Molecular Embryology of the Mammalian Inner Ear

哺乳动物内耳的分子胚胎学

基本信息

批准号：
7857466
负责人：
JOHN Vincent BRIGANDE
金额：
$ 18.33万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7857466
关键词：
Alkaline Phosphatase Anterior Auditory Avian Leukosis Virus Base Pairing Birds Carbocyanines Cell Line Cells Cellular Structures Complex Development Developmental Gene Dorsal Dyes Educational process of instructing Electroporation Embryo Embryology Endolymphatic duct Epithelial Epithelium Fiber Optics Fibroblasts Gene Expression Gene Transfer Gene Transfer Techniques Genetic Genus Alpharetrovirus Goals HMG Domain HMG-Box Hair Cells Heterogeneity Homologous Gene Human In Situ In Vitro Injection of therapeutic agent Knockout Mice Label Laboratories Labyrinth Lateral Libraries Maps Medial Mediating Medical Microinjections Mitotic Molecular Molecular Probes Morphogenesis Movement Mus Mutant Strains Mice Oligonucleotides Organ Organ of Corti Otic Vesicle Pattern Pattern Formation Plasmids Positioning Attribute Reagent Regulation Research Personnel Role Roller Bottle Sensory Signal Transduction Specific qualifier value Staging Stem cells Supporting Cell System Testing Time Tracer Transgenic Mice Viral Virus Virus Receptors Wild Type Mouse cell fate specification embryo culture equilibration disorder gain of function hearing impairment in utero in vivo insight mammalian embryology murine retroviral vector mutant otoconia postnatal progenitor programs receptor expression regenerative tool transcription factor vector

项目摘要

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.

描述（由申请人提供）：我们实验室的长期目标是了解导致发育中的哺乳动物内耳中细胞命运规范和感觉斑的形成的分子机制。在子宫内小鼠胚胎的无法访问，理解内耳发育的这些基本方面的进展受到了阻碍。同样令人困惑的是缺乏分子工具来原位动态操纵发育基因表达。我们应用实验性胚胎学来研究小鼠体外和体内的内耳。我们的目的：1）在野生型和突变小鼠中命运小鼠耳杯闭合； 2）确定内耳中组成细胞之间的谱系关系； 3）探测哺乳动物感觉斑块形成的分子机制。小鼠整个胚胎培养物支持内耳的发育，从斑点到早期耳囊阶段。命运图将通过将荧光示踪剂染料的离子遗迹注射到杯子的边缘和凹度中的指定位置进行。命运映射将教会我们在杯闭合期间耳朵上皮祖细胞的形态发生运动，并允许它们与基因表达的知识结构域的相关性。生物活性试剂对早期耳尾阶段小鼠的胚胎体内的三耐氨酸微注射可以在发育中和产后内耳中进行广泛的研究。谱系分析将通过对编码碱性磷酸酶的逆转录病毒构建体和复杂的24碱基对库中的逆转录病毒构建体进行谱系分析进行，并在成熟的，产后的内耳中进行后续克隆分析。谱系分析将向我们展示细胞命运选择的类型和时机，这些细胞命运选择会导致感觉和非感官细胞。感觉斑的形成将通过将表达质粒的显微注射到早期的耳鼻细胞中进行研究，然后进行体内电穿孔以进行功能增益。我们将知道Misexpress的转录因子与野生型和突变小鼠内耳的感觉贴片形成有关，以洞悉其在建立听觉感觉上皮的机械作用中。对耳囊的形态发生，谱系关系和感觉斑的形成的清晰理解对于定义人类的再生医学方法的定义至关重要。