Probing Regulatory Networks setting Notch signaling levels in vivo in Drosophila and Zebrafish

Development of organisms and maintenance of healthy tissues depends on proper control of cell to cell communication networks that control cell fate and behaviour. It is remarkable how robust developmental processes are to genetic variation and environmental perturbation. To study mechanisms of developmental robustness we are using Notch signalling in Drosophila and Zebrafish as model systems. Notch is an important cell signalling protein controlling normal development and in the healthy maintenance of adult stem cells. Notch is activated at the cell surface by membrane bound ligands, which results in proteolytic release of the Notch intracellular domain. The latter translocates to the nucleus to regulate specific target gene expression. Notch signalling levels are maintained within critical thresholds by an endosomal network that can tune signalling up or down (Baron 2012, Shimizu et al. 2014). This involves regulation of a second activation mechanism that releases the active Notch intracellular domain from internalised Notch located in the endosomal membrane, an activation pathway promoted by an intracellular interacting protein called Deltex (Dx). In both Drosophila and Zebrafish Dx mutation has relatively mild developmental phenotypes but has crucial roles to control Notch signalling in stem cell regulation reflected in age-dependent disruption of tissue homeostasis in the intestine. Interestingly morpholino knockdown of Zebrafish Dx-2 expression displays defects in vascular development not observed in mutations that remove Dx-2 function. This surprising result is indicative of compensatory mechanisms that have previously been shown to act in response to gene mutations but not gene knockdowns (Rossi et al. 2015). Such compensatory mechanisms have been proposed to act through alterations of gene expression through interacting gene networks. Compensation may also occur at the level of the trafficking networks controlling Notch activity themselves since parallel mechanisms of endocytic routes may allow endocytic flux to be redirected in dx mutants. This project will investigate developmental compensation mechanisms by understanding the differences in gene expression resulting from altered copy number of Dx in Drosophila and Zebrafish. WT and Dx mutant Drosophila wing epithelial imaginal disc and Zebrafish endothelial tissues to compare gene and protein expression profiles by RNA-seq and Proteomics. Comparison of Notch target gene expression in Drosophila and Zebrafish Dx mutants using in situ, reporter gene assays and real time imaging of target gene and protein expression profiles. Use CRISPR/CAS9 to generate Drosophila and Zebrafish mutants of candidate genes to test hypotheses of compensatory gene and protein regulatory network mechanisms. The work provides new ways of working going across genetic model systems and provided world class underpinning bioscience by focussing on a crucial signalling pathway with important implications for health aging.