|ZFIN ID: ZDB-IMAGE-161111-7|
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:GFPlo;Gata1:dsRed- HSCs (5 embryos/sample x 3 replicates/ condition); error bars, mean ± SD).
Scale bar 100μm.
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