Regulation of Supporting Cell Development in the Mammalian Cochlea

National Institute On Deafness And Other Communication Disorders

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Abstract

During the last year we have undertaken a detailed study of the signaling pathway that regulates the formation of one specific type of supporting cell, the pillar cell. Pillar cells are only found in mammalian inner ears and the presence of these cells is required for normal auditory function. An examination of expression of members of the fibroblast growth factor signaling pathway indicated that one fgf ligand, Fgf8 is expressed in a limited pattern of cells within the organ of Corti and that one of the fgf receptors, Fgfr3 is expressed in an adjacent population of cells. Interestingly, the cells that express Fgfr3 include cells that will develop as pillar cells. Previous work from the laboratory has demonstrated that deletion of Fgfr3 leads to a specific loss of pillar cells while over activation of Fgfr3 leads to increased pillar cell formation. These results suggest that expression of Fgfr3 plays a role in regulating the temporal and spatial position of pillar cells. To begin to understand how expression of Fgfr3 is regulated we examined the effects of thyroid hormone on Fgfr3 expression. Thyroid hormone signaling is known to regulate some aspects of Fgfr expression in other systems and the inner ear phenotype in thyroid hormone receptor mutants closely resembles the phenotype in Fgfr3 mutants. Initial studies have demonstrated that induction of either hyper- or hypo-thyroid conditions in mouse embryos results in specific changes in Fgfr3 expression with increased thyroid hormone signaling resulting in a down regulation of Fgfr3. Moreover, analysis of the Fgfr3 promoter indicates the presence of several thyroid hormone receptor binding sites, suggesting potential direct regulation of Fgfr3 expression by thyroid hormone. Ongoing experiments will determine whether these binding sites are valid and can regulate Fgfr3 expression. During an analysis of the effects of deletion of Fgfr3 we observed that Fgfr3-mutant cochleae contain a greater number of hair cells suggesting that some of the cells that would have developed as pillar cells have undergone a fate change to become additional hair cells. A screen for genes with altered expression in Fgfr3 mutants indicated that bone morphogenetic protein 4 (Bmp4) is up-regulated. Since Bmp4 has been shown to influence cell fate, we wanted to determine whether the increase in hair cells might be a result of the increase in Bmp4 signaling. To examine this possibility, Bmp4 signaling within the cochlea was modulated in vitro. Results indicated that increased Bmp4 leads to an increase in hair cells while inhibition of Bmp4 leads to hair cell loss. Moreover, the increased hair cell number in Fgfr3 mutant cochlea can be inhibited if Bmp4 signaling is blocked. These results suggest that a balance between Fgf and Bmp signaling may play a role in regulating the number of pillar cells versus hair cells within the developing organ of Corti. To begin to examine the specific role of Bmp4 in the formation of hair cells versus supporting cells, we first determined the expression of a family of transcription factors, referred to as Smads, that are activated in response to Bmp4 signaling. We found that multiple Smads are expressed in the same region of the ear as Fgfr3, suggesting that these two signaling pathways are active within the same cells. Moreover, using an antibody against the phosphorylated (activated) form of Smads1/5/8, we were able to demonstrate that Smads are activated in the same region of the inner ear in which Fgfr3 is activated. This result suggests that the balance of activated Bmp versus activated Fgf signaling pathways plays a key role in regulating the choice between hair cell and supporting cell. In order to test this hypothesis directly, we used and endogenous inhibitor of Bmp signaling, Noggin, to antagonize the amount of Bmp signaling within the inner ear. Preliminary results indicate that inhibition of Bmp signaling results in a dose dependent elimination of hair cells. These results support the hypothesis that the Bmp and Fgf signaling pathways interact within individual cells to determine whether those cells will form as hair cells or supporting cells.