Lineage analysis of the developing mammalian inner ear

描述(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指定的分子机制。对于眼球上皮祖细胞在发育过程中做出的细胞命运选择，人们几乎一无所知。然而，血统分析可以告诉我们这些选择的类型和时机。确定血统关系是至关重要的，因为哺乳动物拥有一系列独特的内耳细胞类型，这是低等脊椎动物所没有的。不幸的是，哺乳动物内耳发育的形成阶段发生在植入后早期胚胎被包裹在母源组织的复杂排列中时。因此，在移植后晚期，可用于将生物活性试剂引入耳道上皮的经微量注射技术在材料组织的不透明阻碍传统成像努力的早期阶段是无效的。为了解决哺乳动物内耳形成和功能背后的分子遗传机制的基本问题，必须克服这种发育中的内耳在子宫中不可及的问题。 这项应用旨在通过以下方式建立实验操作子宫内发育的小鼠内耳的模型系统：1)应用超声生物显微镜对子宫内小鼠内耳进行成像；2)在超声生物显微镜引导下向耳部胎盘、耳杯和耳囊内注射经子宫肌素显微注射；以及3)利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。超声生物显微镜实时生成高分辨率的胚胎组织图像。我们将在超声引导下将复制缺陷逆转录病毒注射到耳道胎盘、杯状和早期囊泡中，该逆转录病毒编码谱系标签、碱性磷酸酶以及作为每个整合事件的标签的寡核苷酸文库。克隆身份的明确分配将通过扩增和测序成熟后内耳中单个碱性磷酸酶阳性细胞的整合标签来进行。在内耳发育的最早阶段引入谱系病毒应该会产生大量复杂成分的克隆，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和指定细胞命运的潜在机制的理解。此外，在子宫内获得发育中的小鼠内耳的实验胚胎研究应该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.