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

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

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

Number of fish sampled during each stage in a swim-experiment.

<p>Number of fish sampled during each stage in a swim-experiment.</p

Swim-training had a differential effect on on the age at appearance of bone structures between control and trained fish.

<p><b>A,B</b>) BF50<i>_age</i> values visualized in the corresponding structures in control fish (A) and trained fish (B). The branchial region is indicated separately, ventral view. <b>C</b>) Differences in BF50<i>_age</i> values between control and trained fish. Positive values indicate that structures appear earlier in the trained fish. Structures with a difference less than twice the standard error are indicated in grey.</p

Swim-training had a differential effect on the order of appearance of cartilage and bone structures between control and trained fish.

<p><b>A,B</b>) Difference in the rank of cartilage (A) and bone (B) structures between control and trained fish. Red structures indicate a forward shift in the order of appearance, blue structures a delay. Structures which did not show a difference are indicated in grey.</p

Schematic representation of the swim-training set-up in lateral view and critical flow velocity.

<p><b>A</b>) Water was pumped (1) to the top aquarium and flowed back into the reservoir via the training tubes (4), outflow tubes (5) and outflow hoses (6) due to gravity. The difference in water level between the top aquarium and the outflow tubes (5) (indicated with up down black arrow) determined the flow velocity in the training tubes (4). Control fish were kept in similar tubes in the same set-up (7). Both the training and control section consisted of five tubes placed parallel to each other (not visible in drawing). Each tube had its own outflow tube and hose. <b>B</b>) Critical flow velocity (<i>U_crit</i>) over time and during swim-training experiments (<i>U_critse</i>). Zebrafish were subjected to 50% (<i>U_crit</i>_50%) of the moving average <i>U_crit</i> (<i>U_critma</i>).</p

Swim-training increased burst frequency and growth in trained fish.

<p><b>A</b>) Average number of bursts per second ± standard deviation as a function of age in days post fertilization (dpf). <b>B</b>) Average standard lengths (dots) ± standard deviation (with quadratic regression fit) as a function of age (dpf).</p

Proteomics Analysis of the Zebrafish Skeletal Extracellular Matrix

<div><p>The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.</p></div