EMBO/soda-nel huggingface dataset:
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 3

Figure 3. Maternal SCT promotes iWAT browning in offspring.

A, B Time-resolved oxygen consumption (A) and heat production (B) in 48-hour light/dark cycle measured by CLAMS in M-SCT floxed and M-SCT KO mice with HFD (n = 6 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (A) or heat production (B) analysis of body weight are shown in the right part.

C Body temperature of 8-week-old M-SCT floxed and M-SCT KO male offspring with chow diet during cold exposure (4℃; n = 8).

D Representative HE staining and UCP1 staining of Ing from M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or 3 days of cold exposure (4℃). Scale bar, 100 μm.

E Relative mRNA levels of UCP1, CIDEA, PRDM16, PGC1α and DIO2 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet (n = 8 per group).

F Representative western blots of UCP1, PRDM16, PGC1α and HSP90 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet after 3 days of cold exposure (4℃).

G Total UCP1 protein per mg iWAT of M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d (4℃) (n = 3).

H Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in SVF isolated from M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group).

I, J OCR of Oligomycin, FCCP and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD, and the AUC of OCR as indicated were calculated (n = 5 per group).

K Glucose oxidation in adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group).

Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel A and B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 4

Figure 4. SCT participates in methylation-dependent reprogramming of offspring.

A MeDIP analyses genomic features of iWAT DNA methylation. Shown is the number of total DMR peaks (left) or total DMR peaks within promoters (right), proportions of up- vs down- regulated DMR peaks (M-SCT KO vs M-SCT floxed) (FDR < 0.1).

B Stacked bars showing the Proportional distribution of hypo- and hypermethylated DMRs (M-SCT KO vs M-SCT floxed) in different regions.

C Heat map of DMR methylation level in mRNA between M-SCT floxed and M-SCT KO. The color bar indicates the normalized tag counts in M-SCT floxed or M-SCT KO. Positive z scores, shown in the table, indicate hypermethylated regions, while negative z scores imply hypomethylated regions.

D As in (C), for enhancer region.

E Scatter plot representation of the most significant difference of methylation in mRNA (left) and enhancer (right) regions between M-SCT floxed and M-SCT KO (p < 0.005).

F Volcano plot DNA methylation represents all DMRs in mRNA (left) and enhancer (right) regions. Cutoffs (vertical and horizontal dashed lines) used to identify DMRs between M-SCT floxed and M-SCT KO mice. The vertical lines correspond to twofold up and down, respectively, and the horizontal line represents a P value of 0.01.

G Selected pathways of genes with increased DNA methylation through KEGG analysis.

H Enrichment of gene ontology (GO) biological processes of DMRs showing significant different methylation levels (p < 0.01) GO terms are plotted against negative log of corrected p-value.

I, J Schematic diagram showing the CpG islands of Mus musculus PRDM16 (I) and CIDEA (J) genes. The conditions to be met by CpG islands identified were: 200-300 bp length, CG content of > 50% and observed CpG content vs expected CpG content > 0.6. Methylation levels of each CpG islands in PRDM16 (I) and CIDEA (J) are presented as the fold change relative to the average value of the M-SCT floxed group and β-actin promoter was used as a negative control (n = 3 per group).

Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 5

Figure 5. SCT inhibits DNMT1 protein levels by activating AMPKα.

A Western blots analysis of DNMT1, DNMT3a, DNMT3b and HSP90 in Ing from M-SCT KO and M-SCT floxed mice with HFD.

B Relative mRNA levels of DNMT1, DNMT3a and DNMT3b in Ing from M-SCT KO and M-SCT floxed mice with HFD (n = 8). Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. ns, not statistically significant.

C Western blots analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in embryo of M-SCT KO and M-SCT floxed mice at E18.5.

D Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in mouse embryo fibroblast (MEF) cells treated with 10 nM or 100 nM SCT.

E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C (C.C.) for 24 h.

F Western blot analysis of p-AMPKα, AMPKα, DNMT1, SCTR and HSP90. MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist (AICAR) for 24 h.

G Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in AMPK+/+ or AMPKα1/α2 double knockout (AMPK-/-) MEF cells treated with 10 nM SCT for 24 h.

H, I Western blot analysis of Flag-DNMT1 in Flag-DNMT1-transfected HEK293T cells treated with AICAR with indicated dose or time periods.

