Collaborative Research: Identifying osmosensitive molecular targets using a unique vertebrate model

本研究探讨了特化细胞如何感知和响应外部环境的变化，进而控制维持稳定内部环境的生理系统。盐和水平衡的调节，或渗透调节，是包括人类在内的大多数脊椎动物的基础。检测渗透浓度或盐度的变化是成功渗透调节的必要的第一步。然而，由于渗透受体细胞在许多动物大脑中的复杂结构和排列，对渗透受体细胞如何工作的理解仍然不完整，这使得开发有效的实验方法变得困难。这个问题通过罗非鱼催乳素细胞的研究得到了解决，因为这些细胞在脑垂体中排列成一个几乎均匀的团块。罗非鱼是一种河口鱼类，可以在从淡水到高于全强度的海水的盐度范围内茁壮成长，而催乳素激素参与了罗非鱼的渗透调节。本项目以罗非鱼催乳素细胞模型为手段，在机体水平上独特地将渗透受体细胞的运作与环境适应联系起来。这项合作研究将夏威夷大学马诺阿分校和斯基德莫尔学院的研究、教学和推广项目联系起来，并将以适用于所有脊椎动物的方式揭示支持渗透作用的机制。它将使来自未被充分代表的群体的研究生和本科生沉浸在合作机构严格的跨学科教育项目中。本研究的科学成果将通过机构外展教育计划、研讨会和专题讨论会、海报、外行新闻文章、指导参观研究设施和网站传达给广泛的受众。这个合作项目的总体目标是进一步了解渗透接受的分子机制，渗透接受是一种基本的，尽管尚未得到充分研究的感觉模式。罗非鱼催乳素细胞允许识别渗透接受的一些机制，如细胞外Ca2+通过机械敏感的瞬时受体电位香草蛋白通道进入和cAMP积累。尽管如此，人们对泌乳素（prl）基因如何被低渗刺激激活知之甚少。通过在表达含有罗非鱼prl启动子的载体的胚胎肾细胞（HEK293）中使用双荧光素酶报告基因检测系统，对细胞外渗透压变化激活的次级信使作出反应的特定启动子区域将被表征，从而允许鉴定介导prl基因低渗诱导的新型转录因子。在发现催乳素特异性调节因子的基础上，可以在其他脊椎动物基因组中寻找响应细胞外渗透压变化的保守基因序列。该项目将通过鉴定在脊椎动物中介导低氧诱导基因转录调控的启动子序列来推进该领域。研究生和本科生将受益于参与研究项目和计划的教育活动。研究结果将广泛传播，包括通过网站和公共宣传项目，包括与夏威夷大学海洋生物研究所和海洋基金合作的项目。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。

This research addresses how specialized cells sense and respond to changes in the external environment and, in turn, control the physiological systems that maintain a stable internal environment. The regulation of salt and water balance, or osmoregulation, is fundamental to most vertebrates, including humans. Detection of a change in osmotic concentration or salinity is the necessary first step in successful osmoregulation. Nevertheless, understanding of how osmoreceptor cells work remains incomplete because of their complex structure and arrangement among other cells in the brain of many animals, making it difficult to develop effective experimental approaches. This problem is solved by study of the prolactin cells of the tilapia fish, because these cells are arranged into a nearly homogeneous mass in the pituitary gland. The hormone prolactin is involved in osmoregulation in tilapia, an estuarine fish that can thrive in salinities ranging from fresh water to greater than full-strength seawater. This project uses the tilapia prolactin cell model as a means to uniquely connect the operation of osmoreceptor cells with environmental adaptation at the organismal level. The collaborative study links the research, teaching, and outreach programs at the University of Hawai'i at Manoa and Skidmore College and will reveal the mechanisms supporting osmoreception in a manner that is applicable to all vertebrates. It will immerse graduate and undergraduate students from underrepresented groups in rigorous and interdisciplinary educational programs at the collaborating institutions. The scientific outcomes of this research will be conveyed to a broad audience through institutional outreach educational programs, workshops and symposia, posters, lay press articles, guided visits to research facilities, and a website.The overarching goal of this collaborative project is to further the understanding of the molecular mechanisms of osmoreception, a fundamental, albeit understudied, sensory modality. Tilapia prolactin cells have allowed for the identification of some of the mechanisms underlying osmoreception, such as extracellular Ca2+ entry through mechanosensitive transient-receptor-potential vanilloid channels and cAMP accumulation. Nonetheless, little is known about how the prolactin (prl) gene is activated by hyposmotic stimuli. By employing a dual luciferase reporter assay system in embryonic kidney cells (HEK293) expressing a vector containing the tilapia prl promoter, the specific promoter regions that respond to secondary messengers activated by changes in extracellular osmolality will be characterized, thereby allowing for the identification of novel transcription factors that mediate hyposmotic induction of the prl gene. Based on the discovery of specific regulators of prolactin, other vertebrate genomes can be searched for conserved gene sequences that respond to changes in extracellular osmolality. The project will advance the field by identifying promoter sequences that mediate transcriptional regulation of hyposmotically-induced genes in vertebrates. Graduate and undergraduate students will benefit from involvement in the research project and the planned educational activities. The results will be broadly disseminated, including via a website and public outreach programs, including those in association with the University of Hawai'i Institute of Marine Biology and Sea Grant.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.