media containing FGF2, corresponding to elevated LEC prolifer- ation under these conditions (Figure 6b). We next set out to investigate the requirement for FGFR1 in FGF2-mediated proliferation. Due to the reported inhibition of FGFR3 by SU5402 [38], we sought to specifically ablate FGFR1 activity using siRNA to knockdown FGFR1 levels in primary LEC. Transfection of primary embryonic dermal LEC with Fgfr1, but not control siRNA, reduced Fgfr1 mRNA levels by ,70% (Figure 6c) within 48 h of transfection. Fgfr3 mRNA levels were not affected following transfection of LEC with Fgfr1 siRNA (Figure S6), demonstrating specificity of Fgfr1 siRNA for its intended target. A corresponding decrease in FGFR1 protein levels by ,70% was also observed in Fgfr1 siRNA treated cells (Figure 6d, e). Importantly, transfection of primary embryonic LEC with Fgfr1 siRNA inhibited LEC proliferation in full media containing FGF2 (Figure 6f) to a similar extent as treatment with SU5402 (Figure 6g). These data reveal that FGFR1 plays a key role in LEC proliferation and suggest that signal transduction via FGFR1 is likely to be important for lymphangiogenesis in vivo. Discussion Though primary mouse endothelial cells have traditionally proven difficult to culture, we have developed robust methodology for the measurement of primary embryonic mouse LEC prolifer- ation, migration and three-dimensional tube forming ability in response to pro-lymphangiogenic stimuli in vitro. Moreover, we have utilised these techniques to demonstrate that FGF2 potently promotes LEC proliferation, migration and tube formation and that these activities rely on signal transduction via FGFR1. Our ability to dissect FGF signalling in a cell autonomous manner has revealed that FGF2 promotes LEC proliferation independently of VEGF receptors and moreover, that FGF2 and VEGF-C play distinct roles in lymphatic vascular morphogenesis. The assays we have developed fill a longstanding gap in the field and provide the opportunity to rapidly and precisely determine gene function and delineate signalling pathway activity in primary embryonic LEC isolated from both wild-type and genetically modified mice ex vivo. While long recognised as a pro-lymphangiogenic factor, the mechanisms by which FGF2 acts on LEC have, to date, been poorly understood. In contrast to previous work suggesting that FGF2 promotes lymphangiogenesis indirectly, via increasing VEGF-C and VEGF-D production and VEGFR-3 signalling [31,32], our assays revealed that FGF2 potently promotes the proliferation of primary embryonic mouse LEC directly, via FGFR1. Though our studies focussed on embryonic mouse LEC, previous work has shown that FGF2 stimulates the proliferation and migration of postnatal bovine, human and rat LEC in vitro [4,15,36] and promotes lymphangiogenesis when ectopically introduced to adult mouse tissues in vivo [31,32]. On the basis of these data, we predict that primary adult mouse LEC would also be responsive to FGF2. Whether FGF2 primarily activates Ras/ MAPK, Plcc/Ca2+ or PI3K/Akt pathways (the 3 major pathways activated by FGF signalling [39]) in primary embryonic mouse LEC downstream of FGFR1 remains to be established. Our assays revealed distinct effects of FGF2 and VEGF-C in cellular functions important for lymphangiogenesis. While FGF2 potently promoted LEC proliferation, VEGF-C primarily pro- moted cell sprouting and together, these growth factors cooper- atively induced the formation of lymphatic vascular tubes. The effect of the small molecule inhibitor of VEGFR-3, MAZ51, on the morphology of primary LEC in a three dimensional environment confirmed that signalling via VEGFR-3 is particu- larly important for LEC sprouting and elongation. Taken Lymphangiogenesis Assays Using Primary Mouse LEC together, these data demonstrate that the assays we have developed are useful tools with which to dissect pro-lymphangio- genic signalling pathways in primary embryonic mouse LEC. Further elucidating the downstream effectors of FGFR1 and VEGFR-3 signalling pathways that are responsible for LEC proliferation versus sprouting will provide important new insights into how morphogenetic events important for lymphangiogenesis are regulated. Moreover, our data pave the way for future experiments to characterise the interplay between FGF and VEGF signalling pathways in primary lymphatic endothelial cells. Our discovery that FGFR1 is the predominant FGF receptor in primary, non-cultured embryonic mouse LEC and that FGFR1 receptor levels are substantially higher in LEC than BEC, provides an explanation for the potent stimulation of LEC proliferation by FGF2. Furthermore, our finding that embryonic LEC prolifera- tion is dependent on FGFR1 provides an explanation for the observation that knockdown of FGFR3, identified as a gene induced in response to ectopic Prox1 expression in BEC, does not completely block FGF2 stimulated proliferation of human LEC [36]. Further evidence of a predominantly pro-proliferative role for FGFR1 in LEC was provided by our finding that in response to the addition of growth media containing FGF2, FGFR1 accumu- lated in the nucleus of LEC. Nuclear translocation of FGFR1 has previously been shown to correlate with transcriptional activation and cell proliferation [37,40]. Defining the proteins that interact with nuclear FGFR1, together with the transcriptional targets of FGFR1 in primary LEC will no doubt shed further light on the mechanisms by which FGFR1 activation promotes LEC prolifer- ation. The early embryonic lethality of Fgfr12/2 mice [41,42] has, to date, precluded analysis of the role of this receptor in blood and lymphatic vascular development, though the recent generation of Fgfr1flox/flox mice [43], together with blood and lymphatic vascular specific Cre lines, now enable this question to be addressed. Our data provide compelling evidence in support of a crucial role for FGF signalling in lymphangiogenesis and pave the way for further analysis of FGFR1 function in developmental and disease stimulated lymphangiogenesis in vivo. Supporting Information Figure S1 Vascular morphology and marker expres- sion. (a) Whole mount immunostaining of E14.5 skin illustrating that the calibre of lymphatic capillaries (Prox1-positive, Nrp2- positive, CD31-positive) is substantially larger than that of blood vascular capillaries (Prox1-negative, Nrp2-negative, CD31-posi- tive). (b) LYVE-1 levels are heterogeneous on lymphatic capillaries, while Nrp2 levels appear uniform. Scale bars represent 120 mm. (TIF) Figure S2 Purity of isolated primary embryonic mouse LEC. Immunostaining of purified primary embryonic LEC cultured in EGM-2MV demonstrating that the majority of DAPI-positive nuclei are positive for the lymphatic endothelial cell marker Prox1. (TIF) Figure S3 VEGF-C stimulated proliferation of primary mouse LEC is inhibited by small molecule inhibitors of VEGFR-2 and VEGFR-3. Primary LEC were cultured in EBM+0.5 mg ml21 Albumax (Control) or EBM+0.5 mg ml21 Albumax containing VEGF-C (200 ng ml21) and the small molecule tyrosine kinase inhibitors SU5416 (5 mM, VEGFR-2) or MAZ51 (5 mM, VEGFR-3) for 48 h. LEC proliferation was measured using the CellTiter 96H AQueous One Solution Cell PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40497