Hair Cell Development in the Mammalian Cochlea

听觉和前庭功能依赖于功能性内耳的形成。虽然这两个系统都有多个组成部分，但本实验室专注于感觉上皮的发育，其中包含机械感觉毛细胞和相关细胞，称为支持细胞，以及来自VIIIth(听前庭)脑神经的神经元对这些毛细胞的神经支配。这三种细胞类型都来自耳囊，耳囊是一种胎盘结构，在发育早期形成于后脑附近。确定指定每种细胞类型的因素，然后将它们组装成功能单位是发育神经科学科的一个关键目标。在过去的一年里，实验室的不同成员检查了这些发育过程的几个不同方面。 内耳发育的一个关键步骤是形成机械感觉毛细胞。我们实验室以及其他几个实验室之前的工作已经证明，转录因子Atoh1在诱导毛细胞命运中发挥关键作用。基于这些结果，Atoh1被认为是一种可能的候选基因，用于开发基因治疗方法来潜在地修复听觉或前庭功能的缺陷。然而，一个重要的悬而未决的问题是，强制表达Atoh1是否足以产生功能正常的毛细胞。为了解决这个问题，我们将表达Atoh1的腺病毒载体导入成年胞果的外植体培养中。结果表明，虽然大多数支持细胞会对Atoh1的强制表达做出反应，但这些细胞的形态和基因表达的变化与未成熟毛细胞的形成一致，但这些细胞无法表达与成熟毛细胞一致的基因和蛋白质。虽然这些实验是在体外进行的，而不是在体内进行的，但它们表明Atoh1可能不足以诱导具有功能的毛细胞。 许多感觉系统，包括听觉系统，都是基于将复杂的感觉输入分离成更基本的组件来组织的。在听觉系统中，这种分离是基于频率的。结果，声音根据个别频率被分离，然后刺激科尔蒂器官的不同区域。这种被称为强直性复制的组织通过听觉脑干被保存下来，进入听觉皮质，在那里成分频率被重新组装，以允许感知原始声音。Corti器官的频率分离是基于细胞的多种特征和耳蜗物理结构的分级变化，导致沿其长度的不同的最佳共振。在鸟类功能相当于哺乳动物的Corti器官，称为基底乳头的地方也观察到了类似的结构变化。由于胚胎阶段的操作更容易，我们选择了在鸡的基底乳头这个鸟类系统中初步研究这种现象。在进行了一系列实验以确定沿基底乳头的细胞何时开始形成频率特异性特征之后，进行了基因表达谱分析，以确定可能影响音调识别的潜在信号分子。结果表明，Bmp7这一可溶性分子沿基底乳头的正位轴呈渐进性表达。随后的体外和体内实验表明，Bmp7的梯度变化会导致调谐梯度的变化，从而使沿基底乳头长度的细胞都调整到相同的频率。随后的实验证明，Bmp7的作用是通过激活下游信号通路Tak1来实现的。这些结果提供了有关影响听觉外周组织的因素的第一批信息，并将导致与整个听觉系统的立位组织有关的有价值的发现。 听觉和前庭感觉上皮均含有毛细胞和支持细胞。电生理学数据表明，这两个群体并不是同质的，而是由服务于不同功能的不同亚型组成。然而，我们对这些不同类型的毛细胞和支持细胞如何发育的理解受到这样一个事实的限制，即我们没有允许我们区分不同类型的标记。因此，为了解决这个问题，我们利用最新可用的分离和分析单个细胞的协议来生成单个毛细胞和支持细胞的转录图谱。为了做到这一点，产生了转基因小鼠，其中毛细胞和支持细胞表达独特的荧光标记。然后用Fluidigm微流控芯片分离和分离来自椭圆区(前庭)或耳蜗区(听觉)感觉上皮的细胞。单个细胞被可视化，然后从这些细胞中提取RNA，反转录，然后用来生成转录本的文库。根据这些实验的结果，我们能够识别毛细胞和支持细胞的几种不同的亚型。我们现在正在努力将这些细胞的分子特征与独特的电生理特征联系起来。

Auditory and vestibular function are dependent of the formation of a functional inner ear. While there are multiple components for both of these systems, this laboratory focuses on the development of the sensory epithelia, which contain mechanosensory hair cells and associated cells called supporting cells and on the innervation of those hair cells by neurons from the VIIIth (acousticovestibular) cranial nerve. All three of these cell types are derived from the otocyst, a placodal structure that forms adjacent to the hindbrain early in development. Identifying the factors the specify each of these cell types and then direct their assembly into functional units is a key goal of the Section on Developmental Neuroscience. 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 inner ear 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. Based on these results, Atoh1 has been proposed as a possible candidate gene for the development of gene therapy approaches to potentially repair defects in auditory or vestibular function. However, an important un-answered question was whether forced expression of Atoh1 alone is sufficient to generate functional hair cells. To address this question, adenoviral vectors expressing Atoh1 we introduced into explant cultures of adult utricles. Results indicated that while most supporting cells will respond to forced expression of Atoh1 with changes in morphology and gene expression that are consistent with the formation of immature hair cells, these cells fail to express genes and proteins consistent with mature hair cells. While these experiments were performed in vitro rather than in vivo, they suggest that Atoh1 may not be sufficient to induce functional hair cells. 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. Both the auditory and vestibular sensory epithelia contain hair cells and supporting cells. Electrophysiological data suggest that both of these populations are not homogenous and, instead are made of up different subtypes that serve different functions. However, our understanding of how these different types of hair cells and supporting cells develop is limited by the fact that we do not have markers that allow us to discriminate the different types. Therefore, to address this problem, we utilized newly available protocols for the isolation and profiling of individual cells to generate transcriptional profiles for individual hair cells and supporting cells. To accomplish this, transgenic mice were generated in which hair cells and supporting cells express unique fluorescent tags. Then cells from the utriclar (vestibular) or cochlear (auditory) sensory epithelia were dissociated and separated using Fluidigm microfluidics chips. Individual cells were visualized and then RNA was isolated form those cells, reverse-transcribed and then used to generate libraries for profiling of their transcriptomes. Based on the results of these experiments we were able to identify several different subtypes of both hair cells and supporting cells. We are now working to correlate the molecular profiles of these cells with unique electrophysiological characteristics.