Research resource: whole transcriptome RNA sequencing detects multiple 1α,25-dihydroxyvitamin D(3)-sensitive metabolic pathways in developing zebrafish

Abstract

The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)(2)D(3)-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)(2)D(3). Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)(2)D(3) vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)(2)D(3)-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)(2)D(3) functions in vivo in developing eukaryotes.

Fig. 1.

Experimental design. Experiment 1: zebrafish embryos were treated with 1α,25(OH)₂D₃ (300 pm) or vehicle beginning at 24 hpf. Medium and additives were changed to every 24 h (black arrows). Five batches of 25–30, 1α,25(OH)₂D₃-treated, and five batches of 25–30 vehicle-treated zebrafish embryos/larvae were collected at 2, 4, 6, and 7 dpf (red arrows) for isolation of RNA, construction of cDNA libraries, and sequencing. Experiment 2: zebrafish larvae were treated with 1α,25(OH)₂D₃ (300 pm) or vehicle beginning at 144 hpf; 24 h later, three batches of 25–30 1α,25(OH)₂D₃-treated and three batches of 25–30 vehicle-treated zebrafish were collected (red arrow) for isolation of RNA, construction of cDNA libraries, and sequencing.

Fig. 2.

PPAR signaling pathway is significantly altered by 1α,25(OH)₂D₃ treatment on dpf 6 [120-h 1α,25(OH)₂D₃ treatment]. RNAs increased after 1α,25(OH)₂D₃ treatment (compared with vehicle) are indicated by red up arrows, and RNAs decreased after 1α,25(OH)₂D₃ treatment are indicated by blue down arrows. RXR, Retinoid X receptor; UCP, uncoupling protein; VLDL, very-low density lipoprotein.

Fig. 3.

Venn diagram showing the developmental distribution of the genes altered by 1α,25(OH)₂D₃ treatment. Genes were grouped based on their altered expression at single or multiple stages. Differential expression analyses were evaluated between ethanol and 1α,25(OH)₂D₃ treatments on dpf 2, 4, 6, and 7. In total, 2553 mRNA transcripts were differentially expressed in at least one the four studied developmental stages.

Fig. 4.

Alcian blue-stained, dpf 7 (168 hpf) zebrafish larvae treated with ethanol (vehicle, EtOH) or 1α,25(OH)₂D₃ (300 pm) continuously for 6 d (144 h). The length of the palatoquadrate (PQ) and ceratohyal (CH) cartilages was assessed in each embryo. The palatoquadrate and ceratohyal cartilages were larger in 1α,25(OH)₂D₃-treated zebrafish compared with vehicle-treated zebrafish (palatoquadrate P = 0.0005; ceratohyal P = 0.015). A1, B1, and C1 represent ethanol-treated zebrafish, lateral view; A2, B2, and C2 represent ethanol-treated zebrafish, ventral view. D1, E1, and F1 represent 1α,25(OH)₂D₃-treated, lateral view; D2, E2, and F2 represent 1α,25(OH)₂D₃-treated, ventral view. VitD, Vitamin D.

Fig. 5.

Alizarin red-stained, dpf 7 (168 hpf) zebrafish larvae treated with ethanol (vehicle, EtOH) or 1α,25(OH)₂D₃ (300 pm) continuously for 6 d (144 h). The mineralized skeleton appears darker in the 1α,25(OH)₂D₃-treated zebrafish. n, Notochord. A1, B1, and C1 represent ethanol-treated zebrafish, lateral view; A2, B2, C2 represent ethanol-treated zebrafish, ventral view. D1, E1, and F1 represent 1α,25(OH)₂D₃-treated, lateral view; D2, E2, and F2 represent 1α,25(OH)₂D₃-treated, ventral view. VitD, Vitamin D.