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：
9147423
负责人：
Doris Wu
金额：
$ 154.25万
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/9147423
关键词：
Abdominal colic Affect Apical Area Auditory Basilar Papilla Birds Brain Cell physiology Cells Chickens Clinical Cochlea Cochlear duct Complex Coupled Crista ampullaris Development Disease Ear Embryonic Development Epithelium Equilibrium Erinaceidae Etiology Floor Frequencies Gait Ganglia Gene Expression Generations Genes Genetic Goals Hair Cells Head Movements Height Holoprosencephaly Human Kinocilium Knockout Mice Labyrinth Lateral Lead Length Maps Medial Mediating Modeling Molecular Morphogenesis Mus Mutant Strains Mice Mutation Neurons Operative Surgical Procedures Organ Pathway interactions Patients Pattern Phenotype Physiological Play Positioning Attribute Process Property Publishing Reflex action Relative (related person)Replacement Therapy Reporting Role Saccule and Utricle Sensory Sensory Hair Source Specific qualifier value Staging Stereocilium Structure Surface Techniques Testing Time Transplantation Type I Hair Cell Utricular macula Vestibular ganglion Vestibule base cellular microvillus deafness design differential expression ear development emx2 protein gain of function gaze hearing impairment homeodomain insight macula malformation millisecond mouse model mutant neuroblast neuronal circuitry notochord relating to nervous system response smoothened signaling pathway sound sound frequency transcription factor vestibular reflex

项目摘要

This years major accomplishments are in the following areas: 1) Conserved role of Sonic Hedgehog in tonotopic organization of the avian basilar papilla and mammalian cochlea Mutations in Sonic Hedgehog (Shh) causes holoprosencephaly and cyclopia in humans. The cyclopia phenotype is attributed to the loss of Shh secreted by the ventral midline structures - floor plate and notochord - during embryogenesis. This same source of Shh has also been shown to be important for the ventral patterning of the inner ear. The cochlea is tonotopically organized such that the base of the cochlear duct is most sensitive to high frequency sounds and the apex to low frequencies. Many cellular and physiological features facilitated this property such as changes in the length of hair cells as well as the height and number of stereocilia of hair cells along the cochlear duct. The molecular mechanisms that give rise to these important cellular features are not clear. Here, we show that Shh signaling from the ventral midline induces differential expression of various genes along the apical-basal axis of the cochlear anlage in both chicken and mice. The expression domain of genes associated with the cochlear apex are expanded in the presence of ectopic Shh signaling. More importantly, these gene expression changes resulted in hair cells in the basal region of the chicken basilar papilla acquiring cellular features that are typical of apical hair cells. Hence, these results suggest that the early regional patterning of the cochlear anlage by Shh pre-stages the tontopic organization of the cochlea. 2) The specification of neural and macular fates within the chicken inner ear are temporally coupled The cochleo-vestibular ganglion (CVG), which relays sensory information from the inner ear to the brain, is derived from the same placodal epithelium that gives rise to the inner ear. During inner ear development, the neural-fated cells exit from the otic epithelium and coalesce to form the CVG. These neural-fated cells originate from the neural-sensory competent region of the otic cup, which also gives rise to some of the sensory organs of the inner ear in later stages. Deciphering the molecular pathways that mediate the neural versus sensory fate during normal development may provide insights into alternatives for hair cell replacement and therapy in the long run. Previously published results using genetic fate mapping techniques in mice suggest that the neuronal fates and the two vestibular sensory organs, the maculae, share a common lineage. Based on the physical locality of these sensory organs within the mature inner ear and the positions where neuroblasts initially delaminate from the otic cup, we hypothesize that specification of maculae of the utricle and saccule occur early during development coupled in timing to the specification of the vestibular and auditory neurogenic fates, respectively. We tested this hypothesis by inverting