Lineage analysis of the developing mammalian inner ear

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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.