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依赖于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有多么强大。为了研究发育稳健性的机制，我们使用果蝇和斑马鱼的Notch信号作为模型系统。Notch是控制成体干细胞正常发育和健康维持的重要细胞信号蛋白。Notch在细胞表面被膜结合配体激活，导致细胞内Notch结构域的蛋白水解释放。后者易位到细胞核以调节特定靶基因的表达。Notch信号水平通过内体网络维持在临界阈值内，该网络可以调节信号的上调或下调（Baron 2012, Shimizu et al. 2014）。这涉及第二种激活机制的调节，即从位于内体膜的内化Notch释放活性Notch胞内结构域，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，但在控制Notch信号在干细胞调节中起关键作用，反映在肠道组织稳态的年龄依赖性破坏中。有趣的是，斑马鱼Dx-2表达的morpholino敲低显示血管发育缺陷，而在去除Dx-2功能的突变中没有观察到这种缺陷。这一令人惊讶的结果表明，补偿机制先前已被证明对基因突变起作用，但对基因敲低不起作用（Rossi et al. 2015）。这种补偿机制被认为是通过相互作用的基因网络来改变基因表达。补偿也可能发生在控制Notch活性的贩运网络本身的水平上，因为在dx突变体中，内吞途径的平行机制可能允许内吞通量重定向。本项目将通过了解果蝇和斑马鱼Dx拷贝数改变导致的基因表达差异来研究发育补偿机制。通过RNA-seq和蛋白质组学比较WT和Dx突变果蝇翅膀上皮、影像盘和斑马鱼内皮组织的基因和蛋白表达谱。利用原位、报告基因检测和实时成像技术对果蝇和斑马鱼Dx突变体中Notch靶基因的表达进行比较。利用CRISPR/CAS9构建果蝇和斑马鱼候选基因突变体，验证代偿基因和蛋白质调控网络机制假说。这项工作提供了跨遗传模型系统工作的新方法，并通过关注对健康衰老具有重要意义的关键信号通路，提供了世界级的基础生物科学。

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.