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Fig. 7 Egfl7 interacts with integrin αvβ3 to regulate BLECs formation. a, b, g Schematic diagram showing the lateral view, the lower middle layer, and the top layer of the vessels in the dorsal view, respectively. Black frames indicate the orientation and the image area of corresponding panels. c–f In Tg(lyve1b: GFP; kdrl: DenNTR) transgenic background, FISH and antibody staining show itgb3a and itgb3b are expressed in CVPs at 54 hpf (c, n = 20/21 embryos, e n = 20/20 embryos, arrowheads), and expressed in BVs and departed BLECs at 3 dpf (d, n = 20/20 embryos, f, n = 19/20 embryos). h Double labeling of FISH-itgav and anti-GFP in Tg(lyve1b: GFP) transgenic background at 4 dpf, arrowheads points the itgav expressed in BLECs. n = 15/16 embryos. i HEK293T cells were transfected with Egfl7-Myc and Itgb3b-Flag or Itgav-HA. Egfl7 was immunoprecipitated using anti-Myc antibody and the immunoprecipitants were analyzed for the presence of Itgav-HA or Itgb3b-Flag by Western blot. j–l Inhibition of integrin αvβ3 by Cilengitide treatment from 54 hpf to 5 dpf phenocopy the devoid of the lymphatic loop in the brain (k). Arrows point to the BLECs in the control group (j). The statistics show the number of BLECs in the double loops after Cilengitide treatment (l, n = 15 embryos; two-tailed unpaired t-test. Data are represented as mean ± SD). m–p The itgb3b transcript elongation inhibition causes BLECs reduction in the brain. Dorsal view of BLECs loop-like structure of uninjected siblings and samples co-injected with dCas9-KRAB mRNA and itgb3b gRNAs (m, n). The co-injection of gRNA and CRISPRi results in decreased expression of itgb3b and the number of BLECs (o, n = 3 replicants; p, n = 23 embryos; two-tailed unpaired t-test. Data are represented as mean ± SD). Scale bar, 50 μm.