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Transcriptional regulation in early development: identification of downstream targets through time series perturbation in Xenopus tropicalis

Cooper, BRD; (2016) Transcriptional regulation in early development: identification of downstream targets through time series perturbation in Xenopus tropicalis. Doctoral thesis , UCL (University College London). Green open access

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Abstract

This thesis aims to improve our understanding of zygotic gene regulation during early vertebrate development using Xenopus tropicalis as a model system. The aim of the first part of the work was to differentiate between maternal polyadenylation and zygotic transcription as mechanisms of gene activation in the early embryo. This work used non-polyA+ selective gene expression analysis to systematically discriminate de novo zygotic transcription from polyadenylation of maternal transcripts. It concludes that immediate post-fertilisation transcripts are activated by polyadenylation and shortly before mid-blastula transition (MBT) transcription becomes the dominant mechanism of activation. The motivation then was to explore the gene regulatory network downstream of early activated transcription factors. A morpholino screen of early, transiently activated transcription factors was carried out and Mix1 was selected for its early, penetrant phenotype. The aims of the following work were to identify the targets of Mix1, and to evaluate the use of knockdown time-series RNA-seq as a means of determining transcription factor targets. To identify downstream targets of Mix1, the transcriptomes of knockdown and control embryos were compared over a time course. This work concludes that the time-series RNA-seq approach can be used to identify candidate Mix1 targets, but that these may include morpholino-specific off-target effects. The final aim was to control for off-target morpholino effects and to validate candidate Mix1 targets. To do so, two additional knockdown time-series were generated using two different translation blocking morpholinos targeting Mix1 and Mixer. In addition, mix1-expressing animal cap explants were transcriptionally profiled to validate candidate Mix1 targets. This work concludes that nine Mix1 validated activatory targets are found, most of which are transcription factors which are enriched for functions in neural development indicating a novel role for Mix1, as well as functions in antero-posterior and dorsal-ventral patterning, supporting previous publications linking Mix1 to dorso-anterior development.

Type: Thesis (Doctoral)
Title: Transcriptional regulation in early development: identification of downstream targets through time series perturbation in Xenopus tropicalis
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: Xenopus tropicalis, Mix1, Mixl1, MBT, polyadenylation, transcription
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: http://discovery.ucl.ac.uk/id/eprint/1473875
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