DEVELOPMENTAL ORIGINS OF THE INNER EAR SENSORY ORGANS

内耳发育过程中的问题可能会导致听力和平衡障碍。几乎每1000名婴儿中就有一名出生时耳聋，而每1000名婴儿中有六名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。平衡障碍可能更难诊断，但同样会使人虚弱。考虑到人们对了解新生儿耳聋和平衡障碍的原因的兴趣，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发育中的内耳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。拟议的实验将确定胎盘感觉器官的起源，研究单个细胞S谱系以确定毛细胞分化的时间和位置(感官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。非洲爪蛙的生命染料、活体成像和基因功能分析的进展，为在活的胚胎和蝌蚪中解决这一假设提供了独特的机会。特异目标号1。在耳道胎盘发育的不同阶段生成内耳结构的命运图谱，并将这些图谱与基因表达模式相关联。用一种荧光活性染料标记胎盘内的小群细胞，并跟随这些细胞，直到大多数内耳结构分化，这将使我们能够直接解决我们的假设。特定目的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。通过在不同和逐渐较晚的发育阶段用荧光谱系示踪剂标记单个细胞，可以确定所产生的生殖力限于一种细胞类型的时间，例如毛细胞。具体目的3：利用功能获得和功能丧失的基因结构来干扰Notch/Delta信号，并确定其对内耳发育的影响。已知有三种跨膜蛋白，即受体Notch及其配体Delta和Serrate，在其他感觉组织中参与细胞间信号传递和细胞命运决定，但它们在耳朵发育中的作用尚不清楚。通过向4-细胞胚胎的一个细胞中注射含有这三个基因之一的mRNA或质粒DNA，表达可以针对一个内耳，而留下另一侧作为对照。其中两个结构将通过增加Notch/Delta信令来提供功能增益效应，而第三个结构将通过减少Notch/Delta信令来提供功能损失效应。

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.