Fig. 1

Endothelial Nrarp Expression Is Regulated by Notch Signaling

(A–C) In situ hybridization (ISH) of mouse Nrarp in mouse embryonic (E10.5) intersomitic vessels (arrows in [A]) and limb bud vessels ([B and C]; Endomucin, red). (C) Single plane confocal LSM image of limb bud vessels outlined with dotted red line (ISH reflection signal, blue; transmitted light; endomucin, green). Nrarp is more strongly expressed by ECs at branch points (filled arrowheads in [C]) compared to ECs in between (stroked arrowheads in [C]).

(D–G) Notch signaling positively regulates endothelial Nrarp expression. Whole-mount retina ISH shows high Nrarp expression (blue) in ECs (stained with Isolectin-B4, green) often located at vessel branch points in vehicle-treated P5 retinas (filled arrowheads, [D]) and absent in between branch points (stroked arrowheads). Endothelial stalk cells with Nrarp expression are indicated by arrows. Endothelial Nrarp expression decreased after 6 hr of DAPT treatment (E). (F) qPCR, relative expression of Nrarp in the retinas of P5 C57Bl6/J mice treated with DAPT (6 hr) or Dll4-Fc (24 hr). ^***, p ≤ 0.0004. Vehicle and DAPT treatments, n ≥ 7; hFc, n = 4; Dll4-Fc, n = 6. Data show mean ± SEM, n = animals. (G) HUVECs were stimulated with rDll4 for 6 or 24 hr. Fold increase in NRARP, HEY1 and HEY2 expression are shown. n = 3.

(H–J) nrarp-a ISH in 24 hpf zebrafish embryos. (H) and (H′) are lateral views of the same region in the zebrafish trunk taken at different focal planes. The boxed region in (H) is magnified in (I). (J) is a transverse section of the zebrafish trunk. nrarp-a is expressed in ECs of ISV (arrowhead, [I]) and in cells located between somite boundaries (empty arrowheads, [H]). nrarp-a is also expressed in the DA and in the neural tube (H′, J). Arrows in (I) indicate somite boundary. NT, neural tube; NC, notochord; DA, dorsal aorta; PCV, posterior cardinal vein.