Fig. 5.

grb2b mutants show a defect in PCV polarization at 32hpf and exhibit decreased numbers of Prox1 and pERK positive endothelial cells. a–c The number of Prox1⁺ endothelial cells is significantly reduced in grb2b^mu404 mutants at 32hpf. a–bʺ Confocal projections of the PCV at 32hpf in wild-type and mutant embryos. Prox1 antibody staining is shown in red and flt4:mCitrine in green, with arrows pointing at cells that co-express Prox1 and Flt4. c Quantification of Prox1⁺ endothelial cells in the PCV at 32hpf across 9 segments. wt: n = 38; het: n = 72; mut: n = 33. *Between wt and mut: P value = 0.0373 (t test, two-tailed); *Between het and mut: P value = 0.0412 (t test, two-tailed). d–f pERK⁺ endothelial cells are reduced in numbers in grb2b^mu404 mutants at 32hpf. d–eʺ Confocal projections showing pERK antibody staining in red and endothelial cells in green (flt4:mCitrine) in the PCV at 32hpf. Arrows point at cells that express both pERK and Flt4. f Quantification of the number of pERK⁺ cells across 9 segments, demonstrating a decreased number in grb2b mutants. wt: n = 8; het: n = 14; mut: n = 6. **Between wt and mut: P value = 0.0013 (Mann–Whitney); ** between het and mut: P value 0.0036 (Mann–Whitney). g Moreover, pERK⁺ cells in wild type and heterozygotes are equally distributed between dorsal and ventral hemispheres of the PCV, whereas in mutants they are more concentrated in the ventral side. wt: n = 8; het: n = 14; mut: n = 6. **Between dorsal side and ventral side in mut: P value = 0.0022. h, i Confocal projections of fli:nEGFP wild-type and mutant embryos, highlighting the PCV between the more prominent dashed lines. The thinner dashed lines divide the PCV in two equal parts: the dorsal and the ventral side. j Quantifications of nuclei in the PCV at 32hpf showing that in grb2b^mu404 mutants, endothelial cells are equally distributed between the ventral and the dorsal side. In siblings, endothelial cells are enriched in the dorsal part of the PCV. wt: n = 10; het: n = 21; mut: n = 11. *Between wt dorsal and wt ventral: P value 0.0327 (t test, two-tailed); *Between het dorsal and het ventral: P value  = 0.044 (t test, two-tailed). ns not significant. Scale bars: a–bʺ: 10 µm; d–eʺ: 10 µm; h, i 20 µm. Data in c, f, g are mean ± s.d. Data in j represent the mean with individual data points. All quantifications have been done blindly