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Fig. 1 Blood flow promotes the proliferation of brain pericytes (A) Representative images of the pericytes and blood vessels in the brain of Ki(pdgfrb:GAL4-VP16);Tg(4×nrUAS:GFP);Tg(fli1a:DsRedEx) larvae at 4.5 DPF. EC is in red and pericyte in green. (B) Graph showing the numerical changes of the blood vessel segments in the midbrain, the pericytes in the brain, and the pericytes on the pericerebral vessels, including basal communicating artery (BCA), posterior communicating segment (PCS), choroidal vascular plexus (CVP), basilar artery (BA), and primordial hindbrain channel (PHBC). Zebrafish larvae during 1.5–6.5 DPF for pericyte number analysis and the larvae during 1.0–7.5 DPF for blood vessel number analysis in midbrain. N = 9 for pericytes, and n = 8 for midbrain vessel segments. (C) Graphic showing the event number of the ingress of pericerebral pericytes into the brain, and the brain pericyte proliferation occurred over a 12-h period each day during the early embryonic stage. (D) Graphic showing the number of the cases that a photoconverted single brain pericyte proliferated for varying times that occurred during 2.5–4.5 DPF, based on the images for brain pericyte fate tracking on Ki(pdgfrb:GAL4-VP16);Tg(UAS:Kaede) larvae as Figure S4D shows. (E) Representative images of the pericytes and blood vessels in the brain of the DMSO-, NB-, and BDM-treated Ki(pdgfrb:GAL4-VP16);Tg(4×nrUAS:GFP);Tg(fli1a:DsRedEx) larvae at 4.5 DPF. The images of pericytes in the midbrain are highlighted in (E′)–(E‴). EC is in red and pericyte in green. (F–H) Linear regression analysis for the effects of pharmacological modulations of blood flow on pericyte coverage of brain vessels (F), brain pericyte proliferation (G), and ingress of the pericerebral pericytes into the brain (H). Images are shown from a top view and partially z-projected. Scale bar, 50 μm for (A) and (E). Images of 4.5-DPF larvae are used for counting the number of brain pericytes and the pericyte coverage of brain vessels; time-lapse images of 3.0–3.5 DPF are used for counting the mean proliferation frequency of brain pericytes and the event number of ingress of pericerebral pericytes into the brain. Data are represented as mean ± SEM. The N values are shown above the bars in (C). See also Figures S1–S3. To study the dynamics of brain pericytes during cerebrovascular development, we carried out long-term serial confocal imaging of Ki(pdgfrb:GAL4-VP16);Tg(4×nrUAS:GFP);Tg(fli1a:DsRedEx) larvae during 1.5–6.5 days post-fertilization (DPF) (Figure S2A). We found that, at 1.5 DPF, no pericyte was present in the brain or on the pericerebral vessels, although the cerebral vessels had already begun forming (Figures 1B and S2A). Pericytes started to appear on pericerebral vessels at around 2.0 DPF and were initially observed in the brain at around 2.5 DPF (Figures 1B and S2A). After 2.5 DPF, the number of pericytes markedly increased in both the midbrain and hindbrain, and the brain vasculature was fully covered by pericytes at 6.5 DPF (Figures 1B and S2A). To determine how the number of pericytes increase in the brain, we performed in vivo time-lapse imaging with the interval of 2–4 h during 2.0–6.5 DPF. We found that pericytes on pericerebral vessels, including the basal communicating artery (BCA), choroidal vascular plexus (CVP), posterior communicating segment (PCS), and basilar artery (BA), migrated into the brain along perfused blood vessels (Figure S2B and Video S1). The ingress of pericerebral pericytes was the major contributor to the growth of brain pericyte population during 2.0–2.5 DPF (Figure 1C). After ingress into the brain, pericytes underwent proliferation to expand their population (Figures 1C and S2C and Video S2), while the proliferation of pericytes in the brain took a dominant role after 3.0–3.5 DPF, and the ingress of pericerebral pericytes into the brain could hardly be observed after 5.0 DPF (Figure 1C). Therefore, brain pericytes expand their population mainly through proliferation after ingress into the brain during development.