Fig. 7

Tie1 regulates lymphatic development at least in part through the modulation of Vegfc/Vegfr3 signaling. (A) Whole-mount immunofluorescence staining for pERK (green) and DsRed (magenta) in WT and tie1−/− Tg(lyve1:DsRed) embryos. Arrowheads point to EC nuclei of secondary sprouts assessed by the DAPI staining. Secondary sprouts migrate towards the HM around 39 hpf in WT and 44 hpf in the tie1 mutant. (B) Quantitative analyses of the data shown in A. Fluorescence intensity of nuclear pERK staining (DAPI+ area, not shown in A) was quantified in the lyve1:DsRed+ ECs budding from the PCV of WT (39 hpf) and tie1−/− (44 hpf) embryos. Data are mean±s.d. (WT, n=32 ECs in 5 embryos; tie1−/−, n=38 ECs in 7 embryos). (C) Trunk of Tg(kdrl:EGFP);Tg(lyve1:DsRed) tie1−/− embryos with or without Tg(flt1:Gal4FF);Tg(10×UAS:vegfc) (53 hpf). Formation of PLs (arrowheads) and venous ISVs (V) are partially recovered by overexpression (o/e) of Vegfc in the tie1 mutant. (D) Percentage of PL coverage at 54 hpf. Data are mean±s.d. (tie1−/−, n=20 embryos; tie1−/− with Vegfc o/e, n=12 embryos). (E) Number of secondary sprouts scored across six segments at 54 hpf. Data are mean±s.d. (tie1−/−, n=20 embryos; tie1−/− with Vegfc o/e, n=12 embryos). (F) Number of A-V connections across six segments at 54 hpf. Data are mean±s.d. (tie1−/−, n=20 embryos; tie1−/− with Vegfc o/e, n=12 embryos). Scale bars: 50 μm. P-values were determined by two-tailed Mann–Whitney test (B) and by two-tailed Student's t-test (D,E).