Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A ) Schematic representation of facial lymphatic network at 5 dpf and maximum intensity projection of confocal images of flt4:mCitrine positive svep1 mutants ( n = 10) and siblings ( n = 6), highlighting facial lymphatic structures at 5 dpf. Scale bar = 100 µm. Note the absence of the FCLV (red dotted line) in svep1 mutants whereas other facial lymphatic structures are less strongly affected (OLV, LFL, MFL, and LAA marked by blue dotted lines). ( B ) Confocal images of flt4:mCitrine positive facial lymphatics in vegfc ( n = 19), ccbe1 ( n = 5), and adamts3;adamts14 ( n = 2) mutants at 5 dpf. Scale bar = 100 µm. ( C ) Confocal images of svep1 and vegfc expression domains during sprouting from the PHS at 2 dpf, with schematic representation of different lymphatic progenitor populations. svep1 is expressed in close proximity to sprouting PHS-LPs, while vegfc expressing cells are more concentrated on the LECs arising from the CCV. Arrows point to sprouting PHS-LP. Scale bar = 50 µm. Expression patterns were confirmed in six embryos each . CCV, common cardinal vein; dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel; FLS, facial lymphatic sprout; hpf, hours post-fertilization; LAA, lymphatic branchial arches; LEC, lymphatic endothelial cell; LFL, lateral facial lymphatic; MFL, medial facial lymphatic; OLV, otolithic lymphatic vessel; PHS, primary head sinus; PHS-LP, primary head sinus lymphatic progenitor; VA, ventral aorta; VA-A, ventral aorta angioblast; VA-L, ventral aorta lymphangioblast; WT, wildtype.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Expressing

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A–D ) Confocal images of siblings and svep1 mutant embryos at 5 dpf, expressing the flt4:mCitrine transgene. Asterisk indicates reduced lymphatic vessels. Scale bar = 100 µm.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Mutagenesis, Expressing

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A–D ) Facial lymphatic phenotype of svep1; ccbe1 double mutants ( n = 6) at 5 dpf expressing flt4:mCitrine . Note the complete lack of all lymphatic structures in double mutant embryo ( D ). OLV, LVL, MFL, marked by blue dotted lines. FCLV marked by red dotted lines. Scale bar = 100 µm.; FCLV, facial collecting lymphatic vessel; LFL, lateral facial lymphatic; MFL, medial facial lymphatic; OLV, otolithic lymphatic vessel.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Expressing, Mutagenesis

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: Confocal images of svep1:Gal4; UAS:RFP; flt4:mCitrine and vegfc:Gal4; UAS:RFP; flt4:mCitrine transgenic embryos 2 dpf. Scale bar = 100 µm.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Transgenic Assay

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A ) Confocal images of sprouting BLECs, marked by flt4:mCitrine , at 3 dpf in svep1 mutants and siblings. Asterisks mark missing BLECs in svep1 mutants. Scale bar = 100 µm. ( B ) Quantification of BLECs at 3 dpf on each side of the embryo showed that svep1 mutants have significantly less BLECs on one or both sides of the brain hemispheres compared to siblings. For statistical analysis, no BLECs were counted as 0, BLECs being present on only one hemisphere as 1, whereas BLECs being detectable on both brain hemispheres were included as 2, for each embryo ( svep1+/+ : n = 10; svep1+/− : n = 12; svep1−/− : n = 12). Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD), ****p < 0.0001, ns = not significant. Scale bar = 100 µm. ( C ) Confocal images of svep1:Gal4; UAS:RFP , showing svep1 expression immediately adjacent to BLECs, marked by arrowheads, at 3 dpf. Scale bar = 100 µm. ( D ) Magnification and reduced stack numbers of boxed area in ( C ). Arrowhead marks BLEC. Scale bar = 50 µm. BLEC, brain lymphatic endothelial cell; dpf, days post-fertilization; MsV, mesencephalic vein; PHS, primary head sinus;.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: MANN-WHITNEY, Standard Deviation, Expressing

