VDR Is Necessary for Zebrafish Definitive Hematopoiesis

(A) Representative runx1 WISH images and qualitative phenotype distribution (high/medium/low, n ≥ 30 embryos/condition) of vdra MO-injected embryos and sibling controls at 36 hpf. Error bars, mean ± SD.

(B) WISH example and phenotype distribution for flk1 in vdra morphants (n ≥ 20 embryos/condition).

(C) qPCR analysis quantifying the impact on HSPCs (runx1, ^∗∗p < 0.01) and hemogenic endothelium (flk1, ephrinb2a, p = N.S.) in control and vdra morphants (30 embryos/sample × 3 replicates).

(D-F) Representative WISH (D), phenotype distributions (E), and qPCR quantification (F) of vdra mutant embryos (runx1, ^∗∗p < 0.01; flk1, ephrinb2a, p = N.S.) phenocopied the impact on HSPCs but not on vessels.

(G) FACS analysis of vdra morphants indicated significant reductions in Flk1:dsRed⁺/cMyb:GFP⁺ HSPCs (^∗p < 0.05) at 48 hpf (5 embryos/sample × 4 replicates/condition). Error bars, mean ± SD.

(H) WISH samples and phenotype distribution of runx1/cmyb expression in cyp27b1 MO-injected embryos at 36 hpf (n ≥ 30 embryos/condition).

(I) FACS analysis showed a significant decrease in HSPCs (^∗∗p < 0.01) with cyp27b1 MO injection (n value and error bars as in C).

Scale bars, 100 μm. See also Figure S1.

1,25(OH)D3 Regulates HSPC Numbers via Vitamin D Receptor Signaling

(A) Representative runx1/cmyb WISH images and qualitative phenotype distribution following 25(OH)D3 exposure from 12–36 hpf (n ≥ 40 embryos/condition).

(B) FACS analysis of 25(OH)D3-treated (10 μM) embryos showed a significant increase in HSPCs (^∗∗p < 0.01) (5 embryos/sample × 4 replicates/condition). Error bars, mean ± SD.

(C) qPCR analysis indicated a significant induction in the VDR transcriptional target cyp24a1 at 36 hpf after exposure to 25(OH)D3 (^∗p < 0.05) or 1,25(OH)D3 (^∗∗∗p < 0.001) (12–36 hpf) (30 pooled embryos/condition × 3 replicates).

(D) Phenotype distribution of runx1/cmyb expression in embryos exposed to increasing concentrations of 1,25(OH)D3 from 12–36 hpf (n ≥ 30 embryos/condition).

(E) FACS analysis of 1,25(OH)D3-treated (10 μM) embryos showed a 20% increase in HSPCs at 48 hpf (^∗∗p < 0.01) compared with controls (n value and error bars as in B).

(F) runx1/cmyb WISH and phenotype distribution after exposure to the non-calcemic vitamin D3 analog calcipotriol (n ≥ 30 embryos/condition).

(G) FACS analysis of calcipotriol-treated embryos showed a significantly more HSPCs (^∗p < 0.05) at 36 hpf (n value and error bars as in B).

Scale bars, 100 μm. See also Figure S2.

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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.

Vdra knockdown affects HSPCs expansion (related to figure 1).

A. qPCR for vdra in FACS-sorted Flk1-/cMyb- (double negative), Flk1+/cMyb- (endothelium), and Flk1⁺/cMyb⁺ (HSPCs) fractions (cells sorted from ≥1000 embryos/condition x 3 replicates) populations at 36hpf.

B. Representative ephrinb2a WISH images and qualitative phenotype distribution (High (green)/ Med (red)/ Low (blue); n≥40 embryos/condition) of vdra morpholino (MO) injected embryos and sibling controls at 36hpf.

C. WISH and phenotype distribution of vdrb MO injected embryos showing no effect on runx1 expression at 36hpf (n≥35 embryos/condition).

D. FACS analysis of vdra morphants showed a significant decrease in Lmo2:GFP⁺/Gata1:dsRed⁺ EMPs (*p<0.05) at 30hpf (5 embryos/sample x 4 replicates/condition); error bars, mean ± SD.

E. Schematic representation of the predicted exon 7 truncation caused by the vdra splice MO, and DNA gel analysis showing the smaller vdra band following MO injection (n>20 embryos/condition).

