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：
8745648
负责人：
Doris Wu
金额：
$ 194.7万
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/8745648
关键词：
Adult Affect Apical Area Auditory Behavior Binding Brain C-terminal Cell Cycle Cell Differentiation process Cells Chickens Cochlea Cochlear duct Complex Crista ampullaris Defect Development Ear Electroporation Embryo Embryonic Development Eph Family Receptors Ephrin-B2 Epithelial Epithelium Equilibrium Erinaceidae Fetus Fibroblast Growth Factor Ganglia Gene Expression Genes Genetic Genetic Recombination Genetic Transcription Goals Growth Hair Cells Head Movements Heterozygote Homeostasis Integral Membrane Protein Ion Transport Knock-out Labyrinth Ligands Link Manuscripts Maps Mediating Mitochondria Mitosis Mitotic Molecular Morphogenesis Mus Mutant Strains Mice Mutation Neuronal Differentiation Neurons Organ Pathway interactions Pattern Phosphotransferases Plant Roots Preparation Proteins Publishing Reporting Retina Role Second Messenger Systems Semicircular canal structure Sensory Signal Transduction Signaling Molecule Source Specific qualifier value Staging Structure Surveys Techniques Testing Time Tissues Up-Regulation Vestibular ganglion Vestibule Work base bone morphogenetic protein 4 deafness design endolymphatic sac fetal hindbrain malformation mutant neuroblast neurogenesis otoconia overexpression premature receptor relating to nervous system second messenger spiral ganglion

项目摘要

This years major accomplishments are in the following areas: 1)Role of Bmp2 in mediating semicircular canal formation (manuscript in preparation) The vestibular apparatus that is responsible for detecting angular head movements consists of three semicircular canals and their associated sensory organs, cristae ampullaris. Results from our previous studies suggest that signals such as Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factors (Fgfs) secreted from the sensory tissue, crista, are important for canal formation by inducing Bmp2 in the canal pouch, which gives rise to the canals. To test this hypothesis further, we investigated the role of Bmp2 in canal formation by generating conditional knockout of Bmp2 in the developing mouse inner ear. Our results show that Bmp2 conditional knockout inner ears are devoid of canals but the cristae are intact. Furthermore, our results show that Bmp2 mediates canal formation by maintaining expression of genes such as Dlx5 and Lmo4 at the rim and negatively regulating genes such as Netrin1 in the resoprtion domain of the canal pouch. 2)Role of Lmx1a in the developing inner ear (manuscript in preparation) Previously, we have shown that Lmx1a is important for inner ear development based on inner ear analyses of the spontaneous mouse mutant, dreher, in which mutations in Lmx1a generated a functional null protein. However, in addition to the otic epithelium, Lmx1a is also expressed in the hindbrain adjacent to the developing inner ear, which has been shown to be important in providing other signaling molecules for inner ear development. To distinguish which source of Lmx1a, expressed in the developing inner ear or hindbrain, is more important for inner ear development, we generated conditional knockout of Lmx1a in the inner ear and our results suggest that inner ear defects reported in dreher mutants are largely due to the loss of Lmx1a functions within the otic epithelium. 