The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro. (Blood. 2012;119(7):1747-1756) IntroductionRecently, several mutant mouse models have shown a defect in the separation of the lymphatic vasculature from the blood vasculature typically resulting in the appearance of blood-filled lymphatic vessels in the skin at embryonic day (E) 14.5 (review in Tammela and Alitalo 1 ). Mice deficient in the tyrosine kinase Syk show this phenotype during gestation and die around the time of birth. 2-4 A similar defect is found in mice deficient in the adapter protein SLP76 (Lcp2) 4 or in PLC␥2, 5 which play vital roles downstream of Syk in immunoreceptor tyrosine-based activation motif (ITAM) and integrin signaling cascades, providing circumstantial evidence that the Syk-SLP76-PLC␥2 pathway is required for normal lymphatic development.The C-type lectin-like protein type 2 (CLEC-2, encoded by the Clec1b gene) is highly expressed on platelets and at lower levels on other hematopoietic cells [6][7][8][9] and signals through a cytosolic YxxL sequence known as a hemITAM. 10,11 These receptors signal through a similar pathway used by ITAM receptors which have a dual YxxL/I sequence. HemITAM receptors activate Syk, initiating a signaling cascade partially dependent on SLP76 that leads to activation of PLC␥2. 6,12,13 The role of CLEC-2 in hemostasis and thrombosis is debatable because some lines of evidence suggest that it is required 14,15 and others show that it has no significant involvement in these processes. 16 CLEC-2 has been recognized as a receptor for the transmembrane protein podoplanin. 17,18 Podoplanin is expressed on lymphatic endothelial cells (LECs), lung type-1 alveolar cells, and kidney podocytes but not in blood endothelial cells (BECs). Podoplanin-deficient mice die shortly after birth because of an inability to inflate their lungs and, like Syk-deficient mice, show dilated, tortuous blood-filled lymphatics in mid-gestation. 19,20 A similar phenotype is seen in mice lacking megakaryocytes/...

SummaryCLEC-2 is a C-type lectin receptor which is highly expressed on platelets but also found at low levels on different immune cells. CLEC-2 elicits powerful platelet activation upon engagement by its endogenous ligand, the mucin-type glycoprotein podoplanin. Podoplanin is expressed in a variety of tissues, including lymphatic endothelial cells, kidney podocytes, type I lung epithelial cells, lymph node stromal cells and the choroid plexus epithelium. Animal models have shown that the correct separation of the lymphatic and blood vasculatures during embryonic development is dependent on CLEC-2-mediated platelet activation. Additionally, podoplanin-deficient mice show abnormalities in heart, lungs, and lymphoid tissues, whereas absence of CLEC-2 affects brain development. This review summarises the current understanding of the molecular pathways regulating CLEC-2 and podoplanin function and suggests other physiological and pathological processes where this molecular interaction might exert crucial roles.

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.