Stem Cell Arrival in Sites of Hematopoiesis Triggers Supportive Endothelial Niche Formation

Abstract 218

Hematopoietic stem and progenitor cells (HSPCs) self-renew and give rise to all blood cell types throughout adulthood. In mammals, definitive HSPCs first arise from the hemogenic endothelium of the dorsal aorta, are released into circulation, and then seed the fetal liver as an intermediate tissue before colonizing the adult bone marrow. We have used the zebrafish and mouse as complementary model systems to follow HSPC migration throughout embryonic development. The first HSPCs in the zebrafish embryo emerge from the dorsal aorta and migrate to the caudal hematopoietic tissue (CHT), a vascular plexus in the ventral tail of the embryo. To directly track HSPCs in the live zebrafish embryo, we created a novel transgenic line using the known mouse Runx1 +23 kb intronic enhancer that delineates stem cells, as described by de Bruijn and colleagues (Nottingham et al. Blood 2007). A double transgenic zebrafish embryo for HSPCs (Runx1+23:eGFP) and endothelial cells (flk1:dsRed) allowed us to perform live imaging of stem cells during migration. Upon arrival in the CHT, HSPCs adhere to the endothelial wall of the vessel, cross through the wall by extravasation, and trigger niche formation—endothelial cells actually remodel around the HSPC to create a niche. Once in this niche, the HSPC: 1) remains quiescent; 2) divides symmetrically to give rise to a second progenitor; or 3) divides asymmetrically to give rise to a myeloid progenitor that migrates out of the endothelial niche. To determine if this mechanism of endothelial niche formation in the embryo was conserved in mammals, we dissected fetal livers from E11.5 mouse embryos, the stage that marks the beginning of fetal liver seeding by dorsal aorta HSPCs. Staining these fetal livers with anti-ckit and PECAM antibodies, we could perform live imaging on cultured explants. Strikingly, we observed PECAM+ endothelial cells adhere to and form a rosette around a single ckit+ HSPC—similar to the de novo niche formation we observed in the zebrafish. To better understand the mechanism of how HSPCs navigate their migration from dorsal aorta to fetal liver/CHT, a chemical genetic screen was performed to detect changes in expression of hematopoietic progenitors in the CHT. Surprisingly, we found that sphingosine-1-phosphate (S1P), a signal known to regulate adult progenitor and lymphocyte trafficking, changed the migration pattern of HSPCs after embryo treatment. Knockdown of the s1pr1 receptor, which is required for S1P-dependent HSPC trafficking, resulted in reduction of HSPC markers in the CHT. S1PR1 receptor function was tested using small molecules. A highly specific S1PR1 antagonist (W146), or depletion of endogenous S1P using a sphingosine kinase inhibitor (SKI-2), reduced HSPC markers in the CHT. Endogenous S1P depletion was dose-dependently rescued by addition of exogenous S1P or S1PR1-specific agonist (AUY954). S1PR1 receptor antagonist (W146) effects were also rescued by addition of exogenous S1P. To assess if the effects of the S1P pathway were autonomous to the HSPC, a dominant-negative S1PR1 driven in HSPCs by the Runx1 +23 kb enhancer was injected into fish and followed by time-lapse live imaging. The expressing cells had difficulty engrafting in the CHT. We tested possible synergism between S1P and CXCR4 pathways in the embryo by using combination chemical genetics. Embryos were treated in a dose matrix of two different chemicals, one against CXCR4 and the other against the S1P pathway. Specific dose ratios could synergistically increase hematopoiesis in the CHT to a maximum engraftment, demonstrating an interaction between CXCR4 and S1P signaling. Doses of CXCR4 and S1P signaling could also be found that decreased engraftment, suggesting that the concentration of the two ligands and receptors is critical. We used these same dose ratios to treat whole bone marrow from adult mouse. The combination that gave maximum CHT engraftment also increased cell surface receptor levels of both CXCR4 and S1PR1. Together, our data demonstrate a novel and conserved role for S1P during migration of HSPCs between successive niches during development.