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Fig. 5. Manipulation of Barhl2 levels changes the number of chordin- and Xshh-expressing cells. In situ hybridization of embryos injected in either two (A-E) or one (F) dorsal blastomere with mouse Barhl2, Xbarhl2AS or human BCL2. Embryos are shown at (A) stage 10, (B,C) stage 15, (D) stage 18, (E) stage 12, (F) stage 15. (A-D) In situ hybridization using chordin as a probe. (A,B) control (a), mouse Barhl2 (b), Xbarhl2AS (c), human BCL2 (d). (C) Serial sections. (a) Scheme of a stage 14 embryo showing position of cuts. (b-i) Sections of Xbarhl2AS-injected embryo. α level: (b) control; (c) Xbarhl2AS. β level: (d) control; (e) Xbarhl2AS. γ level: (f) control; (g) Xbarhl2AS. Thinβ level sections in the prospective notochord area: (h) control; (i) Xbarhl2AS. (D) β level sections of stage 18 embryos: (a) control; (b) mouse Barhl2; (c) Xbarhl2AS. (E) In situ hybridization using Xvent2 as a probe: (a) control; (b) mouse Barhl2; (c) Xbarhl2AS. (F) In situ hybridization using Xshh as a probe (a-i). (a) Control; (b) mouse Barhl2; (c) Xbarhl2AS; (d) human BCL2. (e-h) Magnified views of a-d, respectively. (g) β level section of Xbarhl2AS-injected embryo. (h,l) In situ hybridization using gli1 as a probe. (i) In situ hybridization using gli3 as a probe. The line in e-h is shown as a scale indicator. |
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Fig. 4. Barhl2 pro-apoptotic activity is normally involved in regulating endogenous apoptosis. (A) TUNEL staining of stage 12.5 (a), stage 15 (b) and stage 18 (c,d) embryos. (d) Magnified view of the boxed region in c. (e,f) Double in situ hybridization of stage 18 embryos using digoxigenin- and fluorescein-labeled RNA probes: Xbarhl2 (dark blue) and Xshh (light blue) (e); Xbarhl2 (dark blue) and Xpax6 (light blue) (f). (g) In situ hybridization of Xbarhl2AS-injected embryo using Xpax6 as a probe. (a,b) Dorsal views, anterior upwards. (c-f) Anterior views, dorsal upwards. (B) Comparison of the percentage of TUNEL-positive embryos (defined as exhibiting more then 30 apoptotic nuclei in the axial midline, i.e. where endogenous apoptosis normally occurs) between wild-type embryos and embryos injected in the two dorsal blastomeres at the two-cell stage with Xbarhl2AS. Each experimental batch (n=60) was assessed independently. Three different experiments were performed and the results are shown as mean±s.e.m. (C) Kinetics of Xbarhl2 induced apoptosis between stage 8 and stage 13. Embryos were injected in one dorsal blastomere at the two-cell stage with GFP (dark blue), Xbarhl2 (red), Xbarhl2 together with Xbarhl2AS (green) or Xpax6 (light blue) and collected at indicated developmental stages. Cell death was measured by cell death detection ELISA. The apoptotic EF is calculated using GFP-injected embryos as a control. We carried out three different experiments and the results are shown as mean±s.e.m. (D) Western blot analysis on 3 μg of protein extracted from stage 12 embryos injected with Xbarhl2 (100 pg) together with Xbarhl2ASIII or Xbarhl2 (100 pg) together with Xbarhl2ASII, Xpax6 (100 pg), as indicated. The arrow indicates the large (19 kDa) cleaved caspase 3 fragment. The broken arrow shows a nonspecific band used as an internal control. |
