The broad goal of this research is to identify and mutate genes involved in otic development and function in Xenopus (frog). The well-characterized external embryological development of the frog allows simple access to specific developmental stages by rapid visual inspection. We will conduct an insertional mutagenesis screen in Xenopus using a gene-trap vector containing a splice acceptor (SA) sequence followed by a marker gene, green fluorescent protein (GFP). Since the marker gene lacks a promoter, it can only be transcribed and translated if it integrates properly into an exon or intron of an endogenous gene. Expression of the bright GFP following insertional mutagenesis is visible in living embryos. In addition, since the trapped (and prospectively mutated) gene is always marked by GFP, one can easily distinguish between embryos carrying the mutation from ones that do not. We will use visual inspection to screen for tadpoles with insertions of the GFP- transgene into genes expressed during otic development. We propose the following specific aims: (1) We will use insertional mutagenesis to generate stable GFP- expressing lines of the true diploid, Xenopus tropicalis, carrying transgenes expressed in the otic regions. These stable lines will be used for studying the mutant phenotype generated by the insertion. (2) We will clone and characterize otic genes into which the GFP transgene has inserted. (3) We will characterize the development of the tissue in which the GFP-transgene is expressed using fluorescence video microscopy in live heterozygotic embryos or in fixed tissues using immunohistochemistry (both light and EM). Results of these investigations will provide essential knowledge of the specific genes involved in otic development as well as mutant lines that can be used for future studies of the specific function of these genes in acoustic or vestibular functions.
