Molecular embryology of the mammalian inner ear

描述（由申请人提供）：我们实验室的长期目标是确定基于基因和细胞的策略，以恢复功能失调的内耳的听力和平衡。短期目标是促进我们对小鼠内耳的形态发生、模式形成和细胞命运规范的理解。我们使用小鼠作为模型系统，因为存在不断扩大的自然和诱导基因突变阵列，这些突变可作为人类内耳功能障碍的准确范例。我们的主要技术方法依赖于实验胚胎学、外科手术、成像、显微注射和分子技术，这些技术允许在子宫内进入发育中的小鼠内耳，并对产生听觉和前庭感觉结构的前体细胞进行遗传操作。在本研究中，我们的目标是：1)在体内绘制小鼠耳囊的命运图谱；2)确定耳前体细胞命运选择的类型和选择的时间；3)确定神经上皮祖细胞对分化的内耳的克隆贡献。一张命运图描述了随着内耳的成熟，不同的细胞群会变成什么样子。发育生物学家使用命运图来了解发育中的组织或器官中的祖基因表达如何驱动成熟并导致形态和功能的获得。不可磨灭标记的前体与已知的基因表达域的交集可以告诉我们设置感觉器官模式和指定细胞命运所需的分子信号。谱系分析，另一方面，确定分化细胞的一个单独的听觉前体细胞，并揭示其位置在成熟的感觉器官。克隆关系将告诉我们，基因定义的前体池是否在发育早期就被编程，以产生内耳的大体解剖结构和感觉补丁。命运图谱和谱系分析的结果是对祖细胞身份和行为的遗传调控有了更深入的了解。而且，这种监管可能比最初设想的要复杂得多。最近的遗传命运图谱数据显示，来自神经管和神经嵴的神经上皮祖细胞为耳上皮提供前体细胞，并在分化过程中产生感觉细胞和非感觉细胞。这些新数据有力地挑战了内耳仅由placodal外胚层发育的观点，并暗示神经外胚层可能独特地参与了模式和细胞命运规范。克隆分析这些遗传上可分离的神经上皮前体细胞群将推进我们对小鼠内耳胚胎起源的核心知识。更全面地了解内耳祖细胞的命运、谱系和行为，将有助于设计以基因和细胞为基础的策略，旨在恢复听力和平衡。

DESCRIPTION (provided by applicant): The long term goal of our laboratory is to define gene- and cell-based strategies that restore hearing and balance in the dysfunctional inner ear. The short term goals are to advance our understanding of morphogenesis, pattern formation, and cell fate specification in the mouse inner ear. We use the mouse as a model system because an ever-expanding array of natural and induced genetic mutations exist that serve as accurate paradigms for human inner ear dysfunction. Our overarching technical approach relies on experimental embryology, a palette of surgical, imaging, microinjection and molecular techniques that permit access to the developing mouse inner ear in utero and enable genetic manipulation of precursor cells that give rise to the auditory and vestibular sensory structures. I this proposal, we aim: 1) to fate map the mouse otic vesicle in vivo~ 2) to define the types of cel fate choices otic precursors make and the timing of those choices~ and 3) to define the clonal contributions of neuroepithelial progenitors to the differentiated inner ear. A fate map describes what distinct populations of cells become as the inner ear matures. Developmental biologists use fate maps to understand how progenitor gene expression in a developing tissue or organ drives maturation and results in the acquisition of form and function. Intersection of indelibly labeled precursors with known domains of gene expression can teach us the molecular signals required to pattern sensory organs and specify cell fate. Lineage analysis, on the other hand, identifies the differentiated cells an individual otic precursor makes and reveals their location i the mature sensory organ. Clonal relationships will teach us if genetically defined pools of precursors are programmed early on in development to give rise to gross anatomical structures in the inner ear and to the sensory patches. The output of fate mapping and lineage analysis is a deeper understanding of the genetic regulation of progenitor cell identity and behavior. And that regulation promises to be far more complex that originally envisioned. Recent genetic fate mapping data show that neuroepithelial progenitor cells from the neural tube and likely the neural crest contribute precursor cells to the otic epithelium that generate sensory and nonsensory cells upon differentiation. These novel data firmly challenge the precept that the inner ear develops exclusively from placodal ectoderm and imply that neural ectoderm may uniquely participate in patterning and cell fate specification. Clonal analysis of these geneticall separable neuroepithelial precursor populations will advance our core knowledge about the embryonic origins of the mouse inner ear. A more complete understanding of the fate, lineage, and behavior of inner ear progenitor cells will inform the design of gene- and cell-based strategies aimed at the restoration of hearing and balance.