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.

今年的主要成就在以下领域： 1）听觉神经节分泌的声音刺猬调节哺乳动物耳蜗中细胞周期出口的时间和感觉毛细胞的分化（手稿提交）大多数神经和感觉系统都遵循发展程序，其中首个经历末端有丝分裂的神经前体是第一个分化为神经元的神经前体。相反，哺乳动物耳蜗中的感觉毛细胞的调节不同。毛细胞前体的细胞周期出口是在人工耳蜗的顶端启动的，并朝向底部，而毛细胞分化开始在细胞周期出口后的导管底部开始。这种非常规的细胞调节可以预示耳蜗的调子组织。在这项研究中，我们表明在听觉神经节中表达的一个分泌因子Sonic Hedgehog（SHH）调节了终末有丝分裂的时间和耳蜗毛细胞的分化。 SHH的听觉神经节源的缺乏会导致毛细胞前体的过早细胞周期出口，然后迅速在顶点伸向基础方向的毛细胞分化。 2）Sox2在内耳神经发生中的作用（手稿提交） SOX2是神经发育多个阶段所必需的。首先，它保持神经祖细胞的扩散并赋予神经能力。然后，为了使神经元分化，SOX2需要下调。这些过程的分子机制尚不清楚。在这项研究中，我们表明在发育中的鸡内耳中过表达SOX2会诱导神经蛋白素1（Neurog1），这是神经发生所需的基因。然而，这些神经1阳性细胞不会从上皮分层以形成耳蜗 - vestibular神经节的神经细胞，并且无法表达下游神经源基因神经od1的键。然而，过表达Neurog1或NeuroD1很容易诱导耳囊中的神经细胞分层。我们假设，当Sox2是由外源，不受监管的启动子驱动时，神经发生无法进行。此外，我们证明了Neurog1和NeuroD1都抑制了体内Sox2的系统发育保守的增强子。总而言之，我们建议SOX2通过促进Neurog1表达来介导神经能力，而神经前体的进展形成神经元需要负面反馈抑制SOX2通过Neurog1和Neurod1。 3）神经和感觉命运的规范在发育中的内耳中有关（手稿准备）在脊椎动物的内耳中，人们认为各种感觉器官及其支配神经元源自在耳杯和耳状阶段发育中的内耳中常见的神经感知统一域（NSD）。几条证据表明，在神经细胞从NSD中退出之前，前庭和听觉神经元的命运已经在眼膜上皮上确定。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出现到乌尔特克里亚的黄斑和糖果大黄斑中。此外，遗传命运图研究表明，这两个黄斑与前庭和听觉神经节神经元共享谱系。总之，这些结果表明，在发育过程中，建立前庭和听觉神经元以及两个黄斑的命运可能是相关的。我们通过首先使用亲脂染料来绘制发育中的耳杯前侧区域的推定前庭神经源区域的首次命运来检验了这一假设。然后，我们跟随OVO中耳杯的内侧反转后这些细胞的命运。如果在移植时已经指定了前庭神经源性命运，则应固定前庭神经元的命运，但在移植后可以将其位置倒置。除了分析神经元的命运外，我们还研究了产生前庭神经元的耳射区域的命运，并确定它是否在这些轴向倒置的标本中发展为乌里克拉尔黄斑。我们的结果表明，前庭神经源性和肾小黄斑黄斑命运似乎在关闭耳环囊肿之前早期是指定的，这表明神经元和感觉器官类型的规范可能是发展内耳中的相关事件。 4）与Ephrinb2突变相关的平衡干扰的发展机制（手稿中的手稿） Ephrin-B2（EFNB2）编码I型跨膜蛋白，并用作各种EPH受体酪氨酸激酶的配体。 EFNB2的C末端细胞内结构域本身被辅助激酶与受体结合时磷酸化，从而导致含有配体细胞中的第二信使活性激活。先前的工作发现成年小鼠中K+稳态的菌株特异性圆圈和失调，以删除EFNB2 C末端，但导致该的细胞和分子机制尚不清楚。我们已经通过分析来自Cre介导的重组去除整个EFNB2基因的胎儿耳朵，以及来自efnb2 c-C-t末端的圆圈和非循环菌株的胎儿耳朵，研究了这种干扰的发育基础。这些研究揭示了EFNB2对耳囊中的增殖和细胞凋亡以及内淋巴管和囊的初始生长和模式的要求。在后来的胎儿阶段，EFNB2突变会影响内聚囊离子转运蛋白的细胞水平上的丰度及其已知的转录激活剂FOXI1。零和C末端缺失纯合表型之间的强烈相似性表明，活化的EFNB2（而不是通过激活的EPH受体）引发的信号传导在内耳离子转运上皮的发展中具有至关重要的作用。当前的工作旨在确定在圆圈菌株的C末端半合子胎儿中是否以衰减的方式存在纯合突变体特征。