Loss of E2f7/8 impaired venous sprouting and lymphangiogenesis.

A, In situ hybridisation and B, qPCR (** P<0.05; two independent experiments with n = 10 per condition and experiment) for flt4 and ccbe1 in zebrafish embryos 32 hpf, un-injected control (nic) or injected with e2f7/8 MOs or mRNA. C, Flt4:YFP transgene level of 36 hpf uninjected or e2f7/8 MOs injected embryos, lateral view (n = 30 per condition). D–G Lateral images and quantification of Tg(fli1a:gfp;flt1^enh:rfp) embryos treated as indicated and imaged at 52 hpf or 5 dpf. H–J Lateral images and quantification of Tg(fli1a:gfp;flt1^enh:rfp) embryos treated as indicated and imaged at 52 hpf or 5 dpf. Concentrations: e2f7/8 MOs (10 ng each); e2f7/8 mRNA (100 pg each); ccbe1 mRNA (100 pg). Open arrow heads indicate missing intersegmental vessels or dorsal longitudinal anastomotic vessel. Closed arrow heads indicate PLs (upper panel) or the TD (lower panel). Arrows depict PLs that have connected to ISVs. Stars indicate missing TD fragments. All scale bars are 100 μm. Data presented as the average (±s.e.m.) compared to the control condition in three independent experiments (*** P<0.001). At least n = 150 embryos per condition in three independent experiments were used for D–J.

Figure 3. E2f7/8 rescued Ccbe1dependent lymphangiogenesis phenotype.

A, Representative images of Tg(fli1a:gfp;flt1^enh:rfp) un-injected control embryos (nic) or embryos injected with e2f7/8 MOs or mRNA. B, C, D, Quantification of the indicated parameters. Concentrations: e2f7/8 MOs (10 ng each); ccb1 MO (5 ng); e2f7/8 mRNA (100 pg each); ccbe1 mRNA (100 pg).Open arrow heads indicate in (A; upper panel) missing dorsal longitudinal anastomotic vessels. Closed arrow heads indicate (upper panel in A) PLs or (lower panel, A) presence of the TD. All scale bars are 100 μm. Stars indicate missing TD fragments. Data presented as the average (±s.e.m.) compared to the control condition in three independent experiments (*** P<0.001). At least n = 150 embryos per condition in three independent experiments were used for A–D.

E2f7/8 rescued Flt4-dependent lymphangiogenesis phenotypes.

A, Representative images of Tg(fli1a:gfp;flt1^enh:rfp) un-injected control embryos (nic) or embryos injected with e2f7/8 MOs or mRNA. B, C, D, E Quantification of the indicated parameters. Concentrations: e2f7/8 MOs (10 ng each); dll4 MO (3 ng); e2f7/8 mRNA (100 pg each); dll4 mRNA (100 pg).Open arrow heads indicate in (A; upper panel) hyper-branching of intersegmental vessels. Closed arrow heads indicate (upper panel in A) PLs or (lower panel, A) presence of the TD. All scale bars are 100 µm. Stars indicate missing TD fragments. Data presented as the average (±s.e.m.) compared to the control condition in three independent experiments (*** P<0.001). At least n = 150 embryos per condition in three independent experiments were used for A–E.

Hyperbranching and venous sprouting is dependent on proper Flt4 signaling. S2A Lateral images and quantification of Tg(fli1a:gfp;flt1^enh:rfp) embryos treated as indicated and imaged at 52 hpf. S2C Lateral images and quantification of Tg(fli1a:gfp;flt1^enh:rfp) embryos treated as indicated and imaged at 52 hpf. S2D, S2E, S2F, quantification of the indicated parameters. Concentrations: dll4 MO (low 1.5 ng in S2A and 3 ng in S2C–F); e2f7/8 mRNA (100 pg each); flt4 mRNA (100 pg); ccbe1 mRNA (100 pg). Arrows depict PLs that have connected to ISVs (S2C). Closed arrow heads indicate (upper panel in S2C) PLs or (lower panel, S2C) presence of the TD. Open arrowheads indicate hyperbranching ISVs. All scale bars are 100 μm. Stars indicate missing TD fragments. Data presented as the average (±s.e.m.) compared to the control condition in three independent experiments (*** P<0.001). At least n = 150 embryos per condition in three independent experiments were used for S2A–S2F.