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Figure 6. Effects of Yap knockdown in the neural tube.(A) Immunostaining with anti-YAP antibody on stage 42 sections. The left side of the neural tube is delineated with yellow dotted line. A higher magnification of the ventricular zone (white dotted line) is provided in the right panel. YAP labeling is most strongly detected in this region where progenitor cells reside (arrows). (B) Analysis of EdU-labeled replication foci (1 hr-pulse) in the neural tube of stage 40 tadpoles following two-cell stage microinjection of Yap-5-mismatch-MO (control) or Yap-MO. Enlargements (dotted lines) are shown on the right. Early (red arrows) and mid/late profiles (white arrows) were distinguished. (C) Corresponding quantification. The number of analyzed tadpoles per condition is indicated in each bar. Data are represented as mean ± SEM. (D, E) In situ hybridization analysis of c-Myc or p53 expression in the neural tube of stage 40 tadpoles injected as in (B). Note the strong upregulation in the ventricular zone of the neural tube (black arrows). Scale bars = 40 µm.DOI:
http://dx.doi.org/10.7554/eLife.08488.013 |
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Figure 7. Yap loss of function induces DNA damage.(A) γ-H2AX immunolabeling in the CMZ of retinal sections from stage 40 tadpoles following two-cell stage microinjection of Yap-5-mismatch-MO (control) or Yap-MO. Arrows point to γ-H2AX-positive cells. (B) Corresponding quantification. (C) TUNEL assay on retinal sections from stage 40 tadpoles injected as in (A). Images on the right show higher magnifications of the CMZ delineated with dotted lines. (D) Quantification of TUNEL-positive cells in the different compartments of the CMZ as illustrated on the schematic. (E) 2 days-chase of EdU-labeled cells in the CMZ of stage 42 tadpoles injected as in (A). EdU-positive cells inside the zone encircled with a red dotted line have exited the CMZ (white dotted lines) and integrated the different retinal layers. GCL: ganglion cell layer; INL: inner nuclear layer; PR: photoreceptor layer. (F) Quantification of EdU-positive cells in the neural retina layers. (G) mRNA expression levels of cell cycle genes as measured with the NanoString nCounter system in heads from stage 40 tadpoles following two-cell stage microinjection of Standard MO (control) or Yap-MO. Data are the mean of four independent experiments. (H) In situ hybridization analyses of p53 and p21 expression on retinal sections from stage 40 tadpoles injected as in (A). The number of analyzed retinas per condition is indicated in each bar (B, F) or on the graph (n in D). Data are represented as mean ± SEM. Scale bar = 40 µm.DOI:
http://dx.doi.org/10.7554/eLife.08488.014 |