the position of the vestibular and auditory neurogenic regions in ovo. This was accomplished by surgically inverting the medial-lateral axis of the otic cup relative to the body axis at the time when neuroblasts started to delaminate from the epithelium. We then asked whether formation of these various neuronal and sensory fates were affected as a result of this surgical manipulation. We reasoned that cell fates that were already specified at the time of transplantation should maintain their identities though their positions might be displaced within the body. Our results showed that the formation of the vestibular ganglion was not affected by the surgery and appeared specified at the time of transplantation. Coincidently, the identity of the utricular macula, the sensory organ postulated to be related to the vestibular ganglion was also maintained in the transplanted ears. In contrast, both the auditory ganglion and its presumed-associated sensory organ, the saccular macula, only maintained their identities partially in the transplants. The correlation in the timing of specification among the two postulated ganglion - macula pairs supported our hypothesis that specifications of the neuronal and macular fates are temporally coupled during inner ear development. 3) Stereocilia polarity of sensory hair cells The stereociliary bundle on the apical surface of a sensory hair cell is comprised of a kinocilium and a number of specialized microvilli arranged in a staircase pattern. The asymmetric positioning of the staircase dictates the directional sensitivity of its hair cell; only deflections of the stereociliary bundle towards the kinocilium open the mechanotransduction channels located on the tip of the stereocilia and lead to activation of the hair cell. The proper positioning and formation of the kinocilium are essential for normal hair cell functions and these failings are associated with deafness in humans. As predicted by the importance of stereocilia orientation in hair cell function, each sensory organ of the inner ear displays a unique and defined pattern of stereocilia orientation. Thus, understanding the molecular mechanisms that establish stereocilia pattern in each sensory organ is important from both a functional and clinical perspective. A knockout mouse mutant of Emx2, which encodes a homeodomain transcription factor, was reported to show disrupted stereocilia pattern in the maculae of the inner ear. Both of these vestibular sensory organs lack the normal line of polarity reversal (LPR) and all the stereocilia are pointing unidirectionally in the mutants, in contrast to the wildtype maculae, each consists of two regions of hair cells with stereocilia that are arranged in opposite orientation across the two regions. We investigated these mutant phenotypes in more detail and we showed that the unidirectional orientation of stereocilia in the mutant maculae is caused by 180 degrees inversion of stereocilia in the region that normally expresses Emx2. To test the role of Emx2 in mediating stereocilia polarity, we generated several gain-of-function mouse models, in which Emx2 can be activated in nascent hair cells. Our preliminary results indicate that hair cells that do not normally express Emx2 show a 180 degrees reversal of their stereocilia orientation in the presence of ectopic Emx2. Taken together, these results suggest that Emx2 is necessary and sufficient to invert stereocilia orientation in sensory hair cells. 4) Functions of striola of the maculae and central zone of the cristae within the inner ear Reflexes initiated from the vestibular organs of the inner ear such as vestibule-ocular reflex (VOR) and vestibular-colic reflex (VCR) are extremely quick responses operating in millisecond range to help us maintain gaze and gait during head movements, respectively. While the neuronal circuits for these reflexes are well defined, the type of sensory hair cells that are responsible for initiating these reflexes in the vestibular organs are not clear. Presumably, type I hair cells residing in the central zone of the cristae and the striola of the maculae are most likely to play a direct role in mediating these reflexes since they are more sensitive to frequency changes. To test this hypothesis, we are in the process of generating mouse mutants that are devoid of the central zones and striola. Generation of these mouse mutants will serve as good models to study vestibular disorders in humans.

