Molecular Embryology of the Mammalian Inner Ear

描述(申请人提供)：我们实验室的长期目标是了解在发育中的哺乳动物内耳中负责细胞命运指定和感觉斑块形成的分子机制。在理解内耳发育的这些基本方面方面的进展受到了小鼠胚胎在子宫内无法获得的阻碍。同样令人困惑的是，缺乏动态操纵原位发育基因表达的分子工具。我们应用实验胚胎学的方法对小鼠内耳进行了体外和体内研究。我们的目标是：1)在野生型和突变型小鼠身上定位小鼠耳杯闭合的命运；2)确定内耳组成细胞之间的谱系关系；3)探索哺乳动物感觉斑块形成的分子机制。小鼠全胚胎培养支持从胎盘到耳囊早期的内耳发育。命运图将通过离子导入将荧光示踪染料注入到杯子边缘和凹陷的指定位置。命运图谱将告诉我们耳廓上皮祖细胞在闭杯过程中的形态发生运动，并允许它们与基因表达的已知领域相关联。通过宫内微量注射生物活性物质到小鼠耳囊早期胚胎体内，可以对发育中和出生后的内耳进行广泛的研究。谱系分析将通过经子宫内膜微量注射编码碱性磷酸酶的逆转录病毒构建体和复杂的24b碱基对文库到耳囊中进行，随后在成熟的出生后内耳进行克隆分析。谱系分析将向我们展示由听觉上皮祖细胞做出的细胞命运选择的类型和时机，这些选择产生感觉和非感觉细胞。感觉斑块的形成将通过经子宫肌内微量注射表达质粒到早期耳囊中进行研究，然后在体内进行功能获得研究。我们将在野生型和突变小鼠内耳中错误表达已知参与感觉斑块形成的转录因子，以深入了解它们在建立听觉感觉上皮中的机制作用。清楚地了解耳泡的形态发生、谱系关系和感觉斑块的形成对于定义改善人类听力损失和平衡障碍的再生医学方法是至关重要的。

DESCRIPTION (provided by applicant): The long term goals of our laboratory are to understand the molecular mechanisms responsible for cell fate specification and sensory patch formation in the developing mammalian inner ear. Progress toward understanding these essential aspects of inner ear development have been hampered by the inaccessibility of the mouse embryo in utero. Equally confounding is the lack of molecular tools to dynamically manipulate developmental gene expression in situ. We apply experimental embryology to study the mouse inner ear in vitro and in vivo. We aim: 1) to fate map mouse otic cup closure in wild type and mutant mice; 2) to determine the lineage relationships among constituent cells in the inner ear; and 3) to probe the molecular mechanisms underlying mammalian sensory patch formation. Mouse whole embryo culture supports inner ear development from the placode through early otocyst stages. The fate map will be conducted by iontophoretic injection of fluorescent tracer dye into designated positions in the rim and concavity of the cup. Fate mapping will teach us the morphogenetic movements of otic epithelial progenitors during cup closure and permit their correlation with know domains of gene expression. Tranuterine microinjection of bioactive reagents into the early otocyst stage mouse embryo in vivo enables a broad range of studies in the developing and postnatal inner ear. Lineage analysis will be performed by transuterine microinjection of a retroviral construct encoding alkaline phosphatase and a complex 24 base pair library into the otocyst with subsequent clonal analysis in the mature, postnatal inner ear. Lineage analysis will show us the types and timing of cell fate choices made by otic epithelial progenitors that give rise to sensory and nonsensory cells. Sensory patch formation will be investigated by transuterine microinjection of expression plasmid into the early otocyst followed by in vivo electroporation for gain-of-function studies. We will misexpress transcription factors know to be involved in sensory patch formation in wild type and mutant mouse inner ears to gain insight into their mechanistic roles in establishing the auditory sensory epithelium. A clear understanding of otic vesicle morphogenesis, lineage relationships, and sensory patch formation is essential for the definition of regenerative medical approaches to ameliorate hearing loss and balance disorders in humans.