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.

项目摘要 动物中的多能干细胞整合了来自各种细胞外信号的信息，以在命运之间进行选择。 信号集成可以实现强大的，上下文特定的决策，而此过程中的错误是发展的基础 疾病和癌症。为了有效，信号积分必须与命运之间的协调紧密相关， 即，目标命运计划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，基因调节 基本螺旋 - 环螺旋（BHLH）转录因子的电路使两个信号集成到 类似地激活星形胶质细胞分化并抑制替代命运。转录相互作用 在BHLH中，这是该电路的重要组成部分，但仅它们就无法解释信号积分。 我假设BHLHS的其他两个关键特征起着至关重要的作用：蛋白质级二聚体和 BHLHS的振荡动力学。在这里，我将研究这些功能如何促进信号集成 NSC电路使用二聚化和动力学的定量测量通过精确完成 扰动。此外，我将分析BHLH电路在发育中的斑马鱼脊髓中的作用 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AIM 1中，我将通过设计基于新型二聚体突变体来研究BHLH二聚体的作用 在计算序列共同进化分析（与Debora Mark博士合作）上，使用 我开发的基于酵母的定量测量平台，并分析了它们对信号的影响 在AIM 2中，我将在NSC中使用及时的成像和多路复用RNA-FISH的组合 分析BHLH HES1中的振荡动力学如何由上游信号控制，然后 影响电路中的其他BHLH以及下游命运结果。我还将评估异位 调节HES1动力学会影响电路行为。在AIM 3中，我将确定BHLH电路是否以及如何 斑马鱼神经板的干细胞整合了两个发育信号，以使早期的命运选择 这个组织。具体而言，我首先要表征单个单元中的信号活动如何使用A影响其命运 体内时间解体显微镜和靶向信号扰动的组合。然后我将调查如何 BHLH电路中的相互作用介导信号对命运选择的影响。 BHLH因子在发育和成人组织中的大多数干细胞中均表示。 BHLH电路 因此，可以在整合信号信息以实现细胞脂肪决策中发挥无处不在的作用。这项工作 试图广泛了解它们的功能，利用体外和体内的定量方法 我的导师Galit Lahav博士和Sean Megason博士的指导。这项调查将阐明命运的基础 选择多样性的时机，并为“重新连接”此过程提供机会，以改善所需的产生 组织工程的细胞类型或在疾病环境中处理病理命运的选择。