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Fig. 4
Enhanced membrane accumulation of phosphatase-active Pten induces stalled intersegmental vessels.
(A-J) Zebrafish embryos from a tg(kdrl:eGFP) ptena+/-ptenb-/- incross were microinjected at the one-cell stage with 300 pg synthetic mRNA encoding either Ptenb-mCherry WT, K13R, K13A or K13E. At 3dpf the embryos were analyzed for the hyperbranching vessel phenotype by confocal live imaging on a Leica TCS-SPE microscope (anterior to the left, 20x objective, 2?m z-stacks). Pictures show the trunk region distal from the urogenital opening of representative, genotyped embryos. Non-injected control embryos (NIC) were included for reference. (A?-J?) A close-up is added on the right side of each image. Stalled vessels are indicated with white arrows. (K) Quantification of the embryos expressing the indicated Ptenb mutants, showing the typical ptena-/-ptenb-/- hyperbranching vessel phenotype (blue bars) or the stalled vessel phenotype (red bars) at 3dpf. In the non-injected control (NIC), approximately 25% of the embryos showed the characteristic hyperbranching phenotype (Mendelian segregation). The statistical significance of each of the conditions compared to the non-injected control was determined using two-tailed Fisher?s exact test and is indicated in the bar graph (ns = not significant, * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001). (L) Embryos from a ptena+/-ptenb-/- incross were microinjected at the one-cell stage with wild type Ptenb or Ptenb K13 mutants as indicated. At 4dpf, single embryos were cut in half. The trunk region was used for genotyping and the anterior half was lysed and processed for immunoblotting. Lysates from siblings and ptena-/-ptenb-/- embryos were run side by side on gels and blotted. The membranes were probed with phosphospecific anti-pAkt antibody (directed against pSer473), stripped and probed with Akt-specific antibody, stripped and probed with Tubulin-specific antibody as a loading control. Representative blots are shown.