Dissecting the role of Erk signaling dynamics in positioning and coordinating germ layer fates

剖析 Erk 信号动力学在定位和协调胚层命运中的作用

• 批准号: 10537311
• 负责人: Naomi Jessie Baxter
• 金额: $ 3.91万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537311
• 关键词: Address; Animal Model; Animals; BMP4; Biological; CRISPR/Cas technology; Cell Line; Cell Proliferation; Cell physiology; Cells; Characteristics; Complex; Cues; Defect; Development; Ectoderm; Embryo Research; Embryology; Embryonic Development; Engineering; Ethics; Etiology; Exhibits; Fibroblast Growth Factor; Gene Expression Profile; Genes; Germ Layers; Homeostasis; Human; Human Development; Image; Knowledge; Lead; Light; Link; MAP Kinase Gene; MAPK Signaling Pathway Pathway; Measures; Mesoderm; Modeling; Molecular; Mutation; Outcome; Pathway interactions; Pattern; Pattern Formation; Phosphotransferases; Play; Positioning Attribute; Process; Reporter; Reproducibility; Role; Signal Pathway; Signal Transduction; Stains; Study models; System; Technology; Therapeutic Intervention; Time; Variant; Visualization; Work; cell behavior; cell type; developmental disease; engineered stem cells; experimental study; gastrulation; genome editing; human embryonic stem cell; human model; insight; migration; optogenetics; small molecule inhibitor; spatiotemporal; stem cell fate; stem cells; tool; transmission process

Project Summary: It is becoming increasingly clear that one of the ways that cells interpret and encode information into multiple cell fates is by multiplexing information through dynamic encoding. This is especially true for the MAPK/Erk pathway, that governs many cell processes including cell proliferation, differentiation and migration. For years, how such diverse outcomes were controlled by the same pathway remained elusive, but the advent of single cell studies and optogenetics has elucidated the many ways in which Erk activity can be interpreted into distinct cell fates, and even more recently, the role of dynamics in these complex decisions. The role of developmental Erk dynamics in determining and coordinating human gastrulation, however, has not yet been investigated. We will combine live cell kinase activity reporters and optogenetic control over intracellular signaling pathways to probe the role of ERK dynamics in positioning and coordinating the three germ layers. Additionally, we will uncover whether RASopathy causing mutations influence human gastrulation to pace the way for potential therapeutic intervention. This proposal brings together recent advances in stem cell and molecular engineering. We utilize advances in 2D micropatterning, cellular optogenetic control, live cell kinase activity reporters and CRISPR Cas9 genome editing. Together, these technologies give us unprecedented control over and visualization of microengineered models of human gastrulation, thereby enabling us to investigate the principles of dynamic information transmission. Our platform does not face the same ethical barriers that have limited human embryo research, allowing us to provide otherwise unavailable information about human embryonic development. In this proposal we focus on the role of Erk signaling dynamics in coordinating the three germ layers, as well as uncover impact of RASopathy mutations. In Aim 1 we will image and quantify Erk signaling dynamics using the Erk kinase translocation reporter during the process of gastrulation and determine which features of signaling dynamics are predictive of germ layer fate. Aim 2 will allow us to identify which of these dynamical features are sufficient to determine the cell fate outcome using cellular optogenetics. Finally, in Aim 3 we will uncover whether RASopathy mutations lead to gastrulation defects and investigate whether these are linked to disruption to Erk dynamical signatures using CRISPR Cas9 gene editing and our 2D gastruloid model. Approaching this cell biological question from a systems level perspective, using reproducible precisely controllable tools that are otherwise unavailable without optogenetics and microengineered platforms, has the potential to shine new light on the field.

项目概要： 越来越清楚的是，细胞将信息解释和编码到多个细胞中的方式之一是 命运是通过动态编码来复用信息。对于 MAPK/Erk 通路尤其如此， 它控制着许多细胞过程，包括细胞增殖、分化和迁移。多年来，如何如此 由相同途径控制的不同结果仍然难以捉摸，但单细胞研究的出现 光遗传学已经阐明了 Erk 活性可以解释为不同细胞命运的多种方式， 甚至最近，动态在这些复杂决策中的作用。发育性 Erk 的作用 然而，确定和协调人类原肠胚形成的动力学尚未得到研究。我们将 结合活细胞激酶活性报告基因和对细胞内信号通路的光遗传学控制来探测 ERK 动力学在定位和协调三个胚层中的作用。此外，我们还将揭开 引起突变的 RAS 病是否会影响人类原肠胚形成，从而为潜在的治疗铺平道路 干涉。 该提案汇集了干细胞和分子工程的最新进展。我们利用 二维微图案、细胞光遗传学控制、活细胞激酶活性报告基因和 CRISPR Cas9 方面的进展 基因组编辑。这些技术共同为我们提供了前所未有的控制和可视化 人类原肠胚形成的微工程模型，从而使我们能够研究动态原理 信息传输。我们的平台不会面临限制人类胚胎的道德障碍 研究，使我们能够提供有关人类胚胎发育的其他信息。在这个 该提案我们重点关注 Erk 信号动力学在协调三个胚层中的作用，并揭示 RAS 病突变的影响。在目标 1 中，我们将使用 Erk 激酶对 Erk 信号传导动力学进行成像和量化 原肠胚形成过程中的易位记者并确定信号动力学的哪些特征 预测胚层的命运。目标 2 将使我们能够确定哪些动态特征足以 使用细胞光遗传学确定细胞命运结果。最后，在目标 3 中，我们将揭示 RAS 病是否 突变会导致原肠胚形成缺陷，并研究这些缺陷是否与 Erk 动力学破坏有关 使用 CRISPR Cas9 基因编辑和我们的 2D 原肠胚模型进行签名。接近这种细胞生物学 从系统级的角度来看问题，使用可重复的精确控制工具，否则 没有光遗传学和微工程平台就无法实现，有潜力在该领域发出新的光芒。