An organ-specific angiogenic control mechanism for endothelial tailoring

To address the role of endothelial Wnt/β-catenin signaling in CNS angiogenesis, we compared the bulk transcriptomes of WT and Wnt/β-catenin signaling-deficient PHBC endothelial cells, prior to CNS vascular invasion. To this end, we used three approaches to abrogate endothelial Wnt/β-catenin signaling: Morpholino-mediated knock-down of gpr124, reck or wnt7aa. We find that the expression of mmp25b is decreased in PHBC endothelial cells of all Wnt/β-catenin signaling deficient conditions as compared to the WT controls. Tg(fli1:Gal4)ubs3;(UAS:Kaede)rk8 embryos were injected at the single cell stage with Morpholinos targeting gpr124, reck or wnt7aa. Prior to CNS vascular invasion (30 hpf), PHBC endothelial cells expressing the Kaede fluorescent protein, were photoconverted (shifting green towards red fluorescence) by exposere to a 405 nm laser nanobeam, specifically at the level of the PHBC. Subsequent embryo dissociation and FACS allowed for isolation of PHBC endothelial cells. RNA extraction and sequencing was performed on 4 samples per condition, each containing photoconverted PHBC endothelial cells of 80 embryos.