Extended Data Fig. 4: mrc1a+ microglia are dependent on lymphangiogenesis.
(A) Representative images of two orthogonal rotations of confocal z-projection (left) and IMARIS 3D surface rendering (right) of 6 dpf Tg(mrc1a:egfp) animals showing a secondary sprout of a growing lymphatic vessel. White arrows indicate vessel secondary sprout site. Blue arrowheads indicate hollow vessel center. (B) Representative confocal z-projections of Tg(mrc1a:egfp) animals stained with 4C4 showing the reduction of mrc1a+;4C4+ microglia in cinnarizine, flunarizine, and leflunomide treated animals compared to DMSO control animals. Blue arrowheads represent mrc1a+;4C4+ microglia. (C) Representative confocal z-projections of 5 dpf Tg(mrc1a:egfp) animals showing the disruption of vessel growth and development in animals treated with A77-1726, cinnarizine, flunarizine, or leflunomide compared to control DMSO animals. (D) Quantification showing the reduced average length of brain lymphatic vessels in lymphatic inhibitor treated animals compared to DMSO control animals (one-way ANOVA/Dunnett’s multiple comparisons: DMSO vs. A77-1726 p = 0.0444, Mean diff=22.94, DF-358, q = 2.622, SE of diff=8.749; DMSO vs. cinnarizine p = 0.2378, Mean diff=16.62, DF = 358, q = 1.937, SE of diff=8.592; DMSO vs. flunarizine p = 0.0060, Mean diff=35.94, DF = 358, q = 3.263, SE of diff=11.02; DMSO vs. leflunomide p = 0.0008, Mean diff=25.83, DF = 358, q = 3.81, SE of diff=6.78) (n = 80 animals) (E) Quantification showing the reduced average number of secondary sprouts in lymphatic inhibitor treated animals compared to DMSO control animals (one-way ANOVA/Dunnett’s multiple comparisons: DMSO vs. A77-1726 p = 0.0207, mean diff =1.8, DF = 63, q = 2.966, SE of diff=0.6068; DMSO vs. cinnarizine p = 0.0012, Mean diff=2.371, DF = 63, q = 3.908, SE of diff=0.6068; DMSO vs. flunarizine p = 0.0178, Mean diff=2.336, DF = 63, q = 3.019, SE of diff=0.7736; DMSO vs. leflunomide p = 0.0004, Mean diff=2.308, DF = 63, q = 4.213, SE of diff=0.5478)(n = 80 animals). (F) Quantification showing the reduced average number of lymphatic vessels surrounding the brain in lymphatic inhibitor treated animals compared to DMSO control animals (one-way ANOVA/Dunnett’s multiple comparisons: DMSO vs. A77-1726, DMSO vs. cinnarizine p = 0.0081, Mean diff=3.242, DF = 64, q = 3.29, SE of diff=0.9854; DMSO vs. cinnarizine p = 0.0124, Mean diff=3.099, DF = 64, q = 3.145, SE of diff=0.9854; DMSO vs. flunarizine p = 0.0883, Mean diff=3.028, DF = 64, q = 2.408, SE of diff=1.257; DMSO vs. leflunomide p = 0.1368, Mean diff=1.972, DF = 64, q = 2.218, SE of diff=0.889)(n = 80 animals). (G) Quantification of the number of 4C4+ only microglia in DMSO control animals compared to leflunomide and flunarizine treated animals (t-test: DMSO vs. leflunomide, DMSO vs. flunarizine (Ordinary one-way ANOVA/Dunnett’s multiple comparisons: DMSO vs. leflunamide p = 0.9997, Mean diff = −0.04274, DF = 25, q = 0.01816, SE of diff=2.353; DMSO vs. flunarizine p = 0.2097, Mean diff=4.346, DF = 25, q = 1.623, SE of diff=2.678)(n = 29 animals). (H) Quantification of the number of pu1+ only microglia in DMSO control animals compared to leflunomide and flunarizine treated animals (Ordinary one-way ANOVA/Dunnett’s multiple comparisonst: DMSO vs. leflunamide p = 0.9932, Mean diff = −0.2222, DF = 36, q = 0.09986, SE of diff=2.225; DMSO vs. flunarizine p = 0.6213, Mean diff = −2.167, DF = 25, q = 0.8554, SE of diff=2.533)(n = 29 animals). (I) Quantification of the number of pu1+;4C4+ microglia in DMSO control animals compared to leflunomide and flunarizine treated animals (Ordinary one-way ANOVA/Dunnett’s multiple comparisons: DMSO vs. Leflunamide p = 0.7997, Mean diff = −1.342, DF-25, q = 1.19, SE of diff=1.167; DMSO vs. Flunarizine p = 0.4353, Mean diff=0.7692, DF = 25, q = 0.5789, SE of diff=1.329)(n = 29 animals). Imaging window equals 0.0027 mm3 (A-F), 0.0081 mm3 or 3000 µm (G-I). Scale bar equals 10 µm (A-C).
