Accumulation of the Vitamin D Precursor Cholecalciferol Antagonizes Hedgehog Signaling to Impair Hemogenic Endothelium Formation

• Vitamin D3 metabolites exert differential effects on embryonic HSPC formation
• D3 inhibits specification of the hemogenic endothelial niche
• D3 blocks Hedgehog signaling by interaction at the Smoothened sterol-binding domain
• Cyp2r1 loss disrupts of Hedgehog-mediated fate specification via D3 accumulation

SummaryHematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.