Nkd在果蝇幼虫发育阶段抑制Wg信号通路及其分子机制

The inhibition of Wnt signaling is one of the research focuses in biomedical area. Physiologically, one way to prevent Wnt signaling from overacitivation is achieved by the feedback inhibitory loop. In Drosophila, Nake cuticle (Nkd) is one of such feedback inhibitors thus far been identified for Wingless (Wg, a major fly Wnt) signaling. Previous studies show that the action of Wg signaling appears to perfectly match the sufficient and necessary conditions for Nkd expression in all development stage from egg to adult. However, for a long time, Nkd is thought to only antagnize Wg in certain embryonic development stage and its role in larvae is dispensible. We have recently published a story describing a novel model in which Dpp (a fly BMP) inhibits Wg signaling through brinker-nkd negative circuit in fly wings (Yang et al., 2013), which has certainly shown in one aspect of the essential physiological role of nkd in later devlopmental stage. In this grant we propose to examine the role of nkd on Wg signaling in larval development in more detail aiming to clearify the role of nkd in larval development. In addition to using fly genetics as a major tool, we planed to dig in detail the molecular mechanism by which Nkd influence Wg transcriptional activation based on our newly deviced quantitative Wg activation analytical system. We have constructed Armadillo (Arm, fly beta-catenin) fused to fluoresent protein and Wg reporters from several direct Wg targets. By transient turning on Wg signaling using Wg molecules and manupilation of nkd gene dosage we expect to look into the Nkd nulear localization, Arm stability and the dynamics of Wg transcriptional activation in a more careful way than previously and gain further insights into Nkd function.

抑制Wnt信号是生物医学领域关注的焦点问题。反馈抑制是体内抑制Wnt信号的重要方式，而果蝇Nkd即是Wg信号通路的一种反馈抑制因子。尽管Wg信号广泛诱导Nkd的表达，但一般认为Nkd对Wg的拮抗作用仅限于胚胎发育阶段。我们最近在果蝇翅发育模型中阐明了Dpp信号通过Brk-nkd的负调控环路抑制Wg通路的新模型(Yang et al., 2013)，说明nkd在发育后期存在必要作用。本课题将用果蝇遗传学对nkd拮抗Wg信号通路进行深入研究，试图澄清其幼虫发育阶段的作用。同时，我们在果蝇Kc细胞建立了Wg信号通路的定量观察系统，经Wg配体瞬时激活Wg通路对荧光蛋白标记的Nkd和Arm/beta-Catenin进行含量和胞内定位的动态观察，同时利用基于多种Wg靶基因构建的荧光蛋白报告基因实时记录Wg的转录调控活性。通过动力学分析，我们预期可以获得解读Nkd功能的新线索。

抑制Wnt信号是生物医学领域关注的焦点问题。本课题利用果蝇遗传学对nkd拮抗Wg信号通路进行深入研究，试图澄清其幼虫发育阶段的作用。鉴于传统的nkd突变体在分子水平未得到精确地鉴定，我们通过Crispr/Cas9构建了新的nkd突变果蝇。我们的研究表明，过表达nkd可以抑制Wingless靶基因Distal-less的表达，以及wing notch表型；RNAi及突变体克隆则显示Distal-less和Sensless表达的增加，同时，也在成虫翅膀表现典型的Wingless信号通路增强的表型：ectopic bristles。这些结果清楚表明nkd在幼虫发育抑制Wingless信号通路。我们还探讨了nkd不同突变体对Wingless信号的影响以及nkd在细胞水平对Wingless靶基因表达动力学的影响。总之，根据这些结果我们得出结论，nkd对Wingless信号通路的抑制作用是广泛的，对其分子作用机制的研究将丰富我们对Wg信号通路调控机制的认识。