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

项目摘要/摘要 这项拟议的工作将研究通过ERK通路对信号进行转录解释，这是 在动物发育中起关键作用，在人类疾病中通常被解除管制。我们将使用 果蝇作为实验模型为研究ERK基因调控提供了无与伦比的机会 在从特定ERK底物到整个胚胎的多个生物组织水平上的信号转导。目标1 重点关注Capicua(CIC)，一种在果蝇中发现的转录抑制因子，最近 在发育和病理背景下成为ERK激活的关键传感器。我们将确定 并研究其对ERK依赖的调控的影响。 CIC蛋白稳定性、核定位和DNA结合。目标2旨在弥合基因和基因之间的鸿沟 通常只确定少数ERK底物的研究，以及组学水平的研究，它们表明 ERK通过大的底物队列发挥作用。我们将使用已有的工作来评估这两个场景 急性光遗传扰动、定量磷蛋白质组学和功能活体成像的组合 ERK信号的显著转录反应。最后，目标3将研究ERK的转录效应 信号，这通常是通过同时激活一些细胞命运和抑制其他细胞命运来起作用的。我们会 使用定量光遗传扰动和实时成像来检验激活和抑制 ERK信号的作用需要不同水平的ERK激活。我们对这一假设的实验测试将 解决发育中ERK信号转导的关键问题，并将提供预测所需的定量数据 计算建模。初步数据支持了拟议工作的可行性，这些数据包括 体内发现ERK的磷酸化蛋白质组学方法--CIC亚磷酸盐(AIM 1)的特性 底物(AIM 2)，以及预测计算模型数据驱动设计的光遗传方法(AIM 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).