Figure 2—figure supplement 2.

(A, B) Diagrams of the GAL4-responsive constructs used for forced endothelial expression in plxnd1^fov01b; Tg(fli1a:GAL4FF)^ubs4; Tg(flt1:nls-mCherry)^skt7 embryos. (A) Construct for expression of the green fluorescent marker EGFP (negative control). (B) Construct for bicistronic coexpression of 2xHA-Plxnd1 (2xHA-Plxnd1^WT or 2xHA-Plxnd1Δ^GBM) and EGFP (to fluorescently label cells with exogenous 2xHA-Plxnd1 expression). (C–I) Confocal lateral images of 32 hpf embryo trunks. Anterior, left; dorsal, up. Scale bars (white horizontal lines), 50 μm. Image colors: Cells with exogenous gene expression, green (EGFP⁺); arterial nuclei, red; somite boundaries, blue. The position of EGFP⁺ clones within the arterial tree indicated as follows. DLAV (Dorsal Longitudinal Anastomotic Vessel), Se (Segmental vessel), DLAV-Se (both Se and DLAV), DAd (dorsal side of the Dorsal Aorta), and DAv (ventral side of the Dorsal Aorta). White asterisks mark clones with non-endothelial, ectopic expression. (C) Expression of EGFP alone fails to rescue the vascular defects of plxnD1^fov01b mutants. EGFP⁺ arterial cells form mispatterned, ectopic and over branched Se vessels and aberrantly shaped DLAVs. (D–I) EGFP⁺ cells expressing 2xHA-Plxnd1^WT (D–F) or 2xHA-Plxnd1Δ^GBM (G–I) rescue the vascular defects of plxnd1^fov01b mutants. These cells displayed a WT-like phenotype. Briefly, they were not found within ectopic Se sprouts, displayed normal shapes according to their position within Se and DLAVs and, when found at the base of a sprout, were properly positioned just anterior to a somite boundary; see (Zygmunt et al., 2011). (J) Bar graph. Quantification of the vascular phenotype (WT-like or plxnd1 null-like) of EGFP⁺ angiogenic endothelial cells (those at the DLAV, DLAV-Se, and DAd positions but not the DAV position) with exogenous expression of 2xHA-Plxnd1^WT (top) or 2xHA-Plxnd1Δ^GBM (bottom) in plxnd1^fov01b mutants. Both 2xHA-Plxnd1 forms rescue the vascular defects of plxnd1^fov01b mutants with similar efficiency. Quantifications. We scored the vascular phenotype of angiogenic endothelial cells in plxnd1^fov01b mutants coexpressing EGFP and the following 2xHA-Plxnd1 forms: 2xHA-Plxnd1^WT (21 clones, 15 embryos), 2xHA-Plxnd1Δ^GBM (nine clones, 17 embryos). Note that embryos harboring only DAV clones were excluded from this analysis. The significance value (p=1) was calculated using a two-sided Fisher’s Exact test. The significant difference value is p<0.05. The proportions were not significantly different. For additional supplementary information related to this figure, see Supplementary file 1, Supplementary file 3 and Supplementary file 8. This figure is related to Figure 2.