In Vitro Assays Using Primary Embryonic Mouse Lymphatic Endothelial Cells Uncover Key Roles for FGFR1 Signalling in Lymphangiogenesis Jan Kazenwadel1., Genevieve A. Secker1., Kelly L. Betterman1,2, Natasha L. Harvey1,2* 1 Division of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia, 2 School of Medicine, University of Adelaide, Adelaide, Australia Abstract Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo. Citation: Kazenwadel J, Secker GA, Betterman KL, Harvey NL (2012) In Vitro Assays Using Primary Embryonic Mouse Lymphatic Endothelial Cells Uncover Key Roles for FGFR1 Signalling in Lymphangiogenesis. PLoS ONE 7(7): e40497. doi:10.1371/journal.pone.0040497 Editor: Tsutomu Kume, Feinberg Cardiovascular Research Institute, Northwestern University, United States of America Received March 30, 2012; Accepted June 8, 2012; Published July 6, 2012 Copyright: 2012 Kazenwadel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Health and Medical Research Council of Australia (Project Grant 626959, NLH) and Cancer Council South Australia (NLH), a MedVet Pty Ltd Research Fellowship (NLH) and National Breast Cancer Foundation PhD Scholarship (KLB) (http://www.nhmrc.gov.au/, http://www.cancersa.org.au/aspx/home.aspx, http://www.nationalbreastcancer.org/, http://www.medvet.com.au/corporate/index.php). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: natasha.harvey@health.sa.gov.au . These authors contributed equally to this work. Introduction The cellular processes underpinning the growth and develop- ment of lymphatic vessels (lymphangiogenesis) include prolifera- tion, migration, adhesion and lumen formation. All of these events need to be precisely orchestrated in order to build a lymphatic vascular network able to function optimally to maintain tissue fluid homeostasis, coordinate immune cell trafficking and absorb lipids from the digestive tract. Aberrant lymphangiogenesis is associated with a spectrum of human disorders including vascular malfor- mations, lymphoedema, inflammatory diseases and cancer [1,2]. Deciphering the genes and signalling pathways that control lymphangiogenesis is crucial in order to uncover targets to which new therapeutics able to combat these diseases can be designed. While the study of lymphangiogenesis in vivo using animal models including the mouse, frog and fish has yielded invaluable information regarding the genetic pathways important for lymphatic vascular development [1,2], the dearth of established assays to manipulate primary lymphatic endothelial cells (LEC) isolated from mouse tissue in culture has restricted our ability to dissect lymphangiogenic signalling pathways ex vivo. Models established to study lymphangiogenesis in vitro include the culture of LEC isolated from bovine [3,4], canine [5], human [5], rat [6] and ovine [7] collecting mesenteric lymphatic vessels or thoracic duct, and the culture of LEC isolated from adult mouse [8] or human skin [9]. Approaches to yield greater numbers of cells for analysis have included the culture of immortalised LEC from human [10,11] or mouse lymphangiomas [12,13], the introduc- tion of telomerase to human LEC [14] and the isolation of SV40 large T antigen immortalised LEC from the thoracic duct of rats [15] and from various organs of mice [16,17]. Conditions that promote the differentiation of LEC from embryonic stem cells have also been established [18,19,20]. Three dimensional models employed to study sprouting lymphangiogenesis in vitro include the culture of small segments of rat [21] or mouse [22] thoracic duct in collagen gels, culture of human LEC subjected to flow in collagen [23] or fibrin gels [24] and the culture of beads coated with human LEC or LEC spheroids in fibrin gels [25]. Each of these PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40497