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Fig. 5
1,25(OH)D3 Mediates HSPC Expansion through CXCL8 Signaling
(A) WISH for cxcl8 showing elevated expression with 1,25(OH)D3 treatment at 36 hpf (n ≥ 30 embryos/condition).
(B) Whole-embryo qPCR confirmed increased cxcl8 expression at 36 hpf after 1,25(OH)D3 exposure (∗p < 0.05) (40 pooled embryos/condition × 3 replicates). Error bars, mean ± SD.
(C) FACS analysis of embryos injected with cxcl8 mRNA (100 ng/embryo) showed increased Flk1:dsRed+/cMyb:GFP+ HSPCs at 36 hpf (∗p < 0.05) (5 embryos/sample × 4 replicates/condition). Error bars, mean ± SD.
(D) WISH analysis and phenotype distribution indicated that the effect of 1,25(OH)D3 on runx1/cmyb expression was diminished in cxcl8 morphants (n ≥ 35 embryos/condition).
(E) FACS analysis of MO-injected embryos treated with 1,25(OH)D3 (con versus 1,25(OH)D3, ∗p < 0.05; con versus cxcl8 MO, N.S.; cxcl8 MO versus cxcl8 MO + 1,25(OH)D3, N.S.; 1,25(OH)D3 versus 1,25(OH)D3 + cxcl8 MO, N.S.)(5 embryos/sample × 4 replicates/condition; error bars, mean ± SD) showed that loss of cxcl8 blocked vitamin D-mediated HSPC expansion.
(F) Western blot analysis for pAKT, with AKT as loading control, following 1,25(OH)D3 stimulation (12-36 hpf) in cxcl8 mutants versus WT siblings (60 embryos/condition).
(G) WKM stromal cell assay documenting changes in proliferation by 1,25(OH)D3 with and without co-incubation with SB25002 (con versus 1,25(OH)D3, ∗∗∗∗p < 0.0001; con versus SB225002, p < 0.05; con versus 1,25(OH)D3 + SB25002, N.S.; 1,25(OH)D3 versus 1,25(OH)D3 + SB25502, ∗∗∗∗p < 0.0001) (n = 4; error bars, mean ± SD).
(H) WKM methylcellulose CFU assay showing fold increase with and without co-treatment of 1,25(OH)D3 with SB25002 (con versus 1,25(OH)D3, ∗∗p < 0.01; con versus calcipotriol, ∗∗p < 0.01; con versus SB225002, ∗p < 0.05; con versus 1,25(OH)D3 + SB25502, N.S.; con versus calcipotriol + SB25502, N.S.). n = 4 (SB25503 is n = 6); error bars, mean ± SD.
Scale bars, 100 μm. See also Figure S5.