The role of feedforward loops in regulating the dynamics of cell-cell signaling

项目概述：基因表达调控在动物发育中至关重要， 而不适当的调控会导致发育缺陷和疾病状态。在组织发育过程中，基因 通常通过被称为形态致病因子的远程信号启动，并由基因调节来精炼/调节 网络。最近，人们发现形态梯度的动态变化会影响基因调控。作为签名- NALING动态通常由前馈环路(FFL)调节，作者的目标是确定A 果蝇早期胚胎基因表达动态的前馈环 通过开发能够实时测量蛋白质和信使核糖核酸表达的新方法。 我们的长期目标是确定细胞如何解释信号并做出实时转录决定。 因此，我们在这项提案中的总体目标是开发新的方法来测量活的mrna和 在果蝇早期胚胎中检测FFL对蛋白质表达的影响。我们的中央- 假设是FFL对于稳定动态信号的活动是必不可少的。我们的假设是基于 关于文献、初步数据和FFL的性质，这通常是为了调节动力学而设计的。如果 正确，这一假设将使我们深入了解前馈回路在以下动力学背景下的作用 多细胞有机体中的活组织。建议进行这项研究的理由是，我们对 时变信号和转录反应之间的关系目前很弱，受到Cur-2基因的限制。 目前可用的实时成像工具。我们计划检验我们的中心假设，从而达到目标 通过提出以下两个具体目标，对这一申请提出建议： 具体目标1：建立基于蛋白结合子的mRNA动态检测平台和新的 合成蛋白质。在果蝇早期胚胎的情况下，GFP成熟时间排除了研究 最早的构图阶段。拟议的平台将包括基于蛋白质的粘合剂，旨在促进- DUCE在与蛋白质/RNA结合后产生明亮的FRET信号，不会有高密度的常见缺点 背景、微弱信号和送货问题。具体目标2：使用输入验证来自目标1的传感器 Vivo FFL系统。野生型胚胎的测量将验证我们的新型传感器。我们的FFL系统 选择研究也显示出快速的动态，所以用我们的传感器测量这个系统将有助于阻止- 我的角色是FFL。我们还将通过检查突变体和基因来改变活性梯度 通过突变关键的调控DNA位点。具有遗传扰动信号动力学的胚胎的测量- ICS或活性将揭示信号与基因表达边界稳定性之间的因果关系。 瑞斯。因此，它将以定量的方式测试FFL的作用。最后，我们将把我们的数据合成一个 该FFL系统的数学模型。我们预计这些结果将产生积极影响，通过设定 决定细胞如何做出决定的阶段，如迁移、分化或经历凋亡。

PROJECT SUMMARY: Regulation of gene expression is of paramount importance in animal development, while improper regulation results in developmental defects and disease states. In developing tissues, genes are often initiated through long-range signals, called morphogens, and refined/regulated by gene regulatory networks. Recently, the dynamics of morphogen gradients have been found to impact gene regulation. As sig- naling dynamics are often regulated by feedforward loops (FFLs), the authors aim to determine the role of a feedforward loop in gene expression dynamics in early fruit fly (Drosophila melanogaster) embryos, specifically through development of novel methodologies that enable live measurements of protein and mRNA expression. Our long-term goal is to determine how cells interpret signals and make real-time transcriptional decisions. Therefore, our overall objective in this proposal is to develop novel methodologies to measure live mRNA and protein expression in order to measure the effect of a FFL in the early Drosophila embryo. Our central hy- pothesis is that the FFL is essential to stabilize the activity of a dynamic signal. Our hypothesis is based on literature, preliminary data, and the nature of FFLs, which are typically designed to regulate dynamics. If correct, this hypothesis would give insight into the role of feedforward loops in the context of the dynamics of living tissues in a multicellular organism. The rationale for the proposed research is that our knowledge of the relationship between time-variant signaling and transcriptional response is currently weak, limited by the cur- rently available real-time imaging tools. We plan to test our central hypothesis, thereby attaining the objective of this application, by proposing the following two Specific Aims: Specific Aim 1: Establish a protein-binder based platform to measure dynamics of mRNA and newly- synthesized proteins. In the case of the early Drosophila embryo, GFP maturation time precludes studying the earliest patterning stages. The proposed platform will comprise protein-based binders engineered to pro- duce a bright FRET signal upon binding to the protein/RNA, and will not have the common drawbacks of high background, faint signal, and delivery issues. Specific Aim 2: Validate the sensors from Aim 1 using an in vivo FFL system. Measurements in wildtype embryos will validate our novel sensors. The FFL system we chose to study also exhibits rapid dynamics, so measurements of this system with our sensors will help deter- mine the role of the FFL. We will also genetically perturb the activity gradient by both examining mutants and by mutating crucial regulatory DNA sites. Measurements of embryos with genetically perturbed signal dynam- ics or activity will reveal a causative relationship between signaling and stability of gene expression bounda- ries. Thus, it will test the role of the FFL in a quantitative fashion. Finally, we will synthesize our data into a mathematical model of this FFL system. We expect these outcomes to have a positive impact by setting the stage to determine how cells make decisions – such as to migrate, differentiate, or undergo apoptosis.