Problems during inner ear development can result in hearing and balance disorders. Almost one in 1,000 infants is born deaf while six in 1,000 have some form of hearing impairment. This makes hearing impairment one of the more common birth defects. Balance disorders can be more difficult to diagnose but are no less debilitating. Considering the interest in understanding the causes of newborn deafness and balance disorders, relatively little is known about ear development, especially at the cellular and molecular levels. The long-term goal of this study is to understand the cellular and molecular processes involved in the early patterning of the developing inner ear, in particular the origin and differentiation of the sensory organs. The experiments proposed will determine from where in the placode sensory organs arise, study a single cell s lineage to determine the timing and location of hair cell differentiation (specialized mechanosensory cells of the sensory organs), and test the role of the genes Notch and Delta in this differentiation. Advances in vital dyes, intravital imaging and gene function assays in the African clawed frog, Xenopus laevis, offer the unique opportunity to address the proposed hypothesis in living embryos and tadpoles.
SPECIFIC AIM NUMBER 1. Generated fate maps of inner ear structures from different stages during otic placode development and correlate these maps with gene expression patterns. Labeling small groups of cells within the placode with a fluorescent vital dye and following these cells until most of the inner ear structures have differentiated, will allow us to address our hypothesis directly.
SPECIFIC AIM NUMBER 2. Use single cell labeling to assess the lineage relationship between hair and supporting cells. By labeling a single cell with a fluorescent lineage tracer at different and progressively later developmental stages, the time that the resulting progency become restricted to one cell type, such as a hair cell, can be determined.
SPECIFIC AIM NUMBER 3. Use gain and loss of function genetic constructs to perturb Notch/Delta signaling and determine the effects on inner ear development. Three transmembrane proteins, the receptor Notch and its ligands Delta and Serrate, are known to be involved in cell-to-cell signaling and cell fate determination in other sensory tissues, but their roles in ear development are still unknown. By injecting either mRNA or plasmid DNA containing one of these three genes into one cell of a 4-cell embryo, expression can be targeted to one inner ear, leaving the other side as a control. Two of the constructs will provide gain-of-function effects by increasing Notch/Delta signaling while the third will provide a loss-of-function effect by decreasing Notch/Delta signaling.