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A ) Facial lymphatics at 3 dpf in flt4:mCitrine positive tie1 , svep1 and tie2 mutants and sibling embryos (lateral view). Arrowheads point to FCLV and asterisks indicate the absence of FCLV. Scale bar = 100 µm. ( B ) flt4:mCitrine; flt1:tdTomato positive dorsal head vasculature in tie1 , svep1 , and tie2 mutants and in siblings at 3 dpf (dorsal view). In svep1 and tie1 mutants (but not in tie2 mutants) the presence of BLECs is strongly reduced. Arrowheads point to BLECs and asterisks indicate areas lacking BLECs. Scale bar = 100 µm. ( C ) Confocal images of PL cells, indicated by arrowheads, at 2 dpf in flt4:mCitrine; flt1:tdTomato positive tie1 , svep1 , and tie2 mutants and siblings, showing reduced PL numbers in svep1 and tie1 mutants. Asterisks indicate missing PLs. Scale bar = 100 µm. ( D ) Quantification of the presence of BLECs in tie1 mutants compared to siblings. ( tie1+/+ : n = 6; tie1+/− : n = 16; tie1−/− : n = 10) Mann–Whitney test was applied for statistical analysis. ***p = 0.001, ns = not significant. ( E ) Quantification of PL cell numbers in tie1 ( tie1+/+ : n = 9; tie1+/− : n = 23; tie1−/− : n = 14) , svep1 ( svep1+/+ : n = 16; svep1+/− : n = 31; svep1−/− : n = 19), and tie2 ( tie2+/+ : n = 17; tie2+/− : n = 27; tie2−/− : n = 16) mutants compared to siblings. Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD), ****p < 0.0001, ns = not significant; BLEC, brain lymphatic endothelial cell; dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel; PL, parachordal lymphangioblast.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: MANN-WHITNEY, Standard Deviation

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: Confocal images of lyve1:DsRed transgenic svep1 ( n = 9) and tie1 mutant ( n = 6) embryos and siblings ( n = 19) at 3 dpf. Scale bar = 100 µm. Arrowhead indicates FCLV in siblings and asterisk marks loss of FCLV in svep1 and tie1 mutants. dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Transgenic Assay, Mutagenesis

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A–L ) Still frames from confocal time-lapse imaging of embryos in a flt4:mCitrine; flt1:tdTomato transgenic background. ( A–D ) PL migration (indicated by arrowheads) of sibling embryo along aISV from 2.5 to 3.5 dpf. ( E–H ) Failed PL migration (indicated by asterisk) of svep1 mutants and ( I–L ) tie1 mutants along artery from 2.5 to 3.5 dpf. ( M, N ) Classification of PL migration along arteries. Statistical analysis was performed using Mann–Whitney test comparing the % of PL migration along arteries in each sibling and mutant embryo ( sibling : n = 96 PLs in 18 embryos; svep1−/− : n = 36 PLs in 15 embryos; siblings: n = 52 PLs in 14 embryos; tie1−/− : n = 28 PLs in 10 embryos); ****p < 0.0001, ***p = 0.0003. ( O, Q ) Representative cell tracking routes (tracks centred to origin) of single PL cells marked by different colours in siblings ( n = 17 PLs in 4 embryos; n = 7 in 2 embryos) , tie1−/− ( n = 5 PLs in 2 embryos) and svep1−/− ( n = 6 PLs in 3 embryos). ( P, R ) Quantification of dorsal and ventral PL migration (delta Y migration distance), mean velocity and total migration distance in svep1 and tie1 mutants compared to sibling embryos excluding apoptotic PLs quantified in ( M, N ) revealed decreased migration in dorsal and ventral direction in svep1 (*p = 0.0148) as well as tie1 mutants (**p = 0.0023). ns = not significant; aISV, arterial intersegmental vessel; dpf, days post fertilization; HM, horizontal myoseptum; PL, parachordal lymphangioblast. Scale bar = 100 µm (D, H, L = 25 µm).

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Imaging, Transgenic Assay, Migration, MANN-WHITNEY, Mutagenesis, Cell Tracking Assay

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: Additional cell tracking routes of PL cells in svep1 ( n = 12 PLs in 5 embryos) from 2.5 to 3.5 dpf compared to siblings ( n = 72 PLs in 14 embryos) tracked with manual tracking tool in each of three individual experiments. dpf, days post fertilization; PL, parachordal lymphangioblast.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Cell Tracking Assay

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A ) apelin:eGFP and flt1:tdTomato expression at 2 dpf in UIC compared to ( B ) svep1 morphants. ( C, D ) Quantification of ISVs with apelin expression in dorsal and ventral parts of the ISVs. Dorsal part was counted from dorsal longitudinal anastomotic vessel (DLAV) until midline region. Lateral region was counted from midline region onwards in ventral direction. svep1 morphants showed significant increase of apelin positive ECs compared to siblings (UIC: n = 21; svep1 MO: n = 21). Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD). ****p < 0.0001, ***p = 0.0002. Scale bar = 100 µm. dpf, days post-fertilization; hpf, hours post-fertilization; ISV, intersegmental vessel; UIC, uninjected control.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Expressing, MANN-WHITNEY, Standard Deviation

