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，以恢复听力和功能失调的内耳的平衡。短期目标是促进我们对小鼠内耳的形态发生、图案形成和细胞命运规范的理解。我们使用小鼠作为模型系统，因为存在一系列不断扩大的自然和诱导基因突变，这些突变可以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，这是一种手术、成像、显微注射和分子技术的调色板，允许进入子宫中发育中的小鼠内耳，并能够对形成听觉和前庭感觉结构的前体细胞进行遗传操作。在这项建议中，我们的目标是：1)在活体内对小鼠耳囊进行命运图谱~2)确定耳前体细胞命运选择的类型和选择的时机~3)确定神经上皮祖细胞对分化内耳的克隆贡献。命运图描述了随着内耳的成熟，不同的细胞群会变成什么。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细胞基因表达如何推动成熟并导致形式和功能的获得。不可磨灭的标记前体与已知基因表达区域的交集可以教会我们形成感觉器官模式和确定细胞命运所需的分子信号。另一方面，谱系分析识别单个耳科前体产生的分化细胞，并揭示它们在成熟感觉器官中的位置。克隆关系将告诉我们，如果在发育早期就对基因定义的前体细胞池进行编程，以产生内耳和感觉补丁中的大体解剖结构。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理解。而这一监管承诺将比最初设想的要复杂得多。最近的遗传命运图谱数据显示，神经上皮祖细胞来自神经管，很可能是神经脊，它们为耳廓上皮贡献前体细胞，在分化过程中产生感觉和非感觉细胞。这些新的数据有力地挑战了内耳完全由胎盘外胚层发育的观点，并暗示神经外胚层可能独特地参与了模式形成和细胞命运的指定。对这些基因可分离的神经上皮细胞前体群体的克隆分析将促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，将有助于设计旨在恢复听力和平衡的基于基因和细胞的策略。

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.