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. 2023 Apr 25;12:e82969. doi: 10.7554/eLife.82969

Figure 5. Reduced blood flow leads to vascular anastomosis defects in tie1 mutants, similar to the defects in svep1 mutants.

(A, B) Confocal images of sibling and tie1 mutant embryos at 2 dpf in a flt4:mCitrine and flt1:tdTomato transgenic background. (B’) Magnification and reduced stack of boxed area in (B). (C, D) Confocal images of sibling and tie1 mutant embryos treated with 0.014% tricaine from 30 until 48 hpf. Asterisks indicate incompletely formed DLAV segments. (D’) Magnification and reduced stack numbers of boxed area in (D). (E) Quantification of gaps in the DLAV in sibling and tie1 mutants that were either untreated or treated with 0.014% tricaine revealed significant increase of gaps in the DLAV in tie1 mutants. (F) Quantification of lumenized trunk segments of the DLAV in siblings and tie1 mutants, either untreated or treated with 0.014% tricaine (siblings untreated: n = 16; tie1−/− untreated: n = 20; siblings treated with 0.014% tricaine: n = 20; tie1−/− treated with 0.014% tricaine: n = 22), revealed significant decrease of lumenized segment numbers in the DLAV in tie1 mutants. Mann–Whitney test was applied for statistical analysis. (G, H) apelin:eGFP and flt1:tdTomato expression in 48-hpf-old embryos after tricaine treatment from 30 to 48 hpf and (I, J) in untreated conditions. (K) Maximum intensity projection of an aISV at 48 hpf, highlighting the ventral and dorsal region used for further quantifications in (J) adapted from Figure 5J of Coxam et al., 2022. (L) Quantification of ISVs with apelin expression in dorsal and ventral parts of the ISVs. Dorsal part was counted from DLAV until midline region. Lateral region was counted from midline region onwards in ventral direction. tie1 mutants showed significant increase of apelin positive ECs compared to siblings in untreated (dorsal: ***p = 0.0001; ventral: **p = 0.0028) and treated with 0.014% tricaine conditions (dorsal: **p = 0.0033; ventral: ***p = 0.0002) (siblings untreated: n = 53; tie1−/− untreated: n = 21; siblings treated with 0.014% tricaine: n = 66; tie1−/− treated with 0.014% tricaine: n = 28). Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD). ****p < 0.0001. Scale bar = 100 µm. hpf, hours post-fertilization; ISV, intersegmental vessel; DLAV, dorsal longitudinal anastomotic vessel; dpf, days post-fertilization.

Figure 5.

Figure 5—figure supplement 1. vegfc and tie2 mutants do not show defects in DLAV formation upon tricaine treatment.

Figure 5—figure supplement 1.

(A, B) Confocal images of sibling and tie2 mutant embryos, expressing flt4:mCitrine and flt1:tdTomato at 2 dpf treated with 0.014% tricaine from 30 until 48 hpf. (C) Quantification of gaps in the DLAV in sibling and tie2 mutants that were either untreated or treated with 0.014% tricaine (siblings untreated: n = 8; tie2−/− untreated: n = 7; siblings treated with 0.014% tricaine: n = 24; tie2−/− treated with 0.014% tricaine: n = 11). (D) Quantification of lumenized trunk segments of the DLAV in siblings and tie2 mutants, either untreated or treated with 0.014% tricaine. (E, F) Confocal images of sibling and vegfc mutant embryos, expressing flt4:mCitrine and flt1:tdTomato at 2 dpf treated with 0.014% tricaine from 30 until 48 hpf. (G) Quantification of gaps in the DLAV in sibling and tie2 mutants that were either untreated or treated with 0.014% tricaine (siblings untreated: n = 22; vegfc−/− untreated: n = 9; siblings treated with 0.014% tricaine: n = 26; vegfc−/− treated with 0.014% tricaine: n = 10). (H) Quantification of lumenized trunk segments of the DLAV in siblings and tie2 mutants, either untreated or treated with 0.014% tricaine. Mann–Whitney test was applied for statistical analysis. Scale bar = 100 µm. DLAV, dorsal longitudinal anastomotic vessel; dpf, days post-fertilization; ISV, intersegmental vessel.
Figure 5—figure supplement 2. svep1 morphants show increased apelin expression in ISVs.

Figure 5—figure supplement 2.

(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.
Figure 5—figure supplement 3. apelin expression is reduced in svep1 and tie1 mutants.

Figure 5—figure supplement 3.

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.