Molecular Basis For The Morphogenesis Of The Inner Ear

内耳形态发生的分子基础

基本信息

批准号：
8565493
负责人：
Doris Wu
金额：
$ 189.24万
依托单位：
NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8565493
关键词：
Adult Affect Apoptosis Area Attenuated Auditory Binding C-terminal Carrier Proteins Cell Cycle Cell Differentiation process Cells Chickens Cochlea Cochlear duct Complex Development Duct (organ) structure Dyes Ear Endolymphatic duct Enhancers Eph Family Receptors Ephrin-B2 Epithelium Equilibrium Erinaceidae Event Feedback Fetus Ganglia Genes Genetic Genetic Recombination Goals Hair Cells Homeostasis Homozygote Integral Membrane Protein Ion Transport Labyrinth Lateral Ligands Location Manuscripts Maps Medial Mediating Mitosis Molecular Morphogenesis Mus Mutation Neurons Organ Pattern Phenotype Phosphotransferases Preparation Process Role Second Messenger Systems Sensory Sensory Hair Signal Transduction Source Specific qualifier value Specimen Staging Structure Testing Time Transcription Coactivator Transplantation Utricular macula Vestibular ganglion Work base cell growth regulation deafness design endolymphatic sac fetal in vivo macula malformation mutant nerve stem cell neuroblast neurodevelopment neurogenesis otoconia overexpression premature programs promoter receptor relating to nervous system second messenger sensory system

