High-throughput RNA sequencing reveals the effects of 2,2',4,4' -tetrabromodiphenyl ether on retina and bone development of zebrafish larvae

Abstract

Background: 2,2',4,4'-Tetrabromodiphenyl ether (BDE47) is a prevalent environmental pollutant and has been demonstrated to be a serious toxicant in both humans and animals, but little is known about the molecular mechanism underlying its toxic effect on the early development of vertebrates. BDE47-treated zebrafish larvae were found to present the light-related locomotion reduction in our previous study, therefore, we aimed to use high throughput sequencing to investigate the possible reasons from a transcriptomic perspective.

Results: By exposing zebrafish embryos/larvae to 5 μg/l and 500 μg/l BDE47, we measured the influence of BDE47 on the mRNA expression profiles of zebrafish larvae until 6 days post-fertilization, using Illumina HiSeq 2000 sequencing. Differential expression analysis and gene enrichment analysis respectively revealed that a great number of genes, and gene sets based on two popular terminologies, were affected by the treatment of 500 μg/l BDE47. Among them, BDE47 caused changes in the retinal metabolism and related biological processes involving eye morphogenesis and visual perception, as confirmed by disordered photoreceptor arrangement and thickened bipolar cell layer of larval retina from histological observations. Other altered genes such as pth1a and collaborative cathepsin family exhibited disrupted bone development, which was consistent with the body curvature phenotype. The transcriptome of larvae was not significantly affected by the treatment of 5 μg/l BDE47, as well as the treatment of DMSO vehicle.

Conclusions: Our results suggest that high BDE47 concentrations disrupt the eye and bone development of zebrafish larvae based on both transcriptomic and morphological evidences. The abnormal visual perception may result in the alteration of dark adaption, which was probably responsible for the abnormal larval locomotion. Body curvature arose from enhanced bone resorption because of the intensive up-regulation of related genes. We also proposed the larval retina as a novel potential target tissue for BDE47 exposure.

Figure 1

Venn diagram showing the effects of BDE47 exposure on the detected genes. Genes were grouped on the basis of their expression changes under different BDE47 concentrations. Expressions were evaluated on the basis of the number of reads per gene model. The shaded areas represent the existence of no intersection between two subsets.

Figure 2

Functional enrichment analysis based on different gene expression patterns. (A) Expression profiles in color indicate significant ones (p < 0.05). Green, up-regulated; red, down-regulated. Profile number (up left), gene number (bottom left), and trend (line) in each profile were labelled. The significant terms were graphed using (B) GO annotation (p < 0.001, FDR < 0.05), and (C) KEGG pathway annotation (p < 0.05). Tawny block, pattern 4; teal block, pattern 3; red block, pattern 0.

Figure 3

Gene regulatory network of 6 dpf zebrafish larvae under BDE47 treatments. The abbreviations on arrows between two nodes reflect their regulatory relationship. A, activation; b, binding/association; c, compound; dep, dephosphorylation; e, expression; ind, indirect effect; inh, inhibition; p, phosphorylation. Node colors indicate gene expression pattern. Red, pattern 0; violet, pattern 1; green, pattern 2; teal, pattern 3; tawny, pattern 4; yellow, pattern 5; lilac, pattern 6; lavender, pattern 7.

Figure 4

Morphological observation of 6 dpf zebrafish larvae under BDE47 treatment and control. (A) Normal histological patterns of larval retina (40X). (B) Larvae exposed to 500 μg/l BDE47 had retinal morphology distinct from the control (40X). (C) Normal body type of zebrafish larvae in the control group (4X). (D) Body curvature phenotype after 500 μg/l BDE47 exposure (4X). RPE, retinal pigment epithelium; PCL, photoreceptor cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 5

Genes differentially transcribed related to vision formation and eye development in zebrafish larvae. Hierarchical-clustering-analysis-based transcription levels were performed on 25 related genes showing significant differential expression (p < 0.05) in larvae. Gene tree (left) and condition tree (top) were obtained using Pearson’s uncentered distance metric calculated from all log10 transcription ratios (exposed/controls). Color scale from green to red indicate log10 ratios from −1.00 (10-fold down-regulation) to +1.00 (10-fold up-regulation). C: control; S: vehicle (0.1% DMSO); 5: 5 μg/l BDE47 treatment; 500: 500 μg/l BDE47 treatment.