J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h, followed by IP and IB analysis.

K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed, followed by IP and IB analysis.

L AMPK+/+ or AMPK-/- MEF cells were transfected with indicated plasmids and then lysed, followed by IP and IB analysis.
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 6

Figure 6. SCT improves metabolic homeostasis in offspring of HFD-fed pregnant mice.

A Serum SCT levels in M-HFD and M-HFD+SCT mice with HFD (n = 8 per group).

B Change of body weight of M-HFD (offspring of dams with high-fat pregnant diets) and M-HFD+SCT (offspring of dams with high-fat pregnant diets supplied with SCT) mice upon HFD feeding (n = 8 per group). Representative photograph of M-HFD and M-HFD+SCT mice after HFD feeding.

C, D Time-resolved oxygen consumption (C) and heat production (D) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (C) or heat production (D) analysis of body weight are shown in the right part.

E GTT (1 g glucose/kg body weight) in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group) and AUCs were calculated in right part. Mice were fasted for 18h before assays.

F ITT (0.75 U insulin/kg body weight) in M-HFD and M-HFD+SCT mice with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice were fasted for 6h before assays.

G Serum TG, TC and NEFA levels in M-HFD and M-HFD+SCT mice with HFD. (n = 8 per group).

H Representative HE staining and UCP1 staining of iWAT from M-HFD and M-HFD+SCT mice with HFD. Scale bar, 100 μm (n = 5 per group).

I Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in iWAT from M-HFD and M-HFD+SCT mice with HFD (n = 5 per group).

J Schematic diagram of the working model: maternal SCT reprogrammed inguinal adipose tissue development in offspring during pregnancy.

Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel C and D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Legends of Expanded View figures
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 1

Figure 1 : Sir3 overexpression inhibits Sae2 and increases error-prone NHEJ

A. Schematic representation of the assay used to estimate error-prone NHEJ at euchromatic DSB.

B. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments.

C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments.

D. Schematic representation of the LYS2 locus with primers located at 1 kb from the I-SceI site for the DNA measurements (blue arrows).

E. DNA levels measured at 0.2 kb from the I-SceI cut site at LYS2 after 2h DSB induction by qPCR in WT and sae2Δ cells expressing or not high levels of Sir3p (oeSir3 and WT respectively). DNA levels were normalized to DNA levels at the OGG1 locus and corrected for differences in DSB cleavage efficiency (see Materials and Methods for details). Error bars represent the standard deviation (SD) of three independent experiments.

F. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm.

G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments.

H. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD (Hocher et al 2018) is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments.

Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 2

Figure 2: Sir3 and Sae2 physically interact

A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.

B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).

C. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.

D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 3

Figure 3: Direct physical interaction between Sir3SaID and Sae2C domains

A. Schematic representation of Sir3 and Sae2 protein domains.

B. Delineation of the Sir3 domain responsible for interaction with Sae2 by two-hybrid assays. The GAL4-BD fusions with indicated Sir3 fragments were tested in combination with a GAL4-AD-Sae2 fusion; "+" indicates an interaction.

C. Delineation of the Sae2 domain responsible for interaction with Sir3 by two-hybrid assays. The GAL4-BD fusions with indicated Sae2 fragments were tested in combination with a GAL4-AD-Sir3 fusion; "+" indicates an interaction.

D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.

E. Comassie-stained gel of purified GST-Sir3SaID and Sae2C peptides.

F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 4).
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 4

Figure 4: The T557I point mutation in Sir3 abolishes Sae2-Sir3 interaction

A. Schematic representation of the assay used to screen for SIR3 mutants deficient for Sae2 interaction while maintaining interaction with Sir4. The SIR3SaID fragment (464-728) was mutagenized by PCR, cloned in the pACT2 two hybrid plasmid and transformed into the reporter strain along with plasmids expressing LexA-BD-SAE2C and GAL4-BD-SIR4C fusion proteins. The reporter strain (yKD1991) bears a Gal4 binding sequence (Gal4BD) upstream of a HIS3 reporter gene, and a LexA binding sequence (LexABD) precedes a LacZ reporter gene. Transformants in which the gal4-BD-Sir4C and Sir3SaID-gal4-AD fragments interact were selected for HIS3 expression on -HIS + 3AT medium and subsequently screened for LacZ expression upon X-gal coloration. Cells showing no LacZ expression were collected and the mutated sir3SaID-GAL4-AD was retrieved and sequenced.

