DEVELOPMENTAL ORIGINS OF THE INNER EAR SENSORY ORGANS

内耳发育过程中的问题可能导致听力和平衡障碍。 几乎每1,000名婴儿中就有一名天生失聪，而每1,000名婴儿中就有6名患有某种形式的听力障碍。 这使得听力障碍成为最常见的出生缺陷之一。平衡障碍可能更难诊断，但同样会使人衰弱。 考虑到对了解新生儿耳聋和平衡障碍的原因的兴趣，对耳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发育中内耳早期模式的细胞和分子过程，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的起源，研究单细胞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Notch和Delta基因在这种分化中的作用。 在非洲爪蛙，非洲爪蟾，活体染料，活体成像和基因功能测定的进展，提供了独特的机会，以解决所提出的假设在活胚胎和蝌蚪。 具体目标1.在耳基板发育的不同阶段，从内耳结构的产生的命运地图，并将这些地图与基因表达模式。 用荧光活体染料标记基板内的小细胞群，并跟踪这些细胞，直到大多数内耳结构分化，将使我们能够直接解决我们的假设。 具体目标编号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。 通过用荧光谱系示踪剂在不同的和逐渐后期的发育阶段标记单个细胞，可以确定所产生的子代变得限于一种细胞类型（例如毛细胞）的时间。 具体目标编号3.使用功能基因结构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。 已知三种跨膜蛋白，受体Notch及其配体Delta和Serrate，参与其他感觉组织中的细胞间信号传导和细胞命运决定，但它们在耳发育中的作用仍然未知。 通过将含有这三种基因之一的mRNA或质粒DNA注射到4细胞胚胎的一个细胞中，表达可以靶向一个内耳，留下另一侧作为对照。 两种构建体将通过增加Notch/Delta信号传导提供功能获得效应，而第三种构建体将通过减少Notch/Delta信号传导提供功能丧失效应。

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.