ZFIN ID: ZDB-FIG-150923-19
Esain et al., 2015 - Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via PGE2 and P-selectin Activity. Stem Cells   33(8):2596-612 Full text @ Stem Cells
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage Range: 14-19 somites to Prim-25
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagent:
Observed In:
Stage: Prim-25

Fig. 1

CNR2 modulates hematopoietic stem cell (HSC) formation during embryogenesis.

(A): Embryonic exposure to endogenous (2-AG, 5 µM; AEA, 5 µM) or synthetic (AM1241, CNR2-selective agonist, 10 µM) cannabinoids during HSC formation (12–30 hpf) increased runx1 expression in the AGM (n e 90 per condition).

(B): In vivo imaging of flk1:dsRed;cmyb:egfp embryos indicated the number of Flk1:dsRed+;cMyb:GFP+ HSCs (arrowheads) was increased in the AGM at 36 hpf following exposure to endogenous and synthetic cannabinoid compounds (n e 7 per condition).

(C): Qualitative phenotypic distribution of embryos from panel (A) and Supporting Information Figure S1A scored with low, medium, or high runx1 expression in the AGM (O2545: dual CNR1/2 agonist, 5 µM, ACEA: CNR1-selective agonist, 5 µM).

(D): Absolute counts of Flk1:dsRed+cMyb:GFP+ HSCs in embryos from panel (B) and Supporting Information Figure S1B (DMSO: 5.4 ± 0.5, 2-AG: 7.3 ± 1.7, AEA: 7.5 ± 2.4, O2545: 7 ± 1.5, ACEA: 6.2 ± 1.2, AM1241: 7.9 ± 1.9; *, p d .05; **, p d .01; ****, p d .0001, two-tailed t test).

(E): Whole-mount in situ hybridization of wild type embryos at 18 and 24 hpf showed cnr2 was expressed throughout the embryo and enriched in the AGM region at the time of HSC specification.

(F): MO-mediated knockdown of cnr1 or cnr2 revealed that CNR2, but not CNR1, was required for normal runx1 expression (n ≥ 150 per condition).

(G): Qualitative phenotypic distribution of embryos from panel (F) scored with low, medium, or high runx1 expression in the AGM.

(H): In vivo imaging confirming the number of Flk1:dsRed+;cMyb:GFP+ HSCs (arrowheads) was decreased in the AGM at 36 hpf following cnr2, but not cnr1, MO-mediated knockdown (n ≥ 10 per condition).

(I): Absolute counts of Flk1:dsRed+cMyb:GFP+ HSCs from embryos in panel (B) (uninjected: 5.3 ± 0.7, cnr1 MO: 5.3 ± 0.3, cnr2 MO: 3.6 ± 0.3; *, p < .05, two-tailed t test). Scale bars: (A, B, F, H) = 100 µm, (E) =500 µm. Abbreviations: 2AG, 2-arachidonoylglycerol; AEA, anandamide; AGM, aorta-gonad-mesonephros; MO, morpholino.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
cnr2 WT standard conditions 14-19 somites whole organism ISH
Prim-5 ventral wall of dorsal aorta ISH
Prim-5 whole organism ISH
DsRed2 pd27Tg; zf169Tg control Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: 2-arachidonoylglycerol Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: anandamide Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: cannabinoid receptor agonist Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg + MO2-cnr2 standard conditions Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg + MO5-cnr1 standard conditions Prim-25 trunk vasculature IFL
Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
EGFP pd27Tg; zf169Tg control Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: 2-arachidonoylglycerol Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: anandamide Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg chemical treatment: cannabinoid receptor agonist Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg + MO2-cnr2 standard conditions Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
pd27Tg; zf169Tg + MO5-cnr1 standard conditions Prim-25 ventral wall of dorsal aorta IFL
Prim-25 ventral wall of dorsal aorta hematopoietic stem cell IFL
runx1 WT control Prim-15 ventral wall of dorsal aorta ISH
WT chemical treatment: 2-arachidonoylglycerol Prim-15 ventral wall of dorsal aorta ISH
WT chemical treatment: anandamide Prim-15 ventral wall of dorsal aorta ISH
WT chemical treatment: cannabinoid receptor agonist Prim-15 ventral wall of dorsal aorta ISH
WT + MO2-cnr2 standard conditions Prim-15 ventral wall of dorsal aorta ISH
WT + MO5-cnr1 standard conditions Prim-15 ventral wall of dorsal aorta ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
pd27Tg; zf169Tg chemical treatment: 2-arachidonoylglycerol Prim-25 ventral wall of dorsal aorta hematopoietic stem cell increased amount, abnormal
pd27Tg; zf169Tg chemical treatment: anandamide Prim-25 ventral wall of dorsal aorta hematopoietic stem cell increased amount, abnormal
pd27Tg; zf169Tg chemical treatment: cannabinoid receptor agonist Prim-25 ventral wall of dorsal aorta hematopoietic stem cell increased amount, abnormal
pd27Tg; zf169Tg + MO2-cnr2 standard conditions Prim-25 ventral wall of dorsal aorta hematopoietic stem cell decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Stem Cells for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Stem Cells