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FIGURE 2
Nxhl regulates vascular development in vivo. (A–F) Gross morphology at 2-dpf and 3-dpf. Compared with control zebrafish, nxhl knock-down causes pericardial oedema (B,C,E,F, red arrowheads) and circulation defects. Heart beat and circulation in caudal vein (CV) is visible in the control fish, but is abnormal in nxhl morphants (Supplementary Videos 1, 2). (G) A time-course plot of percent survival in control vs. nxhl morphants for 3 days. (H) Shows the percentage of embryos with development defects. (I) Quantification of the pericardial area of embryos. Error bars, mean ± SEM; ***p < 0.0001 (n = 10; ANOVA). (J–R) Representative fluorescent images of Tg(fli1a:EGFP)y1 embryos at 2-dpf. (J,M) Image of trunk regions taken at 2-dpf, with the vascular structures visualized by eGFP fluorescence and labeled ISV and DLAV showed regular development in the embryo injected with control MO. The boxed regions are shown at higher magnification in the bottom panels. (K,L,N,O) Compared with control MO, embryos injected with nxhl-MO present a lower number of incomplete ISVs and thinner ISVs (N,O, yellow arrows) and ectopic sprouts (asterisk) of dorsal aorta. In control embryos, the parachordal vessels (PAV) form normally (M, red arrows). Compared with control, MO knock down nxhl prevents the parachordal vessels (PAV) formation, the precursor to the lymphatic system. In control embryos, caudal vein plexus (CVP) were formed honeycomb-like structures at the tail around 2-dpf (P, white arrows). In contrast, nxhl knock down resulted in specific defects in caudal vein plexus (CVP) formation (Q,R). (S–U) Quantification of the mean diameter ISVs (S,T) and loop formation at CVP (U) shows significantly decrease in nxhl morphants. Columns, mean; SEM (n = 10; ANOVA) ***p < 0.0001. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessel; DA, dorsal aorta; PCV, posterior cardinal vein; CVP, caudal vein plexus; CA, caudal artery; CV, caudal vein; dpf, days post fertilization. Scale bar, 100 μm.