Whole Transcriptome RNA Sequencing Detects Multiple 1a,25-Dihydroxyvitamin D3-Sensitive Metabolic Pathways in Zebrafish Embryos

Vitamin D receptors (VDR) are abundantly expressed in developing zebrafish as early as 48 hours post-fertilization, and prior to the development of a mineralized skeleton, and mature intestine and kidney. We probed the role of VDR in 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 D3, 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3). We observed significant changes in RNAs encoding proteins of fatty acid, amino acid, and xenobiotic metabolism pathways, and RNAs of transcription factors, leptin, peptide hormones, receptor-activator of NFkB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early small, and subsequent massive changes in >10% of expressed cellular RNAs were observed. At day 2 (24h 1a,25(OH)2D3-treatment), only 5 RNAs were differentially expressed (hormone vs. vehicle). On day 4 (72h-treatment), 78 RNAs; on day 6 (120h-treatment) 1040 RNAs; and on day 7 (144h-treatment), 1755 RNAs were differentially expressed in response to 1a,25(OH)2D3. Fewer RNAs (n = 482) were altered in day 7 embryos treated for 24h with 1a,25(OH)2D3 vs. those treated with hormone for 144h. At 7 days, in 1a,25(OH)2D3-treated embryos, 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 1a,25(OH)2D3 functions in vivo in developing eukaryotes. Zebrafish embryos were obtained from mating of Segrest wild-type (SWT) parents under controlled barrier conditions, in the Mayo Clinic Zebrafish Core Facility, in Instant Ocean media . Zebrafish embryos (25-30) were placed in 20 mL embryo medium (pH 7.2) containing 1-phenyl-2-thiourea (PTU) (0.003% (w/v) and were maintained at 28-30 oC. At 24 hpf (1 day post fertilization, dpf), 10 microliters of 1a,25(OH)2D3 in ethanol was added to embryos maintained in 20 mL fresh embryo medium with PTU. The final concentration of 1a,25(OH)2D3 was 300 pM. Control zebrafish were treated with 10 microliters ethanol alone (vehicle controls). The medium containing either 300 pM 1a,25(OH)2D3 or vehicle was changed every 24 h . In experiment 1, at 2, 4, 6 and 7 dpf embryos/larvae were removed and immediately frozen at -80 0C for later RNA preparations. 25-30 embryos per set were used for preparation on RNA. At the same times, 7-12 embryos were fixed in 4% paraformaldehyde in 0.75 X Dulbecco’s phosphate buffered saline (DPBS). In experiment 2, 6 dpf larvae were treated with 1a,25(OH)2D3 (300 pM) or vehicle for 24 h. RNA was prepared from three sets of larvae.