Monteiro, Rita S; (2018) Dorso-ventral differences in gene expression and Brachyury chromatin binding in Xenopus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The main question developmental biologists pursue is the understanding of the processes involved in transforming a single cell into a whole organism with distinct tissues. In this thesis, I addressed this question, using the frog embryo and focusing on tissue-specific regulation by transcription factors. As an approximation for dorsal and ventral tissues, I used UV and LiCl treatments, that resulted in ventralised and dorsalised embryos, respectively. In a first instance, I did a whole poly(A)-transcriptome comparison of LiCl- and UV-treated embryos which validated the use of this approach as a proxy for dorsal and ventral cell types. Furthermore, I characterised these cell types, identified and characterise new genes, with a special focus in a dorsally expressed gene activated by the canonical Wnt signalling. To study cell-specific regulation at the chromatin level I compared the chromatin landscape of dorsalised and ventralised embryos. I observed that both RNAPII and the putative enhancer marker p300 bind the chromatin differently in a way that correlated with gene expression. I also compared the binding of Brachyury, a zygotic transcription factor expressed in both cell types. Although the majority of sites were bound similarly, a small subset was differentially bound in the two condition. I showed evidence that these differences could be due to absence/presence of co-factors that mediated the binding to these sites. Finally, I compared Brachyury morpholino(MO)-mediated KD (knockdown) and KO (knockout) embryos and showed, that whilst phenotypically are similar, transcriptionally there was a group of genes misregulated only in MO-injected embryos. I further explored the nature of these genes and revealed new insights into the use of morpholinos in loss of gene function experiments.

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