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了解发育中哺乳动物内耳中细胞命运特化和感觉贴片形成的分子机制。对内耳发育的这些基本方面的理解进展受到子宫内小鼠胚胎不可接近性的阻碍。同样令人困惑的是缺乏分子工具来动态操纵原位发育基因表达。本文应用实验胚胎学方法对小鼠内耳进行了离体和体内的研究。我们的目标是：1）在野生型和突变型小鼠中对小鼠耳杯闭合进行命运作图; 2）确定内耳中组成细胞之间的谱系关系;以及3）探索哺乳动物感觉斑形成的分子机制。小鼠全胚胎培养支持内耳从基板到早期耳囊阶段的发育。通过将荧光示踪剂染料离子电渗注射到髋臼杯边缘和边缘的指定位置，进行结局图。命运映射将教我们的形态发生运动的耳上皮祖细胞在杯关闭，并允许其与已知的基因表达领域的相关性。将生物活性试剂经子宫显微注射到体内早期耳囊期小鼠胚胎中，可以在发育和出生后的内耳中进行广泛的研究。将通过经子宫显微注射编码碱性磷酸酶的逆转录病毒构建体和复杂的24个碱基对文库到耳囊中进行谱系分析，随后在成熟的出生后内耳中进行克隆分析。谱系分析将向我们展示耳上皮祖细胞产生感觉细胞和非感觉细胞的细胞命运选择的类型和时间。将通过经子宫显微注射表达质粒到早期耳囊中，然后通过体内电穿孔进行功能获得研究来研究感觉斑的形成。我们将错误表达已知参与野生型和突变型小鼠内耳感觉片形成的转录因子，以深入了解它们在建立听觉感觉上皮中的机制作用。清楚地了解耳泡形态发生、谱系关系和感觉斑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至关重要。

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.