Platelets Play an Essential Role in Separating the Blood and Lymphatic Vasculatures During Embryonic Angiogenesis
Rationale: Several mutations that impair the development of blood lineages in the mouse also impair the formation of the lymphatic vasculature and its separation from the blood vasculature. However, the basis for these defects has remained unknown because the mutations characterized affect more than one blood lineage.
Objective: We tested the hypothesis that megakaryocytes/platelets are required for the formation of the lymphatic vasculature and its separation from the blood vascular system.
Methods and Results: We characterized the vascular patterning defects of mice deficient for the homeodomain transcription factor Meis1 (myeloid ecotropic viral integration site 1), which completely lack megakaryocyte/platelets. Meis1 null embryos fail to separate the blood and lymphatic vasculature, showing blood-filled primary lymphatic sacs and superficial lymphatic vessels. To test the involvement of megakaryocytes/platelets in this phenotype, we generated megakaryocyte/platelet-specific deficient mice by targeted lineage ablation, without affecting other blood lineages. This model reproduces the lymphatic/blood vasculature separation defects observed in Meis1 mutants. A similar phenotype was induced by antibody-mediated ablation of circulating platelets in wild type mice. Strong association of platelets with vascular endothelium at regions of contact between lymphatic sacs and veins confirmed a direct role of platelets in the separation of the 2 vasculatures.
Conclusions: In addition to their known protective function in the response accidental vascular injury, platelets are also required during embryonic lymphangiogenesis for the separation of the nascent lymphatic vasculature from blood vessels.
The mammalian lymphatic vasculature develops at midgestation from lymphendothelial precursors produced from embryonic veins.1 Endothelial cells in different regions of the cardinal veins start to express the lymphatic endothelial cell master gene Prox1 at embryonic development day (E)10.5.2 This activates expression of lymphatic-specific molecules and the formation of lymphatic sacs from the vein-derived lymphendothelial precursors, stimulated by vascular endothelial growth factor-C/vascular endothelial growth factor receptor 3.3,4 The mature postnatal lymphatic vasculature is completely independent of the blood vascular system, except for the connections of the thoracic and right ducts with the subclavian veins, through which lymph drains into the blood circulation. At these junctions, specialized valves prevent blood reflux into the lymphatic vessels.5 However, during development, connections between the venous system and the forming lymphatic vasculature lack valves and therefore additional mechanisms must ensure their separation. Mutations in Syk and Slp-766,7 and Runx1,1 all affecting the development of blood lineages, block separation of the blood and lymphatic vasculature; however, the nature of the cell lineages involved and the mechanisms by which they control blood/lymphatic vessel separation remains unclear, because all mutations studied affect more than one blood lineage. Meis1 (myeloid ecotropic viral integration site 1) encodes a homeodomain transcription factor important for definitive hematopoietic stem cell development in the embryo and essential for megakaryocyte lineage development.8 Here, through characterization of Meis1-deficient mice and targeted ablation of megakaryocytes/platelets, we demonstrate an essential role for platelets in the separation of the blood and lymphatic vasculature.
Immunohistochemistry and immunofluorescence were performed as previously described.8 Primary antibodies were as follows: anti-CD31 (553370) and anti-CD41 (553847) (BD Biosciences-Pharmingen); anti-CD61 (NB100–79980CC, Novus Biologicals); anti–Lyve-1 (103-PA50S ReliaTech); and anti-Meis1.8 Secondary antibodies were as follows: biotinylated goat antirat (Ab7096, Abcam) and goat anti-rabbit biotin (111-066-003, Jackson Immunoresearch), followed by Streptavidin-Alexa 488 (s-11223, Invitrogen) or -Cy3 (016-160-084, Jackson Immunoresearch). For immunohistochemistry, we used Vectastain ABC (Vector Laboratories) with alkaline phosphatase (AK-5000) and developed with FastRed (11496549001, Roche Diagnostic).
Whole mount in situ hybridization was performed using standard procedures. Riboprobes were obtained by SP6 polymerase transcription from cDNA PCR-amplified fragments.
Meis1 mutant mice8 were backcrossed to C57BL/6J mice for more than 20 generations. Platelet factor (PF)4-Cre9 and R26:lacZbpAfloxDTA10 were backcrossed to the C57BL/6J background for more than 10 generations.
To transiently deplete platelets during gestation, pregnant C57BL/6J females received IP injections twice, at E11.5 postcoitum and 9 hours later, with 125 μg (100 μL) of rabbit anti-mouse thrombocyte antibody (AIA31440 Accurate Chemical).
