Figure 2.
PP2A regulatory subunit Bα controls vascular lumen integrity in zebrafish. (A) Tg(fli1a:eGFP)y1 embryos were injected with control morpholino (CTL Mo) or with the combination of morpholinos targeting both zebrafish Bα orthologues (MoA+MoB). Confocal pictures of the trunk vasculature were taken at 55 hpf. Whereas Ctl embryos show normal ISVs and DLAVs with open lumen, these vessels appear thin and collapsed in the morphant embryos. Scale bars represent 50 μm. DA, dorsal aorta; PCV, posterior cardinal vein; ISV, intersegmental vessel; DLAV, dorsal longitudinal anastomotic vessels. (B) Tg(fli1a:eGFP)y1 embryos were injected with control Mo, MoA (MoA), MoB (MoB) or MoA and MoB (MoA+MoB). Vascular defects were classified into four categories: normal, mild, medium or severe depending on whether circulation was detected in all, the majority, a minority or none of the ISVs, respectively. For each condition of injection, the distribution of each phenotype among the population was quantified (n=number of embryos; Pearson’s chi-squared test: P<0.001). (C) Microangiographies of control and MoA/MoB-injected Tg(fli1a:eGFP)y1 embryos at 55 hpf: confocal pictures showing normal quantum dots perfusion (red) in ISVs (green) of Ctl embryos, whereas perfusion is blocked in most of the ISVs of morphant embryos. Arrow indicates trapped perfusion dye in the mid-portion of an ISV indicating partial collapse of a previously open lumen. Scale bars represent 50 μm. (D) Tg(fli1a:RFP) embryos were transplanted with cells from control or MoA/MoB-injected Tg(fli1a:eGFP)y1 embryos. At 2 dpf, angiography was performed with quantum dots and confocal pictures were taken in order to monitor perfusion (blue) in ISVs having incorporated transplanted cells from the donor (green) and/or the host (red). Scale bars represent 50 μm. (E) Quantification of the transplantation experiments from (D) was performed by evaluating the perfusion of ISVs containing transplanted cells from control versus morphant embryos. The graph shows the percentage of ISVs that are collapsed and perfused (n=number of ISVs; Pearson’s chi-squared test: P<0.001). (F) Formation of lumen in control and MoA/MoB Tg(fli1a:eGFP)y1 embryos is illustrated by still images from Supplementary Movies 3 and 4. Asterisks indicate closed lumen whereas arrows indicate open lumen.