Analysis of genetic enhancers of Ras/MAPK signaling in C. elegans

从单个细胞发育成多细胞动物是一个非常复杂的过程，需要细胞的协调和沟通。一些高度保守的信号转导通路被反复用于调节发育的许多方面。在线虫中，Ras/MAPK信号调节着多种发育事件，包括生殖细胞的减数分裂进程、卵母细胞的生长、细胞命运的指定和细胞的迁移。为了让RAS/MAPK信号引发不同的生物事件，必须有独特的调节器和下游靶点来定制信号，以便达到适当的结果。*我们在排泄管细胞(EDC)的规范过程中发现了几个RAS/MAPK信号的正调控因子。EDC是组成排泄系统的三个管状细胞之一。缺少EDC的动物会积聚废液，幼虫会死亡。这些正性调节因子大多促进RAS/MAPK信号转导，特别是在EDC中。然而，UBC-25似乎通过RAS/MAPK信号通路在促进卵母细胞生长方面发挥了第二个作用。我们NSERC研究计划的目标是了解这些细胞类型特定的调节因子促进RAS/MAPK信号转导的机制。本研究的目的是：1)研究UBC-25和RAS/MAPK信号在卵母细胞生长中的作用；2)研究Ekl-7在EDC调控中的作用；3)确定未克隆的调控因子的分子特性。*UBC-25是一种保守的E2泛素结合酶。除了决定EDC的命运外，UBC-25还通过RAS/MAPK信号调节卵母细胞的生长，而不是减数分裂进程，这为确定RAS/MAPK促进卵母细胞生长的机制提供了独特的机会。我们假设UBC-25拮抗RAS/MAPK信号的负调控因子来调节卵母细胞的生长和EDC的命运。EKL-7缺乏已知的功能结构域，但有一簇MAPK ERK磷酸化位点。Ekl-7在基因上与已知的ERK底物相互作用。我们假设Ekl-7是RAS/MAPK信号的直接靶点，与其他ERK底物协同作用来决定EDC的命运。最后，我们将完成在EDC中促进RAS/MAPK信号转导的两个新基因突变的遗传图谱，以确定它们的分子身份，以确定它们如何提供信号特异性。这项研究将对UBC-25和Ekl-7蛋白的分子功能、调节Ras/MAPK信号的机制以及Ras/MAPK如何调节卵母细胞生长提供新的见解。这项工作将为HQP在生物科学和工程领域的职业生涯提供批判性思维、模型构建、科学写作以及分子、遗传和细胞生物学技能和知识方面的培训。

Development of a multicellular animal from a single cell is a highly complex process requiring cell coordination and communication. Several highly-conserved signal transduction pathways are repeatedly used to regulate many aspects of development. In the nematode C. elegans, Ras/MAPK signaling regulates several developmental events including meiotic progression of germ cells, oocyte growth, cell fate specification and cell migration. For Ras/MAPK signaling to elicit different biological events, there must be unique regulators and downstream targets that tailor signaling such that the appropriate outcomes are reached. ******We identified several positive regulators of Ras/MAPK signaling during the specification of the excretory duct cell (EDC). The EDC is one of three tubular cells that make up the excretory system. Animals missing the EDC accumulate waste fluid and die as larvae. Most of these positive regulators promote Ras/MAPK signaling specifically in the EDC. However, UBC-25 appears to have a second role in promoting oocyte growth with Ras/MAPK signaling. The objective of our NSERC research program is to understand the mechanisms by which these cell type specific regulators promote Ras/MAPK signaling. The objectives of this proposal are 1) characterize the role of UBC-25 and Ras/MAPK signaling in oocyte growth, 2) characterize the role of EKL-7 in specification of the EDC and 3) determine the molecular identities of uncloned regulators. ******UBC-25 is a conserved E2 ubiquitin-conjugating enzyme. In addition to specifying the EDC fate, UBC-25 functions with Ras/MAPK signaling to regulate oocyte growth, but not meiotic progression providing a unique opportunity to determine the mechanisms by which Ras/MAPK promotes oocyte growth. We hypothesize that UBC-25 antagonizes a negative regulator of Ras/MAPK signaling to regulate oocyte growth and the EDC fate. EKL-7 lacks known functional domains, but has a cluster of MAPK Erk phosphorylation sites. EKL-7 genetically interacts with known Erk substrates. We hypothesize that EKL-7 is a direct target of Ras/MAPK signaling that cooperates with other Erk substrates to specify the EDC fate. Finally, we will complete genetic mapping of two novel mutations in genes that promote Ras/MAPK signaling in the EDC to determine their molecular identities to determine how they provide specificity to signaling. This research will provide new insights into the molecular functions of UBC-25 and EKL-7 proteins, the mechanisms that regulate Ras/MAPK signaling, and how Ras/MAPK regulates oocyte growth. This work will train HQP in critical thinking, model building, scientific writing, and molecular, genetic, and cell biological skills and knowledge for careers in the biological sciences and engineering.