Hair Cell Development in the Mammalian Cochlea

听觉功能依赖于功能性耳蜗的形成，其包括听觉感觉上皮（称为Corti器官）和相关的螺旋神经节神经元，所述螺旋神经节神经元向Corti器官提供传入神经元神经支配。 Corti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的有限区域，耳囊是一种胚胎结构，发育于后脑附近。 耳囊的其他区域通常继续发展为内耳内的非感觉结构。 现有的数据表明，单个细胞被指定为神经母细胞，这将产生耳蜗的传入神经元，或者是一群前感觉细胞，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一些调节这些发育事件的分子信号通路，但我们的理解仍然相当有限。 在过去的一年里，实验室的不同成员已经检查了这些发展过程的几个不同方面。 首先，我们证明了三种不同的转录因子Neurog 1，Neurod 1和Sox 2都是螺旋神经节发育所必需的，并且可以诱导内耳的非感觉区域发育为神经元。 实验室先前的工作已经证明，一种不同的转录因子Atoh1可以诱导类似的非感觉细胞发育为毛细胞。 有趣的是，这些细胞作为神经元发育的能力在小鼠胚胎晚期丧失，而这些细胞作为毛细胞发育的能力持续了相当长的时间。 在一系列单独的实验中，研究了不同的转录因子Pou3F4在神经元发育中的作用。 Pou3F4的突变导致小鼠和人类耳聋，尽管该基因在毛细胞或神经节细胞中不表达。 为了确定听觉缺陷的基础，我们研究了Pou3F4突变对神经元神经支配的影响。 结果表明，Pou3F4在神经节细胞神经突周围的间充质细胞中表达，因为它们向毛细胞延伸，并且在Pou3F4突变体中，这种延伸被破坏。 在一个单独的系列实验中，我们研究了正常表达Atoh1转录因子的细胞的发育潜力。 之前的结果表明Atoh 1仅在将发育为毛细胞的细胞中表达。 然而，使用基于遗传的谱系追踪方法，我们能够证明最初表达Atoh1的一些细胞将继续发展为支持细胞。 这些结果表明，其他因素有助于确定哪些Atoh1阳性细胞将继续发展为毛细胞。 实验室以前的工作表明，Rbpj是notch通路的一个组成部分，是前感觉细胞形成所必需的。 这些实验的结果之一表明，胰岛素生长因子（IGF）信号通路是Rbpj的靶点。 在一系列的后续实验中，我们已经证明，干扰的Igf信号阻止前感觉细胞表达Atoh1，因此，从分化为毛细胞。 在实验室内的一系列新的实验中，我们研究了耳蜗生长对Corti器官内单个毛细胞和支持细胞的图案化的作用。 如果耳蜗生长受到抑制，那么细胞模式就会被破坏，这表明发育中的毛细胞在形成过程中会主动移动或重新排列。 为了更仔细地研究这一点，我们已经开始制作迁移毛细胞的延时电影。 结果表明，第一次积极的迁移运动的发展毛细胞。 最后，最近的几项研究已经确定了一类新的基于遗传的综合征，这是由与纤毛或基体相关的基因突变引起的。 这些综合征被称为纤毛病，其特征在于许多不同的生物学缺陷，包括在某些情况下的听力损失。 与NEI的Anand Swaroop实验室合作，我们研究了两种已知的纤毛病基因Cep290和Mkks在眼睛和耳朵纤毛形成中的作用。 Mkks的缺失导致纤毛的缺失，这在Cep290的一些突变形式的存在下令人惊讶地得到纠正。 这些结果对纤毛形成的基础生物学研究以及现有的患者群体都有有趣的影响。

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 demonstrated that three different transcription factors, Neurog1, Neurod1 and Sox2 are both required for development of the spiral ganglion and can induce non-sensory regions of the inner ear to develop as neurons. Previous work from the laboratory had demonstrated that a different transcription factor, Atoh1, could induce similar non-sensory cells to develop as hair cells. Interestingly, the ability of these cells to develop as neurons is lost during the late embryonic period in mice while the ability of these cells to develop as hair cells persists for a considerably longer period of time. In a separate series of experiments the role of a different transcription factor, Pou3F4, in neuronal development was examined. Mutations in Pou3F4 lead to deafness in mice and humans even though this gene is not expressed in either hair cells or ganglion cells. Te determine the basis for the auditory defect, we examined the effect of mutations in Pou3F4 on neuronal innnervation. Results indicate that Pou3F4 is expressed in the mesenchymal cells that surround ganglion cell neurites as they extend towards the hair cells and in Pou3F4 mutants this extension is disrupted. In a separate series of experiments we have examined the developmental potential of cells that normally express the Atoh1 transcription factor. Previous results had suggested that Atoh1 is only expressed in cells that will develop as hair cells. However, using a genetically based lineage tracing method we were able to demonstrate that some cells the initially express Atoh1 will go on to develop as supporting cells. These results suggest that other factors contribute to the determination of which Atoh1-positive cells will go on to develop as hair cells. Previous work from the laboratory demonstrated that Rbpj, a component of the notch pathway, is required for the formation of prosensory cells. One of the results of those experiments indicated that the insulin growth factor (IGF) signaling pathway was a target of Rbpj. In a series of follow up experiments we have demonstrated that perturbation of Igf signaling prevents prosensory cells from expression Atoh1, and therefore, from differentiating as hair cells. In a new series of experiments within the laboratory we have examined role of cochlear outgrowth on patterning of individual hair cells and supporting cells within the organ of Corti. If cochlear outgrowth is inhibited, then cellular patterning is disrupted, suggesting that developing hair cells actively move or rearrange during their formation. To examine this more closely, we have begun generating time-lapse movies of migrating hair cells. Results indicate for the first time active migratory motion of developing hair cells. Finally, several recent studies have identified a new class of genetically-based syndromes, that arise from mutations in genes that are associated with cilia or basal bodies. These syndromes, referred to as ciliopathies, are characterized by a number of different biological defects, including, in some cases, hearing loss. In collaboration with the laboratory of Anand Swaroop at NEI, we have examined the effects of two known ciliopathy genes, Cep290 and Mkks, in ciliary formation in both the eye and ear. Deletion of Mkks leads to an absence of cilia that is surprisingly corrected in the presence of some mutated forms of Cep290. These results have intriguing implications for both basic biological studies of cilia formation as well as for existing patient populations.