Fig 2: RND1 is required for Notch-mediated suppression of sprout number and length. (A) Representative images of Fibrin bead angiogenesis (FiBA) assays using lentivirally-transduced HUVECs with full-length RND1 overexpression vectors (RND1OE) or empty vector (Ctrl). HUVEC were coated on cytodex beads, embedded in a fibrin gel with fibroblasts (D551) grown on the surface of the gel to provide growth factors, and allowed to develop for 5 days. (B) After 5 days, overexpression of RND1 reduces the number of sprouts, length of sprouts, and number of tip cells in a highly significant manner. (C) Lentivirally-transduced HUVECs with RFP or DLL4 expression constructs and were transfected with siCNT or siRND1 used for FiBA assays as above. Representative images of FiBA under each condition, scale bar indicates 320 µm. (D) After 5 days, Notch activation by DLL4 suppresses the number of sprouts from each bead, but knockdown of RND1 partially rescues sprout number (left). Notch activation by DLL4 suppresses the length of sprouts from each bead, but knockdown of RND1 partially rescues sprout length (right).

Fig 3: MPDZ recruits DLL1 and DLL4 to Nectin-2.(A) DLL1 and DLL4 full length and such lacking the PDZ-binding site (?PDZ) constructs containing a mCherry tag were expressed in HUVEC. Cells were stained with antibodies against DLL1 or DLL4. DLL1 and DLL4 surface expression of mCherry positive cells was analyzed by flow cytometry. n = 3; *, p<0.05; **, p<0.01 unpaired Student’s t-test. (B) Endothelial cells were isolated from Mpdz+/+ embryos and Mpdz-/- littermates at embryonic day E11.5. Cells were purified by CD31 magnetic dynabeads and stained with anti-CD34 and anti-Dll4 antibodies for flow cytometric analysis. n = 4; *, p<0.05; unpaired Student’s t-test. (C, D) HEK293T control cells (293T sh-ctrl) as well as MPDZ-silenced HEK293T cells (293T sh-MPDZ) were transfected with Nectin-2 and HA-tagged DLL1 or Flag-tagged DLL4. For immunoprecipitation a Nectin-2 antibody was used and HA-tagged DLL1, Flag-tagged DLL4 and MPDZ were detected by western Blotting. Input, 10% of immunoprecipitate. IB, Immunoblot; IP, Immunoprecipitation; neg.ctrl., negative control.10.7554/eLife.32860.010Figure 3—source data 1.Source data of FACS analysis related to Figure 3A and B.

Fig 4: Global and mosaic loss of DLL4 promote LEC proliferation through inhibition of Notch signaling in neighboring cells. a, b DAPT induced effect on the proliferation of control (siCTRL) or VEGFR3 (siVEGFR3) siRNA-treated LECs. a Tile scan immunofluorescence images and (b) quantification of proliferating EdU+ LECs after 3 h of incorporation (mean (n = 3 biological replicates) ± s.e.m). c Western blot analysis of cell lysates from LECs treated with siCTRL or NOTCH1 siRNA (siNOTCH1) and (d) quantification of proliferating EdU+ LECs cultured with or without VEGF-C (data are normalized to the proliferation rate in siCTRL alone and represent mean (n = 3 biological replicates) ± s.e.m). e Western blot analysis of cell lysates from LECs treated with siCTRL or DLL4 siRNA (siDLL4) and (f) quantification of proliferating EdU+ cells in co-cultures of siCTRL and siDLL4 LECs mixed in different ratios (data are normalized to the proliferation rate in siCTRL alone and represent mean (n = 6 biological replicates) ± s.e.m). g Model of cell-autonomous and non-cell-autonomous effects of VEGFR3 expression defining LEC responses during sprouting lymphangiogenesis. VEGF-C-VEGFR3 signaling positively regulates DLL4 expression leading to activation of Notch and cell cycle arrest in neighboring LECs. VEGFR3 deficiency (green cell) consequently leads to downregulation of DLL4 and inhibition of Notch in neighboring LECs, but only VEGFR3 expressing (red) cells respond by proliferation and sprouting. When VEGFR3- cells are in excess, VEGFR3+ cells interact predominantly with LECs with low DLL4 and respond by proliferation (dashed line) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-tailed unpaired Student’s t test (b, d, f) or one-way ANOVA with Tukey’s post hoc test (d; comparison between VEGF-C untreated and treated groups). Scale bars: 200 µm (a). ns: not significant

Fig 5: DNA methylation pattern of DLL4 gene promoter in LFS, breast and brain cancer cell linesA. Schematic representation of DLL4 promoter and CpG islands; B. Methylation status of the DLL4 promoter in LFS cell lines and cancer cell lines as detected by MS-PCR; C. Methylation status of the DLL4 promoter in human normal tissue and tumor tissue samples including colon, stomach and lung. D. Marked Reactivation of DLL4 gene expression in MCF7 but neither in the two DLL4-negative LFS cell lines nor in the normal cell line, HS27 by the DNA methylation inhibitor, 5-aza-dC.