Maintenance of homeostasis of body fluids is one of most important topics in vertebrate physiology, and transepithelial transport is the major process in this mechanism. My laboratory has been focusing on fish transepithelial transport physiology with diversified approaches. The current models of ion regulation in fish gill mitochondria-rich cells (the major ionocyte) have been proposed mainly based on studies in traditional model species like salmon, trout, tilapia, eel, and killifish, but the mechanisms are still being debated due to the lack of convincing molecular physiological evidence. Recently, my lab took zebrafish’s advantages in plentiful genetic databases and various established molecular physiological approaches to open several new windows for the ion regulation mechanisms of fish.

 

(1) Identification and functional analysis of ionocytes  Based on our recent molecular physiological studies, a new model of ion regulatory mechanisms in zebrafish gill/skin ionocytes is proposed. In this new model, there are at least three subtypes of ionocytes (NaR cells, HR cells, and NCC-expressing cells) in zebrafish gill/skin, and relevant transporters and enzymes are thought to achieve the transport of different ions in the ionocyte subtypes, respectively. Although many unknown points deserve more exploration in the future, NaR cells and HR cells have been demonstrated to be responsible for Ca²⁺ uptake and H⁺ secretion/Na⁺ uptake, respectively. Serial studies have also been conducted to demonstrate how these transporters in ionocytes are differentially regulated in response to fluctuated environments.

(2) Differentiation of ionocytes  Effects of environmental factors and hormones on cell renewal and proliferation of fish gill MR cells have been another important issue in fish osmoregulation for a long time, but nothing was known about the molecular mechanisms behind the differentiations of MR cells and their subtypes until our recent study on zebrafish. In zebrafish embryos, two duplicated forkhead transcription factor, foxi3a and foxi3b, were identified as forming a positive regulatory loop for specification and differentiation of epidermal ionocyte. Knocking-down translation of the upstream regulatory genes for ionocyte differentiation provides a convincing molecular physiological approach for studying the in vivo functions of ionocytes.

(3) Energy metabolism for ion regulation  A sufficient energy supply is a prerequisite for the operation of ion regulation mechanisms in fish gill MR cells. Using subtractive PCR, a novel gill glycogen phosphrylase (GP) isoform was identified to specifically express in a group of gill cells, glycogen-rich (GR) cells, which surround ionocytes. Based on serial molecular physiological studies, we found that the spatial and functional relationships between mammalian neurons and astrocytes for rapid mobilization of local energy stores may also occur in gill MR and GR cells; GP expression in GR cells is stimulated by an acute salinity challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.

(4) Functional genomic studies on fish physiology  Microarray analysis was used to study the gene expression profiles in gills of zebrafish after acclimation to low temperature. The results showed that gill ionocytes may extend their lifespan by delaying natural cell death, and gills may sustain their functions by yielding mature ionocytes from preexisting undifferentiated progenitors during acclimation to low temperature, providing new insights into the cellular physiological mechanisms of survival and growth of ectothermic vertebrates in low-temperature environments.