Fig 2: Normal molecular and cellular lung development in late gestation Clp1K/K embryos. Immunostaining for CC10, SPC, PDPN, PDGFRa, PDGFRß, NG2, a-SMA, Desmin, Vimentin, PECAM1, VEGFR3, PROX1, LYVE1, and DAPI nuclear staining are shown in late gestation Clp1+/+ and Clp1K/K embryonic lungs at E18.5. Representative images are shown of three to five independent experiments per group. Bars, 50 µm.

Fig 3: Characterization of the developmental program of meningeal lymphatics. (A) Expression of GFP and LYVE-1 adjacent to the venous sinuses of whole-mount immunostained meninges of Prox1GFP lymphatic reporter mice at P0 and P21 (P0 n = 9; P21 n = 6). Bars, 500 µm. (B) Expression of GFP and PDPN adjacent to the venous sinuses of whole-mount immunostained meninges of Prox1GFP lymphatic reporter mice at P0 and P21 (P0 n = 6; P21 n = 6). Bars, 500 µm. (C) Expression of YFP and LYVE-1 in the dura mater of Flt4YFP lymphatic reporter mice detected by whole-mount immunostaining at P0 and P21 (P0 n = 6; P21 n = 3). Bars, 500 µm. (D) LYVE-1 and PDPN expression shown in the meninges of P0 and P21 wild type mice (P0 n = 8; P21 n = 8). Bars, 500 µm. (E) PECAM and LYVE-1 expression adjacent to the superior sagittal sinus at P0, P3, P5, P8, P13 and P21 in wild type mice (P0 n = 6; P3 n = 5; P5 n = 2; P8 n = 2; P13 n = 3; P21 n = 7). Bars, 500 µm. (F) GFP signal detected by fluorescent stereo microscopy (n = 18) in meninges of Prox1GFP lymphatic reporter mice at P0. Bars, 500 µm. (G) GFP and LYVE-1 expression detected by confocal microscopy after whole-mount immunostaining of meninges of Prox1GFP lymphatic reporter mice at P0. Bars, 200 µm. (H,I) LYVE-1 and F4/80 expression shown in the meninges of wild type mice at P0 and P21 imaged by fluorescent stereo microscopy (P0 n = 9; P21 n = 6; bars, 500 µm) (H) or confocal imaging (bars, 200 µm) (I). (J) GFP and F4/80 expression in the meninges of P0 and P21 Prox1GFP reporter mice (P0 n = 3; P21 n = 3). Bars, 500 µm. Representative images are shown in all panels.

Fig 4: Expression of lymphatic markers in meninges of young adult mice. (A) Expression pattern of PECAM and LYVE-1 shown in the dura mater of young adult wild type mice (n = 7). Bars, 500 µm. (B,C) LYVE-1 and PDPN immunostaining of mouse meninges imaged by fluorescent stereo microscopy (n = 12; bars, 500 µm) (B) or confocal imaging (n = 3; bars, 100 µm) (C). (D) LYVE-1 and PROX-1 immunostaining of meninges (n = 4). Bars, 500 µm. (E–G) Expression pattern of GFP, LYVE-1 and PECAM adjacent to the venous sinuses of the meninges of Prox1GFP lymphatic reporter mice at P21. Images are shown for native GFP signal (n = 5; bars, 500 µm) (E), GFP and LYVE-1 immunostaining detected by fluorescent stereo microscopy (n = 6; bars, 500 µm) (F) or confocal microscopy (n = 3; bars, 200 µm) (G). (H) Expression pattern of GFP and PECAM shown by fluorescent stereo microscopy after whole-mount immunostaining of wild type meninges (n = 5). Bars, 500 µm. (I) Expression of YFP and LYVE-1 adjacent to the venous sinuses of the meninges of young adult Flt4YFP lymphatic reporter mice shown by GFP and LYVE-1 immunostaining (n = 3). Bars, 500 µm. Representative images are shown in all panels.

Fig 5: Administration of VEGFC mRNA-LNPs results in non-significant blood vessel proliferation and immune response activation compared to the Poly(C) RNA-LNP control.a 1 µg of Poly(C) or VEGFC mRNA-LNPs was intradermally injected into the ear of C57Bl/6 mice and the growth of blood and lymphatic vessels were examined 22 days after the injection. Representative images of anti-CD31, anti-LYVE1, anti-Podoplanin, and anti-vWF stained paraffin-embedded ear samples are shown of 5 mice in each group (bars, 25 µm (anti-CD31, anti-LYVE1), 50 µm (anti-Podoplanin, anti-vWF)). Yellow arrows indicate LYVE1 positive lymphatic vessels, white arrows indicate CD31 positive and LYVE1 negative blood vessels. b The number of vWF high and LYVE1 negative blood vessels was determined 22 days after the administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs. Data are represented as mean and SEM from slides of ears of 10 mice per group (two-tailed, paired T-test, P = 0.6344 for 10 mice). c Monitoring the effect on lymphatic endothelial cells and immune cells after intradermal injection of 1 µg of Poly(C) or VEGFC mRNA-LNPs into ears of Prox1GFP mice shown by flow cytometry analysis. Quantitative data for LYVE1+ , CD45+ , Ly6G/C (GR1)+, CD206+, CD3+, B220+ and CD11b+ Ly6G/C- cell numbers are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 5–7 mice in each group, 22 days after mRNA-LNP injection (two-tailed, unpaired T-test, P = 0.0450 for LYVE1+ for 5 mice injected with Poly(C) and for 5 mice injected VEGFC mRNA-LNP, P = 0.5365 for CD45+ for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP, P = 0.1689 for Ly6G/C+ and CD45+ for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP, P = 0.7211 for CD206+ for 5 mice injected with Poly(C) and for 5 mice injected VEGFC mRNA-LNP, P = 0.8947 for CD3+ for 6 mice injected with Poly(C) and for 7 mice injected VEGFC mRNA-LNP, P = 0.7748 for B220+ for 6 mice injected with Poly(C) and for 7 mice injected VEGFC mRNA-LNP and P = 0.6922 for CD11b+ Ly6G/C- for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP comparing cell number). d Ly6G/C (GR1) positive immune cells were visualized after intradermal administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs into the ears shown by anti-GR1 and anti-LYVE1 22 days after the injection. Representative images of anti-GR1 stained paraffin-embedded ear samples are shown of 3 mice per group (bars, 50 µm). Arrows indicate LYVE1 positive lymphatic vessels. Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.