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。 基于这种表达模式，我们想确定Fgf 8和Fgfr 3之间的信号传导是否可能在柱细胞发育中发挥作用。 作为第一步，我们使用Cre-Lox方法在耳中特异性灭活Fgf 8。 对这些小鼠耳蜗的分析表明柱细胞的特定损失。 类似地，当体外Fgfr 3活化水平增加时，发育为柱细胞的细胞数量增加。 这些结果表明，Fgf 8通过Fgfr 3的诱导激活来诱导柱状细胞。 此外，Fgfr 3活化增加导致柱细胞过度产生的事实表明，上皮内Fgf 8的水平通常是有限的，并且这种限制在确定Corti器官内柱细胞的数量和位置中起关键作用。 在第二系列实验中，我们分析了受体Fgfr 3缺失的影响。 正如预期的那样，Fgfr 3的缺失导致柱细胞发育中的类似缺陷。 该结果证实了在柱状细胞的发育中Fgf 8和Fgfr 3之间的诱导相互作用。 然而，除了缺乏柱细胞外，我们在Fgfr 3突变小鼠中观察到许多其他缺陷。 特别是，Fgfr 3突变耳蜗含有更多数量的毛细胞，这表明一些原本作为柱状细胞发育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。 最后，我们对Fgfr 3在哺乳动物内耳中作用的研究结果表明，激活的Fgfr 3的作用之一是阻止细胞发育为毛细胞。 这一假说对毛细胞再生的研究具有启示意义，因为毛细胞形成的抑制可能在阻止哺乳动物毛细胞再生中起关键作用。 为了检验这种可能性，我们选择检验鸡内耳中Fgfr 3活性抑制的效果。 选择小鸡是因为小鸡耳蜗，也称为基底乳头，能够广泛的毛细胞再生。 此外，Fgfr 3在鸡耳蜗内的支持细胞中以与其在哺乳动物内耳中的表达模式非常相似的模式表达。 在发育中的鸡内耳中阻断Fgfr 3活化导致更多数量的细胞发育为毛细胞，支持Fgfr 3起阻断毛细胞形成的作用的假设。 此外，当在成熟鸡内耳中阻断Fgfr 3信号传导时，观察到新的毛细胞在现有的毛细胞之间发育，这表明Fgfr 3的激活起到防止毛细胞自发形成的作用，至少在鸡中是这样。 这些结果表明，它可能是可能的，以诱导一定程度的毛细胞再生在哺乳动物的耳朵，通过调制的Fgfr 3信号通路。

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. Based on this pattern of expression, we wanted to determine whether signaling between Fgf8 and Fgfr3 might play a role in pillar cell development. As a first step, we inactivated Fgf8 specifically in the ear using a Cre-Lox approach. Analysis of the cochleae from these mice indicated a specific loss of pillar cells. Similarly, when the level of Fgfr3 activation was increased in vitro, the number of cells that developed as pillar cells increased. These results suggest that Fgf8 acts to induce pillar cells through an inductive activation of Fgfr3. Moreover, the fact that increased activation of Fgfr3 leads to an over-production of pillar cells suggests that the level of Fgf8 within the epithelium is normally limiting and that this limitation plays a key role in determining the number and position of pillar cells within the organ of Corti. In a second series of experiments, we analyzed the effects of deletion of the receptor, Fgfr3. As expected, loss of Fgfr3 leads to a similar defect in pillar cell development. This result confirms the inductive interaction between Fgf8 and Fgfr3 in the development of pillar cells. However, in addition to a lack of pillar cells, we observed a number of other defects in Fgfr3 mutant mice. In particular, the 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. Finally, the results of our work on the role of Fgfr3 in the mammalian inner ear suggested that one of the effects of activated Fgfr3 is to prevent cells from developing as hair cells. This hypothesis has implications for the study of hair cell regeneration, since the inhibition of hair cell formation could play a key role in blocking hair cell regeneration in mammals. To examine this possibility, we chose to examine the effect of inhibition of Fgfr3 activity in the chick inner ear. Chicks were selected because the chick cochlea, also called the basilar papilla, is capable of extensive hair cell regeneration. In addition, Fgfr3 is expressed in supporting cells within the chick cochlea in a pattern that is very similar to its pattern of expression in the mammalian inner ear. Blockade of Fgfr3 activation in a developing chick inner ear resulted in a greater number of cells developing as hair cells, supporting the hypothesis that Fgfr3 acts to block hair cell formation. Moreover, when Fgfr3 signaling was blocked in a mature chick inner ear, new hair cells were observed to develop in between the existing hair cells, suggesting that activation of Fgfr3 acts to prevent the spontaneous formation of hair cells, at least in chickens. These results suggest that it may be possible to induce some level of hair cell regeneration in mammalian ears through modulation of the Fgfr3 signaling pathway.