DEVELOPMENTAL ORIGINS OF THE INNER EAR SENSORY ORGANS

内耳感觉器官的发育起源

基本信息

批准号：
6523471
负责人：
ANDRES COLLAZO
金额：
$ 25.54万
依托单位：
HOUSE RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-29 至 2004-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6523471
关键词：
Xenopus cell differentiation cell migration developmental genetics developmental neurobiology dyes ear hair cell early embryonic stage gene expression labyrinth single cell analysis video microscopy

项目摘要

Problems during inner ear development can result in hearing and balance disorders. Almost one in 1,000 infants is born deaf while six in 1,000 have some form of hearing impairment. This makes hearing impairment one of the more common birth defects. Balance disorders can be more difficult to diagnose but are no less debilitating. Considering the interest in understanding the causes of newborn deafness and balance disorders, relatively little is known about ear development, especially at the cellular and molecular levels. The long-term goal of this study is to understand the cellular and molecular processes involved in the early patterning of the developing inner ear, in particular the origin and differentiation of the sensory organs. The experiments proposed will determine from where in the placode sensory organs arise, study a single cell s lineage to determine the timing and location of hair cell differentiation (specialized mechanosensory cells of the sensory organs), and test the role of the genes Notch and Delta in this differentiation. Advances in vital dyes, intravital imaging and gene function assays in the African clawed frog, Xenopus laevis, offer the unique opportunity to address the proposed hypothesis in living embryos and tadpoles. SPECIFIC AIM NUMBER 1. Generated fate maps of inner ear structures from different stages during otic placode development and correlate these maps with gene expression patterns. Labeling small groups of cells within the placode with a fluorescent vital dye and following these cells until most of the inner ear structures have differentiated, will allow us to address our hypothesis directly. SPECIFIC AIM NUMBER 2. Use single cell labeling to assess the lineage relationship between hair and supporting cells. By labeling a single cell with a fluorescent lineage tracer at different and progressively later developmental stages, the time that the resulting progency become restricted to one cell type, such as a hair cell, can be determined. SPECIFIC AIM NUMBER 3. Use gain and loss of function genetic constructs to perturb Notch/Delta signaling and determine the effects on inner ear development. Three transmembrane proteins, the receptor Notch and its ligands Delta and Serrate, are known to be involved in cell-to-cell signaling and cell fate determination in other sensory tissues, but their roles in ear development are still unknown. By injecting either mRNA or plasmid DNA containing one of these three genes into one cell of a 4-cell embryo, expression can be targeted to one inner ear, leaving the other side as a control. Two of the constructs will provide gain-of-function effects by increasing Notch/Delta signaling while the third will provide a loss-of-function effect by decreasing Notch/Delta signaling.

内耳发育过程中的问题可能会导致听力和平衡障碍。几乎千分之一的婴儿出生时耳聋，而千分之六的婴儿患有某种形式的听力障碍。这使得听力障碍成为更常见的出生缺陷之一。平衡障碍可能更难以诊断，但同样会导致身体衰弱。考虑到了解新生儿耳聋和平衡障碍的原因的兴趣，人们对耳朵发育的了解相对较少，特别是在细胞和分子水平上。这项研究的长期目标是了解内耳发育早期模式所涉及的细胞和分子过程，特别是感觉器官的起源和分化。提出的实验将确定基板感觉器官从何处产生，研究单个细胞的谱系以确定毛细胞分化的时间和位置（感觉器官的专门机械感觉细胞），并测试基因 Notch 和 Delta 的作用在这种分化中。非洲爪蛙（Xenopus laevis）活体染料、活体成像和基因功能测定的进展，为在活体胚胎和蝌蚪中解决所提出的假设提供了独特的机会。具体目标号 1. 生成耳基板发育过程中不同阶段的内耳结构的命运图，并将这些图与基因表达模式相关联。用荧光活体染料标记基板内的一小群细胞，并跟踪这些细胞，直到大部分内耳结构分化，这将使我们能够直接解决我们的假设。具体目标号 2. 使用单细胞标记来评估毛发和支持细胞之间的谱系关系。通过在不同且逐渐较晚的发育阶段用荧光谱系示踪剂标记单个细胞，可以确定所产生的后代仅限于一种细胞类型（例如毛细胞）的时间。具体目标号 3. 使用功能遗传构建体的获得和丧失来干扰 Notch/Delta 信号传导并确定对内耳发育的影响。三种跨膜蛋白，受体Notch及其配体Delta和Serrate，已知参与其他感觉组织中的细胞间信号传导和细胞命运决定，但它们在耳朵发育中的作用仍然未知。通过将含有这三个基因之一的 mRNA 或质粒 DNA 注射到 4 细胞胚胎的一个细胞中，可以将表达靶向一个内耳，而将另一侧作为对照。其中两种构建体将通过增加Notch/Delta信号传导来提供功能获得效应，而第三种构建体将通过减少Notch/Delta信号传导来提供功能丧失效应。