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Figure 5. Yap loss of function affects the temporal program of retinal stem cell DNA replication.(A) In situ hybridization analysis of c-Myc expression on stage 40 retinal sections following two-cell stage microinjection of Yap-5-mismatch-MO (control) or Yap-MO. Images on the right show higher magnifications of the CMZ (dotted lines). Note the strong expansion of c-Myc expression area (bracket). (B) Quantification of the staining in the CMZ. The number of analyzed retinal sections per condition is indicated in each bar. (C) qPCR analysis of c-Myc expression in the retina of tadpoles injected as in (A). (D) Schematic representation of replication foci observed during S-phase progression, as inferred from EdU labeling. Pictures illustrate two examples of EdU-labeled foci observed in control CMZ cells, one homogenous (early S-phase) and one with large dots (mid/late S-phase). (E) Analysis of EdU-labeled replication foci (1 hr-pulse) in the CMZ of stage 40 tadpoles injected as in (A). Enlargements of the CMZ tip (dotted lines) are shown on the right. Early (red arrows) and mid/late profiles (white arrows) were distinguished. (F) Corresponding quantification. The number of analyzed retinas per condition is indicated in each bar. Data are represented as mean ± SEM. Scale bar = 40 µm.DOI:
http://dx.doi.org/10.7554/eLife.08488.012 |
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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 9.
Functional interaction between YAP and PKNOX1.
(A) In situ hybridization analysis of pknox1 expression on stage 40 retinal sections. The right panels shows an enlargement of the CMZ region delineated with dotted lines. (B) Lateral views (left panels), head dorsal views (middle panels) and dissected eyes (right panels) of stage 40 tadpoles following two-cell stage microinjection of pknox1-5-mismatch-MO (control) or pknox1-MO. The asterisk indicates the injected side. (C) Quantification of dissected eye area. (D) Analysis of EdU-labeled replication foci (45 min-pulse) in the CMZ of tadpoles injected as in (B). Enlargements of the CMZ tip (dotted lines) are shown on the right. Early (red arrows) and mid/late profiles (white arrows) were distinguished. (E) Corresponding quantification. (F) In situ hybridization analysis of c-Myc expression on stage 40 retinal sections from tadpoles injected as in (B). (G–L) EdU incorporation assays (3-hr pulse) analyzed on retinal sections from stage 40 tadpoles. (G, H) shows the effect of pknox1 knockdown (injection of pknox1-5-mismatch-MO (control) or pknox1-MO). (I, J) shows the synergistic effects of pknox1 and Yap (injection of GFP mRNA and either ß-gal mRNA (control), Yap + ß-gal mRNA (Yap), pknox1 + ß-gal mRNA (pknox1), Yap + pknox1 mRNA (Yap + pknox1)). (K, L) shows the rescue of Yap overexpression by pknox1 knockdown (injection of either pknox1-5-mismatch-MO + ß-gal mRNA (control), pknox1-MO + ß-gal mRNA (pknox1-MO), pknox1-5-mismatch-MO + Yap mRNA (Yap), pknox1-MO + Yap mRNA (Yap + pknox-MO)). Of note, a suboptimal dose of pknox1-MO was used for the rescue experiment so that it does not alone give any eye phenotype. The total number of analyzed retinas per condition is indicated in each bar. Scale bar = 1 mm in (B) and 40 µm for all other panels.
DOI: http://dx.doi.org/10.7554/eLife.08488.016 |