Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing
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abstract
All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
