3D imaging and single-cell analysis reveal cellular heterogeneity of lymphatic valve endothelial cell types.

Lymphatic vasculature has specialized vessels to support unidirectional interstitial fluid transport and immune surveillance. Although the lymphatic system is a critical actor in skin homeostasis, the spatial organization of the lymphatic network from the reticular dermis to the hypodermal adipose tissue remains elusive. Here, we used 3D imaging to determine the lymphatic architecture within the adult skin microenvironment. We showed that lymphatic vasculature forms a highly defined hierarchical organization from the dermal capillary to collecting lymphatic vessels located exclusively in the dermal adipose layer. Further examination revealed multiple lymphatic valves located at the intersection between capillaries and precollecting vessels. In agreement with our 3D imaging, single-cell transcriptomics uncovered two clusters of lymphatic valve endothelial cells with distinct gene signatures and signaling pathways. In summary, we provide a better understanding of the spatial organization and heterogeneity of lymphatic endothelial cells that underline the importance of lymphatic valve in adult skin.