Vitamin D3 Modulates Definitive Hematopoiesis by Two Distinct Mechanisms in the Developing Zebrafish Embryo

Abstract 763

Studies of the vitamin D3 signaling pathway have revealed a broad role for this hormone in tissue homeostasis and as a result there is great interest in exploring the therapeutic potential of vitamin D3 for the treatment of various diseases including cancer. Studies investigating the role of vitamin D3 in hematological malignancies have shown that in vitro vitamin D3 has anti-proliferative and pro-differentiation effects resulting in differentiation of leukemic cell lines towards the monocytic lineage. Surprisingly, the role of vitamin D3 in HSC homeostasis and leukemia progression in vivo is not well understood. To elucidate the mechanisms of action of vitamin D3 in vivo during HSC self-renewal and differentiation, we utilized the zebrafish (Danio rerio) as vertebrate model due to their evolutionary conserved blood system and their amenability for genetic and chemical manipulation. Vitamin D3 was identified in a chemical screen performed in our laboratory as a regulator of runx1 and c-myb expression in the aorta-gonad mesonephros (AGM), the first site of definitive hematopoiesis. Treatments of zebrafish embryos with active vitamin D3 (1,25OH D3) between 12–36 hours post fertilization (hpf) resulted in increased expression of HSC markers (runx1, c-myb, CD41) as determined by whole mount in situ hybridization (WISH). In contrast, treatment with the non-hydroxylated vitamin D3 precursor cholecalciferol (D3) resulted in decreased runx1 and c-myb expression. D3 treatment during HSC induction and expansion from 24–36 hpf did not affect runx1 and c-myb expression, suggesting that D3 is acting early during the establishment of the vascular niche. To quantify the difference in HSC progenitors observed by WISH, FACS analysis was performed on double positive Tg(Lmo2:dsRed), Tg(c-myb:gfp) embryos. 1,25OH D3 treated embryos had a 20% increase in the double positive cell population corresponding to HSCs, while treatment with D3 resulted in a 25% decrease in the number of HSCs. The differential effect of 1,25OH D3 and D3 suggest that these two compounds act via two distinct mechanisms. To determine if the HSC enhancement observed by 1,25OH D3 was acting through the canonical vitamin D3 receptor (VDR), loss of function experiments were performed by injecting morpholinos targeting the vitamin D receptors. Morpholino knockdown of VDRA and VDRB (zebrafish VDR orthologs) resulted in decreased expression of runx1 c-myb, and CD41 via WISH. The decrease in HSC positive cells was confirmed by fluorescence microscopy using the Tg(Runx1P2:gfp) and Tg(-6.0itga:gfp) reporter lines, further supporting our hypothesis. To address the decrease in HSCs by D3, we postulated that D3 was inhibiting hedgehog signaling, as vitamin D3 has been previously shown to act as a negative regulator of the hedgehog pathway. In support of our hypothesis, treatment with D3 resulted in decreased ptch2 mRNA expression a downstream target of hedgehog signaling. In addition, FACS analysis using the hedgehog reporter Tg(6xGli:mCherry) line showed a 15% reduction in the number of mCherry positive cells in D3 treated embryos compared to controls. Co-treatments of zebrafish embryos with the hedgehog antagonist cyclopamine and D3 resulted in additive loss of reporter activity (45%) and loss of runx1 positive cells in the AGM, revealing synergy between these two compounds. In addition, co-exposure with the hedgehog agonist SAG rescued the HSC defect in D3-treated embryos. Hedgehog signaling is known to regulate vein and artery specification through the activation of notch signaling. Consistent with our hypothesis that D3 reduces hedgehog signaling, an expansion of the venous marker flt4 and a reduction in the expression of the arterial marker ephrinb2a was observed by WISH on D3-treated embryos. Furthermore, treatment of the notch reporter line Tg(Notch:gfp) with D3 resulted in lowered notch activity. In summary, these studies reveal that the active 1,25OH D3 positively regulates HSC progenitors in vivo during the onset of definitive hematopoiesis. In contrast, the non-hydroxylated vitamin D3 precursor acts as negative regulator of hematopoiesis by inhibiting hedgehog signaling and affecting vascular niche formation. Based on our studies, the differential effect between D3 and 1,25OH D3 should be considered when investigating the therapeutic potential of vitamin D3 in the context of hematological malignancies and other cancers.