Stochastic Events Regulating a Notch-Mediated Cell Fate Decision in Caenorhabditis elegans

随机事件调节秀丽隐杆线虫介导的细胞命运决定

• 批准号: 8907446
• 负责人: Michelle Andrea Attner
• 金额: $ 5.42万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8907446
• 关键词: Address; Adopted; Adult; Affect; Alleles; Animals; Bacteria; Biological; Birth Order; C. elegans genome; Caenorhabditis elegans; Cancer Etiology; Cell Cycle Progression; Cell Cycle Stage; Cells; Decision Making; Defect; Development; Developmental Biology; Developmental Process; Drosophila genus; Embryo; Event; Family member; Feedback; Fertility; Fluorescent in Situ Hybridization; Generations; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome engineering; Goals; Gonadal structure; Human; Image; Lateral; Ligands; Lobular Neoplasia; Malignant Neoplasms; Measures; Mediating; Methods; Microfluidics; Molecular Genetics; Motor Neurons; Nature; Notch Signaling Pathway; Oncogenes; Process; Quantitative Microscopy; RNA Interference; Regulation; Reporter; Research; Retinal; Signal Transduction; Sister; Spinal Cord; Technology; Testing; Time; To specify; Tumor Suppressor Proteins; cell fate specification; cell type; egg; fascinate; granulysin; human disease; improved; in vivo; mRNA Expression; notch protein; novel; novel therapeutic intervention; precursor cell; protein expression; public health relevance; receptor; single molecule; tool; tumor progression; tumorigenesis

 DESCRIPTION (provided by applicant): This proposal is concerned with how stochastic, initial differences arise in the activity of Notch, which must be properly regulated in development and which, when aberrantly regulated, causes cancer. Stochastic events are utilized from bacteria to humans to mediate myriad developmental processes including cell-fate switching and the generation of cell type diversity. Understanding how stochastic events influence cell fate specification is important to understand how reproducible cell fates are generated from groups of equivalent cells during development. The paradigmatic anchor cell (AC)/ventral uterine (VU) cell fate decision in Caenorhabditis elegans incorporates a stochastic mechanism as part of the process by which initially equivalent cells acquire different fates. The two cells undergoing the AC/VU decision each have the potential to become either an AC or a VU. They interact with each other, mediated by LIN-12/Notch, such that only one becomes the AC and one becomes the VU. During the course of the decision, a stochastic event causes these cells, which initially express both the transmembrane receptor LIN-12/Notch and the transmembrane ligand LAG-2 (a Delta/Serrate/LAG-2 family member), to engage feedback mechanisms that restrict transcription of lin-12 to the presumptive VU and lag-2 to the presumptive AC. The goal of this research is to understand how small, early differences in Notch activity are generated between initially equivalent cells. Birth-order of the AC and VU precursors, a random event, is highly correlated with the subsequent cell fate, suggesting that there is a relationship between cell-cycle progression, Notch signaling, and cell fate specification in the AC/VU decision. Here, we will study this relationship to probe the nature of the stochastic cellular events leading to the specification of the AC and VU by combining the powerful genetics of C. elegans with recent advances in C. elegans genome engineering and in vivo fluorescent imaging. First, we will test whether birth-order of the AC and VU precursors creates or reflects a difference between them by genetically manipulating birth-order and lin-12/Notch level. Second, we will quantify lin-12/Notch expression and activation during the AC/VU decision. We will quantify endogenous lin-12/Notch mRNA and protein expression and develop a novel reporter for lin-12/Notch activity. Third, we will conduct genetic screens to identify novel regulators that control Notch activation i the AC/VU decision and their sister cells by directly imaging the somatic gonad at high magnification. In addition to addressing a central question in developmental biology, studies of Notch-mediated cell fate decisions are highly relevant to cancer: Notch can be an oncogene or tumor suppressor depending on cell context, and studies of how Notch activity is regulated in this cell fate paradigm in C. elegans have identified genes involved in tumorigenesis. Furthermore, illuminating the potential intersection between Notch activity, cell cycle progression, and cell fate specification may suggest novel therapeutic approaches.

 描述（由申请人提供）：该提案涉及Notch活性如何产生随机的、初始的差异，Notch活性必须在发育过程中得到适当的调节，并且当调节异常时，会导致癌症。从细菌到人类，随机事件被用来介导无数的发育过程，包括细胞命运的转换和细胞类型多样性的产生。了解随机事件如何影响细胞命运规范对于理解发育过程中等效细胞群如何产生可重复的细胞命运非常重要。秀丽隐杆线虫中典型的锚细胞（AC）/腹侧子宫（VU）细胞命运决定纳入了随机机制，作为最初相同细胞获得不同命运过程的一部分。进行 AC/VU 决策的两个单元都有可能成为 AC 或 VU。它们在 LIN-12/Notch 的介导下相互作用，使得只有一个成为 AC，一个成为 VU。在决策过程中，随机事件导致这些细胞（最初表达跨膜受体 LIN-12/Notch 和跨膜配体 LAG-2（Delta/Serrate/LAG-2 家族成员））参与反馈机制，将 lin-12 转录限制为假定的 VU，将 lag-2 转录限制为假定的 AC。这项研究的目的是了解最初相同的细胞之间 Notch 活性的早期差异有多小。 AC 和 VU 前体的出生顺序是一个随机事件，与随后的细胞命运高度相关，表明细胞周期进程、Notch 信号传导和 AC/VU 决策中的细胞命运规范之间存在关系。在这里，我们将通过将线虫强大的遗传学与线虫基因组工程和体内荧光成像的最新进展相结合，研究这种关系，以探讨导致 AC 和 VU 规范的随机细胞事件的本质。首先，我们将通过基因操纵出生顺序和 lin-12/Notch 水平来测试 AC 和 VU 前体的出生顺序是否造成或反映了它们之间的差异。其次，我们将量化 AC/VU 决策过程中 lin-12/Notch 的表达和激活。我们将量化内源 lin-12/Notch mRNA 和蛋白质表达，并开发一种新的 lin-12/Notch 活性报告基因。第三，我们将通过直接对体细胞性腺进行高倍放大成像，进行遗传筛选，以确定控制 AC/VU 决策及其姐妹细胞中 Notch 激活的新型调节因子。除了解决发育生物学中的一个核心问题外，对Notch介导的细胞命运决定的研究与癌症高度相关：根据细胞背景，Notch可以是癌基因或肿瘤抑制基因，并且对秀丽隐杆线虫的这种细胞命运范式中Notch活性如何调节的研究已经确定了参与肿瘤发生的基因。此外，阐明Notch活性、细胞周期进程和细胞命运规范之间的潜在交叉点可能会提出新的治疗方法。