Abstract
The colonial ascidian Botryllus schlosseri provides a unique model to study germ cell migration, as long-lived germline stem cells (GSCs) constantly migrate to new germline niches as they develop during repetitive cycles of asexual reproduction. We have previously shown that migration of germ cells in Botryllus is directed by sphingosine-1-phosphate (S1P) signaling. Here, we show that the ABC-transporters abcc1 and abcb1 are highly expressed in germ cells, and inhibition of ABC-transporter activity leads to a reduced migratory response towards S1P in vitro and failure of germ cell homing in vivo. ABC-transporters are highly conserved in the animal kingdom and are involved in the export of lipid-signaling molecules. Phospholipase A2 (PLA2) produces arachidonic acid, which is further metabolized by cyclooxygenases (COX), or by Lipoxygenases (LOX) to eicosanoid signaling molecules. We show that in Botryllus, PLA2 activity is required for migration of germ cells towards low concentrations of S1P in vitro, as well as homing of germ cells in vivo. Botryllus lipoxygenase (bslox) has homology to the 3 human lipoxygenases 5-LOX, 12-LOX and 15-LOX. In humans, 12-LOX metabolizes arachidonic acid to12-Hydroxyeicosatetraenoic acid (12-S-HETE), which stimulates migration of cancer cells and smooth muscle cells. We have identified a g-protein coupled receptor in Botryllus that is closely related to the human receptor for 12-S-HETE, GPR31. BSgpr31 is expressed in vasa-positive germ cells. In vitro, an inhibitor of all 3 types of lipoxygenases significantly reduces migration of Botryllus germ cells towards S1P, whereas specific inhibitors of COX1, COX2 or 5-LOX have no effect. 12-S-HETE rescues migration towards S1P in the presence of inhibitors of ABCC1, ABCB1, PLA2 or LOX. In vivo, inhibition of LOX inhibits homing of germ cells to secondary buds. We conclude that autocrine stimulation by production and ACB-transporter-mediated export of 12-S-HETE is required for migration of germ cells towards a chemotactic gradient of S1P. We hypothesize that signaling of 12-S-HETE through BSgpr31 enhances migratory activity during chemotaxis induced by low-level activation of the S1P-receptor. This is the first report of an eicosanoid signaling molecule regulating germ cell migration.