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Fig. 5 EC-intrinsic Notch signaling functions downstream of the blood flow-Piezo1 axis (A) Representative images of the pericytes and blood vessels in the midbrain of control, Tg(kdrl:H2B-mScarlet-P2A-dnMAML), and Tg(kdrl:H2B-mScarlet-P2A-NICD) larvae with Ki(pdgfrb:GAL4-VP16);Tg(4×nrUAS:GFP);Tg(fli1a:DsRedEx) background at 4.5 DPF. EC and the nucleus of dnMAML/NICD -expressing cell is in red and pericyte in green. (B–D) Effects of EC-specific dnMAML/NICD overexpression on midbrain pericyte number (B), pericyte coverage of midbrain vessels (C), and brain pericyte proliferation (D). All experiments were independent for DMSO controls in (B–D). (E) Effects of DMSO, NB, and Yoda1 treatment on pericyte coverage of midbrain vessels in Ki(pdgfrb:GAL4-VP16);Tg(4×nrUAS:GFP);Tg(fli1a:DsRedEx);Tg(kdrl:H2B-mNeoGreen-P2A-dnMAML) larvae. (F) Graphical summary of our work. Blood flow activates Piezo1 in ECs, which in turn activates EC-intrinsic Notch signaling. Then, Notch activation in ECs might lead to autonomous expression of Notch ligands, which in turn activates Notch signaling in pericytes and promotes brain pericyte proliferation. The arrows with a solid line are demonstrated by our experiments, and the arrows with a dashed line are supported by the literature and inference. Images are shown from a top view and partially z-projected in (A). Scale bar, 50 μm for (A). Images of 4.5-DPF larvae are used for counting the number of pericytes and the pericyte coverage of vessels in the midbrain, and time-lapse images during 3.0–3.5 DPF are used for counting the proliferation frequency of brain pericytes. Data are represented as mean ± SEM. The N values are shown above the aligned plots. Stars represent the results of unpaired two-tailed Student’s t test between groups (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). See also Figures S6 and S7.