Fig. 3

GJA4 regulates vascular remodeling. a GJA4 expression was predominantly detected in the remodeling plexus at the R-M boundary, in addition to expression in SMA-invested arteries. (Colors: PECAM1 (red), SMA (cyan), GJA4 (green); scale bar: 250 µm (lower magnification), 100 µm (higher magnification); symbols: a = artery, v = vein). b At higher magnification, GJA4 expression was observed in vessels beyond the R-M boundary, including in large vessels not invested with SMA, as well as in adjacent smaller vessels. (Colors: PECAM1 (red), SMA (cyan), GJA4 (green); Scale bar = 50 µm; Symbols: a = artery). c GJA4 was absent from tip cells at the vascular edge. (Colors: PECAM1 (red), GJA4 (green); Scale bar = 50 µm). d In P6 retinas lacking GJA4 (Gja4 ^−/−) or NOTCH (WT + DAPT, or Notch1 ^iECKO−Cre+), the developing vasculature was hyperdense in comparison to wild-type (Gja4 ^+/+) tissues. (Colors: IB4 (red); scale bar = 100 µm; symbols: a = artery, v = vein). e Expression of NOTCH target genes HES1, HEY1, and HEY2 were significantly reduced in FACS-sorted endothelial cells (PECAM1+/PTPRC−, commonly CD31+/CD45–) with DAPT treatment (mean relative mRNA expression ± SEM vs. vehicle-treated controls; n = 4 for all groups; paired Students’ t-test: p = 0.0002 (Hes1), p = 0.04 (Hey1), p = 0.047 (Hey2)). f Vessel (PECAM1+ or IB4+) area was significantly increased in NOTCH-inhibited (WT + DAPT or Notch1 ^iECKO−Cre+) and GJA4-deleted (Gja4 ^−/−) mice compared to associated controls (mean % vessel area per retina ± SEM vs. control; n = 6 (WT + Veh, WT + DAPT), n = 3 (Notch1 ^{iECKO−Cre−}), n = 11 (Notch1 ^iECKO−Cre+), n = 4 (Gja4 ^+/+), n = 10 (Gja4 ^−/−); Students’ t-test: p = 0.001 (WT + Veh vs. WT + DAPT), p = 0.004 (Notch1 ^{iECKO−Cre−} vs. Notch1 ^iECKO−Cre+), p = 0.0006 (Gja4 ^+/+ vs. Gja4 ^−/−)). g Branchpoint number was also significantly increased in WT + DAPT, Notch1 ^iECKO−Cre+, and Gja4 ^−/− animals (mean % branchpoint number per retina ± SEM vs. control; n = 7 (WT + Veh), n = 9 (WT + DAPT), n = 3 (Notch1 ^{iECKO−Cre−}), n = 11 (Notch1 ^iECKO−Cre+), n = 4 (Gja4 ^+/+), n = 9 (Gja4 ^−/−) Students’ t-test: p = 0.03 (WT + Veh vs. WT + DAPT), p = 0.049 (Notch1 ^{iECKO−Cre−} vs. Notch1 ^iECKO−Cre+), p = 0.002 (Gja4 ^+/+ vs. Gja4 ^−/−)). h GJA4 expression was significantly reduced in remodeling vessels of DAPT-treated WT animals, as well as in Notch1 ^iECKO−Cre+ animals. (Colors: PECAM1 (red), GJA4 (white); scale bar = 50 µm; symbols: a = artery)