GFP-Positive Elements Can Drive Endothelial Gene Expression

(A) Average read depth, in counts per million (cpm), at each element used for reporter assays. Data are represented as mean ± SD from triplicate ATAC-seq libraries. All elements display GFP⁺/GFP^– log₂ fold change >1 (p < 0.0001; FDR < 0.05; biological triplicates). Name of adjacent endothelial gene shown on x axis with distance (in kilobases) and direction (“+” = downstream; “–” = upstream) of enhancer relative to TSS.

(B) Tol2 plasmid backbone used for reporter assays.

(C, E, G, and I) Overlays of green and red fluorescent images from embryos injected with reporter constructs. Lateral views: dorsal is up, and anterior is to the left. Ratios in left bottom denote number of embryos with GFP expression over number of cryaa:mcherry-expressing embryos from replicate injections. Embryos injected with (C) reporter with only basal promoter driving EGFP, or reporter with elements (E) downstream of mafbb, (G) upstream of nrp1b or (I) tmem88a. (D, F, and H) Mapped reads flanking indicated genes from GFP-positive and -negative nuclei isolated from Tg(fli1a:egfp)^y1 embryos.

(E, G, and I) White arrowheads denote low-level expression in trunk endothelial cells. Black boxes are elements used in reporter assays. Scale bar, 250 μm.

Pairing Cognate Promoters and Enhancers Improves Endothelial Expression

(A, E, and I) GFP-positive (green) and GFP-negative (black) ATAC-seq read density in nuclei at (A) lmo2, (E) clec14a, and (I) dll4 loci. Black boxes are putative enhancer elements; gray boxes denote region used as a promoter.

(B–D, F–H, and J–L) Overlays of green and red fluorescence from embryos injected with enhancer reporter constructs. Ratios in left bottom are number of embryos with endothelial GFP over the total number of cryaa:mcherry-expressing embryos from replicate injections. Lateral views, dorsal is up, anterior to the left. Embryos injected with reporter construct containing (B, F, J) gene-specific promoter, (C, G, K) enhancer and basal promoter, or (D, H, L) enhancer and cognate promoter for indicated genes upstream of EGFP. Scale bar, 250 μm.

Diagnostic assessment of FANS-assisted ATAC-Seq, relating to Figure 1. (A) Transmitted light image of nuclei prior to fluorescence-based sorting. (B) Nuclei in (A) stained with Hoechst and imaged by fluorescence. (C) Higher magnification image of Hoechststained nuclei isolated from Tg(fli1a:egfp)^y1 embryos. Selected GFP-negative nuclei are denoted by arrowheads while a GFP-positive nucleus is indicated by a yellow arrow. (D) Diagnostic fluorescence profile of nuclei from Tg(fli1a:egfp)^y1 or non-transgenic wild type embryos prior to sorting. (E) Diagnostic fluorescence profile of GFP-positive and GFP-negative nuclei from Tg(fli1a:egfp)^y1 after FANS. Y-axis is fluorescence, X-axis is forward scatter. (F) Fluorescence micrograph of fli1a:egfp-positive nuclei after sorting.

Reporter assays for putative endothelial enhancers, relating to Figure 5. (A) Proportion of cryaa:mcherry-positive embryos with endothelial Egfp expression following injection with indicated construct. (B) dusp5 locus. Black box indicates element used for reporter assay with preferential mapping of ATAC-Seq reads in GFP-plus, but not GFP-minus nuclei. Grey box indicates fragment of dusp5 promoter used for promoter assays. (C-J) Epifluorescent overlay images of green and red fluorescence in embryos injected with indicated reporter constructs. “Enh-basP” – indicated enhancer upstream of basal promoter; Prom – promoter element from indicated gene; Enh/Prom – composite element with enhancer and cognate promoter from indicated gene. Ratios in left bottom corner denote number of embryos with endothelial green fluorescents over the number of cryaa:mcherry expressing embryos. Scale bar is 250 μm.