Animals were housed in accordance with Spanish bioethical regulations for laboratory animals.
Results and Discussion
Meis1-deficient embryos accumulate blood at ectopic foci, resulting eventually in generalized edema with internal hemorrhage.8 The distribution of ectopic blood foci matches the pattern of nascent lymphatic vessels at E12.5 and E13.5 (Figure 1a through 1d),1 and analysis of endothelial and lymphendothelial markers identified the ectopic blood foci as the recently formed lymphatic sacs, which were filled with erythrocytes (Figure 1e through 1g and 1i through 1k). Meis1 is not expressed in blood vascular endothelial cells8 or lymphatic endothelium (Figure 1h and 1i), suggesting a nonautonomous involvement of nonvascular cells. We therefore explored the involvement of Meis1 blood lineage defects in this phenotype. At the stage when the first lymphatic defects are observed in Meis1 mutant embryos the only blood cells severely affected are the megakaryocytes.8 Expression of the megakaryocyte-specific marker cxcl7 is first detected in WT embryos at E9.5, in cells distributed throughout the embryo, but not in the yolk sac (Figure 2a). From E10.5, the yolk sac and the incipient liver primordium show colonization by cxcl7-expressing cells, which intensifies by E11.5 and is accompanied by expression of cxcl4 (Figure 2b through 2d). Circulating platelets are detected from E10.5, coinciding with megakaryocyte liver colonization.11 By E12.5 megakaryocytes in WT liver have increased considerably in size and intensity of marker expression, including the platelet antigen CD41 (Figure 2e). In contrast, Meis1-deficient embryos lack cells expressing megakaryocyte markers at all stages analyzed (Figure 2f through 2j).
Although these results suggest involvement of megakaryocytes/platelets in the separation of the lymphatic and blood vasculatures, they are not definitive because Meis1 mutants also show defective generation of hematopoietic stem cells.8 We therefore generated megakaryocyte-deficient mice by targeted ablation of this lineage, using the Rosa26R-LacZbpa-DTA mouse line, which conditionally expresses the diphtheria toxin from the Rosa26 locus upon Cre recombination.10 The inducer strain was the PF4-Cre line, which specifically expresses Cre recombinase in the megakaryocyte lineage (Figure 3a).9 Early megakaryocyte development in the DTA model appeared normal up to E11.5 (data not shown), but E12.5 embryos showed strongly reduced megakaryocyte marker expression (Figure 3d and 3g). Megakaryocyte lineage deletion takes place later in the DTA model than in Meis1-deficient mice (Figure 3c and 3f). Megakaryocyte-deficient mice reproduced the blood-filled lymphatic sacs seen in Meis1-deficient mice at E13.5 (Figure 3h, 3i, 3l, and 3p). However, unlike Meis1-deficient embryos, megakaryocyte-depleted fetuses survive to E15.5 and do not show liver hypoplasia (Figure 3h through 3k; Figure 1b and 1d). Blood-filled peripheral lymphatic vessels are still evident at these later stages (Figure 3i through 3k, 3m through 3o, and 3q through 3s).
To test whether platelets were involved in the observed defects, we injected pregnant females twice, at 11.5 and 12 days postcoitum, with anti-thrombocyte antibody. Treated fetuses examined at E12.5 showed ectopic blood foci similar to those observed in Meis1-null and PF4-deleted embryos (Figure 4a through 4c; N=6/8), and histological analysis confirmed correspondence of these foci to the nascent lymphatic sacs (Figure 4d through 4g). Consistently, circulating platelets, which are abundant in untreated E12.5 WT embryos, were undetectable in Meis1-null and PF4-deleted embryos (Figure 4h through 4m). CD41 staining of anti-platelet–treated embryos confirmed that the megakaryocyte cell population was unaffected (Figure 4o), but that circulating platelets were agglutinated in large aggregates. Analysis of the junctions between primary lymphatic sacs and cardinal veins detected platelets adhering specifically to the lymphatic and venous endothelia at the sites where the 2 vasculatures meet (Figure 4r, 4s, and 4u), but not to endothelia outside this region (Figure 4r, 4h, and 4i).
