hu10125 mutants present with lymphatic vascular deficiency and arterial hyperbranching. A,B: The vasculature Tg(fli1a:EGFP; flt1:tomato) in wild-type siblings (A) and hu10125 embryos (B) at 4 dpf (white arrows indicate a thoracic duct, asterisk an absence of thoracic duct, and arrowheads indicate hyperbranching). C,D: The vasculature Tg(fli1a:EGFP; flt1:tomato) in wild-type siblings (n = 53) (C) and hu10125 (n = 12) embryos (D) at 30 hpf. E,F: The arteries Tg(flt1:tomato) in wild-type siblings (C) and hu10125 (D) embryos at 3 dpf. G,H: Endothelial nuclei Tg(fli1:nucEGFP) in wild-type siblings (G) and hu10125 (H) embryos at 3 dpf. I: Quantification of: thoracic duct extent (number of thoracic duct fragments visible across 10 segments in the trunk) in WT (n = 20) and hu10125 (n = 21) (4 dpf); vascular hyperbranching (number of bilateral paired ISVs presenting with extra branches across 10 segments in the trunk) in WT (n = 32) and hu10125 (n = 32) (4 dpf); parachordal lymphangioblasts (number of PLs visible at the level of the horizontal myoseptum, across 10 segments in the trunk) in WT (n = 28) and hu10125 (n = 21) (56 hpf); and nuclei count (in ISVs, scored dorsal to the horizontal myoseptum across 10 segments) in WT (n = 12) and hu10125 (n = 10) embryos (3 dpf).

hu10125 is a cad (perplexed) mutant. A–E: Overview of the genetic mapping of cad^hu10125 mutants. Recombinant embryos at multiple simple sequence length polymorphism markers identified the critical genetic interval indicated (A). Sequencing of the cad gene identified the presence of a mutation (P1850T) in hu10125 cDNA (B,C), affecting a conserved residue in the linker region of Cad (D,E). F,G: Overall morphology of wild-type siblings (77.5 % of the progeny) and cad^hu10125; cad^hi2694 embryos (22.5% of the progeny) at 4 dpf in a classical complementation assay (n = 165 embryos scored) (F). Quantification of the number of intersegmental arteries per embryo (out of 10 segments) presenting with hyperbranching in WT siblings (n = 21 scored) and cad^hu10125;cad^hi2694 (n = 19 scored) embryos at 4 dpf (G). H–J: In situ hybridization analysis of cad expression shows ubiquitous expression at the 12-somite stage (H), enrichment in the trunk and neural tissues 30 hpf (I) and enrichment within kidney and putative vascular tissues at 48 hpf (J). Arrows: putative vascular endothelial cells. K: Quantitative RT-PCR for markers of the blood vasculature in arterial endothelial cells (AECs) sorted based on TG(flt1:YFP) expression at 36 and 60 hpf: kdrl (expressed in all blood vascular endothelial cells), flt1 and dll4 (restricted to arterial endothelial cells), ccbe1 (not expressed in the vascular endothelium) and cad. Gene expression were normalised to rpl13.

cad artery hyperbranching is Vegfc/Vegfr3 dependent. A,B: The vasculature Tg(fli1a:EGFP) in wild-type sibling (A) and cad^hu10125 (B) embryos at 3 dpf. C–E: The vasculature Tg(fli1a:EGFP) in cad^hu10125/MO-vegfc (C), cad^hu10125/MO-ccbe1 (D), and cad^hu10125/MO-vegfr3 (E) embryos at 3 dpf. F: Quantification of the severity of vascular hyperbranching designated as mild or severe (where mild: 1–7 hyperbranched arteries, and severe: e 8 hyperbranched arteries along the entire embryo body axis) scored in WT (n = 16), MO-vegfc (n = 12), MO-ccbe1 (n = 10) and MO-vegfr3 (n = 14) injected cad^hu10125 embryos. G,H: The thymidylate synthase mutant tyms^hi3510Tg does not present with arterial hyperbranching. G: Overall morphology of wild-type (n = 30/30) and tyms^hi3510Tg (n = 30/30) embryos at 4 dpf. H: The vasculature Tg(fli1a:EGFP) in wild-type (n = 30/30) and tyms^hi3510Tg (n = 30/30) embryos at 4dpf. White arrowhead: thoracic duct, white arrow: example of normal aISV.

Notch signaling is reduced in cad^hu10125 mutants. A–D: The vasculature Tg(flt1:tomato, Tp1-MmHbb:EGFP) in wild-type siblings (n = 43) and cad^hu10125 (n = 12) embryos at 4 dpf, (A,B), Tg(flt1:tomato); (C,D), Tg(Tp1-MmHbb:EGFP; flt1:tomato). E–H Computer assisted segregation of vascular specific expression of (Tp1-MmHbb:EGFP) in Tg(flt1:tomato, Tp1-MmHbb:EGFP) wild-type siblings (n = 13 embryos, 40 aISVs analysed) and cad^hu10125 (n = 13 embryos, 38 aISVs analysed) at 4 dpf. E,G: aISVs, Tg(flt1:tomato, Tp1-MmHbb:EGFP) positive, in wild-type siblings (E) and cad^hu10125 embryos (G) at 4 dpf. F,H: aISVs-restricted expression of Tg(Tp1-MmHbb:EGFP) in wild-type siblings (F) and cad^hu10125 embryos (H) at 4 dpf. Fluorescence intensity is displayed as a heat map, with a low to high color-coded from blue to white. I: Quantification of aISV-restricted Notch-reporter intensity based on analysis displayed in (F) and (H) (P = 0.0012). Each point represents the average aISV signal intensity in individual WT sibling embryos (n = 13 embryos, 40 aISVs analysed) or cad^hu10125 embryos (n = 13 embryos, 38 aISVs analysed) normalised to average WT sibling signal intensity. J: Western blot detection of GFP expression in 48 hpf pooled embryos from WT and cad^hu10125 Notch reporter transgenic embryos. The level of GFP expression is significantly reduced in cad^hu10125 embryos (P = 0.0274). The expression of actin was used to monitor protein input and normalise GFP band intensity. K: Quantitative RT-PCR for the Notch target genes efnb2a, hey1, hey2, and nrarpa in embryos treated with 0.1% DMSO (black columns) or 100 µM DAPT (white columns), shows that the expression of these genes is significantly reduced when Notch signaling is inhibited from 24 hpf to 3dpf) (3 biological replicates, e10 embryos each). L,M: Quantitative RT-PCR for the Notch target genes efnb2a, hey1, hey2 and nrarpa in WT (black columns) and cad^hu10125 (white columns) embryos at 3 dpf (WT, n = 5; cad^hu10125, n = 5 biological replicates, e10 embryos each)(L) and 5 dpf (WT, n = 5; cad^hu10125, n = 4 biological replicates, e10 embryos each) (M). efnb2a reduction at 3 dpf, P = 0.0017, 5 dpf P = 0.0082.; hey1 reduction at 5 dpf, P = 0.0437. Other trends nonsignificant in cad^hu10125 mutants but significant in DAPT treated embryos.

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