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.
| Puligilla, Chandrakala; Feng, Feng; Ishikawa, Kotaro et al. (2007) Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment. Dev Dyn 236:1905-17 |
| Jacques, Bonnie E; Montcouquiol, Mireille E; Layman, Erynn M et al. (2007) Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea. Development 134:3021-9 |