These results demonstrate a morphogenetic role for platelets during the process that separates the blood and lymphatic vasculatures. Adhesion of platelets to the vascular wall suggests that they are activated by contact with the endothelium at the lymphatic/venous interface. Platelets are not components of lymph and therefore might be activated on contact with lymphendothelial-specific surface molecules, thus preventing connections between the lymphatic and venous vasculatures by forming a structural barrier. This model is strongly supported by evidence that platelets are activated by contact with the lymphendothelial molecule podoplanin and by the occurrence of blood–lymphatic separation defects in podoplanin-deficient mice,12 although additional local signaling roles of platelets cannot be discarded. We suggest that the known involvement of Syk and Slp-76 in platelet activation13 and the defective megakaryocyte differentiation in Runx1 mutants14 contribute to the blood/lymphatics separation defects in these mutants. These results indicate that platelets, in addition to their role in repairing accidental vascular injuries, play a morphogenetic role during angiogenesis that allows the proper separation of blood and lymphatic circulation.
We thank Radek Skoda (Basel) for the PF4-Cre mice; Tamara Córdoba and Virginia García for mouse care; Silvia Vela for mouse genotyping; Roisin Doohan for histology; José Manuel Ligos, Mariano Vitón, and Raquel Nieto for FACS; and Simon Bartlett for text editing.
Sources of Funding
The Centro Nacional de Investigaciones Cardiovasculares is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation. This work was supported by Spanish Ministry of Science and Innovation (RD06/0010/0008 and BFU2009-08331/BMC) and the EU COST program (COST-BM0805).
Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, Samokhvalov IM, Oliver G. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev. 2007; 21: 2422–2432.
Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV, Jeltsch M, Jackson DG, Talikka M, Rauvala H, Betsholtz C, Alitalo K. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol. 2004; 5: 74–80.
Oliver G. Lymphatic vasculature development. Nat Rev. 2004; 4: 35–45.
Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R, Mocsai A, Myers EE, Huang B, Jackson DG, Ferrari VA, Tybulewicz V, Lowell CA, Lepore JJ, Koretzky GA, Kahn ML. Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk. Science. 2003; 299: 247–251.
Tiedt R, Schomber T, Hao-Shen H, Skoda RC. Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood. 2007; 109: 1503–1506.
Tober J, Koniski A, McGrath KE, Vemishetti R, Emerson R, de Mesy-Bentley KK, Waugh R, Palis J. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood. 2007; 109: 1433–1441.
Uhrin P, Zaujec J, Breuss JM, Olcaydu D, Chrenek P, Stockinger H, Fuertbauer E, Moser M, Haiko P, Fassler R, Alitalo K, Binder BR, Kerjaschki D. Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation. Blood. January 2010; doi: 10.1182/blood-2009-04-216069.
Samaha FF, Kahn ML. Novel platelet and vascular roles for immunoreceptor signaling. Arterioscler Thromb Vasc Biol. 2006; 26: 2588–2593.
Ichikawa M, Asai T, Saito T, Seo S, Yamazaki I, Yamagata T, Mitani K, Chiba S, Ogawa S, Kurokawa M, Hirai H. AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nat Med. 2004; 10: 299–304.
Novelty and Significance
What Is Known?
The lymphatic vasculature emerges during mammalian embryonic development from preexisting blood vessels through a centripetal sprouting process.
The separation of the blood and lymphatic circulations is linked to the differentiation of the blood lineages, but until now, it was unknown which lineage controls blood/lymphatic vessel separation.
What New Information Does This Article Contribute?
Using 3 independent methods to eliminate the megakaryocyte/platelet lineage in mice, we show that platelets are required for the separation of the blood and lymphatic vasculatures.
This novel action of platelets involves their specific activation and adhesion with the endothelium at the junctions between blood and lymphatic vasculatures.
These findings identify a previously unknown morphogenetic role for platelets and hint at a general role in vascular morphogenesis and remodeling
Separation of the blood and lymphatic vasculatures is disrupted by mutations that affect blood lineage differentiation, but the specific lineages and mechanisms involved were unknown. We show that specific elimination of the megakaryocyte/platelet lineage results in blood-filled lymphatic vessels, indicating a failure to separate the blood and lymphatic circulations. Adhesion of platelets at the junctions between blood and lymphatic vasculatures indicates that local activation of platelets is involved in this process. These results identify a previously unknown morphogenetic role for platelets during lymphangiogenesis and suggest a general role of platelets in vascular morphogenesis and remodelling potentially relevant in vascular disease.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
Original received January 29, 2010; revision received February 15, 2010; accepted February 22, 2010.