Transcriptional Regulation of Cell Differentiation in C. elegans by Tailless

描述(申请人提供)：线虫的NHR-67/无尾基因编码一种核受体转录因子，在调节细胞分化和最终器官发生方面发挥核心作用。人类版本的NHR-67基因TLX已被证明是神经干细胞特性的关键调节因子。当TLX基因在脊椎动物中受到损害时，神经干细胞不能作为分裂干细胞维持。因此，了解TLX如何调控其他基因是理解神经干细胞的核心，也是确定治疗神经退行性疾病的潜在候选者的重要第一步。为此，怀特曼实验室正在研究NHR-67上游和下游的调控途径，以子宫细胞分化为模型。NHR-67最有趣的靶点是细胞-细胞信号受体Notch(LIN-12)及其同源信号Delta(LAG-2)。在人类中，Notch与与主动脉瓣疾病和癌症相关的发育机制有关。虽然Notch也已知在脊椎动物和无脊椎动物的神经发育中发挥作用，但这项研究是第一次将TLX功能与Notch调节联系起来。这项研究试图通过识别NHR-67的上游基因和NHR-67功能的下游靶基因来促进对TLX调控系统的理解。第一个主要目的是确定腹侧子宫NHR-67转录的上游调控因子，将通过体外和体内相结合的方法评估候选上游转录因子(bHLH异源二聚体、Notch反应的LAG-1介导物、性别-1核受体)。在早期腹侧子宫发育中具有明确作用的bHLH转录因子HLH-2，将作为NHR-67表达的候选直接激活剂在体内进行评估。这项研究将调查基于Notch的反馈环如何设置NHR-67的转录水平，并测试性别-1胚胎性别决定功能得到性别特异器官发生中后来功能的补充的假设。第二个主要目的是评估腹侧子宫中NHR-67功能的下游介质，将识别和研究NHR-67在分化事件中的潜在效应。该项目的这一方面将阐明Notch调控作为NHR-67功能的关键特征的作用，检查NHR-67和珠蛋白基因GLB-12之间的关系，并使用新的核纯化和差异表达策略，结合已建立的细胞分选策略，确定意想不到的新靶点 策略。差异表达策略的成功完成将展示一种其他研究人员可以用来研究少数细胞中基因表达的方法。这些分子和遗传策略将提供对支配重要发育事件的调控事件的更好理解。这项研究确定的靶点将为未来控制神经干细胞规格和器官发生的分子途径的研究提供重要的候选对象。

DESCRIPTION (provided by applicant): The nhr-67/tailless gene of C. elegans encodes a nuclear receptor transcription factor that plays a central role in regulating cell differentiation,and ultimately organogenesis. The human version of the nhr-67 gene, Tlx, has been shown to be a key regulator of neural stem cell identity. When the Tlx gene is compromised in vertebrates, neural stem cells cannot be maintained as dividing stem cells. Therefore, an understanding of how Tlx regulates other genes is central to understanding neural stem cells, and an important first step in identifying potential candidates for therapy in neurodegenerative disease. Towards this end, the Wightman Lab is studying the regulatory pathways both upstream and downstream of nhr-67, using the differentiation of uterine cells as a model. Among the most interesting targets of nhr-67 are the cell-cell signaling receptor Notch (lin-12) and its cognate signal delta (lag-2). In humans, Notch has been linked to developmental mechanisms associated with aortic valve disease and cancer. While Notch is also known to function in vertebrate and invertebrate neural development, this study is among the first to link Tlx function to Notch regulation. This study seeks to advance understanding of Tlx-based regulatory systems by identifying both genes that are upstream of nhr-67 and those that are downstream targets of nhr-67 function. The first major aim, to determine the upstream regulators of nhr-67 transcription in the ventral uterus, will evaluate candidate upstream transcription factors (bHLH heterodimers, LAG-1 mediator of Notch response, SEX-1 nuclear receptor) through a combination of in vitro and in vivo approaches. The bHLH transcription factor, HLH-2, which has a well-defined role in early ventral uterine development, will be evaluated in vivo as a candidate direct activator of nhr-67 expression. The study will investigate how a Notch- based feedback loop sets transcriptional levels of nhr-67, and test the hypothesis that the sex-1 embryonic sex determination function is complemented by a later function in sex-specific organogenesis. The second major aim, to evaluate the downstream mediators of nhr-67 function in the ventral uterus, will identify and study potential effectors of NHR-67 for differentiation events. This aspect of the project will clarify the role of Notch regulation as a key feature of nhr-67 function, examine the relationship between nhr-67 and a globin gene, glb-12, and identify unanticipated new targets using a new nuclei purification and differential expression strategy, combined with an established cell-sorting strategy. Successful completion of the differential expression strategy will demonstrate how a method by which other researchers can study gene expression in a small number of cells. These molecular and genetic strategies will provide a better understanding of the regulatory events that govern important developmental events. The targets identified by this study will provide important candidates for future investigations of the molecular pathways that control specification of neural stem cells and organogenesis.