Atypical E2fs control lymphangiogenesis through transcriptional regulation of Ccbe1 and Flt4

Abstract

Lymphatic vessels are derived from venous endothelial cells and their formation is governed by the Vascular endothelial growth factor C (VegfC)/Vegf receptor 3 (Vegfr3; Flt4) signaling pathway. Recent studies show that Collagen and Calcium Binding EGF domains 1 protein (Ccbe1) enhances VegfC-dependent lymphangiogenesis. Both Ccbe1 and Flt4 have been shown to be indispensable for lymphangiogenesis. However, how these essential players are transcriptionally regulated remains poorly understood. In the case of angiogenesis, atypical E2fs (E2f7 and E2f8) however have been recently shown to function as transcriptional activators for VegfA. Using a genome-wide approach we here identified both CCBE1 and FLT4 as direct targets of atypical E2Fs. E2F7/8 directly bind and stimulate the CCBE1 promoter, while recruitment of E2F7/8 inhibits the FLT4 promoter. Importantly, inactivation of e2f7/8 in zebrafish impaired venous sprouting and lymphangiogenesis with reduced ccbe1 expression and increased flt4 expression. Remarkably, over-expression of e2f7/8 rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel in vivo transcriptional regulators of Ccbe1 and Flt4, both essential genes for venous sprouting and lymphangiogenesis.

Figure 1. E2F7/8 directly regulate CCBE1 and FLT4 expression.

A, Genes associated with the gene ontology (GO) term angiogenesis were extracted from the Sox2-cre;E2f7^loxP/−E2f8^loxP/− vs. wild-type E10.5 mouse embryos (P<0.05) database (GEO: GSE30488) and additionally analyzed for E2F binding sites and their presence in GO lymphangiogenesis (GO:0001946). B, E2F binding elements within the CCBE1 and FLT4 promoter. Average and canonical E2F binding consensus. C, E2F7 and E2F8 ChIP performed on CCBE1 and FLT4 promoters in HeLa cells, arrows in (B) indicate primers used for ChIP. A previous reported E2F site within the E2F1 promoter and a non-specific site upstream (+700 bp) served as controls. D, mRNA and protein levels of E2f7-EGFP inducible HeLa cells treated with doxycyclin (0.2 µg /ml) for 24 hours. E, mRNA and protein levels of HeLa cells treated with scrambled or E2f7/8 siRNAs. Fold change in protein were calculated using TUBULIN as reference. Data presented as the average (±s.e.m.) compared to the control condition in three independent experiments.

Figure 2. 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.

Figure 4. 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.