Hair Cell Development in the Mammalian Cochlea

听觉功能依赖于功能性耳蜗的形成，其中包括被称为Corti器官的听觉感觉上皮，以及为Corti器官提供传入神经元神经支配的相关螺旋神经节神经元。Corti的器官包含至少6种不同类型的细胞，包括机械感觉毛细胞和非感觉支持细胞。毛细胞、支持细胞和螺旋神经节细胞都来源于耳囊肿的有限区域，耳囊肿是一种发育在后脑附近的胚胎结构。耳囊肿的其他区域通常会在内耳内发展为非感觉结构。现有的数据表明，单个细胞要么被指定为神经母细胞，从而产生耳蜗的传入神经元，要么被指定为前感觉细胞群，然后再细分为毛细胞和支持细胞。虽然最近的工作已经开始确定调节这些发育事件的一些分子信号通路，但我们的理解仍然相当有限。在过去的一年里，实验室的不同成员研究了这些发展过程的几个不同方面。

Auditory function is dependent on the formation of a functional cochlea, which includes the auditory sensory epithelium, referred to as the organ of Corti, and the associated spiral ganglion neurons that provide afferent neuronal innervation to the organ of Corti. The organ of Corti contains at least 6 different types of cells including mechanosensory hair cells and non-sensory supporting cells. Hair cells, supporting cells and spiral ganglion cells are all derived from a limited region of the otocyst, an embryonic structure that develops adjacent to the hindbrain. Other regions of the otocyst normally go on to develop as non-sensory structures within the inner ear. Existing data suggests that individual cells become specified to develop as either neuroblasts that will give rise to the afferent neurons of the cochlea or a population of prosensory cells that will then become subdivided into hair cells and supporting cells. While recent work has begun to identify some of the molecular signaling pathways that regulate these developmental events, our understanding is still fairly limited. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. First, we examined the role of the transcription factor Pou3f4 in development of spiral ganglion neurons. Mutations in Pou3F4 lead to deafness in mice and humans even though this gene is not expressed in either hair cells or ganglion cells. Rather, Pou3f4 is expressed in the the mesenchymal cells that surround ganglion cell peripheral axons as they extend towards the hair cells. In particular, spiral ganglion peripheral axons form fascicles referred to as inner radial bundles. In Pou3f4 mutants, inner radial bundles are significantly disrupted. As a result, many spiral ganglion neurons fail to reach the hair cells and so the number of synapses between hair cells and spiral ganglion neurons is significantly decreased. An analysis of candidate genes that might be regulated by Pou3f4 identified the axon guidance molecule EphA4 as a possible target. Analysis of EphA4 mutant mice indicated a similar defect in inner radial bundle formation and addition of EphA4 was sufficient to rescue the axon guidance defects observed in Pou3f4 mutants. EphA4 regulates guidance by binding to similar, but distinct, types of molecules on other cells. An analysis of possible binding partners identified EphrB2 as a likely candidtate expressed on spiral ganglion axons. Generation of EphrB2 mutants demonstrated similar path finding defects, indicating that Eph4/EphrB2 complexes are important for axon guidance in the inner ear. Finally, chromatin immunoprecipitation demonstrated that Pou3f4 binds directly to the EphA4 promoter, indicating a direct role for Pou3f4 in the expression of EphA4. In a separate series of experiments we have examined the role of insulin-like growth factors (IGFs) in cochlear development. Previous work has demonstrated a role for Igfs in development of the spiral ganglion, but its role in the cochlea was unknown. To examine this, we generated mouse mutants lacking the primary IGF receptor, IGf1r. While these animals die at birth, we were able to analyze their cochleae at E18.5. Cochleae from Igf1r mutants were shorter than normal, had a reduced number of hair cells and defects in cellular patterning within the cochlea. To determine the bases for this defects, we used an in vitro system that allowed us to specifically examine changes in protein expression in response to inhibition of IGF signaling. Results indicated that many of the defects that occur when IGF signaling is inhibited are a result of a delay in the expression of the transcription factor Atoh1. Since Atoh1 is required for highly stereotyped development of hair cells, a delay in Atoh1 expression could lead to subsequent defects in cellular patterning. Moreover, subsequent experiments demonstrated that IGF signaling acts through the intracellular modulator, AKT, to influence cochlear development. As discussed in the previous paragraph, the transcription factor Atoh1 is important for hair cell formation. However, there has been some debate for several years regarding whether Atoh1 is expressed exclusively in mechanosensory hair cells. To examine this question more closely, we used a newly generated transgenic mouse in which all Atoh1-expressing cells can be permanently labeled at different points during development, to directly label the Atoh1-positive population. Analysis of the fates of Atoh1-positive cells within the inner ear demonstrated that approximately 33% of all cells that become positive for Atoh1 do not develop as hair cells. Instead, these cells develop as supporting cells, the non-sensory cells that surround the hair cells. Further, by modulating the activity of several signaling pathways, we were able to change the number of Atoh1-positive cells that developed as hair cells. These results provide useful data regarding the developmental processes that regulate the formation of the mammalian cochlea as well as identifying signaling pathways that are important for hair cell formation. Previous work from the laboratory had demonstrated that activation of the fibroblast growth factor (Fgf) signaling pathway was sufficient to arrest many cells within the developing mammalian cochlear epithelium in an undifferentiated state. A comparison between mammalian and avian sensory epithelia indicated the potential for a similar role for Fgf signaling in this system. Since birds can regenerate hair cells, the identification of factors that act to inhibit hair cell differentiation has implications for the study of hair cell regeneration in mammals. Using pharmacological inhibitors of the Fgf signaling pathway, we were able to demonstrate that Fgf signaling does act to prevent hair cell formation in the avian auditory organ both during early development and in more mature animals. These results identify Fgf signaling as a potential modulator of hair cell regeneration. One crucial factor in auditory function is the appropriate patterning of connections between mechanosnsory hair cells and the spiral ganglion neurons that convey auditory signals to the brain. Different types of spiral ganglion neurons show unique innervation patterns in terms of which hair cells they synapse with. Despite the importance of these connections, the factors that mediate their formation are unknown. The results of an analysis of different genes that are expressed by developing hair cells indicated that members of the family of Semaphorin molecules could play a role in spiral ganglion guidance. Based on that result, we determined the expression of different Semaphorins within the developing cochlea as well as the expression of the receptors for Semaphorins, Neuropilins, on spiral ganglion neurons. Results indicated potential roles for these interactions in spiral ganglion patterning. To test this hypothesis, we obtained Neuropilin mutant mice and examined spiral ganglion patterning. Preliminary results indicate significant defects in patterning, confirming a role for these molecules in spiral ganglion formation. In collaboration with Richard Chadwick, Section on Auditory Mechanics, we have examined changes in the mechanical properties of developing pillar cells and hair cells. As a mechanosensitive organ that is constantly being vibrated in response to sound, the structural mechanics of the organ of Corti must play an important role in auditory function. However, the factors that mediate cell stiffness within the cochlear are unknown. To address this, atomic force microscopy was combined with pharmacological manipulations of actin and microtubules to demonstrate that hair cell stiffness is dependent on actin and pillar cell stiffness is dependent on microtubules.