PGE₂ production in the AGM is enhanced by calcium flux triggered by WSS. (A) E10.5 AGM-derived cells cultured for 36 h with WSS activate several pathways required for definitive hematopoiesis (n = 5 independent experiments; two-tailed Student’s t test or Mann–Whitney rank sum: *, P < 0.05; **, P < 0.01). Klf2 and Klf4 are WSS-responsive genes that serve as positive controls for transcriptional activity. (B) Transcriptional up-regulation in WSS-exposed cells is apparent in sorted CD144⁺ CD45⁻ hemogenic endothelial cells (n = 5 independent experiments; unpaired Student’s t test: *, P < 0.05; **, P < 0.003). (C) Prostaglandin production as measured by PGE₂ release into media was increased over time by WSS (n = 3 independent experiments; two-way ANOVA: **, P = 0.001). (D) PGE₂ biosynthesis was suppressed by addition of indomethacin or COX2-specific antagonists NS-398 and CAY10404. WSS-treated vehicle control produced significantly higher amounts of PGE₂ than WSS samples treated with COX inhibitors and all static cultured samples at 60 h of culture (n = 3 independent experiments; two-way ANOVA: *, P < 0.02; **, P < 0.001). (E) WSS applied to E11.5 AGM-derived cells triggers elevated levels of intracellular calcium (n = 3 independent experiments, >5 replicates per experiment). WSS was initiated 3 s after image acquisition began. Abbreviated videos are available in Videos 1 and 2. Bar, 50 µm. (F) Quantification of Fluo-4 AM intensity by MetaMorph captures multiple spikes in calcium flux after application of WSS (red arrow on horizontal axis marks initiation of WSS at 3 s). Pastel traces represent calcium levels in individual cells (n = 150 cells), whereas bold traces (blue or red) represent the mean intensity of values collected from individual cells. (G) Ptgs2 transcript level was reduced by an inhibitor of calcium signaling (BAPTA-AM; n = 3 independent experiments at 3 h of WSS; one-tailed unpaired Student’s t test: *, P = 0.036; P > 0.26 for comparison between static vehicle- and BAPTA-AM–treated WSS group). (H) COX2 protein accumulates in response to transient treatment with A23187, a compound which increases intracellular calcium. Cells were pulse treated under static conditions with A23187 for 1 or 3 h, washed, and incubated in fresh medium for a total culture period of 18 h. A23187 was not removed from the 18-h sample (n = 3 independent experiments at 2 µM A23187). (I) COX2 protein levels are decreased in Ncx1^−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). All data are represented as mean ± SEM.