Regulation of Supporting Cell Development in the Mammalian Cochlea

在过去的一年中，我们已经进行了详细的研究信号通路，调节形成一个特定类型的支持细胞，柱细胞。 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传导途径成员的表达的检查表明，一种FGF配体Fgf 8在Corti器官内的有限细胞模式中表达，并且一种FGF受体Fgfr 3在相邻细胞群中表达。 有趣的是，表达Fgfr 3的细胞包括将发育为柱细胞的细胞。 来自实验室的先前工作已经证明，Fgfr 3的缺失导致柱细胞的特定损失，而Fgfr 3的过度激活导致柱细胞形成增加。 这些结果表明，Fgfr 3的表达起着调节柱细胞的时间和空间位置的作用。 为了开始了解Fgfr 3的表达是如何调节的，我们检测了甲状腺激素对Fgfr 3表达的影响。 已知甲状腺激素信号传导调节其他系统中Fgfr表达的某些方面，并且甲状腺激素受体突变体中的内耳表型与Fgfr 3突变体中的表型非常相似。 初步研究表明，在小鼠胚胎中诱导甲状腺功能亢进或甲状腺功能减退导致Fgfr 3表达的特异性变化，甲状腺激素信号传导增加导致Fgfr 3下调。 此外，Fgfr 3启动子的分析表明存在几个甲状腺激素受体结合位点，表明甲状腺激素可能直接调节Fgfr 3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Fgfr 3的表达。 在Fgfr 3缺失的影响的分析过程中，我们观察到Fgfr 3突变耳蜗含有更多数量的毛细胞，这表明一些本来会发育为柱细胞的细胞已经经历了命运的改变，成为额外的毛细胞。 在Fgfr 3突变体中具有改变表达的基因的筛选表明骨形态发生蛋白4（Bmp 4）上调。 由于Bmp 4已被证明影响细胞命运，我们想确定毛细胞的增加是否可能是Bmp 4信号传导增加的结果。 为了检验这种可能性，在体外调制耳蜗内的Bmp 4信号。 结果表明，增加Bmp 4导致毛细胞的增加，而抑制Bmp 4导致毛细胞损失。 此外，如果阻断Bmp 4信号，Fgfr 3突变耳蜗中毛细胞数量的增加可以被抑制。 这些结果表明，成纤维细胞生长因子和BMP信号之间的平衡可能发挥作用，在调节柱细胞与毛细胞内发育的Corti器官的数量。 为了开始研究Bmp 4在毛细胞与支持细胞形成中的特定作用，我们首先确定了一个转录因子家族的表达，称为Smads，其响应于Bmp 4信号传导而被激活。 我们发现，多个Smads在与Fgfr 3相同的耳部区域表达，这表明这两种信号通路在相同的细胞中是活跃的。 此外，使用针对Smads 1/5/8的磷酸化（活化）形式的抗体，我们能够证明Smads在Fgfr 3被活化的内耳的相同区域中被活化。 这一结果表明，活化的BMP与活化的FGF信号通路的平衡在调节毛细胞和支持细胞之间的选择中起着关键作用。 为了直接检验这一假设，我们使用Bmp信号传导的内源性抑制剂Noggin来拮抗内耳内Bmp信号传导的量。 初步结果表明，抑制BMP信号传导导致毛细胞的剂量依赖性消除。 这些结果支持这样的假设，即Bmp和Fgf信号通路在单个细胞内相互作用，以确定这些细胞是否会形成为毛细胞或支持细胞。

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.