Single-cell transcriptomic analysis of neuroepithelial cells and other cell types of the gills of zebrafish (Danio rerio) exposed to hypoxia

Abstract

The fish gill is a multifunctional organ involved in numerous physiological processes, such as gas exchange and sensing of hypoxia by respiratory chemoreceptors, called neuroepithelial cells (NECs). Many studies have focused on zebrafish (Danio rerio) to investigate the structure, function and development of the gills, yet the transcriptomic profile of most gill cells remains obscure. We present the results of a comprehensive transcriptomic analysis of the gills of zebrafish using single-cell RNA sequencing (scRNA‐seq). Gill cells from ETvmat2:EGFP zebrafish were individually labelled before scRNA‐seq library construction using 10× Genomics Chromium technology. 12,819 cells were sequenced with an average depth of over 27,000 reads per cell. We identified a median of 485 genes per cell and 16 cell clusters, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. The identity of NECs was confirmed by expression of slc18a2, encoding the vesicular monoamine transporter, Vmat2. Highly differentially-expressed genes in NECs included tph1a, encoding tryptophan hydroxylase, sv2 (synaptic vesicle protein), and proteins implicated in O2 sensing (ndufa4l2a, cox8al and epas1a). In addition, NECs and neurons expressed genes encoding transmembrane receptors for serotonergic, cholinergic or dopaminergic neurotransmission. Differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of acclimation to hypoxia. NECs in the hypoxia group showed high expression of genes involved in cell cycle control and proliferation. The present article provides a complete cell atlas for the zebrafish gill and serves as a platform for future studies investigating the molecular biology and physiology of this organ.

Neurochemical Signalling Associated With Gill Oxygen Sensing and Ventilation: A Receptor Focused Mini-Review

“…Recent single-cell transcriptomic analysis of chemosensory NECs and other cell types of the gills of adult zebrafish has begun to provide compelling evidence for potential actions of these receptor types by localizing them to, and within, the gill. The gene encoding 5-HT 3A -like receptors shows highest expression in gill neurons ( Pan et al, 2022 ). As NECs are innervated by at least one population of intrabranchial neurons, as well as extrabranchial nerve fibres ( Jonz et al, 2004 ), there is a plausible mechanism for chemotransduction via 5-HT 3A receptors in the gill.…”

“…Interestingly, the gene encoding the inhibitory 5-HT 1A receptor, htr1ab , was found to be most abundant in NECs above any other cell type in the gill ( Pan et al, 2022 ). This may suggest a specific O 2 -sensing role for these receptors, possibly through a negative feedback or autoreceptor mechanism, which has not yet been explored pharmacologically.…”

“…The nicotinic receptor α2b subunit gene, chrna2b , was highly expressed in NECs and neurons; whereas the α6 subunit gene ( chrna6 ) and β3a subunit gene ( chrnb3a ) were expressed primarily in NECs, and the β4 subunit gene ( chrnb4 ) was mainly present in neurons ( Pan et al, 2022 ). The location of these subunits supports a model in which VAChT-positive cells release ACh during hypoxic stimulation, leading to excitatory post-synaptic or paracrine effects on ACh receptors of neurons or NECs ( Pan et al, 2022 ). Like NECs, VAChT-positive cells of the zebrafish gills are closely apposed to nerve fibres ( Zachar et al, 2017 ).…”

“…( Shakarchi et al, 2013 ). RNA sequencing of the adult zebrafish gill detected dopamine receptors in the D 2 -like family, including drd4a in NECs and drd3 highly expressed in neurons ( Pan et al, 2022 ). This recent work using zebrafish suggests an inhibitory dopaminergic mechanism in the gill, possibly via D 2 -like receptors, and is an area for potential future developments.…”

“…RNA sequencing of the adult zebrafish gills has further narrowed down potential ACh receptor subunit targets within the gill. The nicotinic receptor α2b subunit gene, chrna2b, was highly expressed in NECs and neurons; whereas the α6 subunit gene (chrna6) and β3a subunit gene (chrnb3a) were expressed primarily in NECs, and the β4 subunit gene (chrnb4) was mainly present in neurons (Pan et al, 2022). The location of these subunits supports a model in which VAChT-positive cells release ACh during hypoxic stimulation, leading to excitatory post-synaptic or paracrine effects on ACh receptors of neurons or NECs (Pan et al, 2022).…”