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

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific cellular and molecular processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we use single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We show that adult wild-type mice have six subtypes of lymphatic endothelial cells (LECs) in their lungs (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes are specified during development, we integrated our data with data from four stages of development. We show that proliferating and Ptx3 LECs are prevalent during early lymphatic development and that collecting and valve LECs emerge later in development. Additionally, we demonstrate that the proportion of Ptx3 LECs decreases as the lymphatic network matures but remains high in KrasG12D mice. We also show that KrasG12D mice have fewer collecting and valve LECs than wild-type mice. Last, we demonstrate that immature lymphatic vessels in young mice are 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. To evaluate the influence of hyperactive KRAS signaling on the cellular and transcriptional landscape of lymphatic vessels, we bred Prox1-CreERT2, mT/mG, and LSL-KrasG12D mice together to generate Prox1-CreERT2;mT/mG (control) and Prox1-CreERT2;mT/mG;LSL-KrasG12D (KrasG12D) mice. Prox1-CreERT2 mice express a tamoxifen-inducible form of Cre recombinase in lymphatic endothelial cells (LECs), and LSL-KrasG12D mice express an active form of Kras (KRAS.pG12D) from the endogenous Kras locus following Cre-mediated recombination. We included the mT/mG reporter strain in our experiment to label cells that had undergone Cre-mediated recombination with green fluorescence protein (GFP). Newborn mice were fed tamoxifen from postnatal day (P)0 to P2 to induce Cre-mediated recombination in Prox1-expressing cells (Figure 1B). Following tamoxifen administration, lungs from three-week-old control (n= 5 mice) and KrasG12D mice (n= 4 mice) were dissociated into single-cell suspensions, and live GFP-positive cells were sorted by FACS. The cells were then subjected to scRNA-Seq using 10X genomics 3’ chemistry.