Cracking the signaling code: how ERK activity dynamics coordinate gene expression and differentiation in keratinocytes

项目摘要： 上皮器官在细胞水平上的发育和维持依赖于协调一致的 生物化学信号通路，传递环境信号，如生长因子和钙 浓度与细胞中的基因表达水平相关。一个例子是基底角质形成细胞， 在体内起重要的增殖和分化作用。对这些行为的精确调控 建立了构成皮肤的分化细胞的空间轴。增殖和分化 细胞行为与Ras/ERK MAP激酶信号通路有关，然而， 同一途径通过何种途径调节这两种反应仍不清楚。此外，随着身体的不断增加， 研究表明表皮祖细胞有一天可能会用于培养患者特定的皮肤移植物 在培养皿中，对单细胞信号动力学的理解在解剖细胞中变得越来越重要。 紧急组织水平行为。本实验室将ERK活性的活报告基因导入原代小鼠， 发现ERK表现出丰富、复杂的内源性动力学。的目的 我们的建议是了解角质形成细胞中Ras/ERK通路的动力学是否以及如何在细胞水平上被读出。 细胞行为，导致增殖或分化。我将使用活细胞记者的组合 成像，转录动力学的单细胞成像，以及合成报告基因的工程化， ERK活性动力学如何影响ERK下游基因表达程序。控制 生理相关的ERK行为，我将使用器官型细胞培养和光遗传学的组合， 输入。这些方法将被用于探讨ERK动力学对基因的输入-输出功能 表达，以检验不同的动态ERK行为在不同的基因中被读出的假设。 表达程序。成功完成拟议的工作将构成第一个详细的解剖 的ERK动力学在原代细胞的背景下，以及这些动态映射到输出的形式， 生理相关基因表达和细胞反应。全面了解现场信令 分化、增殖皮肤的动力学将可能对组织工程、再生、再生和再生皮肤产生广泛的影响。 医学，以及对上皮器官更深入的基本理解。

Project Summary: The development and maintenance of epithelial organs on a cellular level is reliant upon concerted networks of biochemical signaling pathways, which transmit environmental cues such as growth factor and calcium concentrations to the level of gene expression in the cell. One example is basal keratinocytes, which have important proliferative and differentiation roles in the body. The precise regulation of these behaviors establishes the spatial axis of differentiated cells that makes up the skin. Both proliferative and differentiation cell behaviors have been linked to Ras/ERK MAP kinase pathway signaling; however, the precise mechanisms through which the same pathway regulates both responses remain unclear. Moreover, with an increasing body of work suggesting that epidermal progenitor cells may someday be used to culture patient-specific skin grafts in a dish, an understanding of single-cell signaling dynamics becomes increasingly important in dissecting the emergent tissue level behavior. Our lab has introduced live reporters of ERK activity into primary mouse keratinocytes and found that ERK demonstrates rich, complex endogenous dynamics. The objective of this proposal is to understand if and how Ras/ERK pathway dynamics in keratinocytes are read out at the level of cellular behavior, resulting in either proliferation or differentiation. I will use a combination of live cell reporter imaging, single-cell imaging of transcriptional dynamics, and engineering of synthetic reporter genes to dissect how ERK activity dynamics feed into the ERK-downstream gene expression program. To control physiologically relevant ERK behavior, I will use a combination of organotypic cell culture and optogenetic inputs. These approaches will be used in concert to probe input-output functions of ERK dynamics to gene expression, to test the hypothesis that different dynamic ERK behaviors are read out in different gene expression programs. A successful completion of the proposed work will constitute the first detailed dissection of ERK dynamics in a primary cell context as well as mapping of these dynamics to outputs in the form of physiologically relevant gene expression and cell responses. A comprehensive understanding of live signaling dynamics in differentiating, proliferating skin will likely have a broad impact on tissue engineering, regenerative medicine, and a deeper fundamental understanding of epithelial organs.