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：
7844054
负责人：
JOHN Vincent BRIGANDE
金额：
$ 4万
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：
2009-06-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7844054
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发育中的哺乳动物内耳中负责细胞命运规范和感觉斑块形成的分子机制。由于小鼠胚胎在子宫内难以接近，理解内耳发育的这些重要方面的进展受到了阻碍。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在体外和体内研究小鼠内耳。我们的目标：1）绘制野生型和突变型小鼠耳杯闭合的命运图谱； 2）确定内耳组成细胞之间的谱系关系； 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从基板到早期耳囊阶段的内耳发育。将通过离子电渗疗法将荧光示踪染料注射到杯子边缘和凹面的指定位置来绘制命运图。命运图谱将告诉我们耳上皮祖细胞在耳杯闭合过程中的形态发生运动，并允许它们与已知的基因表达域相关。将生物活性试剂经子宫显微注射到早期耳囊阶段小鼠胚胎体内，可以对发育中和出生后内耳进行广泛的研究。将编码碱性磷酸酶的逆转录病毒构建体和复杂的 24 碱基对文库经子宫显微注射到耳囊中，随后在成熟的产后内耳中进行克隆分析，从而进行谱系分析。谱系分析将向我们展示产生感觉和非感觉细胞的耳上皮祖细胞做出的细胞命运选择的类型和时间。将通过将表达质粒经子宫显微注射到早期耳囊中，然后进行体内电穿孔进行功能获得研究来研究感觉斑的形成。我们将错误表达野生型和突变型小鼠内耳中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。清楚地了解耳囊形态发生、谱系关系和感觉斑块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。