Target Enrichment Transcriptomics of the Chemical Defensome of Zebrafish (Danio rerio)

Abstract

Toxicokinetics describes the movement of foreign chemicals through the body via absorption, distribution, metabolism, and excretion, processes that are mediated by an integrated network of genes collectively referred to as the chemical defensome. While the gene families that comprise the chemical defensome are well defined, the specific genes, their tissue-specific patterns of gene expression, and regulation remain poorly understood in fish. This thesis first examined publicly available RNA-seq datasets from adult zebrafish to assess basal chemical defensome gene expression across tissues and between sexes under unexposed conditions. These analyses provided evidence of tissue- and sex-specific defensome expression where data were available, but also highlighted substantial gaps in existing datasets, particularly in tissues relevant to toxicokinetics and consistent inclusion of both sexes. In this study, a bait target enrichment transcriptomics approach was developed to selectively quantify 595 chemical defensome genes in zebrafish. The approach was validated by direct comparison to unenriched whole transcriptomics using multiple read-mapping and quantification pipelines. Enriched and unenriched datasets were strongly correlated for gene abundance and differential expression, while enriched libraries improved detection of lowly expressed genes and reduced sequencing depth requirements. Enriched basal chemical defensome expression was quantified in the gill, intestine, liver, and kidney of adult male and female zebrafish. Defensome expression patterns were driven primarily by tissue type, with distinct tissue-specific expression patterns reflecting physiological roles in chemical uptake (gills, intestine), metabolism (liver), and excretion (kidney, intestine). Sex-specific differences were observed across all tissues and were pronounced in the liver, including strongly female-biased expression of cyp2k6 and multiple nuclear receptors. Taken together, these findings define tissue- and sex-specific features of basal chemical defensome expression and provide a validated, cost-effective approach for further investigating the coordinated regulation of chemical defense pathways in zebrafish.