Ironfield, H.V.; (2011) Genetic studies of inner ear development in Xenopus tropicalis. Doctoral thesis , UCL (University College London).

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Abstract

Xenopus tropicalis provides an excellent model system for studying inner ear development. At ear-forming stages the head is transparent, so this organ can easily be observed, and the small genome, short generation time, and genomic resources makes this species amenable to genetic studies. A group of recessive mutants displaying inner ear defects were obtained from a pilot mutagenesis and gynogenetic screen. The primary inner ear defect was identified for each mutant and each lesion mapped to a chromosome arm. Two mutants, seasick (ssk) and komimi (kom) were selected for high resolution positional cloning and phenotypic characterization. The ssk mutant was identified due to enlarged otoconial crystals, decreased pigmentation, and balance defects. Positional cloning showed that the ssk phenotype is caused by a premature stop codon in the δ subunit of the adapter protein (AP)-3 complex. AP-3 is required to transport proteins from the endosome and golgi to lysosome-related organelles, a class of organelles that degrade intracellular proteins but also have cell-specific functions such as ionic regulation and pigment synthesis. ap3δ1 is expressed in the endolymphatic sac, which regulates the ionic composition of the fluid filling the inner ear, suggesting that the otoconial defect may be the result of an ionic imbalance. AP-1 also transports proteins from the endosome to the lysosomerelated organelles. Upregulation of AP-1 subunits are observed in response to ssk, suggesting that AP-1 may compensate for loss of or decreased levels of AP-3 function. In kom mutants a single large otoconium forms over each macula, and tadpoles exhibit strong balance defects such as circular swimming. Also, the cells comprising the macula are dysmorphic and lack the columnar structure found in wild type. Positional cloning shows that the kom phenotype is caused by a splicing defect causing partial loss of the otoconin-90 signal peptide and likely stops incorporation of the protein into the core of otoconial crystals. This work successfully demonstrates that X. tropicalis forward genetics is a valuable tool for studying the development of the inner ear.

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