B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C.

C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 5

Figure 5: Sir3-Sae2 interaction prevents Sae2 function and promotes NHEJ

A. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments.

B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.

C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments.

D. Representative images of Sir3-mCherry and Sae2-GFP signal in cells overexpressing Sir3 and sir3T557I . Scale bars are 2 μm.

E. Telomeric silencing assay at TEL7L in WT, sir4Δ, sir3T557I cells, cells overexpressing SIR3 (oeSIR3) or sir3T557I (oesir3T557I). Growth on 5-FOA plates reflects telomeric silencing.

F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).

G. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.

Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 6

Figure 6: Sir3-Sae2 interaction is modulated by Sir4.

A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments.

B. Plasmid cleaved by Xho I was transformed into strains and NHEJ efficiency was measured. Error bars indicate survival standard error (SEM) of at least three independent experiments.

C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments.

D. Representative images of Sae2-GFP in cells overexpressing Sir3 (oeSir3) and expressing or not high levels of Sir4 (oeSir4). Scale bars are 2μm.

E. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).

F. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP cells overexpressing Sir3 and expressing or not high levels of Sir4 (oeSir4).

Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).
Sir3 Heterochromatin Protein Promotes Non-Homologous End Joining by Direct Inhibition of Sae2 (2021)
Figure 7

Figure 7: Modulation of Sae2 recruitment and activity by Sir3

A. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM).

B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM).

C. Representative images of sae2-E267E mutant cells overexpressing or not full-length Sir3. Scale bars are 2 μm.

D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments.

Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).

Expanded View Figure Legends
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 1

Figure 1. Expression of Ena/VASP proteins in postnatal retinal endothelial cells.

(A, B) RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T-cells (CD3E), B-cells (CD19), all leucocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glia cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65). (B) RNA levels of VASP (red), EVL (green) and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***p<0.001, one-way ANOVA with Bonferroni's multi comparison test.

(C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP- (C), EVL- (D), or Mena-specific (E) antibodies (red). Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells. No Mena protein expression was detected in P5 retinal endothelial cells (E). Representative images from three independent experiments are shown. Scale bars, 10 µm.
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 2

Figure 2. EVL expression in mouse endothelial cells.

(A) Wild-type and EVL-/- P5 retinas were digested, labelled with CD31-, CD34- and EVL-specific antibodies and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL-/- (black) cells is shown in the right panel. WT: n=3, EVL-/-: n=2; two retinas per animal. Error bars represent SEM.

(B-C) EVL protein expression in sparse/migrating wild-type (WT) and EVL-/- mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments.

(D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 3

Figure 3. Delayed postnatal retinal angiogenesis in EVL-/- mice.

(A) Isolectin B4 stained vasculature in whole mount retinas of wild-type (WT) and global EVL-/- mice on postnatal days 3, 5 and 7 (P3, P5, P7) assessed by confocal microscopy. Scale bars 200 µm.

(B) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates.

(C) Impact of individual and combined VASP/EVL deletion on sprouting angiogenesis in the postnatal mouse retina at P5 (EVL-deficient animals (E-/-), green; VASP-deficient animals (V-/-), red; VASP/EVL-double deficient animals (EV-/-), orange).

Data information: Error bars represent SEM; *p<0.05, **p<0.01, ***p<0.001, §p<0.001 versus P3 and P5 EVL-/- (one-way ANOVA with Bonferroni's multi comparison test).
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 4

Figure 4. Impaired retinal angiogenesis in endothelial cell specific EVL knockout mice.

The retinal vasculature of endothelial cell specific EVL knockout mice (EVLΔEC) and littermate controls (EVLfl/fl) on P5 was analyzed by Isolectin B4 staining.

(A, B) Radial outgrowth of the retinal vasculature; in (B) global EVL-/- mice are shown for comparison; 4 - 5 independent experiments.

(C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front.

(D) Tipp cells numbers per field of view (387,5 x 387,5 µm).

(E) Filopodia numbers normalized to the length of the angiogenic front.

(F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei.

Data information: (C-F) n = 5 animals per group from 3 different litters. Scale bars 200 µm (A) and 50 µm (B-F), respectively. Error bars represent SEM; **p<0.01, ***p<0.01, unpaired Student's t-test with Welch's correction.