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

 描述（由申请人提供）：该提案涉及Notch活性如何随机产生初始差异，Notch在发育中必须适当调节，并且当异常调节时会导致癌症。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的规范是重要的，以了解可重复的细胞命运是如何产生的等效细胞群在发展过程中。范例锚细胞（AC）/腹侧子宫（VU）细胞的命运决定秀丽隐杆线虫纳入了随机机制的过程的一部分，最初相当于细胞获得不同的命运。经历AC/VU决策的两个细胞各自具有成为AC或VU的潜力。它们通过LIN-12/Notch介导相互作用，使得只有一个成为AC，一个成为VU。在决定的过程中，随机事件导致这些最初表达跨膜受体LIN-12/Notch和跨膜配体LAG-2（Delta/Serrate/LAG-2家族成员）的细胞参与反馈机制，该机制将lin-12的转录限制为推定的VU，将lag-2限制为推定的AC。这项研究的目的是了解最初等同的细胞之间Notch活性的早期差异是如何产生的。AC和VU前体的顺位是一个随机事件，与随后的细胞命运高度相关，这表明在AC/VU决定中细胞周期进展、Notch信号传导和细胞命运规范之间存在关系。在这里，我们将研究这种关系，通过结合C. elegans与最近的进展，在C。线虫基因组工程和体内荧光成像。首先，我们将测试AC和VU前体的出生顺序是否通过遗传操纵出生顺序和lin-12/Notch水平来创建或反映它们之间的差异。第二，我们将在AC/VU决定期间定量lin-12/Notch表达和激活。我们将定量内源性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，我们将进行遗传筛选，以确定新的监管机构，控制Notch激活的AC/VU决定和他们的姐妹细胞直接成像体细胞性腺在高放大率。除了解决发育生物学中的一个中心问题外，Notch介导的细胞命运决定的研究与癌症高度相关：Notch可以是癌基因或肿瘤抑制因子，这取决于细胞背景，并且在C.秀丽线虫已经鉴定出与肿瘤发生有关的基因。此外，阐明Notch活性、细胞周期进展和细胞命运特化之间的潜在交叉可能提示新的治疗方法。

 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.