CPB/CSZ Student Research Grant Winner Yihang Pan

Screening for transgenic zebrafish lines labeling chemoreceptors and ionocytes

In the spring of 2018, I was fortunate to receive the CPB/CSZ Student Research Grant, which allowed me to travel to the National Institute of Genetics in Mishima, Japan to work with Dr. Koichi Kawakami. Our lab has always been interested in examining the roles of chemoreceptors and ionocytes in regulating physiological processes. Because energy supply is a limiting factor constraining these processes, studying the energetic costs for a particular physiological process is highly relevant. Few studies have attempted to ascertain the metabolic cost of chemoreception or ionoregulation at the cellular level in fish, largely due to the lack of reliable methods for identifying the different sub-types of ionocytes, and chemoreceptors in vivo. Luckily for us, transgenic lines labeled for chemoreceptors and ionocytes might already exist in the fish rooms of Dr. Kawakami, awaiting to be screened. Using transposons coupled with gene trap and enhancer trap methods, Dr. Kawakami was able to generate a large number of transgenic fish that express the GFP reporter gene or the yeast Gal4 transcription activator in specific cells, tissues and organs, potentially some labeling chemoreceptors or ionocytes. It was my goal to identify them during my stay in Japan.

The trip turned out to be very rewarding. I was able to identify one fish line that labels neuroepithelial cells (NECs), a cell type that is presumed to be the oxygen chemoreceptor in fish, 4 fish lines that label H+-ATPase rich cells, 1 fish line that labels ionocytes expressing sodium chloride co-transporters (NCC), and 1 fish line that labels both H+-ATPase rich cells and ionocytes expressing NCC which can be differentiated through the intensity of the GFP reporter. Using the lines screed and characterized for chemoreceptors and ionocytes, we now have the ability to identify these cell types in vivo, and can try to measure oxygen consumption and ion fluxes at near cellular level. The metabolic costs of ion regulation and chemoreception would be examined by measuring the O2 consumption of target cells using SMOT, which uses micro-optrodes and computer-driven micro-manipulators to record boundary layer O2 tensions as the electrode is moved either toward or away from the surface of the cell to calculate oxygen fluxes. Ideally, it would be possible to distinguish the specific costs of ion transport and chemoreception from basal metabolism, and provide the first direct measurements of the O2 requirements of any epithelial ion-transporting cell or chemoreceptor at rest or when activated. It is anticipated that this research will lead to significant advances in our understanding of the metabolic costs of ion regulation and chemoreception in fish.