F. FACS analysis of HSPCs at 36hpf for vdra morphants (**p<0.01), with and without co-injection of either vdra or vdrb mRNA; vdra mRNA co-injection rescued the reduced number of HSPCs observed with vdra MO alone (vdra MO vs vdra MO + vdra mRNA, **p<0.01; vdra MO vs vdra MO + vdrb mRNA, N.S.) (5 embryos/sample x 4 replicates/condition); error bars, mean ± SD.

G. Schematic representation on the CRISPR/cas9-mediated deletion in vdra.

H. Gel electrophoresis depicting a mismatched mutation assayed by Surveyor nuclease in vdra heterozygote mutants compared to WT sibling controls.

I. Representative fluorescent microscopy example of CD41:GFP expression in the CHT at 72hpf in control and vdra morphants.

J. FACS analysis of vdra morphants showed a significant decrease (**p<0.01) in the percentage of CD41:GFP^lo;Gata1:dsRed^- HSCs (5 embryos/sample x 3 replicates/ condition); error bars, mean ± SD).

Scale bar 100μm.

Vitamin D treatment expands HSPC number (related to Figure 2).

A. Representative VDRE:mCherry WISH phenotype of embryos treated with 25(OH)D3 and 1,25(OH)D3 compared to DMSO-treated controls (n>10).

B. FACS analysis of Tg(flk1:GFP/VDRE:mCherry) embryos treated with 1,25(OH)D3 from 12-36hpf showed a significant (****p<0.0001) increase in Flk1+ cells responsive to 1,25(OH)D3 (5 embryos/sample x 4 replicates/condition); error bars, mean± SD).

C. FACS analysis of embryos treated with 1,25(OH)D3 (10μM) indicated a 25% increase in EMPs at 30hpf (**p<0.01) compared to controls (n value and error bars as in S2B).

D. FACS analysis of 1,25(OH)D3-treated Tg(flk1:dsred; EF1:mAG-zGEM(1/100) embryos at 36hpf showed a 2-fold increase (***p<0.001) in proliferating endothelial cells, inclusive of the hemogenic population (5 embryos/sample x 4 replicates/condition); error bars, mean ± SD.

E. FACS analysis of vdra MO-injected Tg(flk1:dsred; EF1:mAG-zGEM(1/100) embryos at 36hpf indicated a significant decrease in proliferation (*p<0.05) in the absence of Vitamin D3/VDR signaling (n value and error bars as in S2D).

F. Representative fluorescent microscopy images of Tg(CD41:GFP) embryos treated with 1,25(OH)D3 (1μM) or calcipotriol (1μM) between 12-36hpf depicting a sustained increase in CD41:GFP⁺ expression in the CHT at 72hpf.

G. FACS analysis at 72hpf showed a significant increase in the percentage of CD41:GFP^lo;Gata1:dsRed^- HSPCs after both 1,25(OH)D3 (*p<0.05) and calcipotriol (**p<0.01) treatment (5 embryos/sample x 4 replicates/condition); error bars, mean ± SD.

Scale bar 100μm.

Expression of Cxcl8 receptors cxcr1/cxcr2 in zebrafish HSPCs (related to Figure 5).

A. Expression analysis of FACS sorted populations showed enhanced cxcl8 expression following exposure to 1,25(OH)D3, with the biggest effect in Flk1⁺/cMyb⁺ HSPCs (cells sorted from ≥1000 embryos/condition x 2 replicates); error bars, mean ± SD.

B. qPCR analysis of ZFcxcr1 and Zfcxcr2 expression shows enrichment of ZFcxcr1 in FACS sorted HSPCs (Flk1⁺/cMyb⁺) and the Flk-/cMyb- population (n=2 replicates, cells sorted from ≥1000 embryos/condition); N.D. (not detected).

C. WISH analysis and phenotype distributions of embryos treated with 1,25(OH)D3 with and without the CXCL8 inhibitor SB25002 (1μM) showed co-treatment attenuated Vitamin D-mediated increases in runx1/cmyb expression (n≥50 embryos/condition).

D. FACS quantification of Flk1⁺/cMyb⁺ HSPCs after exposure to 1,25(OH)D3 with and without SB25002 (Con vs 1,25(OH)D3, *p<0.01; 1,25(OH)D3 vs SB25002, *p<0.01; Con vs 1,25(OH)D3 + SB25002, N.S.; (5 embryos/sample x 4 replicates/condition; error bars, mean ± SD ).

Scale bar 100μm.

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