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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 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE272843 | GEO | 2025/01/23

REPOSITORIES: GEO

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Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.

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

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