Harding, J. (2011) Small RNAs as regulators of early vertebrate development. Doctoral thesis, UCL (University College London).
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This thesis explores the role of small RNAs as regulators of early vertebrate development using the frog species Xenopus laevis and Xenopus tropicalis as experimental systems. Firstly, I investigate spatial control of Nodal signalling, which is critical for organisation of the vertebrate body plan during gastrulation, by uncovering the functional relevance and mechanism of spatial regulation of the Nodal coreceptor Cripto-1 in the early X. laevis embryo. Xenopus Cripto-1 (XCR1) mRNA is ubiquitous in the early embryo, but the protein is absent in the prospective endoderm, from which Nodal signalling originates. Cripto-1 is a stem cell marker and upregulated in many cancers and Nodal signalling is reactivated in melanomas and prostate cancer. Therefore understanding how control of Nodal signalling and XCR1 regulation is achieved may have implications for its misregulation in cancer. I show that spatial regulation of XCR1 is critical for the location, timing and magnitude of Nodal signalling in the early Xenopus embryo. Spatial regulation occurs at the level of translational repression, is dependent on the 3’ UTR and the microRNA-processing enzyme Dicer and is abolished by mutating four nucleotides in the minimal regulatory region of the 3’ UTR. I show that XCR1 is negatively regulated by xla-miR-427 in a 3’ UTR-independent mechanism, adding to recent research in Xenopus and zebrafish showing microRNA control of Nodal signalling at the ligand, receptor and antagonist level. Whilst studying Xenopus Cripto-1, it became clear that relatively little was known about microRNAs in early vertebrate development. To fill this void, I performed genome-wide sequencing of all small RNAs expressed in the early X. tropicalis embryo at three stages of development. This revealed dynamic and localised expression of hundreds of microRNAs in the early embryo and the presence of Piwi-interacting RNA sequences. Extensive validation of the small RNA-seq dataset is presented, and novel microRNAs and a novel class of intronic small RNAs are uncovered within the 95 % of the early vertebrate embryo small RNAome that was previously uncharacterised.
|Title:||Small RNAs as regulators of early vertebrate development|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Wolfson Institute and Cancer Institute Administration > Cancer Institute|
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