further investigate the source for spontaneous HC regeneration, we used three CreER lines to fate-map distinct groups of SCs during regeneration. Fate-mapping either alone or combined with a mitotic tracer showed that pillar and Deiters' cells contributed more regenerated HCs overall. However, when normalized to the total fate-mapped population, pillar, Deiters', inner phalangeal and border cells had equal capacity to regenerate HCs, and all SC subtypes could divide after HC damage. Investigating the mechanisms that allow individual SC subtypes to regenerate HCs and the postnatal changes that occur in each group during maturation could lead to therapies for hearing loss.

Spontaneous Hair Cell Regeneration Is Prevented by Increased Notch Signaling in Supporting Cells.

During embryonic development, differentiation of cochlear progenitor cells into hair cells (HCs) or supporting cells (SCs) is partially controlled through Notch signaling. Many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal and drug damaged neonatal mouse cochleae. This mechanism is also implicated during HC regeneration in non-mammalian vertebrates; however, the mechanism of spontaneous HC regeneration in the neonatal mouse cochlea is less understood. While inhibition of Notch signaling can force SCs to convert into HCs and increase the number of regenerated HCs, it is currently unknown whether this pathway is involved in spontaneous HC regeneration observed in vivo. Therefore, we investigated the role of Notch signaling during the spontaneous HC regeneration process using Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+ mice injected with tamoxifen at postnatal day (P) 0 and P1 to ablate HCs and stimulate spontaneous HC regeneration. Expression changes of genes in the Notch pathway were measured using immunostaining and in situ hybridization, with most changes observed in the apical one-third of the cochlea where the majority of HC regeneration occurs. Expression of the Notch target genes Hes1, Hes5, Hey1, HeyL, and Jagged1 were decreased. To investigate whether reduction of Notch signaling is involved in the spontaneous HC regeneration process, we overexpressed the Notch1 intracellular fragment (N1ICD) in cochlear SCs and other non-sensory epithelial cells in the context of HC damage. Specifically, Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+::Sox10rtTA::TetO-LacZ::TetO-N1ICD mice were injected with tamoxifen at P0/P1 to stimulate spontaneous HC regeneration and given doxycycline from P0-P7 to induce expression of N1ICD as well as LacZ for fate-mapping. We observed a 92% reduction in the number of fate-mapped regenerated HCs in mice with N1ICD overexpression compared to controls with HC damage but no manipulation of Notch signaling. Therefore, we conclude that increased Notch signaling prevents spontaneous HC regeneration from occurring in the neonatal mouse cochlea. Understanding which components of the Notch pathway regulates regenerative plasticity in the neonatal mouse cochlea will inform investigations focused on stimulating HC regeneration in mature cochlea and eventually in humans to treat hearing loss.

Cytokine and chemokine responses of lung exposed to surrogate viral and bacterial infections.

The use of in vitro models of complex in vivo systems has yielded many insights into the molecular mechanisms that underlie normal and pathologic physiology. However although the reduced complexity of these models is advantageous with regard to some research questions, the simplification may obscure or eliminate key influences that occur in vivo. We sought to examine this possibility with regard to the lung's response to infection, which may be inherent to resident lung cells or related to the systemic response to pulmonary infection. We used the inbred mouse strains C57BL/6J, DBA/2J, and B6.129S2-IL6(tm1Kopf), which differ in their response to inflammatory and infectious challenges, to assess in vivo responses of lung to surrogate viral and bacterial infection and compared these with responses of cultured lung slices and human A549 cells. Pulmonary cytokine concentrations were measured both after in vivo inoculation of mice and in vitro exposure of lung slices and A549 cells to surrogate viral and bacterial infections. The data indicate similarities and differences in early lung responses to in vivo compared with in vitro exposure to these inflammatory substances. Therefore, resident cells in the lung appear to respond to some challenges in a strain-independent manner, whereas some stimuli may elicit recruitment of peripheral inflammatory cells that generate the subsequent response in a genotype-related manner. These results add to the body of information pointing to host genotype as a crucial factor in mediating the severity of microbial infections and demonstrate that some of these effects may not be apparent in vitro.

In vitro lung slices: a powerful approach for assessment of lung pathophysiology.

This review summarizes the existing literature on the use of in vitro lung slices to study pulmonary physiology, pharmacology, pathogenesis and toxicity. Since in vitro lung slices maintain cell-cell and cell-matrix relationships in a highly controllable and accessible setting, they offer many advantages over both in vivo and single-cell culture systems. With the advent of high-production slicers, lung slices can be rapidly and reproducibly generated, including from animals treated in vivo. Slices can then be treated in vitro and analyzed using high-throughput technology. Therefore, the lung-slice system offers broad, current and unrealized potential for the detection of toxicity and the delineation of pathophysiologic and therapeutic mechanisms.