(Figure 1) can be observed for CXCR7 it is conceivable that the two mechanism work synergistically or at least act in parallel. As a whole our findings are in full agreement with the conclusion that CXCR7 expressed on MDCK cells acts as specific decoy for CXCL12 and CXCL11. In zebrafish contradicting opinions on the role of CXCR7 have been reported, suggesting a signaling [17] versus a scavenger function [19]. To investigate this controversy, we followed the subcellular localization of the receptor in zebrafish cells. CXCR7- EGFP (CXCR7 tagged at the C-terminus with EGFP) was expressed in embryos alone or in combination with a morpholino oligonucleotide suppressing CXCL12 expression [19,21]. Figure 5 shows frames from time-lapse movies (Supplementary Informa- tion, movies S1 (control - MO) and S2 (CXCL12-MO)) which document the spontaneous pinching off from the plasma membrane of CXCR7 containing endosomes and their re- association with the membrane. The movement of CXCR7 appears independent of the presence of CXCL12 indicating that CXCR7 can, in agreement with its proposed scavenger function, spontaneously cycle from and back to the plasma membrane in zebrafish embryonic cells. Boldajipour et al. [19] proposed that by removing CXCL12 at the posterior side CXCR7 sharpens the chemotactic gradient promoting CXCR4-dependent migration of primordial germ cells. The present observation provides an explanation for the efficiency of CXCR7 in supporting CXCR4- CXCR7 Scavenges CXCL12 mediated migration of primordial germ cells and is consistent with the view that CXCR7 that is expressed by somatic cells functions as a scavenger. Targeted deletion of Cxcr7 leads to perinatal death with a marked cardiac phenotype that is associated with septum and heart valve malformation. The phenotype is also manifest upon conditional deletion of Cxcr7 in endothelium [9]. A possible explanation for the phenotype could be that lack of CXCR7 gives rise to CXCL12-stimulated CXCR4-dependent hyperplasia. We therefore tested if mouse heart valves can scavenge CXCL12. Hearts were removed from wild type laboratory (BALB/c) mice and cut transversally. The opened hearts which included all valves were incubated with 150 nM CXCL12 tagged at the C-terminus with the fluorescent protein mCherry. Frozen sections of the hearts reveal endosomes in valve endothelial cells loaded with CXCL12- mCherry. Uptake was marginally impaired in the presence of 10 mM AMD3100, suggesting that the process is mediated by CXCR7 rather than by CXCR4. In line with this a recent report indicates that AMD3100 at 10 mM slightly affects CXCL12 binding to CXCR7 [38]. Addition of 1.5 mM untagged CXCL12 expectedly competed the uptake of CXCL12-mCherry (Figure 6). The results are consistent with a scavenger activity of CXCR7 on valve endothelium in adult mice. Human umbilical cord endothelial cells (HUVECs) express CXCR4 and CXCR7 [7,39]. To test the scavenging activity of Figure 5. Ligand-independent cycling of CXCR7 in zebrafish embryos. In embryos expressing CXCR7-EGFP the receptor localizes at the plasma membrane (grey mark in lower panels) and intracellular vesicles (white dot). Time laps (time stamps) images shows that the vesicles are in contact with the membrane internalize and recycle back to the membrane. Internalization and cycling of CXCR7-EGF is similar in the presence (control–MO) or absence of CXCL12 (CXCL12–MO), underling the ligand independent mechanism. For the complete time laps see supplementary movies S1 cont and S2 CXCL12. doi:10.1371/journal.pone.0009175.g005 PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9175