Terminal Differentiation in C. elegans

线虫的终末分化

• 批准号: 9305208
• 负责人: Ann Rougvie
• 金额: $ 41.18万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1997-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9305208&HistoricalAwards=false
• 关键词: Terminal; Differentiation; C.; elegans

The proper form of a multicellular organism requires precise control of terminal differentiation events. This research will address how a single terminal differentiation event is executed during the development of a simple model organism, the nematode C. elegans. The analysis will focus on the terminal differentiation of lateral hypodermal "seam" cells, which terminally differentiate during the final (4th) molt. Several distinct cellular processes are coordinately controlled during this terminal differentiation event, including cell cycle, cell fusion, and the stage-specific regulation of cuticle gene expression. A gene called lin-29 has been identified as a pivotal component in programming the execution of seam cell terminal differentiation. Loss of lin-29 function causes hypodermal cells of the adult stage to reiterate indefinitely the larval program of cell divisions instead of exiting the cell cycle and differentiating. lin-29 encodes a putative transcription factor of the (Cys)2-(His)2 class, and, thus likely programs these events by regulating the transcription of other genes. The major objective of this work is to identify genes controlled by the lin-29 protein. Three approaches will be employed to identify lin-29-regulated genes. ı1! Interactions of lin-29 protein with predicted target genes: The stage-specific expression of the cuticle collagen genes, col-17 and col-19, will be analyzed. col-17 expression is transcriptionally repressed during the larval/adult switch, whereas col-19 expression is activated. The role of lin-29 in the alternative repression and activation of these genes will be tested. ı2! Differential hybridization screens to identify genes whose expression is influenced by the presence or absence of lin-29 protein: Genes isolated in this fashion will be examined for direct regulation by lin-29, and those directly regulated will be characterized further. ı3! Development of a protein-DNA UV crosslinking method to isolate DNA sequences bound by a specific protein in vivo: In the future, this method will be used to identify genes bound by lin-29 protein in vivo, and should also be applicable to the isolation of genes controlled by other C. elegans gene-regulatory proteins. Genes involved in cell cycle exit and cell fusion, as well as additional stage-specifically expressed cuticle genes, should be identified by these methods. Analysis of the types of genes regulated by lin-29 in vivo will lead to an understanding of how a complex terminal differentiation program is orchestrated at a specific time during development.

多细胞生物的正确形式需要精确控制终末分化事件。本研究将解决在简单模型生物体——线虫——秀丽隐杆线虫的发育过程中如何执行单个终末分化事件。分析将集中于终末分化。外侧皮下“接缝”细胞在最后（第四次）蜕皮过程中最终分化，在此最终分化过程中协调控制几个不同的细胞过程，包括细胞周期、细胞融合和角质层基因的阶段特异性调节。一种名为 lin-29 的基因已被确定为执行缝细胞终末分化的关键成分，lin-29 功能的丧失会导致成虫阶段的皮下细胞无限地重复细胞分裂的幼虫程序。 lin-29 编码 (Cys)2-(His)2 类的假定转录因子，因此可能通过调节其他基因的转录来编程这些事件。这项工作旨在识别 lin-29 蛋白控制的基因，将采用三种方法来识别 lin-29 蛋白与预测靶基因的相互作用： 角质层胶原蛋白的阶段特异性表达。将分析 col-17 和 col-19 基因在幼虫/成虫转换过程中转录受到抑制，而 lin-29 表达则被激活。将测试这些基因的抑制和激活。 ı2! 差异杂交筛选以确定其表达受 lin-29 蛋白存在或不存在影响的基因：将检查以这种方式分离的基因是否受 lin-29 直接调节，以及ı3! 开发蛋白质-DNA UV 交联方法来分离体内特定蛋白质结合的 DNA 序列：将来，该方法将用于鉴定 lin-29 蛋白质结合的基因。体内，并且还应适用于分离由其他秀丽隐杆线虫基因调节蛋白控制的基因。参与细胞周期退出和细胞融合的基因，以及其他阶段特异性表达的角质层基因，应通过这些方法进行鉴定。对体内 lin-29 调节的基因类型的分析将有助于了解复杂的终末分化程序在发育过程中的特定时间是如何编排的。