Investigating how bHLH circuits integrate signals for cell fate decisions

项目摘要 动物体内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。 信号整合实现了稳健的、特定于背景的决策，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， 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？ 我最近的博士后研究表明，在培养的神经干细胞(NSCs)中，一种基因调控 碱性螺旋-环-螺旋(BHLH)转录因子的回路使两个信号能够整合到 同时激活星形胶质细胞分化，抑制另类命运。转录相互作用 在bHLH中，bHLH是该电路的重要组件，但它们单独不能解释信号集成。 我假设bHLH的另外两个关键特征起着至关重要的作用：蛋白质水平的二聚化和 BHLHs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，并辅以精密 微扰。此外，我还将分析bhlh回路在斑马鱼脊髓发育中的作用。 了解使用体外系统获得的电路功能原理如何扩展到活体环境。 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。 关于计算序列共同进化分析(与Debora Marks博士合作)，使用 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 在NSC中的集成。在目标2中，我将在神经干细胞中使用时间流逝成像和多路RNA-FISH的组合 分析bHLHHes1中的振荡动力学如何受到上游信号的控制，并随后 影响电路中的其他bHLH以及下游命运结果。我还将评估如何异地 调制Hes1动态会影响电路行为。在目标3中，我将确定bHLH电路是否以及如何进入 斑马鱼神经板的干细胞整合了两个发育信号，使早期的命运选择成为可能 这块组织。具体地说，我将首先用一个 体内时间流逝显微镜和靶向信号扰动的结合。然后我会调查是如何 BHLH回路中的相互作用在信号对命运选择的影响中起中介作用。 BHLH因子在发育过程中的大多数干细胞和成人组织中都有表达。BHLH电路 因此可以在整合信号信息以决定细胞命运方面发挥无处不在的作用。这部作品 寻求广泛地了解它们是如何发挥作用的，利用体外和体内的定量方法 来自我的导师加里特·拉哈夫博士和肖恩·梅加森博士的指导。这项调查将澄清命运的基础 在不同的组织中进行选择，并提供机会重新连接这一过程，以改进所需的 用于组织工程的细胞类型或在疾病背景下治疗病理命运的选择。

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.