Atypical E2fs control lymphangiogenesis through transcriptional regulation of Ccbe1 and Flt4

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

Atypical E2fs Control Lymphangiogenesis through Transcriptional Regulation of Ccbe1 and Flt4

<div><p>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 <i>CCBE1</i> promoter, while recruitment of E2F7/8 inhibits the <i>FLT4</i> promoter. Importantly, inactivation of <i>e2f7/8</i> in zebrafish impaired venous sprouting and lymphangiogenesis with reduced <i>ccbe1</i> expression and increased <i>flt4</i> expression. Remarkably, over-expression of <i>e2f7/8</i> rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel <i>in vivo</i> transcriptional regulators of <i>Ccbe1</i> and <i>Flt4,</i> both essential genes for venous sprouting and lymphangiogenesis.</p></div

Loss of E2f7/8 impaired venous sprouting and lymphangiogenesis.

<p>A, <i>In situ</i> hybridisation and B, qPCR (** <i>P</i><0.05; two independent experiments with n = 10 per condition and experiment) for <i>flt4</i> and <i>ccbe1</i> in zebrafish embryos 32 hpf, un-injected control (nic) or injected with <i>e2f7/8</i> MOs or mRNA. C, <i>Flt4</i>:YFP transgene level of 36 hpf uninjected or <i>e2f7/8</i> MOs injected embryos, lateral view (n = 30 per condition). D–G Lateral images and quantification of <i>Tg(fli1a:gfp;flt1^enh:rfp</i>) embryos treated as indicated and imaged at 52 hpf or 5 dpf. H–J Lateral images and quantification of <i>Tg(fli1a:gfp;flt1^enh:rfp</i>) embryos treated as indicated and imaged at 52 hpf or 5 dpf. Concentrations: <i>e2f7/8</i> MOs (10 ng each); <i>e2f7/8</i> mRNA (100 pg each); <i>ccbe1</i> 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 (*** <i>P</i><0.001). At least n = 150 embryos per condition in three independent experiments were used for D–J.</p

E2f7/8 rescued Ccbe1dependent lymphangiogenesis phenotype.

<p>A, Representative images of <i>Tg(fli1a:gfp;flt1^enh:rfp</i>) un-injected control embryos (nic) or embryos injected with <i>e2f7/8</i> MOs or mRNA. B, C, D, Quantification of the indicated parameters. Concentrations: <i>e2f7/8</i> MOs (10 ng each); <i>ccb1</i> MO (5 ng); <i>e2f7/8</i> mRNA (100 pg each); <i>ccbe1</i> 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 (*** <i>P</i><0.001). At least n = 150 embryos per condition in three independent experiments were used for A–D.</p

E2f7/8 rescued Flt4-dependent lymphangiogenesis phenotypes.

<p>A, Representative images of <i>Tg(fli1a:gfp;flt1^enh:rfp</i>) un-injected control embryos (nic) or embryos injected with <i>e2f7/8</i> MOs or mRNA. B, C, D, E Quantification of the indicated parameters. Concentrations: <i>e2f7/8</i> MOs (10 ng each); <i>dll4</i> MO (3 ng); <i>e2f7/8</i> mRNA (100 pg each); <i>dll4</i> 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 (*** <i>P</i><0.001). At least n = 150 embryos per condition in three independent experiments were used for A–E.</p

E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1

The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis. The EMBO Journal (2012) 31, 3871-3884. doi:10.1038/emboj.2012.231; Published online 17 August 201