Figure 6. Macrophages mediate the efficient clearance of senescent cells during salamander limb regeneration.
(A) A clodronate-dependent system for effective macrophage depletion in salamanders. Representative images of axolotl limb tissues 36 hr post i.v. injection with fluorescent DiI-liposomes (red) or DiI + clodronate-liposomes. Scale bar: 1000 µm. (B) DiI-liposomes are incorporated specifically by macrophages. Blood cells were extracted from treated animals and stained with antibodies against the specific macrophage marker F4/80. Scale bar: 50 µM. (C) Schematic representation of the implantation experiment. Axolotls with limbs at the midbud stage of regeneration were injected i.v. with either DiI-liposomes or DiI + clodronate(clodro)- liposomes, 36 hr prior to implantation of normal or senescent nGFP+ cells into contralateral regenerating limbs. Liposome treatments were maintained for 2 weeks. (D) Macrophage (DiI, red) recruitment to areas of senescent cells 12 hr following implantation of normal and senescent GFP+ cells within regenerating axolotl limbs. Scale bar: 1000 µm. (E) DiI-labelled macrophages (red) are recruited to sites of implanted nGFP+ senescent cells (green) within regenerating limbs at 16 hs post implantation. Scale bar: 100 µm. Note that in the DiI-liposome treated animals macrophages are recruited to sited of senescent but not normal cells. (F) Quantification of macrophage (DiI-labelled) recruitment to implantation sites at 16 hpi. Note macrophage depletion induced by clodronate-liposome treatment. (G) Clearance of senescent cells is impaired upon macrophage depletion. Quantification of GFP+ cells after 2wpi in DiI-liposome or clodronate-liposome treated animals.