Examining the role of canonical Wnt signalling during developmental vascular patterning

Thesis

The vascular system is composed of heterogeneous endothelial cell (EC) subtypes, each with distinct characteristics and functions. This heterogeneity is shaped, in part, by enhancer-mediated transcriptional regulation. Mapping how enhancers drive gene expression in specific EC subsets is central to understanding the upstream signalling pathways that govern vascular development; however, functional enhancer discovery has proved challenging. The identification of a novel enhancer specifically active in angiogenic ECs therefore provided a powerful tool to investigate the transcriptional networks orchestrating angiogenesis. This enhancer, located 58 kilobases downstream of the SOX7 gene, was termed SOX7-58.

In this thesis, I identify and characterise the transcriptional regulators of the SOX7-58 angiogenic enhancer, combining validated phylogenetic foot-printing with mutational analysis in F0 zebrafish assays. These analyses indicated a potential requirement for Tcell factor/lymphoid enhancer-binding factor (TCF/LEF) binding sites in maintaining the angiogenic specificity of the enhancer. Further in silico analysis suggested similar regulatory logic in another angiogenic enhancer, DLL4in3, where TCF/LEF motifs were likewise essential for correct enhancer activity. Given that TCF/LEF transcription factors act as nuclear effectors of canonical Wnt signalling, I next assessed the role of canonical Wnt in vascular development in vivo using EC-specific Ctnnb1 ablation. Combining immunohistochemistry with a panel of enhancer:LacZ transgenes, I analysed the consequences of disrupted Wnt signalling on EC subpopulations. Arterial EC identity remained intact in the absence of Wnt signalling, whereas angiogenic and venous EC enhancers displayed expanded or ectopic activity, without changes in overall vascular density.

Further, EC-specific Ctnnb1 ablation during coronary vascular development, where VEGFA- and VEGFC-driven angiogenesis are uncoupled, produced no significant phenotypic effects. In contrast, postnatal retinal EC deletion of Ctnnb1 caused both expanded angiogenic enhancer activity and impaired angiogenesis. Collectively, these findings suggest that canonical Wnt signalling may function as a negative regulator of angiogenesis by constraining gene expression in a spatially specific manner.

Authors: Strevens, MAA

• DOI: 10.5287/ora-kzrwg24kw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English