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

这项研究解决了专门的细胞如何感知和响应外部环境的变化，进而控制维持稳定的内部环境的生理系统。盐和水平衡的调节，或渗透调节，对包括人类在内的大多数脊椎动物来说都是基本的。检测渗透浓度或盐度的变化是成功渗透调节的必要第一步。然而，由于渗透压感受器细胞与许多动物大脑中的其他细胞之间的复杂结构和排列，对渗透压感受器细胞如何工作的了解仍然不完整，这使得开发有效的实验方法变得困难。通过对罗非鱼催乳素细胞的研究，这个问题得到了解决，因为这些细胞在脑下垂体中排列成几乎均匀的团块。催乳素激素参与了罗非鱼的渗透调节，罗非鱼是一种河口鱼类，可以在从淡水到高于全强度海水的盐度范围内茁壮成长。该项目使用罗非鱼催乳素细胞模型作为一种手段，将渗透压感受器细胞的运作与生物水平的环境适应独特地联系起来。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在一起，并将以适用于所有脊椎动物的方式揭示支持渗透接受的机制。它将使来自代表性不足群体的研究生和本科生沉浸在合作机构的严格和跨学科教育计划中。这项研究的科学成果将通过机构外展教育计划、研讨会和座谈会、海报、平实的新闻文章、对研究设施的有指导的访问和网站向广大受众传达。这一合作项目的首要目标是进一步了解渗透感受的分子机制，渗透感受是一种基本的、尽管研究较少的感觉模式。罗非鱼催乳素细胞已被用于鉴定渗透感受的一些机制，如通过机械敏感的瞬时受体电位香草素通道进入细胞外钙和cAMP积累。然而，关于催乳素(PRL)基因是如何在低渗刺激下被激活的，人们知之甚少。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系统，将对由细胞外渗透压变化激活的二级信使作出反应的特定启动子区域进行表征，从而能够识别介导低渗诱导PRL基因的新的转录因子。基于催乳素特定调节剂的发现，可以搜索其他脊椎动物基因组，寻找对细胞外渗透压变化做出反应的保守基因序列。该项目将通过识别在脊椎动物中介导低渗诱导基因转录调控的启动子序列来推进这一领域。研究生和本科生将从参与研究项目和计划的教育活动中受益。结果将广泛传播，包括通过一个网站和公共推广计划，包括那些与夏威夷大学海洋生物研究所和海洋赠款有关的计划。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.