Figure 2

G0/G1 cell-cycle arrest in PCV ECs is mediated by p53, p21, and p27

(A–C) Confocal images of Tg(fli1a:Gal4FF;UAS:FUCCI) (red channel) embryos at 48 hpf, showing increased numbers of late-G1 mCherry+ nuclei, which are evenly distributed throughout the PCV (outlined by dashed lines) following roscovitine versus DMSO treatment (A and B); quantified in (C) (n_DMSO = 10, n_rosco = 12). Data show mean ± SEM (unpaired t test).

(D and E) Confocal images of 48-hpf Tg(fli1:EGFP;lyve1b:dsRed2) embryos showing ectopic and mis-patterned PCV sprouts following roscovitine treatment (E, arrowheads), which are not detected in DMSO-treated siblings (D).

(F) Number of ectopic sprouts per segment in roscovitine- versus DMSO-treated embryos (n_DMSO = 11, n_rosco = 12). Data show mean ± SEM (unpaired t test).

(G–J) Confocal images of 3-dfp Tg(fli1:EGFP;lyve1b:dsRed2) embryos treated with flavopiridol (J, n = 14), aphidicolin (G, n = 6), nocodazole (H, n = 11), or etoposide (I, n = 11), showing ectopic and mis-patterned PCV sprouts following flavopiridol treatment (J, arrowheads).

(K and L) Confocal images of Tg(fli1:EGFP;lyve1b:dsRed2;p53^−/−) embryos at 48 hpf, showing normal PACs following DMSO (K) and roscovitine (L, arrowheads) treatments.

(M) Percentage of ectopic sprouts per segment in roscovitine-treated (n = 12) versus DMSO-treated (n = 12) p53^−/− embryos. Data show mean ± SEM (unpaired t test).

(N–P) Confocal images of Tg(fli1:DsRed) embryos 48 after injection with lyve1:nEGFP (N, n = 12), lyve1:p27-EGFP (O, n = 12), or lyve1:p53-EGFP (P, n = 30).

(Q) Confocal images of Tg(fli1:DsRed;lyve1:p21-EGFP) embryos at 48 hpf. Light-blue arrowheads in (N)–(Q) point to GFP+ ECs in the dorsal PCV, white arrowheads denote GFP+ lymphovenous sprouts, and co-localization channel is shown in yellow.

(R) Spatial distribution of GFP+ cells in lyve1:nEGFP-, lyve1:p27-EGFP-, lyve1:p53-EGFP-, and lyve1:p21-EGFP-expressing embryos. Scale bars: 40 μm (A–L), 50 μm (N–Q). ^∗p < 0.05, ^∗∗∗∗p < 0.0001; ns, not significant.