Fig. 3.

Complete ablation of BLECs results in the formation of an mrc1a-, flt4-, tjp1a-positive vessel network and a disrupted arachnoid mater. (A,B) Schematic drawing of the dorsal (A) and ventral (B) head region of an adult zebrafish, indicating the brain areas shown in C-E and C′-E′. (C-E′) Confocal images of 6-month-old mrc1a:mCitrine;kdrl:mCherry-positive zebrafish. (C,C′) In control fish, BLECs (green) are in close contact with the meningeal blood vessels of the dorsal and ventral sides of the brain. (D-E′) T-DM1-injected fish without BLECs show no regeneration of the population but develop a mrc1a-positive endothelial network, which emerges from the ventral side of the brain. In some fish, the network also covers the dorsal side of the brain (E,E′); in others, it remains exclusively ventral (D,D′). Note that the displayed brains underwent identical treatment prior to imaging (see Fig. 6, where samples from C and E are shown with IgG-Alexa-647 uptake indicated in Fig. 6D-G′). (F,G) Confocal images of a 6-month-old flt4:mCitrine;flt1:tdTomato-positive zebrafish (dorsal view). In control fish (F), BLECs are located close to meningeal blood vessels. The ectopic network that develops upon complete BLEC ablation is also positive for flt4:mCitrine (G). (H-I′) Confocal images of 6-month-old mrc1a:mCitrine;tjp1a:tdTomato-positive zebrafish brain (zoomed-in). In control fish (H,H′), BLECs with auto-fluorescent vacuoles are close to tjp1a-positive endothelial cells of the meningeal blood vessels (red arrowheads). The large cells of the arachnoid mater are also positive for tjp1a (green arrowhead). Endothelial cells forming the additional vascular network in BLEC-depleted fish are tjp1a positive (I,I′, red arrows), as is the case for meningeal blood vessels. The arachnoid mater and meningeal blood vessels appear disrupted in T-DM1-injected fish.