项目摘要

This years major accomplishments are in the following areas: 1) Sonic hedgehog secreted by the auditory ganglion regulates timing of cell cycle exit and differentiation of sensory hair cells in the mammalian cochlea (manuscript submitted) Most neural and sensory systems follow a developmental program, in which the first neural precursors to undergo terminal mitosis are the first ones to differentiate into neurons. In contrast, sensory hair cells in the mammalian cochlea are regulated differently. Cell cycle exit of hair cell precursors is initiated at the apex of the cochlear duct and progresses towards the base, whereas hair cell differentiation starts at the base of the duct after cell cycle exit is completed. This unconventional cellular regulation may presage the tonotopic organization of the cochlea. In this study, we showed that a secreted factor, Sonic hedgehog (Shh), expressed in the auditory ganglion regulates the timing of terminal mitosis and differentiation of cochlear hair cells. The lack of the auditory ganglion source of Shh causes a premature cell cycle exit of hair cell precursors, followed promptly by hair cell differentiation in an apex to base direction. 2) The role of Sox2 in neurogenesis of the inner ear (manuscript submitted) Sox2 is required for multiple stages of neural development. First, it maintains proliferation of neural progenitors and confers neural competency. Then, Sox2 needs to be downregulated in order for neurons to differentiate. The molecular mechanisms underlying these processes are not well understood. In this study, we show that overexpressing Sox2 in the developing chicken inner ear induces Neurogenin1 (Neurog1), a gene required for neurogenesis. However, these Neurog1-positive cells do not delaminate from the epithelium to form neuroblasts of the cochleo-vestibular ganglion and they fail to express a key downstream neurogenic gene, Neurod1. Nevertheless, overexpressing either Neurog1 or Neurod1 readily induces neuroblast delamination from the otocyst. We postulate that neurogenesis cannot proceed when Sox2 is driven by an exogenous, unregulated promoter. Furthermore, we demonstrated that both Neurog1 and Neurod1 inhibit a phylogenetically conserved enhancer of Sox2 in vivo. In summary, we propose that Sox2 mediates neural competency by promoting Neurog1 expression, and progression of neural precursors to form neurons requires negative feedback inhibition of Sox2 by Neurog1 and Neurod1. 3) Specification of neural and sensory fates are related in the developing inner ear (manuscript in preparation) In the vertebrate inner ear, various sensory organs and their innervating neurons are thought to derive from a common neural-sensory competent domain (NSD) in the developing inner ear at the otic cup and otocyst stages. Several lines of evidence suggest that the vestibular and auditory neuronal fates are already determined at the otic epithelium before neuroblasts exited from the NSD. Based on the locations, these lateral, vestibular and medial, auditory neurogenic regions within the NSD may subsequently develop into the utricular macula and saccular macula, respectively. Additionally, genetic fate mapping studies indicated that the two maculae share a lineage with neurons of the vestibular and auditory ganglia. Together, these results suggest that establishment of the vestibular and auditory neurons as well as the fates of the two maculae may be related during development. We tested this hypothesis by first fate mapping the putative vestibular neurogenic region in the antero-lateral region of the developing otic cup using lipophilic dyes. Then, we followed the fate of these cells after medial-lateral inversion of the otic cup in ovo. If the vestibular neurogenic fate is already specified at the time of transplantation, the fates of vestibular neurons should be fixed but their location within the host may be inverted after transplantation. In addition to analyzing the neuronal fate, we investigated the fate of the otic region that gave rise to the vestibular neurons and determined whether it developed into the utriclar macula in these axial inverted specimens. Our results showed that both the vestibular neurogenic and utricular macula fates appear to be specified early before the otocyst is closed, suggesting that specification of neuronal and sensory organ types are likely to be related events in the developing inner ear. 4) Developmental mechanisms of balance disturbance associated with mutation of Ephrinb2 (manuscript in preparation) Ephrin-B2 (Efnb2) encodes a Type I transmembrane protein and serves as a ligand for various Eph receptor tyrosine kinases. The C-terminal intracellular domain of Efnb2 is itself phosphorylated by auxiliary kinases upon its binding to a receptor, leading to activation of second messenger activity in the ligand-bearing cell. Previous work found strain-specific circling and dysregulation of K+ homeostasis in adult mice hemizygous for a deletion of the Efnb2 C-terminus, but the cellular and molecular mechanisms responsible for this remain unknown. We have investigated the developmental bases for this disturbance by analyzing fetal ears from mice in which the entire Efnb2 gene is removed by Cre-mediated recombination, as well as from circling and non-circling strains of the Efnb2 C-terminal deletion in hemizygous and homozygous states. These studies reveal requirements for Efnb2 in regulating proliferation and apoptosis in the otocyst, as well as the initial outgrowth and patterning of the endolymphatic duct and sac. At later fetal stages, Efnb2 mutations affect the abundance on a cellular level - of endolympatic sac ion transport proteins and their one known transcriptional activator, Foxi1. Strong similarities between null and C-terminal deletion homozygote phenotypes suggest that signaling initiated by activated Efnb2 (rather than by activated Eph receptors) has critical roles in development of an inner ear ion transport epithelium. Current work seeks to determine whether homozygote mutant features exist in attenuated fashion in the C-terminal hemizygote fetuses of a circling strain.

