TGFβ signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin

Dermal lymphatic endothelial cells (LECs) emerge from the dorsolateral region of the cardinal veins within the anterior trunk to form an intricate, branched network of lymphatic vessels during embryogenesis. Multiple growth factors and receptors are required for specification and maintenance of LECs, but the mechanisms coordinating LEC movements and morphogenesis to develop three-dimensional lymphatic network architecture are not well understood. Here, we demonstrate in mice that precise LEC sprouting is a key process leading to stereotypical lymphatic network coverage throughout the developing skin, and that transforming growth factor beta (TGF beta) signaling is required for LEC sprouting and proper lymphatic network patterning in vivo. We utilized a series of conditional mutants to ablate the TGF beta receptors Tgfbr1 (Alk5) and Tgfbr2 in LECs. To analyze lymphatic defects, we developed a novel, whole-mount embryonic skin imaging technique to visualize sprouting lymphangiogenesis and patterning at the lymphatic network level. Loss of TGF beta signaling in LECs leads to a severe reduction in local lymphangiogenic sprouting, resulting in a significant decrease in global lymphatic network branching complexity within the skin. Our results also demonstrate that TGF beta signaling negatively regulates LEC proliferation during lymphatic network formation. These data suggest a dual role for TGF beta signaling during lymphatic network morphogenesis in the skin, such that it enhances LEC sprouting and branching complexity while attenuating LEC proliferation.