Extended Data Fig. 3: RNA-Seq analysis reveals Wnt–β-catenin-dependent expression of mmp25a/b in CNS ECs.

a, Dorsal views of 30 hpf Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryos injected with control, gpr124, reck, or wnt7aa morpholinos and quantification of 7xTCF:GFP⁺ cells in the PHBCs. b, Left, dorsal views of untreated, DMSO, and IWR-1 (20 µM)-treated Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryos at 36 hpf. Centre, number of 7xTCF:GFP⁺ cells in the PHBCs at 36 hpf. Right, number of CtA sprouts at 36 hpf. In a,b, n ≥ 10 embryos from 3 independent experiments. Data represent median ± interquartile range. p-values were determined using the non-parametric Kruskal-Wallis test. c, Volcano plots illustrating the differential RNA-Seq expression profiles between WT and Wnt-deficient PHBC ECs. d, Bulk RNA-Seq normalized expression values of mmp25a and mmp25b in WT or Wnt-deficient PHBC ECs. n = brain endothelia analysed by RNA-Seq. Data represent mean ± standard deviation. In c,d, p-values were determined using DESeq2 analysis. e, scRNA-Seq t-SNE expression profile of mmp25a and mmp25b and their combined expression score in PHBC-derived EC clusters. f, Expression correlation matrix between Wnt–β-catenin signalling target genes and mmp25a, mmp25b, or their combined score based on the scRNA-Seq transcriptome of the 30 hpf PHBC ECs. Pearson correlation coefficients (r) and p-values were calculated in R. g, Dorsal and dorso-lateral views of WISH for mmp25b in WT and gpr124^−/− embryos at 36 hpf. The white arrowhead points at the PHBC, black arrowheads point at CtA TCs. TG: trigeminal nerve nuclei, CF: craniofacial nerve nuclei, PLL: posterior lateral line nuclei. h, Combined fluorescent in situ hybridization for mmp25b and anti-EGFP immunostaining of 30 hpf WT and gpr124^−/− Tg(kdrl:EGFP) embryos. The magnified view shows expression in the PHBC and the underlying CF. i, Combined fluorescent WISH for mmp25b and anti-EGFP immunostaining of Tg(kdrl:EGFP) embryos in 38 hpf hindbrain when CtAs harbour two cells. The normalized EC signal intensity is shown on the right. n = 10 angiogenic sprouts from 10 embryos examined over ≥ 3 independent experiments. j, Normalized mmp25b fluorescence in PHBC and CtAs during embryogenesis. n ≥ 10 embryos examined from ≥ 3 independent experiments. In i,j, Data represent mean ± SD. p-values were determined using the parametric two-tailed Student’s t-test.

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