Extended Data Fig. 1: Characterization of the pre-angiogenic perineural endothelium.

a, Lateral views and quantifications of angiogenic sprouts in the 36 hpf hindbrain of WT, wnt7aa^−/−, gpr124^−/−, and reck^−/− Tg(kdrl:EGFP) embryos (n ≥ 20 embryos from 3 independent experiments, arrowheads: CtA sprouts). Data represent median ± interquartile range. p-values were determined using the non-parametric Kruskal-Wallis test. b, TC genotype in mosaic angiogenic sprouts during post-invasive secondary branching (42 hpf) of embryos obtained by transplanting wild-type (WT) kdrl:EGFP⁺ donor cells into gpr124 morpholino-injected kdrl:ras-mCherry hosts. The arrowhead points at a TC within the hindbrain. n = number of analysed mosaic vessels from 10 embryos recorded in 5 independent transplantation experiments. c, Heat map of scRNA-Seq transcriptome analysis of 30 hpf PHBC ECs showing the expression levels of the 25 highest ranked (lowest p-value) transcripts for each cell cluster. Ven: venous. d-h, t-SNE expression profiles of arterial markers (d), venous markers (e), BBB-associated transporters (f), tight junction proteins (g), and plvapb, a transcytosis marker (h). i, Lateral view of a WT Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryo at 30 hpf, counterstained with DAPI. The solid and open arrowheads point at some 7xTCF:GFP⁺ and 7xTCF:GFP⁻ PHBC ECs, respectively. j, Immunostaining for β-Galactosidase (LacZ) and Erg1 in a transverse spinal cord section of a WT BAT-GAL E9.5 embryo, counterstained with DAPI. The solid and open arrowheads indicate some LacZ⁺ and LacZ⁻ perineural vascular plexus (PNVP) ECs, respectively.

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