Genetics - Theses

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Author: Badrock, Andrew Paul. × End date: 2019 × Start date: 2010 × Type: PhD thesis ×

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    The genetic and morphological characterisation of the zebrafish intestinal mutant setebos
    Badrock, Andrew Paul. (University of Melbourne, 2010)
    Colorectal cancer (CRC) arises by mutation of tumour suppressors and oncogenes that deregulate the genetic programmes that control intestinal turnover and homeostasis. The zebrafish intestinal epithelium shares extensive phenotypic and genetic homology with the mammalian intestine, providing a genetically tractable model organism for the discovery and characterisation of novel genes required for intestinal development. Increased knowledge of the genetic control of intestinal development may reveal new therapeutic avenues for the treatment of CRC. The Liverplus mutagenesis screen identified zebrafish embryos defective in endoderm organ morphogenesis, including the intestinal mutant setebos, described herein. This study reports the positional cloning of the setebos mutant locus, identifying a premature stop codon as the responsible mutation in the gene nucleolar protein 8 (nol8). Microinjection of wildtype nol8 mRNA rescued setebos developmental abnormalities whereas morpholino oligonucleotide-mediated knockdown of Nol8 phenocopied setebos morphology in wildtype embryos, providing functional proof that nol8 is the mutated locus underlying the setebos phenotype. This study demonstrated that Nol8 is essential for zebrafish survival and that nol8 mRNA is widely expressed in actively proliferating tissues of the zebrafish embryo at 72 hours post fertilisation (hpf), when developmental abnormalities in setebos mutants first become apparent. Northern blot analyses revealed that setebos mutants accumulate an intermediate ribosomal RNA (rRNA) orthologous to mammalian 36S at the expense of downstream 32S rRNA; defects predicted to result in reduced biogenesis of 28S rRNA. Bioanalyzer analyses confirm that setebos mutants are preferentially impaired for 28S biogenesis. 28S, 5.8S and 18S rRNA molecules combine with ribosomal proteins to form functional ribosomes, which are essential for translation and cellular growth. Defects in ribosome biogenesis have been shown to activate the tumour suppressor Tp53, resulting in cell cycle arrest. Tp53 is activated in the setebos mutant, resulting in upregulation of pro-cell cycle arrest genes, including the N-terminally truncated isoform of Tp53, ?113tp53. While this provides a mechanism for the hypoplasia observed in the tissues of the setebos embryo, including the intestinal epithelium, craniofacial cartilages, eyes, liver and pancreas, inactivation of Tp53 signalling in the setebos mutant failed to obviously alter setebos morphology. Nevertheless, activation of Tp53 signalling was found to significantly extend survival of setebos embryos, identifying an important biological role for Tp53 in cells undergoing ribosomal stress. Multiple human syndromes have been linked to abnormal ribosome biogenesis (ribosomopathies), and several predispose to cancer. Zebrafish haploinsufficient for 17 different ribosomal proteins also display increased cancer susceptibility (Amsterdam et al. 2004; Lai et al. 2009). Tp53 is inactivated in these zebrafish tumours, suggesting that inactivation of Tp53 may be a key event in tumour formation in these animals. In summary, setebos provides an ideal in vivo model to further characterise the link between defective ribosome biogenesis, Tp53 signalling and cancer. Since disruption of Nol8 function preferentially impairs the growth of several highly proliferative embryonic zebrafish tissues in the absence of Tp53 signalling, inhibition of Nol8 may provide a therapeutic avenue for the treatment of Tp53 mutant tumours.