Fig. 6.

Crip2 functions upstream of Notch signaling in the dorsal aorta to safeguard HSPC production. (A,B) Confocal images of wild-type (n=25) (A) and crip^DM (n=44) (B) embryos carrying Tg(kdrl:mCherry);Tg(tp1:EGFP) at 28 hpf. Lateral views, anterior to the left. Arrows indicate kdrl:mCherry⁺tp1:EGFP⁺ double positive cells in the floor of the dorsal aorta. Scale bar: 50 µm. (C) Quantification of kdrl:mCherry⁺tp1:EGFP⁺ cells in wild-type and crip^DM embryos. Unpaired two-tailed nonparametric t-test yields a statistically significant difference between wild-type and crip^DM embryos (****P<0.0001). (D) Experimental design schematic depicts the addition of DMSO and DBZ to wild-type and crip^DM embryos at 24 hpf with fixation at 28 hpf for confocal microscopy or 32 hpf for cmyb in situ hybridization (ISH). (E-H) Confocal images of DMSO-treated wild-type (n=21) (E) and crip^DM (n=24) (G) and DBZ-treated wild-type (n=18) (F) and crip^DM (n=15) (H) embryos carrying Tg(kdrl:mCherry);Tg(tp1:EGFP) at 28 hpf reveal normalization of kdrl:mCherry⁺tp1:EGFP⁺ cell number in DBZ-exposed crip^DM embryos. Lateral views, anterior to the left. Arrows indicate kdrl:mCherry⁺tp1:EGFP⁺ double positive cells in the floor of the dorsal aorta. Scale bar: 25 µm. (I) Quantification of kdrl:mCherry⁺tp1:EGFP⁺ cells in the dorsal aorta (DA) from E-H shows a statistically significant reduction in the DBZ-exposed compared to the DMSO-exposed crip^DM embryos. Unpaired nonparametric Mann–Whitney U-test yields ****P<0.0001 between DMSO-exposed wild-type and crip^DM embryos and ****P<0.0001 between DMSO- and DBZ-exposed crip^DM embryos. (J-N) ISH for cmyb at 32 hpf illuminates reappearance of HSPCs in DBZ-exposed crip^DM (n=39) (N) compared to DMSO-exposed crip^DM (n=41) (M) embryos, delineating a trend towards HSPC numbers in DMSO-exposed wild-type (n=40) (K) embryos. Lateral views, anterior to the left. Scale bar: 100 µm. Quantification of cmyb signals in the DA from K-N using pixel intensity analysis (J) shows a statistically significant elevation in the DBZ-exposed compared to the DMSO-exposed crip^DM embryos. Unpaired nonparametric Mann–Whitney U-test yields ***P=0.0007 between DMSO- and DBZ-exposed wild-type embryos, ****P<0.0001 between DMSO-exposed wild-type and crip^DM embryos, *P=0.0111 between DMSO- and DBZ-exposed crip^DM embryos, and *P=0.0278 between DMSO-exposed wild-type and DBZ-exposed crip^DM embryos. Mean and standard error of each dataset are shown. ns, not significant.