FIGURE 5

Loss of ptprb phenocopies nxhl deficiency. (A–F) Gross morphology at 2- and 3-dpf. Compared with control zebrafish, ptprb 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 ptprb morphants (Supplementary Videos 3–8). (G) A time-course plot of percent survival in control vs. ptprb 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 ptprb-MO present thinner ISVs (N,O, yellow arrows). In control embryos, the parachordal vessels (PAV) form normally (M, red arrows). Compared with control, MO knock down ptprb 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, ptprb 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 ptprb morphants. Columns, mean; SEM (n = 10; ANOVA) ***p < 0.0001. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessel; CVP, caudal vein plexus; CA, caudal artery; CV, caudal vein; dpf, days post fertilization. Scale bar, 100 μm. (V) Expression of genes associated with angiogenesis (left) and heart development (right) post injection of ptprb MO 2-dpf using qPCR. The data represent as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.001 represents statistically significant.