今年的主要成就在以下领域： 1）Sonic Hedgehog在禽基基底乳头和哺乳动物耳蜗中的保守作用声音刺猬（SHH）中的突变会导致人类的全脑脑和环形。环境表型归因于胚胎发生过程中腹部中线结构（地板板和脊索）分泌的SHH的丧失。同样的SHH来源也已被证明对于内耳的腹侧图案很重要。耳蜗的组织组织组织，使得耳蜗的底座对高频声音最敏感，而对低频的顶点则是最敏感的。许多细胞和生理特征促进了这种特性，例如毛细胞长度的变化以及沿着人耳蜗的毛细胞的高度和天核数量。产生这些重要细胞特征的分子机制尚不清楚。在这里，我们表明，来自腹部中线的SHH信号传导在鸡肉和小鼠中沿耳蜗的顶端轴沿顶部基因轴诱导各种基因的差异表达。在异位SHH信号的存在下，与人工耳蜗顶点相关的基因的表达结构域扩展。更重要的是，这些基因表达变化导致鸡基底乳头基底区域的毛细胞获得典型的顶毛细胞的细胞特征。因此，这些结果表明，SHH预先进行耳蜗的倾斜组织，对人工耳蜗的早期区域模式。 2）鸡内耳内的神经和黄斑命运的规格是暂时耦合的 Cochleo-vestibular神经节（CVG）将感官信息从内耳转到大脑，源自相同的位置上皮，从而引起内耳。在内耳发育期间，神经果的细胞从耳朵上皮和聚结中退出以形成CVG。这些神经果的细胞源自耳杯的神经感官胜任区，这也会在后期阶段引起内耳的某些感觉器官。从长远来看，在正常发育过程中介导神经与感觉命运的分子途径可以提供替代方案。先前使用小鼠遗传命运映射技术发表的结果表明，神经元的命运和两个前庭感觉器官，黄斑共享一个共同的谱系。基于这些感觉器官在成熟的内耳内的物理位置以及最初与耳杯分层的神经细胞分层的位置，我们假设在发育期间早期出现了在发育期间的早期，在时间上均出现了与前庭和审计神经源性命运的规范。我们通过颠覆OVO中前庭和听觉神经源区域的位置来检验这一假设。这是通过在神经细胞开始从上皮分划分时相对于体轴的手术颠倒耳杯的内侧轴的。然后，我们询问这些各种神经元和感觉命运的形成是否由于这种手术操作而受到影响。我们认为，在移植时已经指定的细胞命运应保持其身份，尽管其位置可能会在体内移位。我们的结果表明，前庭神经节的形成不受手术的影响，并在移植时指定。巧合的是，在尿布状黄斑的身份中，假定与前庭神经节有关的感觉器官也保持在移植的耳朵中。相比之下，听觉神经节及其假定相关的感觉器官糖果大黄斑仅在移植中部分地保持了它们的身份。两个假定的神经节 - 黄斑对之间规范时间的相关性支持了我们的假设，即神经元和黄斑命运的规范在内耳发育过程中暂时耦合。 3）感觉毛细胞的立体胶质极性感觉毛细胞的顶端表面上的立体束由动力学和许多以楼梯模式排列的专业微壁画组成。楼梯的不对称定位决定其毛细胞的方向灵敏度。只有立体扣束向动力学的偏转打开位于立体胶质尖端的机械传输通道，并导致毛细胞的激活。运动核的适当定位和形成对于正常的毛细胞功能至关重要，这些失败与人类耳聋有关。正如立体膜方向在毛细胞功能中的重要性所预测的那样，内耳的每个感觉器官都会显示出独特而定义的立体膜方向模式。因此，从功能和临床角度来看，了解在每个感觉器官中建立立体膜模式的分子机制很重要。据报道，EMX2的敲除小鼠突变体，该突变体编码了同源域转录因子，该突变体在内耳的黄斑中表现出破坏的立体胶质模式。这两种前庭感觉器官都缺乏正常的极性逆转（LPR）线，与野生型斑节相比，所有立体核心都在突变体上单向指向突变体，每个型毛细单元组成的两个区域都与位于两个区域相反方向相反的立体方向组成。我们对这些突变表型进行了更详细的研究，并表明突变珠量中立体尾核的单向取向是由通常表达EMX2的区域的180度反转引起的。为了测试EMX2在介导立体胶质极性中的作用，我们产生了几种功能性的小鼠模型，其中可以在新生的毛细胞中激活EMX2。我们的初步结果表明，通常不表达EMX2的毛细胞在存在异位EMX2的情况下表现出180度的立体胶体取向。综上所述，这些结果表明，EMX2在感觉毛细胞中的立体胶体方向是必要的和足够的。 4）内耳内cristae的马ula和中央区域的Striola功能从内耳的前庭器官（例如前庭 - 眼反射（VOR）和前庭环反射（VCR））发起的反射是在毫秒范围内运行的非常快的响应，以帮助我们在头部运动过程中分别保持凝视和步态。尽管这些反射的神经元电路已经很好地定义了，但导致在前庭器官中启动这些反射的感觉毛细胞的类型尚不清楚。据推测，位于Cristae中心区域和黄斑的striola中的I型毛细胞最有可能在介导这些反射方面发挥直接作用，因为它们对频率变化更敏感。为了检验这一假设，我们正在生成缺乏中央区域和Striola的小鼠突变体。这些小鼠突变体的产生将是研究人类前庭疾病的好模型。