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。基于这种表达模式，我们想确定Fgf8和Fgfr3之间的信号传导是否可能在支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Fgf8。对这些小鼠耳蜗的分析表明，柱状细胞的特异性损失。同样，当体外Fgfr3激活水平升高时，作为支柱细胞发育的细胞数量增加。这些结果表明，Fgf8通过诱导激活Fgfr3来诱导柱细胞。此外，Fgfr3激活增加导致柱状细胞过度产生的事实表明，上皮内Fgf8的水平通常是有限的，这种限制在决定Corti器官内柱状细胞的数量和位置方面起着关键作用。

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.