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 动物体内的多能干细胞整合来自各种细胞外信号的信息，在命运之间做出选择。 信号整合使强大的，特定于上下文的决策成为可能，而这一过程中的错误是发展的基础。 疾病和癌症。为了有效，信号整合必须与命运之间的协调紧密相连， 也就是说，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， 碱性螺旋-环-螺旋（bHLH）转录因子的回路使得两个信号的整合成为可能， 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 在bHLH之间，是该电路的重要组成部分，但它们本身不能解释信号积分。 我假设bHLHs的另外两个关键特征起着重要作用：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有助于信号集成的 NSC电路使用二聚化和动力学的定量测量， 扰动此外，我将分析bHLH回路在发育中的斑马鱼脊髓中的作用， 理解如何使用体外系统获得电路功能的原理扩展到体内环境。 在目标1中，我将通过设计新的二聚化突变体来研究bHLH二聚化的作用， 关于计算序列协同进化分析（与Debora Marks博士合作），使用 我开发的一个基于酵母的定量测量平台，并分析它们对信号的影响。 NSC的整合。在目标2中，我将在神经干细胞中使用时间推移成像和多重RNA-FISH的组合 分析bHLH Hes 1中的振荡动力学是如何由上游信号控制的， 影响回路中的其他bHLH以及下游命运结果。我也会评估异位 调节Hes 1动力学影响电路行为。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， 这个组织。具体来说，我将首先描述单个细胞中的信号活动如何影响它们的命运， 体内时移显微术和靶向信号扰动的组合。然后我会调查 bHLH回路中的相互作用介导了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，在整合信号传导信息以实现细胞命运决定方面可以发挥普遍存在的作用。这项工作 旨在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。这次调查将澄清命运的基础 选择在不同的组织，并提供机会，以'重新布线'这一过程，以改善所需的产生 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。