Mechanisms of signal integration in developmental control of organ size and tissue patterning

项目概要/摘要 拟议的工作将研究通过 ERK 通路进行信号传导的转录解释，该通路 在动物发育中发挥着关键作用，并且在人类疾病中通常不受管制。我们将使用 果蝇作为实验模型，为剖析 ERK 基因调控提供了无与伦比的机会 从特定 ERK 底物到整个胚胎，生物组织多个层面的信号传导。目标1 重点关注 Capicua (Cic)，这是一种在果蝇中发现的转录抑制因子，最近 成为发育和病理环境中 ERK 激活的关键传感器。我们将确定 Cic 中功能重要的磷酸化位点，并研究它们对 ERK 依赖性控制的影响 Cic 蛋白稳定性、核定位和 DNA 结合。目标 2 旨在弥合遗传之间的差距 研究通常只识别出少数 ERK 底物，而组学水平的研究表明 ERK 通过大量底物发挥作用。我们将使用已经工作的模型来评估这两种场景 急性光遗传学扰动、定量磷酸蛋白质组学和功能实时成像的结合 对 ERK 信号传导的显着转录反应。最后，Aim 3 将研究 ERK 的转录效应 信号传导，通常通过同时激活某些细胞命运并抑制其他细胞命运来发挥作用。我们会 使用定量光遗传学扰动和实时成像来检验激活和抑制的假设 ERK 信号传导的影响需要不同水平的 ERK 激活。我们对这一假设的实验测试将 解决发育 ERK 信号传导中的一个关键问题，并将提供预测所需的定量数据 计算建模。拟议工作的可行性得到了初步数据的支持，其中包括功能性数据 Cic 磷酸位点的表征（目标 1），一种用于体内发现 ERK 的磷酸蛋白质组学方法 底物（目标 2），以及预测计算模型的数据驱动设计的光遗传学方法（目标 3）。

PROJECT SUMMARY/ABSTRACT The proposed work will investigate transcriptional interpretation of signaling through the ERK pathway, which plays critical roles in animal development and is commonly deregulated in human diseases. We will use Drosophila as an experimental model that offers unrivaled opportunities for dissecting gene regulation by ERK signaling at multiple levels of biological organization, from specific ERK substrates to the whole embryo. Aim 1 focuses on Capicua (Cic), a transcriptional repressor that was discovered in Drosophila and has recently emerged as a key sensor of ERK activation in developmental and pathological contexts. We will identify functionally significant phosphorylation sites in Cic and investigate their effects on the ERK-dependent control of Cic protein stability, nuclear localization, and DNA binding. Aim 2 is designed to bridge the gap between genetic studies, which commonly identify only a handful of ERK substrates, and omics-level studies, which suggest that ERK functions through large substrate cohorts. We will evaluate these two scenarios using an already working combination of acute optogenetic perturbations, quantitative phosphoproteomics, and live imaging of functionally significant transcriptional responses to ERK signaling. Finally, Aim 3 will study transcriptional effects of ERK signaling, which commonly works by simultaneously activating some cell fates and repressing others. We will use quantitative optogenetic perturbations and live imaging to test the hypothesis that activating and repressing effects of ERK signaling require different levels of ERK activation. Our experimental tests of this hypothesis will address a key issue in developmental ERK signaling and will provide quantitative data needed for predictive computational modeling. Feasibility of the proposed work is supported by preliminary data that include functional characterization of Cic phosphosites (Aim 1), a phosphoproteomics approach for the in vivo discovery of ERK substrates (Aim 2), and an optogenetic approach to data-driven design of predictive computational models (Aim 3).