Fig. S1

Expression of zebrafish endothelial miRNAs (related to Figure 1).

(A) Left, sequencing reads per million for endothelial miRNAs expressed in FAC-sorted Kdrl:GFP+cells. Right, average mature miRNA levels relative to U6 snRNA expression in Kdrl:GFP+ endothelial cells as determined by qRT-PCR for two biological replicates. Developmental stages as indicated.

(B) Clustal Omega alignment of mature sequences for endothelial miRNAs characterized in this study.

(C) Design of miRNA sensor construct to detect miR-139 activity in endothelial cells. Plasmid contains Tol2 direct repeats that flank a bi-directional fli1ep enhancer-basal promoter cassette to drive endothelial-specific expression of i) mCherry regulated by a synthetic 3’UTR with 3 binding sites complementary to the mature miR-139 sequence and ii) control eGFP cassette. Sensor construct was injected into 1-cell zebrafish embryos with Tol2 transposase mRNA. Repression of mCherry expression occurs in the presence of miR-139 activity, while control eGFP expression remains constant.

(D) Representative confocal micrographs of endothelial miRNA sensor expression in 32 hpf wild type and miR-139Δ/Δ embryos. mCherry derepression in ISVs indicates miR-139 loss of function in this vessel.

(E) Quantification of vascular miR-139 activity by flow cytometry. Bar plots depict the fraction of total cells that express mCherry and GFP from the endothelial miR-139 sensor construct. Increased average number of double fluorescent cells in miR-139Δ/Δ embryos indicates loss of miR-139 activity in the endothelium. Error bars show mean + SEM of three biological replicates.

(F) Mature miR-223 localization in the lateral Kdrl:GFP+ trunk vasculature of wild type embryos at 6 dpf. miR-223+ cells are observed within the caudal hematopoietic tissue (CHT) between the dorsal aorta (DA) and caudal vein (CV). White box shows region in the zoomed-in image.