Optogenetic dissection of Erk signal interpretation in early embryogenesis

项目摘要/摘要 在发育中的胚胎中，信号通路决定了细胞命运的选择，但如何只有有限数量的 途径控制着复杂的发育事件？空间信号梯度和时间信号 众所周知，动力学对于用相同的途径引导多个命运是重要的，但其机制 细胞用来解释这些信号事件在很大程度上仍未确定。一条多种多样的道路 在胚胎发生、伤口愈合和癌症进展过程中所扮演的角色是高度保守的细胞外 信号调节蛋白激酶(ERK)信号网络。早期果蝇胚胎是一种经典的体内模型 ERK信号的组织构型：ERK信号在两极的空间梯度指定前部和后部 结构并决定内胚层与外胚层的命运。尽管这些模式是明确定义的， Toettcher实验室使用光遗传学工具在体内操纵ERK信号的最新工作 精致的时间和空间精确度揭示了关于 对ERK信号进行了解释。在这里，我建议定义果蝇胚胎的机制 解释ERK剂量的差异以创建基因表达的空间模式并做出关于细胞的决定 命运。为了解决这个问题，我将使用尖端的光遗传工具、活细胞生物传感器、 基因组分析和经典的遗传扰动。在目标1中，我将研究基因的空间模式 表达对ERK梯度的主要破坏是健壮的。在目标2中，我将探索为什么只有两个转录 在ERK信号强度的大范围内，因子可以控制多个不同的命运。总而言之，使用精度 操作信号输入和量化表达式输出的工具，该提案将揭示ERK是如何 信号由下游基因测量和解释，解决了 发育生物学，它肯定会与其他信号通路和细胞环境相关 ERK信号。通过这项研究和附带的培训计划，我将在以下方面获得特殊培训 光遗传学、高级显微镜和基因组学，为我在 细胞和发育生物学。

PROJECT SUMMARY/ABSTRACT Signaling pathways pattern cell fate choices in the developing embryo, but how do only a limited number of pathways control the complex events of development? Spatial signaling gradients as well as temporal signaling dynamics are known to be important for directing multiple fates with the same pathway, yet the mechanisms cells use to interpret these signaling events remains largely uncharacterized. One pathway which plays diverse roles throughout embryogenesis, wound healing, and cancer progression is the highly conserved extracellular signal-regulated kinase (Erk) signaling network. The early Drosophila embryo is a classic in vivo model of tissue patterning by Erk signaling: a spatial gradient of Erk signal at the poles specifies anterior and posterior structure and dictates endoderm versus ectoderm fate decisions. Although these patterns are well-defined, recent work in the Toettcher laboratory using an optogenetic tool to manipulate the Erk signal in vivo with exquisite spatial and temporal precision has revealed major open questions about the mechanisms by which Erk signals are interpreted. Here, I propose to define the mechanisms by which the Drosophila embryo interprets differences in Erk dose to create spatial patterns of gene expression and make decisions about cell fate. To address this question, I will use a combination of cutting-edge optogenetic tools, live-cell biosensors, genomic analysis, and classical genetic perturbations. In Aim 1, I will examine how spatial patterns of gene expression are robust to major corruption of the Erk gradient. In Aim 2, I will explore how only two transcription factors can control multiple distinct fates over a wide range of Erk signal strength. Altogether, using precision tools for manipulating signaling inputs and quantifying expression outputs, this proposal will reveal how Erk signals are measured and interpreted by downstream genes, addressing a fundamental question in developmental biology which will surely have relevance for other signaling pathways and cellular contexts for Erk signaling. Through this research and the accompanying training plan I will obtain exceptional training in optogenetics, advanced microscopy, and genomics, positioning me for an impactful career at the interface of cell and developmental biology.