Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via Prostaglandin E2 and P-Selectin Activity

Abstract

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb+ HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.

Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization

“…As ECM remodeling is essential in the BM microenvironment (Davis and Senger, 2005) and inflammation influences HSC production, we sought to determine whether inflammation-induced Mmp activity affected the onset or progression of embryonic HSPC development. Consistent with prior observations (Esain et al., 2015), exposure to CAY10397 (10 μM, 12–36 hpf), a selective 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor that stabilizes endogenous PGE 2 levels, enhanced expression of the conserved HSPC markers runx1 and cmyb by whole-mount in situ hybridization (WISH) (Figures S1A and S1B). In addition to a potent effect on runx1 (p < 0.01), CAY10397 also significantly increased the expression of mmp2 (p < 0.05) and  mmp9 (p < 0.01) by qPCR at 42 hpf (Figure 1A).…”

Cannabinoid receptor signaling regulates liver development and metabolism

“…The cnr1, cnr2 and ahcy splice-blocking morpholinos were previously validated (Esain et al, 2015;Matthews et al, 2009). Injections were performed at the onecell stage, with a mismatch standard morpholino used for control.…”

CB1 and CB2 receptors play differential roles in early zebrafish locomotor development

“…First, we determined dose-dependent effects of the antagonists by applying a range of commonly used concentrations (0.05-5 μmol l −1 for AM251 and 0.2-10 μmol l −1 for AM630) (Akhtar et al, 2013;Esain et al, 2015;Fraher et al, 2015;Tran et al, 2016) when testing survival, hatching, heart rates and morphological deficits. Based upon these results, we used a single concentration for…”