Fig 1: CD206hi macrophage-derived PDGF-A increases En1+ fibroblast proliferation to promote wound healing.(A) Dot plots of the ligand–receptor interactions related to PDGF signaling in macrophage subpopulations and Gpnmbhi/Gpnmblo fibroblasts. Macrophage subpopulations that express ligands and fibroblast subpopulations that express receptors are shown on the x axis. Ligand and cognate receptor combinations are shown on the y axis. Circle size denotes P value (permutation test); color denotes average ligand and receptor expression levels in interacting subpopulations. (B) Representative images of wounds from Mrc1-DTR and control mice 5 days post-injury. Wound bed tissue samples were immunostained for PDGF-A (red) and CD206 (green). Scale bar, 50 μm. (C) The percentages of CD206−, CD206+PDGFA−, CD206−PDGFA+ and CD206+PDGFA+ cells out of the total DAPI-positive cells/0.01-mm2 wound area were compared between mouse groups. Data are expressed as means ± SD. *P = 0.0003 (CD206− PDGFA−), 0.0407 (CD206+ PDGFA−), 0.1351 (CD206- PDGFA+), 0.0012 (CD206+ PDGFA+) by unpaired two-tailed t test after applying logit transformation (n = 4 mice/group, biological replicates). (D) Representative images of wounds from control mice 5 days post-injury. Wound bed tissue samples were immunostained for PDGFRA (red) and EN1 (green). Magnified images of orthogonal views of 2.4 μm z-stack images are shown on the right side. Scale bars, 50 μm (left) and 10 μm (right). (E) Comparison of the proportion of EN-negative cells co-expressing PDGFA with the proportion of EN-positive cells co-expressing PDGFA. Data are expressed as means ± SD. *P = 0.0012 by unpaired two-tailed Student’s t test (n = 5 mice/group, biological replicates). Fluorescence images with an IgG-negative primary antibody control are available in Fig. EV5. (F) The mRNA expressions of En1, Col1a1, and Col3a1 in primary mouse fibroblasts treated with PDGF-AA (40 ng/mL) or PBS (control), as determined by qPCR, are shown. Expression levels were first normalized to those of Gapdh and then to the levels of control cells. Data are expressed as means ± SD. *P = 0.0236 (En1), 0.0087 (Col1a1), 0.0059 (Col3a1) by unpaired two-tailed Student’s t test. (n = 3/group, biological replicates). (G) Representative images of wounds of Mrc1-DTR mice treated with PDGF-AA or PBS. Scale bars, 1 mm. (H) Wound area was analyzed at the indicated times after injury. The percentages of wound areas compared to original wound sizes are shown as a bar chart. Data are expressed as means ± SD. *P = 0.0271 (day 5), 0.0072 (day 9) by unpaired two-tailed Student’s t test. (n = 4 mice/group, biological replicates). (I) Representative images of wounds (5 days post-injury) of Mrc1-DTR mice treated with PDGF-AA or PBS for 5 days. Wound bed tissue samples were immunostained for EN1 (green) and Ki67 (red). White arrowheads indicate EN1+Ki67+ cells. Scale bars, 10 μm. Fluorescence images with an IgG-negative primary antibody control are available in the Fig. EV5. (J) The percentages of EN1−, EN1+Ki67−, and EN1+Ki67+ cells out of the total DAPI-positive cells/0.01-mm2 wound area were compared between mouse groups. Data are expressed as means ± SD. *P = 0.0045 (EN1−), 0.3201 (EN1+ Ki67−), 0.0076 (EN1+ Ki67+) by unpaired two-tailed t test after applying logit transformation. (n = 4 mice/group, biological replicates). Source data are available online for this figure.