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. A key step in development of the organ of Corti is the formation of mechanosensory hair cells. Previous work from our laboratory, as well as several others, has demonstrated that the transcription factor Atoh1 plays a key role in the induction of a hair cell fate. However, open questions have been how many progenitor cells within the cochlea initially express Atoh1, and how is Atoh1 expression regulated in those cells. To address these questions we used a newly generated Atoh1-reporter mouse in which it was possible to label all Atoh1-expressing cells at specific developmental stages and then determine the development fates of those cells over time. Analysis of the fates of all Atoh1-expressing cells indicated that only 66% of cells that initially express Atoh1 go on to develop as hair cells. The remaining 33% develop as supporting cells, cells that are located near hair cells but serve very different functions. The number of cells that go on to develop as hair cells is dependent on the ability of those cells to maintain their expression of Atoh1. Using several different manipulations we were able to demonstrate that maintenance of Atoh1 is dependent on several different cellular interactions. First, some Atoh1-positive cells will actively inhibit their neighbors from expressing Atoh1 using the conserved notch-delta signaling pathway. Second, the number of neighboring cells that any given cell can influence is dependent on an active migratory process that allows developing hair cells and supporting cells to move away from one another. When this migration is inhibited, cells that actively inhibit Atoh1 in their neighbors are able to influence a greater number of cells. These results provide new information regarding the types of cell-cell interactions that regulate hair cell number in the cochlea and also, for the first time, demonstrate the importance of cell movement and outgrowth in the development of the cochlea. The previous paragraph discussed the role of migratory processes in the formation of the organ of Corti. Basically, the progenitor cells that will develop as hair cells and supporting cells within the organ of Corti are all generated at an early time point in development of the inner ear. At the time that these cells are formed, the duct may only be about of one turn in length. In contrast by the time the cochlea reaches maturity, it can be 3 to 4 turns in length depending on the species. This observation suggests that the progenitor cells must reposition themselves over time as the duct extends. Work from our lab initially described these movements by examining developing hair cells and supporting cells at different time points in fixed tissue. But to fully understand how this process occurs, it is necessary to develop a system to observe these cells as they extend and migrate. To do so, we combined a new, in vitro culture technique, genetic labeling of progenitor cells and a rapid confocal imaging of living specimens. Using this system we were able to visualize developing cells as they migrate. The results of these experiments revealed active migratory behavior of the progenitor cells allowing us to identify and analyze the processes that occur during cochlear outgrowth. Many sensory systems, including the auditory system, are organized based on a separation of complex sensory input into more fundamental components. In the auditory system, this separation occurs based on frequency. As a result, sounds are separated based on individual frequencies which then stimulate different regions of the organ of Corti. This organization, referred to as tonotopy, is preserved through the auditory brainstem and into the auditory cortex where component frequencies are reassembled to allow perception of the original sound. Separation of frequencies in the organ of Corti is based on graded changes in multiple characteristics of the cells and physical structures of the cochlea, leading to different optimal resonances along its length. Similar structural changes are observed in the avian functional equivalent of the mammalian organ of Corti, referred to as the basilar papilla. Because of greater ease in manipulation at embryonic stages, we opted to initially examine this phenomenon in an avian system, the chicken basilar papilla. Following a series of experiments to determine when cells along the basilar papilla begin to develop frequency-specific characteristics, gene expression profiling was performed to identify potential signaling molecules that could influence tonotopic identity. Results indicated graded expression of the soluble molecule, Bmp7, along the tonotopic axis of the basilar papilla. Subsequent experiments both in vitro and in vivo, using windowed eggs, indicated that changes in the gradient of Bmp7 lead to changes in the tonotopic gradient such that cells along the length of the basilar papilla are all tuned to the same frequency. Subsequent experiments demonstrated that the effects of Bmp7 are mediated through activation of Tak1, a down-stream signaling pathway. These results provide the first information regarding the factors that act to specify tonotopic organization in the auditory periphery and should lead to valuable discoveries related to tonotopic organization throughout the auditory system. In collaboration with Richard Chadwick in the 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 while pillar cell stiffness is dependent on microtubules. Further, based on analysis of mutations in both humans and mice, we have analyzed the role of Fibroblast growth factor signaling in development of pillar cell stiffness. Our results demonstrate a role for activation of Fgf signaling in the assembly of microtubules within developing pillar cells.