Investigating how bHLH circuits integrate signals for cell fate decisions

研究 bHLH 电路如何整合信号以决定细胞命运

• 批准号: 10722452
• 负责人: Nagarajan Nandagopal
• 金额: $ 12.47万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722452
• 关键词: Address; Adult; Affect; Affinity; Animals; Astrocytes; Auxins; BHLH Protein; Behavior; Bioinformatics; Cells; Collaborations; Complement; Complex; Consultations; Cues; Decision Making; Development; Dimerization; Disease; Elements; Embryo; Enabling Factors; Endowment; Environment; Fibroblast Growth Factor; Generations; Genes; Genetic Transcription; Goals; Helix-Turn-Helix Motifs; Human Development; Image; In Vitro; Individual; Investigation; Knowledge; LIF gene; Link; Malignant Neoplasms; Measurement; Measures; Mediating; Mentors; Mentorship; Microscopy; Monitor; Multipotent Stem Cells; Mus; Neurons; Oligodendroglia; Outcome; Pathologic; Phase; Physiological; Play; Postdoctoral Fellow; Process; Protein Analysis; Proteins; RNA; Reagent; Regulator Genes; Reporter; Role; Signal Pathway; Signal Transduction; Spinal Cord; System; Techniques; Testing; Therapeutic; Tissue Engineering; Tissues; WNT Signaling Pathway; Work; Yeasts; Zebrafish; cell type; design; developmental disease; dimer; experimental study; extracellular; fluorescence imaging; improved; in vivo; insight; knockout gene; mutant; nerve stem cell; neural; neural plate; neurodevelopment; novel; programs; repaired; response; signal processing; stem cells; synthetic biology

Project Summary Multipotent stem cells in animals integrate information from various extracellular signals to choose between fates. Signal integration enables robust, context-specific decisions, while errors in this process underlie developmental disorders and cancers. To be effective, signal integration must be tightly linked to coordination between fates, i.e., the activation of a target fate program and inactivation of alternative fates. How is this achieved? My recent postdoctoral work suggested that, in cultured neural stem cells (NSCs), a gene regulatory circuit of basic helix-loop-helix (bHLH) transcription factors enables the integration of two signals to simultaneously activate astrocyte differentiation and suppress alternative fates. Transcriptional interactions among bHLHs are an important component of this circuit, but they alone could not account for signal integration. I hypothesize that two other key features of bHLHs play an essential role: protein-level dimerization and oscillatory dynamics of bHLHs. Here, I will investigate how these features contribute to signal integration by the NSC circuit using quantitative measurements of dimerization and dynamics complemented by precise perturbations. Moreover, I will analyze the role of a bHLH circuit in the developing zebrafish spinal cord to understand how principles of circuit function obtained using in vitro systems extend to an in vivo context. In Aim 1, I will investigate the role of bHLH dimerization by designing novel dimerization mutants based on computational sequence co-evolution analysis (in collaboration with Dr. Debora Marks), validating them using a quantitative yeast-based measurement platform that I have developed, and analyzing their impact on signal integration in NSCs. In Aim 2, I will use a combination of timelapse imaging and multiplexed RNA-FISH in NSCs to analyze how oscillatory dynamics in the bHLH Hes1 is controlled by upstream signals and subsequently impacts other bHLHs in the circuit as well as downstream fate outcomes. I will also assess how ectopically modulating Hes1 dynamics affects circuit behavior. In Aim 3, I will determine whether and how a bHLH circuit in stem cells of the zebrafish neural plate integrates two developmental signals to enable an early fate choice in this tissue. Specifically, I will first characterize how signaling activity in individual cells impacts their fate using a combination of in vivo timelapse microscopy and targeted signaling perturbations. I will then investigate how interactions in the bHLH circuit mediate the effects of signals on fate choice. bHLH factors are expressed in most stem cells during development and in adult tissues. bHLH circuits could therefore play a ubiquitous role in integrating signaling information to enable cell fate decisions. This work seeks to broadly understand how they function, leveraging quantitative approaches both in vitro and in vivo with guidance from my mentors Dr. Galit Lahav and Dr. Sean Megason. This investigation will clarify the basis of fate choice in diverse tissues and provide opportunities to ‘re-wire’ this process to improve the generation of desired cell types for tissue engineering or to treat pathological fate choices in disease contexts.

项目概要 动物中的多能干细胞整合来自各种细胞外信号的信息来选择命运。 信号整合可以实现稳健的、针对具体情况的决策，而这个过程中的错误是发展的基础 疾病和癌症。为了发挥作用，信号整合必须与命运之间的协调紧密相连， 即，目标命运程序的激活和替代命运的失活。这是如何实现的？ 我最近的博士后工作表明，在培养的神经干细胞（NSC）中，基因调控 基本螺旋-环-螺旋（bHLH）转录因子的电路使两个信号能够整合 同时激活星形胶质细胞分化并抑制替代命运。转录相互作用 bHLH 是该电路的重要组成部分，但它们本身并不能解释信号集成。 我假设 bHLH 的另外两个关键特征发挥着重要作用：蛋白质水平的二聚化和 bHLH 的振荡动力学。在这里，我将研究这些功能如何通过 NSC 电路使用二聚化和动力学的定量测量，并辅以精确的 扰动。此外，我将分析 bHLH 回路在斑马鱼脊髓发育中的作用，以 了解使用体外系统获得的电路功能原理如何扩展到体内环境。 在目标 1 中，我将通过设计基于新的二聚化突变体来研究 bHLH 二聚化的作用 计算序列协同进化分析（与 Debora Marks 博士合作），使用以下方法验证它们 我开发的基于酵母的定量测量平台，并分析它们对信号的影响 NSC 中的整合。在目标 2 中，我将在 NSC 中结合使用延时成像和多重 RNA-FISH 分析 bHLH Hes1 中的振荡动力学如何由上游信号控制，随后 影响循环中的其他 bHLH 以及下游的命运结果。我还将评估如何异位 调节 Hes1 动态会影响电路行为。在目标 3 中，我将确定 bHLH 电路是否以及如何在 斑马鱼神经板的干细胞整合了两种发育信号，以实现早期命运选择 这个组织。具体来说，我将首先使用以下方法来描述单个细胞中的信号活动如何影响其命运： 体内延时显微镜和靶向信号扰动的结合。然后我将调查如何 bHLH 回路中的相互作用介导信号对命运选择的影响。 bHLH 因子在发育过程中的大多数干细胞和成体组织中表达。 bHLH 电路 因此，它可以在整合信号信息以实现细胞命运决定方面发挥普遍作用。这部作品 寻求广泛了解它们的功能，利用体外和体内的定量方法 来自我的导师 Galit Lahav 博士和 Sean Megason 博士的指导。这项调查将澄清命运的基础 在不同的组织中进行选择，并提供“重新连接”这一过程的机会，以改善所需的生成 用于组织工程或治疗疾病背景下的病理命运选择的细胞类型。