Regulatory Mechanism for Cellular Dedifferentiation in C. elegans Germline

在它们的整个生命周期中，干细胞要做出几个关键的命运决定：最初的决定是自我更新（保持多能性）还是分化，随后的决定是继续分化还是回到未分化状态，即进行某种形式的去分化。控制分化决定的细胞机制的缺陷可导致特定细胞类型的丧失，未分化细胞的过剩，或癌细胞的明显生长。在过去的十年中，与初始决策相关的分子机制已经在主要模式生物中得到了描述。然而，对后续决策的潜在决定因素的理解充其量是模糊的。长期的研究目标是阐明导致分化细胞回到未分化状态的机制，并建立动物模型来研究这些机制。对这一细胞事件的分子理解有望为干细胞生物学、再生医学和肿瘤治疗开发强有力的工具。该项目还包括一个组成部分，即北卡罗来纳州传统上服务不足的学生群体（少数民族高中生、社区学院和本科生）将积极参与基础研究。该项目解决了关于已知去分化机制的基本分子控制的三个关键问题：(1)分化细胞如何维持其状态或返回到未分化细胞？(2)在体内，这一细胞过程涉及哪些调控网络？(3)该网络的异常调控是否与肿瘤发生有关？研究人员利用线虫的生殖系作为模型系统，先前报道了PUF-8（一种rna结合蛋白）和LIP-1（一种双特异性磷酸酶）需要(a)维持精母细胞的减数分裂（分化状态），(b)通过抑制MPK-1（一种ERK同源物）信号传导阻止它们去分化为有丝分裂细胞（未分化状态）。进一步的研究发现，分化/去分化的决定部分受细胞周期调节剂（CYB-1和x射线）和mRNA衰变因子（CGH-1和CAR-1）的调节。在本项目中，研究人员将研究两个假设：在PUF-8和LIP-1缺失的情况下，(1)精母细胞去分化需要细胞周期阻滞和细胞存活途径的激活（目的1）；(2)mRNA衰变因子的失活诱导mRNA的异位翻译，从而启动精母细胞去分化（目的2）。为了实现这一目标，研究人员将采用多学科方法，包括RNAi筛选、遗传/功能分析、基于CRISPR/ cas9的基因组编辑、基于x射线的生物物理分析和高分辨率多体分析。该研究结果不仅将阐明体内分化/去分化决定的基本机制，而且还将为确定去分化介导的细胞再生和肿瘤发生的治疗靶点提供新的工作平台。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Throughout their lifespan, stem cells make several critical fate decisions: the initial decision between self-renewal (remaining pluripotent) and differentiation, and then the subsequent decision between whether to continue in the differentiation path or to return to an undifferentiated state, i.e., to engage in a form of dedifferentiation. Defects in cellular machineries governing differentiation decisions can result in loss of a specific cell type, overpopulation of undifferentiated cells, or overt growth of cancer cells. Over the past decade, the molecular mechanisms related to the initial decision have been described in major model organisms. However, an understanding of the underlying determinants of the subsequent decisions is vague at best. The long-term research objectives are to elucidate the mechanisms that cause differentiating cells to return to an undifferentiated state, and to develop an animal model to interrogate these mechanisms in the living organism. A molecular understanding of this cellular event holds promise for the development of powerful tools in stem cell biology, regenerative medicine, and oncotherapy. The project also includes a component in which traditionally underserved populations of students in North Carolina (minority high school students, community college and undergraduate students) will be actively involved in the basic research.This project addresses three key questions concerning the fundamental molecular controls of a known dedifferentiation mechanism: (1) How do differentiating cells maintain their state or return to undifferentiated cells? (2) What regulatory network is involved in this cellular process in vivo? (3) Is aberrant regulation of this network associated with oncogenesis? Using the nematode C. elegans germline as a model system, the investigators have previously reported that PUF-8 (an RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) are required to (a) maintain the meiosis (differentiating state) of spermatocytes, and (b) prevent their dedifferentiation into mitotic cells (undifferentiating state) by inhibiting MPK-1 (an ERK homolog) signaling. Further studies found that the differentiation/dedifferentiation decision is regulated in part by cell cycle regulators (CYB-1 and X-ray) and mRNA decay factors (CGH-1 and CAR-1). In this project, the researchers will investigate two hypotheses: In the absence of PUF-8 and LIP-1, (1) spermatocyte dedifferentiation requires cell cycle arrest and the activation of cell survival pathways (Objective 1), and (2) inactivation of mRNA decay factors induces the ectopic translation of mRNAs that initiate spermatocyte dedifferentiation (Objective 2). To accomplish this goal, the researchers will take multidisciplinary approaches, including RNAi screening, genetic/functional analysis, CRISPR/Cas9-based genome editing, X-ray-based biophysical analysis, and high-resolution polysome profiling. Findings from the study will not only elucidate the fundamental mechanisms of the differentiation/dedifferentiation decision in vivo, but they will also provide a novel working platform for the identification of therapeutic targets for dedifferentiation-mediated cellular regeneration and tumorigenesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.