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

生物体的发育和健康组织的维持有赖于对控制细胞命运和行为的细胞间通讯网络的适当控制。值得注意的是，发育过程对遗传变异和环境扰动的影响是多么强劲。为了研究发育稳健性的机制，我们使用果蝇和斑马鱼中的Notch信号作为模型系统。Noch是一种重要的细胞信号蛋白，控制着成体干细胞的正常发育和健康维持。Notch在细胞表面被膜结合的配体激活，导致Notch胞内域的蛋白水解性释放。后者转位到细胞核，调节特定靶基因的表达。缺口信号水平由一个可调高或调低信号的内体网络维持在关键阈值内(Baron 2012，Shimizu等人)。2014年)。这涉及到第二种激活机制的调节，即从位于内膜的内化的Notch释放活性的Notch胞内域，这是一种由称为Deltex(Dx)的细胞内相互作用蛋白促进的激活途径。在果蝇和斑马鱼中，Dx突变具有相对温和的发育表型，但在干细胞调节中对Notch信号具有关键作用，反映在肠道组织内随年龄变化的动态平衡的破坏上。有趣的是，斑马鱼Dx-2表达的吗啉基因敲除显示了血管发育中的缺陷，而在移除Dx-2功能的突变中没有观察到。这一令人惊讶的结果表明，以前已被证明对基因突变起作用的补偿机制，而不是基因敲除(Rossi等人)。2015年)。这种补偿机制被认为是通过相互作用的基因网络改变基因表达而起作用的。补偿也可能发生在控制Notch活性的运输网络本身的水平上，因为平行的内吞途径机制可能允许DX突变体中的内吞通量被重新定向。这个项目将通过了解果蝇和斑马鱼中Dx拷贝数改变导致的基因表达的差异来研究发育补偿机制。WT和Dx突变体果蝇翅膀上皮成像盘和斑马鱼内皮组织，通过RNA-seq和蛋白质组学比较基因和蛋白质表达谱。用原位、报告基因分析和实时成像技术比较果蝇和斑马鱼Dx突变体中Notch靶基因的表达。利用CRISPR/Cas9产生果蝇和斑马鱼的候选基因突变体，以验证补偿基因和蛋白质调控网络机制的假说。这项工作提供了跨越遗传模型系统的新工作方法，并通过关注对健康衰老具有重要影响的关键信号途径，提供了世界级的生物科学基础。

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.