今年的主要成绩有以下几个方面： 1）听觉神经节分泌的Sonic Hedgehog调节哺乳动物耳蜗中感觉毛细胞的细胞周期退出和分化时机（已提交手稿）大多数神经和感觉系统遵循发育程序，其中第一个经历终末有丝分裂的神经前体是第一个分化为神经元的神经前体。相比之下，哺乳动物耳蜗中的感觉毛细胞受到不同的调节。毛细胞前体的细胞周期退出在耳蜗管的顶端开始并向基部进展，而毛细胞分化在细胞周期退出完成后在耳蜗管的基部开始。这种非常规的细胞调节可能预示着耳蜗的音位组织。在这项研究中，我们发现听觉神经节中表达的分泌因子 Sonic humged (Shh) 调节耳蜗毛细胞的终末有丝分裂和分化的时间。缺乏Shh 的听觉神经节源会导致毛细胞前体细胞周期过早退出，随后毛细胞会立即从顶端到基部方向分化。 2）Sox2在内耳神经发生中的作用（已提交手稿） Sox2 是神经发育多个阶段所必需的。首先，它维持神经祖细胞的增殖并赋予神经能力。然后，Sox2 需要下调才能使神经元分化。这些过程背后的分子机制尚不清楚。在这项研究中，我们发现在发育中的鸡内耳中过度表达 Sox2 会诱导神经生成素 1 (Neurog1)，这是神经发生所需的基因。然而，这些 Neurog1 阳性细胞不会从上皮脱落形成耳蜗前庭神经节的神经母细胞，并且它们无法表达关键的下游神经源基因 Neurod1。然而，过度表达 Neurog1 或 Neurod1 很容易诱导神经母细胞从耳囊中剥离。我们假设当 Sox2 由外源的、不受调控的启动子驱动时，神经发生无法进行。此外，我们证明 Neurog1 和 Neurod1 在体内都能抑制 Sox2 的系统发育保守增强子。总之，我们提出Sox2通过促进Neurog1表达来介导神经能力，并且神经前体形成神经元的进展需要Neurog1和Neurod1对Sox2的负反馈抑制。 3）神经和感觉命运的规范与发育中的内耳有关（手稿正在准备中）在脊椎动物内耳中，各种感觉器官及其支配神经元被认为源自发育中的内耳耳杯和耳囊阶段的共同神经感觉主管域（NSD）。多项证据表明，在神经母细胞离开 NSD 之前，前庭和听觉神经元的命运就已经在耳上皮中决定了。根据位置，NSD 内的这些外侧、前庭和内侧听觉神经源性区域随后可能分别发育成椭圆囊黄斑和囊状黄斑。此外，遗传命运图谱研究表明，这两个斑块与前庭神经节和听觉神经节的神经元具有相同的谱系。总之，这些结果表明前庭和听觉神经元的建立以及两个黄斑的命运可能在发育过程中相关。我们通过使用亲脂性染料对发育中的耳杯前外侧区域中假定的前庭神经源区域进行首次命运图谱来测试这一假设。然后，我们追踪了卵内耳杯内外翻转后这些细胞的命运。如果在移植时已经指定了前庭神经元的命运，则前庭神经元的命运应该是固定的，但它们在移植后在宿主内的位置可能会被颠倒。除了分析神经元的命运之外，我们还研究了产生前庭神经元的耳区的命运，并确定它是否在这些轴向倒置标本中发育成椭圆囊黄斑。我们的结果表明，前庭神经源性和椭圆囊黄斑的命运似乎在耳囊闭合之前就已确定，这表明神经元和感觉器官类型的确定可能与发育中的内耳中的相关事件有关。 4）与Ephrinb2突变相关的平衡障碍的发育机制（手稿正在准备中） Ephrin-B2 (Efnb2) 编码 I 型跨膜蛋白，并作为各种 Eph 受体酪氨酸激酶的配体。 Efnb2 的 C 端胞内结构域本身在与受体结合后被辅助激酶磷酸化，导致配体承载细胞中第二信使活性的激活。先前的研究发现，由于 Efnb2 C 末端缺失，成年半合子小鼠中存在品系特异性循环和 K+ 稳态失调，但造成这种情况的细胞和分子机制仍不清楚。我们通过分析小鼠的胎儿耳朵（其中整个 Efnb2 基因通过 Cre 介导的重组被去除）以及半合子和纯合子中 Efnb2 C 端缺失的环状和非环状菌株的胎儿耳朵，研究了这种干扰的发育基础。州。这些研究揭示了 Efnb2 在调节耳囊增殖和凋亡以及内淋巴管和囊的初始生长和模式中的需求。在胎儿后期，Efnb2 突变会影响细胞水平上的丰度 - 内淋巴囊离子转运蛋白及其一种已知的转录激活剂 Foxi1。无效纯合子和 C 端缺失纯合子表型之间的强烈相似性表明，由激活的 Efnb2（而不是由激活的 Eph 受体）引发的信号传导在内耳离子转运上皮细胞的发育中具有关键作用。目前的工作旨在确定纯合子突变体特征是否以减弱的方式存在于循环菌株的 C 端半合子胎儿中。