The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. Previous work done in our laboratory demonstrates that the tubules and duct are specified at stages 12.5 and 14 respectively and the glomus is specified at stage 12.5. The kidney unit functions to control water balance and dispose of wastes. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. Members of this laboratory have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). In this thesis it has been demonstrated that Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype. Depletion of Xanx-4 dsRNA was tested and was shown to specifically reduce levels of Xanx-4 protein in oocytes. Xanx-4 siRNA was also tested for efficacy and was shown to cause a reduced pronephric tubule phenotype. However analysis of the effects of dsRNA and siRNA on mRNA levels have proved inconclusive and cast doubt on the usefulness of dsRNAs in Xenopus laevis. Further studies on RNAi in Xenopus will be required before it can be judged a reliable method for interfering with gene expression. Xanx-4 depletion, using morpholinos produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by coinjection of Xanx-4 mRNA. Thus, Xanx-4 can be successfully depleted in embryos using morpholino oligos.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:524643 |
| Date | January 2001 |
| Creators | Seville, Rachel Alice |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3097/ |
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