Transcriptional Regulation of Cell Differentiation in C. elegans by Tailless

描述（由申请人提供）：秀丽隐杆线虫的nhr-67/无尾基因编码一种核受体转录因子，该因子在调节细胞分化和最终器官发生中起核心作用。nhr-67基因的人类版本Tlx已被证明是神经干细胞身份的关键调节因子。在脊椎动物中，当Tlx基因受损时，神经干细胞不能作为分裂干细胞维持。因此，了解Tlx如何调节其他基因是理解神经干细胞的核心，也是确定神经退行性疾病治疗潜在候选药物的重要第一步。为此，Wightman实验室正在以子宫细胞分化为模型，研究nhr-67的上下游调控途径。nhr-67最有趣的靶标是细胞-细胞信号受体Notch （lin-12）及其同源信号δ (lag-2)。在人类中，Notch与主动脉瓣疾病和癌症相关的发育机制有关。虽然Notch也被认为在脊椎动物和无脊椎动物的神经发育中起作用，但这项研究是第一次将Tlx功能与Notch调节联系起来。本研究旨在通过鉴定nhr-67的上游基因和nhr-67功能的下游目标基因来促进对基于tlx的调控系统的理解。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、LAG-1 Notch反应介质、sex1核受体）。bHLH转录因子HLH-2在子宫早期腹侧发育中具有明确的作用，将作为nhr-67表达的候选直接激活因子在体内进行评估。该研究将探讨基于Notch的反馈回路如何设置nhr-67的转录水平，并验证性别-1胚胎性别决定功能在性别特异性器官发生中由后来的功能补充的假设。第二个主要目的是评估nhr-67在腹侧子宫功能的下游介质，识别和研究nhr-67对分化事件的潜在影响。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，研究nhr-67与珠蛋白基因glb-12之间的关系，并利用新的细胞核纯化和差异表达策略，结合已建立的细胞分选，确定意想不到的新靶点

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.