Lineage analysis of the developing mammalian inner ear

描述（由申请人提供）：拟议研究的长期目标是了解哺乳动物内耳模式形成和细胞命运规范的分子机制。事实上，我们对耳上皮祖细胞在发育过程中的细胞命运选择一无所知。然而，血统分析可以告诉我们这些选择的类型和时间。确定谱系关系是必不可少的，因为哺乳动物拥有一系列独特的内耳细胞类型，这些细胞类型在低等脊椎动物中不存在。不幸的是，哺乳动物内耳发育的形成阶段发生在早期植入后胚胎被包裹在一个复杂的母体来源的组织安排。经子宫显微注射技术可用于在植入后晚期将生物活性试剂引入耳上皮，因此在早期阶段无效，在早期阶段，材料组织的不透明性阻碍了传统的成像努力。这种在子宫内发育的内耳的不可接近性必须被克服，以解决有关哺乳动物内耳形成和功能的分子遗传机制的基本问题。 本申请旨在通过以下方式建立用于子宫内发育小鼠内耳的实验操作的模型系统：1）应用超声生物显微镜对子宫内小鼠内耳进行成像; 2）开创性地将超声生物显微镜引导的经子宫显微注射到耳基板、杯和囊泡中;以及3）使用逆转录病毒介导的谱系分析建立内耳组成细胞之间的克隆关系。超声生物显微镜能够真实的实时生成胚胎组织的高分辨率图像。我们将在超声引导下将编码谱系标签、碱性磷酸酶以及作为每个整合事件标签的寡核苷酸文库的复制缺陷型逆转录病毒微注射到耳基板、杯和早期囊泡中。将通过扩增和测序来自成熟的出生后内耳中的单个碱性磷酸酶阳性细胞的整合标签来进行克隆身份的非环境生物学分配。在内耳发育的最早阶段引入谱系病毒应产生大量复杂组成的克隆，这将是鉴定谱系关系的理想方法。定义这些关系可能会促进我们对哺乳动物内耳模式化和指定细胞命运的潜在机制的理解。此外，在子宫内发育的小鼠内耳的实验胚胎学访问应有助于设计合理的治疗方法，以改善或消除人类先天性耳聋。

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to understand the molecular mechanism underlying pattern formation and cell fate specification in the mammalian inner ear. Virtually nothing is known about the cell fate choices made by otic epithelial progenitors during development. Lineage analysis, however, can teach us about the types and timing of these choices. Defining lineage relationships is essential because mammals possess a unique array of inner ear cell types that are not present in lower vertebrates. Unfortunately, the formative stages of mammalian inner ear development occur when the early postimplantation embryo is encased in a complex arrangement of maternally-derived tissues. Transuterine microinjection techniques useful for introducing bioactive reagents into the otic epithelium at late postimplantation stages are thus ineffective at early stages where the opacity of materal tissues thwarts conventional imaging efforts. This inaccessibility of the developing inner ear in utero must be overcome to address fundamental questions regarding the molecular genetic mechanisrm underlying mammalian inner ear formation and function. This application seeks to establish a model system for the experimental manipulation of the developing mouse inner ear in utero by: 1) applying ultrasound biomicroscopy to image the mouse inner ear in utero; 2) pioneering ultrasound biomicroscopy-guided transuterine microinjection into the otic placode, cup, and vesicle; and 3) establishing the clonal relationships among component cells of the inner ear using retrovirus-mediated lineage analysis. Ultrasound biomicroscopy generates high-resolution images of embryonic tissues in real time. We will microinject into the otic placode, cup, and early vesicle under ultrasotmd guidance a replication-defective retrovirus encoding a lineage label, alkaline phosphatase, as well as an oligonucleotide library that serves as a tag for each integration event. Unambiguous assignment of clonal identity will be conducted by amplification and sequencing of the integration tag from individual alkaline phosphatase-positive cells in the mature, postnatal inner ear. Introduction of lineage virus at the earliest stages of inner ear development should produce a large number of clones of complex composition, which will be ideal for identifying lineage relationships. Defining these relationships may advance our understanding of the underlying mechanisms responsible for patterning the mammalian inner ear and specifying cell fate. Moreover, experimental embryological access to the developing mouse inner ear in utero should assist in the design of rational therapies to ameliorate or eliminate congenital forms of human deafness.