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Fig. 2

ID
ZDB-IMAGE-200615-4
Source
Figures for Wang et al., 2020
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Figure Caption

Fig. 2 Adamts3 and Adamts14 are both able to process pro-Vegfc.

a Schematic depiction of the in vivo Vegfc-processing assay. In wild-type embryos, floorplate expression of full-length Vegfc results in venous hyper-branching at 48 hpf. beflt4:mCitrine; flt1:tdTomato transgenic embryos with arterial structures in red and venous and lymphatic structures in green. b, c While forced expression of full-length zebrafish Vegfc from the floorplate leads to hyper-branching in wild-type embryos (b, arrows), this dominant phenotype is not visible in adamts3; adamts14 double mutants (c, asterisks). Allelic combinations with one remaining wild-type copy of either adamts3 (d) or adamts14 (e) develop the Vegfc-induced hyper-branching phenotype. Blue lines indicate the floorplate position (n = 225 analyzed embryos). fi Mosaic expression of full-length human VEGFC in floorplate cells (marked by tagRFP expression) results in local venous hyper-branching in wild-type embryos (f), but not in adamts3;adamts14 double mutants (g). One functional copy of either gene (h, i) is sufficient to restore the dominant phenotypes (n = 157 injected tagRFP+ embryos). Scale bars: 50 µm. DA dorsal aorta, DLAV dorsal longitudinal anastomotic vessel, hpf hours post fertilization, HM horizontal myoseptum, a/vISV arterial/venous intersegmental vessel, PCV posterior cardinal vein, PL parachordal lymphangioblast. j N-terminal processing of human VEGFC by ADAMTS3 and ADAMTS14 proteases. Conditioned medium from HEK293 cells expressing full-length VEGFC was incubated with ADAMTS3, ADAMTS14, or control buffer, in the presence or absence of EDTA. VEGFC forms were analyzed by Western blotting. In absence of active ADAMTS proteins (lanes 2, 4, and 5), VEGFC can be detected as a 58 kDa (full-length VEGFC without SP) and a 31 kDa form (C-terminally processed VEGFC without SP). In the presence of active ADAMTS14 (lane 1), the 58 kDa form is converted into a 45 kDa polypeptide while the 31 kDa form is processed into the fully mature 21 kDa VEGFC, which is in line with N-terminal processing of VEGFC proteins. A similar shift in VEGFC forms is detectable in the presence of active ADAMTS3 (lane 3) [source data are provided as a Source Data file]. Schematic depicting different VEGFC forms and their molecular weights. SP signal peptide, NT N-terminal propeptide, VHD VEGF homology domain, CT C-terminal propeptide.

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