Fig. 7.

After ablation of BLECs, immune cells show gene signatures indicating a chronic inflammatory response, and the number of microglia is increased in areas covered by the endothelial network. (A) Seurat cluster analysis of clusters 0 (red) and 2 (green), indicating the origin of the cells (control plate, red; T-DM1 plate, blue). (B) Volcano plot of differentially expressed genes. Cluster 0, which is composed of cells from the control and T-DM1 plate, represents dendritic cells with an initial inflammatory response, whereas cluster 2, which is composed of cells from the ‘T-DM1-treated’ plate, represents dendritic cells with a chronic inflammatory response. (C) Schematic overview of the dorsal and ventral head region of a zebrafish, indicating the zoomed-in areas in D,E (left) and F,G (right). (D-G) Maximum projections showing the dorsal (D,E) or ventral (F,G) TeO of lyve1:dsRed;mpeg:GFP-positive brains dissected from 1.5-year-old control (D,F) or T-DM1-injected (E,G) fish. Rectangles indicate the zoomed-in areas of the green channel shown on the right. Control fish have BLECs (red) and microglia (green) (D,F), whereas, in T-DM1-injected fish, BLECs are absent (E,G). The ectopic endothelial network is lyve1 positive, and the number of microglia is increased in T-DM1-injected fish (G). (H) Quantification of the percentage area that was covered with microglia at the dorsal brain surface [n=5 fish per group; P=0.151 (ns, not significant) Mann–Whitney U-test]. (I) Quantification of the percentage area that was covered with microglia at the ventral brain surface (n=5 fish per group; **P=0.00151, unpaired Student's t-test). (J) Dissected brains of T-DM1-treated fish with the ectopic vessel network had a higher relative brain weight compared to control brains (n=3 brains per group, *P=0.0325, unpaired Student's t-test). ns, not significant.