A single-cell atlas of normal and KRASG12D-malformed lymphatic vessels

Abstract

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We identified 6 subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from 4 stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in KrasG12D mice. We also observed that the proportion of collecting and valve LECs was lower in KrasG12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.

Keywords: Angiogenesis; Development; Endothelial cells; Vascular biology.

Figure 1. Pulmonary lymphatic vessels are enlarged in Kras^G12D mice.

(A) Schematics of the Prox1-CreER^T2 and Kras^LSL-G12D alleles. (B) Schematic showing when mice received tamoxifen (50 μg; p.o.). Tissues were collected when mice were 21 days old. (C) Low- and high-magnification views of Vegfr3-positive lymphatic vessels in 100-μm-thick lung sections from control and Kras^G12D mice. The high-magnification images are of the boxed regions. (D) The diameter of lymphatic vessels was significantly greater in Kras^G12D mice (25.65 ± 1.070 μm; n = 5) than in control (Ctrl) mice (14.31 ± 0.9752 μm; n = 5). Data are presented as mean ± SEM. ****P < 0.0001 by 2-tailed, unpaired Student’s t test. Scale bars: 500 μm (low magnification) and 50 μm (high magnification).

Figure 2. Kras^G12D impairs lymphatic valve development.

(A) Schematics of the Flt4-CreER^T2, Rosa26^mT/mG (R26^mT/mG), and Kras^LSL-G12D alleles. (B) Schematic showing when mice received tamoxifen (100 μg; s.c.). Tissues were collected when mice were 21 days old. (C) GFP-positive interlobular lymphatic vessels in control and Kras^G12D mice. Lymphatic valves (arrows) were readily observed in control mice (n = 4 mice), but not in Kras^G12D mice (n = 3 mice).

Figure 3. The cellular and transcriptional profile of pulmonary LECs in control mice revealed by scRNA-Seq.

(A) Schematics of the Prox1-CreER^T2, R26^mT/mG, and Kras^LSL-G12D alleles. (B) Schematic showing when mice received tamoxifen (50 μg; p.o.). Tissues were collected when mice were 21 days old. (C) Violin plots showing the expression of select blood endothelial cell and LEC markers and LEC subtype markers. (D) UMAP showing the clustering of LECs (n = 1,334 cells) from control mice. We identified 6 unique clusters of LECs in the lungs of control mice. (E) Trajectory analysis of LECs from control mice using Monocle 3. (F) Heatmap showing the top 10 most differentially expressed genes for each LEC subtype. (G) GO terms analysis of genes enriched in each LEC subtype.

Figure 4. Temporal mapping of the pulmonary LEC landscape.

(A) UMAPs showing the pulmonary LEC subtypes present at E14.5, E16.5, E18.5, P0, and P21. (B) Graph showing the percentage of the LEC subtypes present at E14.5, E16.5, E18.5, P0, and P21.

Figure 5. Kras^G12D induces changes in the cellular and transcriptional landscape of lymphatic vessels.

(A) UMAPs for pulmonary LECs from control and Kras^G12D mice. (B) Graph showing the percentage of the LEC subtypes in the lungs of control and Kras^G12D mice. (C) Violin plots showing average gene expression module scores for capillary signature genes. (D) Violin plots showing average gene expression module scores for collecting vessel signature genes. (E) GO terms associated with genes upregulated by Kras^G12D. (F) GO terms associated with genes downregulated by Kras^G12D.

Figure 6. Temporal sensitivity of lymphatic vessels to Kras^G12D.

(A) Schematics of the Prox1-CreER^T2, R26^mT/mG, and Kras^LSL-G12D alleles. (B) Schematic showing when mice received tamoxifen (early induction = 50 μg, p.o.; late induction = 2 mg, i.p.). (C) Representative images of ear skin whole mounts stained with an anti-GFP antibody. (D–F) Results for early tamoxifen treatment. (D) Branch points per millimeter vessel for control (2.718 ± 0.07512; n = 6) and Kras^G12D (2.086 ± 0.09636; n = 5) mice. (E) Valves per millimeter vessel for control (1.828 ± 0.1274; n = 6) and Kras^G12D (0.0760 ± 0.01503; n = 5) mice. (F) Vessel diameters for control (38.12 ± 1.212 μm; n = 6) and Kras^G12D (72.89 ± 4.360 μm; n = 5) mice. (G–I) Results for late tamoxifen treatment and early collection. (G) Branch points per millimeter vessel for control (2.122 ± 0.07161; n = 7) and Kras^G12D (2.252 ± 0.1862; n = 7) mice. (H) Valves per millimeter vessel for control (2.299 ± 0.1534; n = 7) and Kras^G12D (2.133 ± 0.1016 n = 7) mice. (I) Vessel diameters for control (44.25 ± 1.164 μm) and Kras^G12D (47.92 ± 0.5579 μm) mice. (J–L) Results for late tamoxifen treatment and late collection. (J) Branch points per millimeter vessel for control (1.947 ± 0.09457; n = 6) and Kras^G12D (1.711 ± 0.08402; n = 5) mice. (K) Valves per millimeter vessel for control (1.937 ± 0.1105; n = 6) and Kras^G12D (1.948 ± 0.1595; n = 5) mice. (L) Vessel diameters for control (43.26 ± 0.6543 μm; n = 6) and Kras^G12D (47.81 ± 1.036 μm; n = 5) mice. Data are presented as mean ± SEM. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed, unpaired Student’s t test (D, E, H, I, J, K, and L) or Mann-Whitney test (F and G). Scale bar: 500 μm.