Spreds Are Essential for Embryonic Lymphangiogenesis by Regulating Vascular Endothelial Growth Factor Receptor 3 Signaling

Vessels of the lymphatic system are highly permeable and specialized for the uptake of fluid and macromolecules from the interstitium and their return to venous circulation (2, 21). Embryonic development of the lymphatic vessels starts when a subset of endothelial cells in the cardinal vein commits to the lymphatic lineage and sprouts to form the primary lymph sacs (2, 21, 22). Recent studies have identified specific transcription factors and growth factors required to regulate the development of lymphatic vessels. In mice, the lymphatic vasculature starts to develop at embryonic day 10.5 (E10.5), when the cardiovascular system is already functioning. After the formation of the initial lymph sacs, the peripheral lymphatics are generated by centrifugal sprouting. Several genes have been shown to play essential roles in embryonic lymphatic vessel development. The Prox1 homeobox transcription factor, vascular endothelial growth factor C (VEGF-C), and its receptor, vascular endothelial growth factor receptor 3 (VEGFR-3), are essential for the generation of lymphatics (2, 21). Hematopoietic intracellular signaling proteins, Syk, SLP-76, and PLCγ2, are important for the separation of lymphatic vessels from blood vessels (1, 30). Furthermore, the final patterning and maturation of lymphatic vasculature require angiopoietin 2, neuropilin 2, and podoplanin/T1 (2, 21). However, regulatory genes for lymphatic vessel development have not been identified yet. Recently, Sprouty/Spred family proteins were identified as negative regulators for growth factor- and cytokine-induced ERK activation (5, 9, 14, 29). In mammals, four Sprouty homologues and three Spred (for Sprouty-related Ena/VASP homology 1 domain-containing proteins) homologues have been identified (6, 14, 29). Spred-1 has been implicated in hematopoiesis, since bone marrow-derived mast cells and eosinophils from Spred-1−/− mice were more sensitive to interleukin 3 and interleukin 5, respectively, than those from wild-type (WT) mice (11, 20). In Spred-2−/− mice, embryonic hematopoiesis was enhanced in the aorta-gonad-mesonephros region compared with WT mice (19). However, except for a shortened face and slight growth retardation, neither Spred-1−/− nor Spred-2−/− mice showed strong developmental abnormalities in most organs (11, 19). To define overlapping functions of Spred-1 and Spred-2, we generated Spred-1/Spred-2 double-knockout (DKO) mice. We found that Spreds are key regulators of embryonic lymphangiogenesis and that they can specifically regulate VEGF-C signaling by suppressing VEGFR-3-mediated ERK and Akt activation.