purify primary LEC and BEC from adult ear skin, except that enzymatic digestion of skin cell suspensions was increased from 30 to 60 min with frequent agitation. FGF2 promotes primary mouse LEC proliferation The proliferation of primary embryonic dermal LEC in culture was assessed using an established colorimetric assay for the quantification of viable cells. To investigate the effect of established pro-lymphangiogenic growth factors on the prolifera- tion of primary embryonic dermal LEC in culture, LEC were seeded in full media (EGM-2MV) for 18 h and then starved in basal media (EBM-2 containing 0.5 mg ml21 Albumax II) for 4 h. Base media was then replaced with media containing either VEGF-C or FGF2 at a range of concentrations and cell proliferation was measured 48 h later. These assays revealed that FGF2 potently stimulated the proliferation of primary embryonic dermal LEC (Figure 3a). In fact, while 2.5 ng ml21 of FGF2 promoted approximately three-fold greater LEC proliferation than did base media, 200 ng ml21 of VEGF-C stimulated LEC proliferation only two-fold (Figure 3a). Previous reports have ascribed a pro-lymphangiogenic role to FGF2, but suggested that this effect is mediated indirectly, via the activity of VEGF-C and VEGF-D [31,32]. We assessed whether this was the case in primary embryonic dermal LEC by treating LEC with FGF2 in combination with small molecule tyrosine kinase inhibitors of FGFR (SU5402) [33], VEGFR-2 (SU5416) [34] and VEGFR-3 (MAZ51) [35]. The addition of SU5402 to LEC media in the presence of FGF2 completely abrogated FGF2 stimulated proliferation (Figure 3b). In contrast, small molecule tyrosine kinase inhibitors of VEGFR-2 (SU5416) and VEGFR-3 (MAZ51) that reduced VEGF-C mediated LEC proliferation (Figure S3), did not significantly inhibit FGF2-mediated LEC proliferation (Figure 3b). These data demonstrate that FGF2 promotes the proliferation of primary LEC directly via FGF receptors and independently of VEGF receptors. FGF2 and VEGF-C promote the migration of primary mouse LEC We next established an assay to quantify the migration of primary LEC in response to lymphangiogenic stimuli. We first assessed the ability of LEC seeded in Boyden chambers to migrate through a filter towards VEGF-C, but found that only a very low proportion of cells were capable of chemotactic migration through the filter, perhaps due to detrimental effects of trypsinization prior to cell seeding. We next established a ‘‘scratch’’ assay to quantify cell migration. For this assay, freshly isolated LEC were allowed to grow to confluence and were then washed and starved for 16 h. Scratches across LEC monolayers were made and LEC migration in response to designated treatments was measured following 8 h. A dose response assay revealed that maximal LEC migration was achieved at a dose of 10 ng ml21 FGF2 (Figure S4) and that this dose of FGF2 promoted the migration of primary embryonic dermal LEC to a similar extent as 200 ng ml21 VEGF-C (Figure 4). Moreover, FGF2 stimulated LEC migration was inhibited by the small molecule FGFR inhibitor SU5402 (10 mM), illustrating that FGF2 promoted LEC migration directly, via cell autonomous signalling through FGF receptors. FGF2 cooperates with VEGF-C to promote tube formation We next sought to establish a three-dimensional assay able to reproduce features of lymphatic vessel growth in vivo, and to this end, developed a protocol to promote the assembly of primary Lymphangiogenesis Assays Using Primary Mouse LEC embryonic dermal LEC into tubes. We initially attempted to promote LEC tube formation in an analogous fashion to methods used to stimulate the formation of human LEC tubes, by plating LEC onto Matrigel [9]. However, primary embryonic mouse dermal LEC did not form tubes in this setting. To promote tube formation, we first established semi-confluent (50–70%) cultures of primary dermal LEC (Figure 5a) and then overlaid LEC monolayers with Matrigel containing selected growth factors and/or small molecule inhibitors. Tube formation was measured 24 and 48 h later. Primary embryonic mouse dermal LEC rapidly organised into aggregates following the addition of Matrigel (Figure 5b). However, in the presence of FGF2 (10 ng ml21), LEC dynamically organised into a network of tubes and exhibited features characteristic of lymphatic vessel growth including proliferation, sprouting, migration and anastomosis (Figure 5b). The effects of growth factor treatment on tube formation were quantified by measuring average vessel diameter and the number of vessel branchpoints per field. Cell proliferation was a striking feature of FGF2-promoted tube formation, reflected in the increased diameter of LEC tubes following treatment with FGF2 compared to Matrigel alone (Figure 5b, d). The addition of VEGF- C to Matrigel also enhanced LEC tube formation, but the morphology of tubes formed in response to VEGF-C treatment was strikingly different to that promoted by FGF2. VEGF-C potently promoted LEC sprouting and elongation, rather than proliferation (Figure 5b); VEGF-C induced tubes contained substantially more branchpoints than FGF2-induced tubes, (Figure 5e), while vessel diameter was significantly lower (Figure 5d). In combination, FGF2 and VEGF-C promoted proliferation and sprouting, resulting in the development of large, interconnected lymphatic vascular tubes (Figure 5b, d, e). Tube formation promoted by FGF2 was inhibited by the small molecule FGFR inhibitor SU5402, but not by the VEGFR-2 inhibitor SU5416 (Figure 5c, d, e). Intriguingly, FGF2 promoted tube formation was significantly affected by treatment with the VEGFR-3 inhibitor MAZ51 (Figure 5 c, d). In particular, vessel diameter and the morphology of cells were altered; LEC assumed a more cuboidal, less elongated morphology (Figure 5c) in response to MAZ51 treatment. FGFR1 is the predominant FGF receptor in primary dermal mouse LEC and is crucial for LEC proliferation In order to investigate which FGF receptors were responsible for transducing FGF2 mediated pro-lymphangiogenic signals, we first quantified the mRNA expression level of each of the four FGF receptors in primary dermal LEC and BEC immediately following cell isolation from the skin. Real-time RT-PCR of embryonic and adult LEC revealed that Fgfr1 was by far the predominant FGF receptor in both embryonic (Figure 6a) and adult (Figure S5) LEC and BEC. Moreover, Fgfr1 levels were more than two-fold elevated in LEC compared to BEC, providing a possible explanation for the sensitivity of LEC to low doses of FGF2. In comparison, Fgfr3, previously suggested to have a pro-lymphangiogenic role [36], was expressed at dramatically lower levels than Fgfr1 in primary embryonic (Figure 6a) and adult (Figure S4) LEC. Investigation of FGFR1 protein levels in primary embryonic LEC revealed that FGFR1 was localised primarily to the nucleus of LEC grown in complete media (Figure 6b). Nuclear FGFR1 has previously been associated with cell proliferation [37]. In order to establish whether this was also the case in primary embryonic LEC, FGFR1 localisation was compared between cells grown in complete media and cells that were serum starved. Substantially more nuclear FGFR1 was observed in LEC grown in complete PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40497