3)Developmental mechanisms of balance disturbance associated with deletion of Ephrin-B2 (manuscript in review) Ephrin-B2 (Efnb2) encodes a Type I transmembrane protein and serves as a ligand for multiple 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 with heterozygous deletion of the Efnb2 C-terminus, but whether these functional deficits are rooted in a disruption of embryonic development has remained unknown. We investigated the potential 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 the heterozygous and homozygous states. Major progress for the prior twelve months includes the following advances. We found a mis-localization of endolymphatic sac mitochondrial rich cells (ion transport cells) in fetuses of the circling Efnb2 C-terminal deletion heterozygote strain but not in non-circling heterozygote strains, thus correlating this feature of the Efnb2 conditional knock-out with circling behavior. Second, we mapped region-specific patterns of reduced proliferative activity at the otocyst epithelium prior to formation of the endolymphatic epithelium, thus linking mis-localization of inner ear ion transport cells to early-stage defects in epithelial growth. Finally, we conducted an expression survey of all relevant Eph receptor tyrosine kinases, and found evidence for the potential involvement of at least four cognate receptors. 4)Auditory ganglion source of Sonic hedgehog regulates timing of cell cycle exit and differentiation of mammalian cochlear hair cells (manuscript published) An unusual feature of the mammalian cochlear development is that hair cell precursors initiate terminal mitosis from an apical to basal direction along the cochlear duct, whereas the wave of hair cell differentiation is in a reverse direction of base to apex. Thus, post-mitotic hair cell precursors at the apex delay differentiation for several days than their counterparts at the base. This dis-synchrony in timing between terminal mitosis and differentiation is in contrast to neurons in the retina and brain, in which neuronal differentiation follows promptly after terminal mitosis. Using conditional knockout approach in mice, we show that Sonic hedgehog (Shh) emanating from the spiral ganglion regulates the timing of cell cycle exit of hair cell precursors and their subsequent differentiation. We show that a subpopulation of Shh expressing spiral ganglion neurons restricts over time towards the apical region of the cochlear duct during cochlear development, which we postulated to delay hair cell differentiation in the apical cochlea. To test this hypothesis, we generated mouse embryos with conditional knockout of Shh in the spiral ganglion. In these mutants, cochlear hair cell precursors undergo premature but similar apical-to-basal wave of terminal mitosis as the wildtype. However, in the absence of spiral ganglion source of Shh, the wave of hair cell differentiation proceeds in the reverse apical-to-basal direction as predicted by our hypothesis. Whether this change in the timing of hair cell differentiation affects the tonotopic organization of the cochlea remains to be determined. 