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: Representative pictures of in situ hybridization of apelin in svep1 ( n = 7) and tie1 ( n = 9) mutants ( B, E ) and siblings ( n = 26 + 23) ( A, D ) at 48 hpf. Prior to genotyping the staining intensity was categorized in three groups (weak, medium, and strong). For statistical analysis ( C, F ), weak staining was counted as 0, medium staining as 1, whereas strong staining was included as 2, for each embryo. Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD), **p = 0.0011; ***p = 0.0001. Scale bar = 100 µm. hpf, hours post-fertilization.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: In Situ Hybridization, Staining, MANN-WHITNEY, Standard Deviation

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A–G ) Confocal images of blood and lymphatic vasculature in the trunk of 2-dpf-old embryos derived from svep1; tie1 double heterozygous fish, showing severely reduced PL numbers in svep1; tie1 double mutants and significant decrease of PL cell numbers in svep1+/−; tie1−/− compared to svep1+/+; tie1−/− (**p = 0.0012). ( H ) Quantification of PL cell numbers at 2 dpf using Mann–Whitney test (siblings: n = 45; svep1+/−; tie1+/− : n = 45; svep1−/−; tie1+/+ : n = 13; svep1+/+; tie1−/− : n = 15; svep1−/−; tie1+/− : n = 20; svep1+/−; tie1−/− : n = 21; svep1−/−; tie1−/− : n = 11). Scale bar = 100 µm. Values are presented as means ± standard deviation (SD), ****p < 0.0001, ***p = 0.007, *p = 0.0163, ns = not significant. dpf, days post-fertilization; PL, parachordal lymphangioblast.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Derivative Assay, MANN-WHITNEY, Standard Deviation

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: flt4:mCitrine; flt1:tdTomato positive dorsal head vasculature in ( A ) siblings and ( B ) svep1+/−; tie1+/− embryos at 3 dpf (dorsal view). svep1+/−; tie1+/− embryos ( n = 9) show normal amount of BLECs. Scale bar = 100 µm. Facial lymphatics at 3 dpf in flt4:mCitrine positive ( D ) svep1+/−; tie1+/− embryos ( n = 6) and in ( C ) siblings ( n = 7) (lateral view). Arrowheads point to BLECs in ( A, B ) and to FCLV in ( C, D ). Scale bar = 100 µm. BLEC, brain lymphatic endothelial cell; dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques:

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: ( A ) 293T HEK cells were transfected with zebrafish Svep1-HIS (zfSvep1) and zebrafish Tie1-HA (zfTie1). zfSvep1 was immunoprecipitated and associated Tie1 was detected by western blot. ( B ) Co-immunoprecipitation of C-terminal human SVEP1 co-transfected in 293T HEK cells with human TIE1. ( C ) Pull-down of recombinant C-terminal human SVEP1-Strep-tag II protein, which was incubated with TIE1 transfected 293T HEK cell lysates, shows binding of TIE1. Protein structure with all domains indicated and C-terminal part used for pull-down assays (adapted from Figure 2F of , published under the CC BY-NC 4.0 license, https://creativecommons.org/licenses/by-nc/4.0/ ). It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need to follow the terms of the CC BY-NC 4.0 license. Ly05-265 indicates position of stop codon in the zebrafish hu6985 allele , suggesting that the protein domains C-terminal to the nonsense allele are critical for function. Red and blue rectangle: signal peptide; blue pentagon: von Willebrand factor type A domain (vWF); orange rectangle: ephrin-receptor like domain; brown rectangle: Hyalin repeat; yellow ovals: SUSHI repeat; green pentagons: epidermal growth factor (EGF)-like and calcium-binding EGF-like domains; and pink hexagon: pentraxin domain (PTX). Figure 8—source data 1. Raw data of western blots.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Transfection, Immunoprecipitation, Western Blot, Recombinant, Strep-tag, Incubation, Binding Assay

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet: In situ hybridization of tie1 in sibling ( n = 14) ( A, B ) and svep1 mutants ( n = 6) ( C, D ) at 24 hpf. Images have been assembled from individual pictures to ensure proper focus of all areas. ( B and D ) are magnifications of boxed area in ( A and C ), respectively. Scale bar = 100 µm. hpf, hours post-fertilization.

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: In Situ Hybridization

Journal: eLife

Article Title: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner

doi: 10.7554/eLife.82969

Figure Lengend Snippet:

Article Snippet: For pull-down of human SVEP1-StrepII, we used Strep-TactinXT 4Flow high capacity resin (2-5030-025, iba-lifesciences).

Techniques: Transfection, Construct, Recombinant, In Situ, Purification, Labeling, Software