Transcriptomic Analysis of Trout Gill Ionocytes in Fresh Water and Sea Water Using Laser Capture Microdissection Combined with Microarray Analysis

Abstract

Fish gills represent a complex organ composed of several cell types that perform multiple physiological functions. Among these cells, ionocytes are implicated in the maintenance of ion homeostasis. However, because the ionocyte represents only a small percent of whole gill tissue, its specific transcriptome can be overlooked among the numerous cell types included in the gill. The objective of this study is to better understand ionocyte functions by comparing the RNA expression of this cell type in freshwater and seawater acclimated rainbow trout. To realize this objective, ionocytes were captured from gill cryosections using laser capture microdissection after immunohistochemistry. Then, transcriptome analyses were performed on an Agilent trout oligonucleotide microarray. Gene expression analysis identified 108 unique annotated genes differentially expressed between freshwater and seawater ionocytes, with a fold change higher than 3. Most of these genes were up-regulated in freshwater cells. Interestingly, several genes implicated in ion transport, extracellular matrix and structural cellular proteins appeared up-regulated in freshwater ionocytes. Among them, several ion transporters, such as CIC2, SLC26A6, and NBC, were validated by qPCR and/or in situ hybridization. The latter technique allowed us to localize the transcripts of these ion transporters in only ionocytes and more particularly in the freshwater cells. Genes involved in metabolism and also several genes implicated in transcriptional regulation, cell signaling and the cell cycle were also enhanced in freshwater ionocytes. In conclusion, laser capture microdissection combined with microarray analysis allowed for the determination of the transcriptional signature of scarce cells in fish gills, such as ionocytes, and aided characterization of the transcriptome of these cells in freshwater and seawater acclimated trout.

Hepatocellular adenoma in a European flatfish (Limanda limanda): Genetic alterations in laser-capture micro-dissected tissue and global transcriptomic approach

“…A total of 82 successfully ligated plasmids were cloned, isolated and sequenced. The success rate for unique sequences obtained from the SSH experiment was 30.5%, which is comparable to similar studies using non-model fish (8%) (Urbatzka et al, 2013); lower relative to studies using fish model organisms (45-80%) (Marchand et al, 2006;Luckenbach et al, 2008;Leguen et al, 2015); and an improvement compared with inveretebrate species such as molluscs (6-22%) (Boutet et al, 2008;Craft et al, 2010;Ciocan et al, 2011). Duplicate sequences accounted for 27% of clones and the remaining 43% of clones could not be identified based on sequence similarities.…”

“…Laser Capture-Microdissection (LCM) allows the ability to view and dissect target cells microscopically, thereby providing a direct link between a specific histopathological lesion and the molecular profile of that lesion (Gillespie et al, 2001). LCM has previously been applied to pathological studies in aquatic organisms including fish (Vinas and Piferrer, 2008;Jorgensen et al, 2009;Kitahashi et al, 2009;Lerebours et al, 2013;Nowak et al, 2013;Leguen et al, 2015;Schaeck et al, 2016), crustaceans (Small et al, 2008), and cnidarians (Wiebring et al, 2010).…”

Carbonic anhydrase expression in the branchial ionocytes of rainbow trout

“…Both H + -ATPase (Galvez et al, 2002) and CAc ( present study) are expressed by at least a subset of PNA + ionocytes. The key element of this model, the apical Cl − /HCO 3 − exchanger, remains uncertain but recently a member of the solute carrier 26 family of anion exchangers, SLC26a6, was cloned from trout (Boyle et al, 2015) and found to be expressed in a subset of ionocytes (Leguen et al, 2015; this study did not distinguish between ionocyte types). However, the relatively low number of PNA + ionocytes that exhibited CAc immunofluorescence under control conditions (i.e.…”

A single nuclei transcriptomic analysis of the Atlantic salmon gill through smoltification and seawater transfer

“…For example, the accessory cell signature included highly restricted expression of Slc26a6, an apical membrane Cl -/HCO3 exchanger, associated with gill function but until now misattributed to expression within MRCs 25,26 . We were interested to note that the markers defining the erythrocyte population, including beta-globin, were expressed widely among all cell types.…”