5)Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by Neurogenin1 and Neurod1 (manuscript published) During inner ear development, a poorly-defined neural-sensory competent region is thought to give rise to neuroblasts, which delaminate from the otic epithelium to form neurons of the cochleo-vestibular ganglion. Cells remaining in the neural-sensory competent region are thought to give rise to various sensory patches of the inner ear over time. The molecular mechanisms that specify the neural versus sensory fate are largely not known. Using an electroporation technique to over-express genes that are associated with the neural fate such as Sox2, Neurogenin1 (Neurog1) and Neurod1 in the developing chicken inner ear in ovo, we discovered a general molecular pathway of neurogenesis. We show that Sox2 is likely to promote neurogenesis by upregulating a neurogenic gene, Neurog1. In turn, Neurog1 inhibits Sox2 transcription, which results in the upregulation of another neurogenic gene, Neurod1, and neurogenesis progresses. When Sox2 is overexpressed and its level cannot be regulated, despite the upregulation of Neurog1, NeuroD is not upregulated and neurogenesis fails to proceed.

今年的主要成就在以下领域： 1）BMP2在介导半圆形管形成中的作用（制备中的手稿）负责检测角头运动的前庭设备由三个半圆形管及其相关的感觉器官Cristae Ampullaris组成。我们先前研究的结果表明，诸如骨形态发生蛋白4（BMP4）和从感觉组织Crista分泌的骨形态发生蛋白4（BMP4）和成纤维细胞生长因子（FGFS）对于通过在运河袋中诱导BMP2诱导BMP2而产生的信号很重要。为了进一步检验这一假设，我们通过在发育中的小鼠内耳中产生BMP2的条件敲除，研究了BMP2在运河形成中的作用。我们的结果表明，BMP2有条件的敲除内耳没有运河，但cristae完好无损。此外，我们的结果表明，BMP2通过在边缘处保持基因（例如DLX5和LMO4）的表达来介导运河的形成，并在运河袋的重置域中对基因（例如Netrin1）进行负调控基因。 2）LMX1A在发育中的内耳中的作用（手稿准备）以前，我们已经表明，基于自发小鼠突变体Dreher的内耳分析，LMX1A对于内耳发育很重要，其中LMX1A中的突变产生了功能性无效蛋白。但是，除了耳朵上皮外，LMX1A还在与发展内耳发展的后脑相邻的后脑中表达，这在为提供内耳发育的其他信号分子中很重要。为了区分在发育中的内耳或后脑中表达的LMX1A的来源对内耳的发育更为重要，我们在内耳中产生了有条件的LMX1A敲除，我们的结果表明，Dreher突变体中报道的内耳缺陷很大程度上是由于Otic上pic峰的LMX1A功能的丧失。 3）与删除以弗林-B2相关的平衡障碍的发展机制（审查中的手稿） Ephrin-B2（EFNB2）编码I型跨膜蛋白，并用作多种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，EFNB2 C末端的杂合缺失的成年小鼠中K+稳态的菌株特异性圆圈和失调，但是这些功能缺陷是否植根于胚胎发育的破坏源于胚胎发育的破坏。我们通过分析来自CRE介导的重组以及杂质和纯净状态的EFNB2 C-末端缺失的圆圈和非循环菌株来消除整个EFNB2基因的胎儿耳朵的潜在发育碱。过去十二个月的重大进展包括以下进展。我们发现，在圆圈EFNB2 C末端缺失杂合子菌株的胎儿中，内淋巴囊线粒体富细胞（离子转运细胞）的错误定位，但在非循环杂合子菌株中，因此与EFNB2条件敲除具有圆圈行为的这种特征相关。其次，我们绘制了在形成内淋巴上皮细胞之前耳尾上皮的增殖活性降低的区域特异性模式，从而将内耳离子离子传输细胞的错误定位与上皮生长的早期缺陷联系起来。最后，我们对所有相关的EPH受体酪氨酸激酶进行了表达调查，并找到了至少四个同源受体的潜在参与的证据。 4）声音刺猬的听觉神经节来源调节细胞周期出口的时间和哺乳动物耳蜗细胞的分化（手稿已发布）哺乳动物人工耳蜗发育的一个异常特征是，毛细胞前体沿着人工耳蜗启动末端有丝分裂，从顶端到基础方向，而毛细胞分化的波在碱基的反向方向向顶点到顶点。因此，与基部相比，在顶点延迟分化几天的有丝分裂后毛细胞前体。在终末有丝分裂和分化之间定时的这种异步与视网膜和大脑中的神经元相反，视网膜和大脑中的神经元在终末有丝分裂后迅速遵循神经元分化。使用小鼠中的条件敲除方法，我们表明从螺旋神经节发出的声音刺猬（SHH）调节毛细胞前体的细胞周期出口及其随后的分化。我们表明，表达螺旋神经神经元的SHH的亚群会随着时间的推移限制在人工耳蜗发育过程中，朝着耳蜗的顶端区域限制，我们假设这是为了延迟顶端耳蜗的毛细胞分化。为了检验这一假设，我们在螺旋神经节中使用SHH的条件敲除产生了小鼠胚胎。在这些突变体中，耳蜗前体经历了末期有丝分裂的过早但类似的根尖到基质波波与野生型。但是，在没有SHH的螺旋神经节来源的情况下，毛细胞分化的波沿我们假设所预测的反向的根尖到基础方向进行。毛细胞分化时间的这种变化是否会影响耳蜗的调动组织，这尚待确定。 5）内耳神经发生的进展需要抑制神经素1和NeuroD1的SOX2转录（手稿）在内耳发育过程中，人们认为一个定义不明确的神经感官胜任区域会引起神经细胞，该神经细胞从类似的上皮分层以形成Cochleo-vestibular神经节的神经元。随着时间的流逝，仍认为仍留在神经感官胜任区域中的细胞会引起内耳的各种感觉斑。指定神经与感觉命运的分子机制在很大程度上尚不清楚。在OVO中发育中的鸡内耳中，使用电穿孔技术与神经命运相关的过表达基因，例如Sox2，Neurogenin1（Neurog1）和Neurod1，我们发现了神经发生的一般分子途径。我们表明，Sox2可能通过上调神经源基因Neurog1来促进神经发生。反过来，Neurog1抑制SOX2转录，这导致了另一个神经源基因，NeuroD1和神经发生的上调。当Sox2过表达并且无法调节其水平时，尽管神经1的上调，神经轨道并未上调，并且神经发生未能进行。