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哺乳动物内耳发育中细胞命运规范和感觉斑块形成的分子机制。了解内耳发育的这些重要方面的进展一直受到小鼠胚胎在子宫内不可接近的阻碍。同样令人困惑的是缺乏分子工具来动态地操纵发育基因的原位表达。应用实验胚胎学方法对小鼠内耳进行体外和体内研究。我们的目标是：1)绘制野生型和突变型小鼠耳杯闭合的命运图谱；2)确定内耳组成细胞间的谱系关系；3)探索哺乳动物感觉斑块形成的分子机制。小鼠全胚培养支持内耳从母耳到早期耳囊肿阶段的发育。命运图将通过离子电泳将荧光示踪染料注射到杯子边缘和凹面的指定位置来进行。命运图谱将告诉我们耳上皮祖细胞在杯闭期间的形态发生运动，并允许它们与已知基因表达域的相关性。经子宫微注射生物活性试剂到早期耳囊肿期小鼠胚胎体内，可以广泛地研究发育和出生后的内耳。谱系分析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物和一个24碱基对的复杂文库到耳囊肿中进行，随后在成熟的、出生后的内耳中进行克隆分析。谱系分析将向我们展示由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。将表达质粒经子宫显微注射到早期耳囊肿中，然后在体内电穿孔进行功能获得研究，以研究感觉贴片的形成。我们将在野生型和突变型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。清楚地了解耳小泡的形态发生、谱系关系和感觉斑块的形成对于确定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.