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

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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.