Inhibitors of CXCR4 affect the migration and fate of CXCR4+ progenitors in the developing limb of chick embryos

We have shown that in the chick embryo CXCR4+ cells invade the chick limb mesenchyme starting from stage HH18‐19. These CXCR4+ cells later contribute to muscle and vessel formation in the limbs. We tested two specific peptidic inhibitors of CXCR4, T140 and its derivative TN14003 to study the functional role of the CXCR4+ population in the limb bud. For our experiments, acrylic beads were soaked in 5–10mg/ml of T140 or TN14003 and placed in the limb at stage HH18 of chick and quail embryos. The embryos were then fixed after different reincubation periods to obtain stages HH21–27. In situ hybridization was performed using specific RNA probes for CXCR4, Pax3, lbx‐1 and MyoD. Immunohistochemistry was done using QH1 and anti‐GFP antibodies. T140 and TN14003 both lead to a decreased number of CXCR4+ cells in the limb and a decreased Pax3 and Lbx‐1 signal. In situ hybridizations with MyoD showed a decreased muscle mass in the operated side. Immunohistochemistry showed that the cells around the beads were QH1 positive. We could show by electroporation that these cells had a dermomyotomal origin. No apoptosis was seen around the bead. CXCR4 inhibitors, prevent the entry of CXCR4+ cells into the limb bud and thus result in the decrease of muscle mass in the operated side and cause an increase of vessel formation around the bead. We conclude that the CXCR4/SDF‐1 axis is important for the migration and preservation of the pluripotency of these precursors, and blocking it results in migration defects with fewer myoblast in the limb bud.