Regulation of Supporting Cell Development in the Mammalian Cochlea

在过去的一年里，我们对调节一种特定类型的支持细胞——柱状细胞形成的信号通路进行了详细的研究。柱状细胞只存在于哺乳动物的内耳中，这些细胞的存在是正常听觉功能所必需的。对成纤维细胞生长因子信号通路成员表达的检查表明，fgf配体Fgf8在Corti器官内的有限细胞模式中表达，而fgf受体之一Fgfr3在邻近的细胞群体中表达。有趣的是，表达Fgfr3的细胞包括将发育为支柱细胞的细胞。先前实验室的研究表明，Fgfr3的缺失会导致柱状细胞的特异性缺失，而Fgfr3的过度激活会导致柱状细胞形成增加。这些结果表明Fgfr3的表达在调节柱细胞的时间和空间位置中起作用。为了开始了解Fgfr3的表达是如何调控的，我们研究了甲状腺激素对Fgfr3表达的影响。已知甲状腺激素信号可以调节其他系统中Fgfr表达的某些方面，甲状腺激素受体突变体的内耳表型与Fgfr3突变体的表型非常相似。最初的研究表明，在小鼠胚胎中诱导甲状腺亢进或甲状腺低下都会导致Fgfr3表达的特异性变化，甲状腺激素信号的增加导致Fgfr3的下调。此外，对Fgfr3启动子的分析表明，存在几个甲状腺激素受体结合位点，提示甲状腺激素可能直接调节Fgfr3的表达。正在进行的实验将确定这些结合位点是否有效，是否可以调节Fgfr3的表达。

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.