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Fig. 3. Barhl2- and Casp3-depleted defects are rescued by inhibition of β-catenin activity. (A–C) Representative IHC. P-3H (red) merged with BB (blue) and GFP (green) images on diencephalic sections of st 26/27 embryos injected with (A) MObarhl2 or MObarhl2/MOßcat, (B) MOcasp3 or MOcasp3/MOßcat, or (C) MOßcat. Inj, injected side. The white arrows indicate GFP+/P-3H+ cells detached from the apical membrane. (D) Normalization of Rinj/ct. The dotted line indicates a Rinj/ct of 1. MOßcat (n = 10); gsk3β (n = 8); gsk3β-dn (n = 4). MObarhl2 (n = 5), MObarhl2/MOßcat (n = 13), MObarhl2/gsk3β (n = 8); MOcasp3 (n = 8), MOcasp3/MOßcat (n = 8); MOcasp3/gsk3β (n = 4); ct vs. MOßcat (P ≤ 0.001); ct vs. gsk3β (P ≤ 0.025); ct vs. gsk3β-dn (P ≤ 0.005); MObarhl2 vs. MObarhl2/MOßcat (P ≤ 0.001); MObarhl2 vs. MObarhl2/gsk3β (P ≤ 0.011); MOcasp3 vs. MOcasp3/MOßcat-10 (P ≤ 0.000); MOcasp3 vs. MOcasp3/gsk3β (P ≤ 0.002). (E) Normalization of the ratio of ectopic mitosis. NI, noninjected. MOßcat (n = 5); gsk3β (n = 6); MObarhl2 (n = 8), MObarhl2/MOßcat (n = 10), MObarhl2/gsk3β (n = 4), MOcasp3 (n = 7) MOcasp3/MOßcat (n = 7), MOcasp3/gsk3β (n = 5); ct vs. MOßcat (P ≤ 0.005); ct vs. gsk3β (P ≤ 0.007); MObarhl2 vs. MObarhl2/MOßcat (P ≤ 0.003); MObarhl2 vs. MObarhl2/gsk3β (P ≤ 0.665); MOcasp3 vs. MOcasp3/MOßcat (P ≤ 0.007); MOcasp3 vs. MOcasp3/gsk3β (P ≤ 0.604); MOßcat vs. MObarhl2/MOßcat (P ≤ 0.01); MOßcat vs. MOcasp3/MOßcat (P ≤ 0.015). (F–I) Normalization of Wnt activity. axin2 expression profile of st 30-injected neural tubes analyzed by ISH. (F and G) Representative embryos are shown either as the diencephalic section or (H and I) the ct (a) and injected (b) sides of one representative dissected neural tube mounted flat. At st 30 in the P2 alar plate, axin2 expression territory is reduced in MOßcat-depleted (F; 73%, n = 15) and gsk3β overexpressing (G; 75%, n = 12) embryos. The P2 alar plate (white rectangle) axin2 expression territory is reduced in both MObarhl2/MOßcat- (compare H a and b; 66%, n = 18) and MOcasp3/MOßcat-injected (compare I a and b; 65%, n = 38) sides compared with the MObarhl2- and MOcasp3-injected and respective ct sides. The black arrow indicates the optic stalk. |
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Fig. S4. Depletion of Barhl2 or Casp3 generates neural tube hyperplasia, an increase in the BrdU LI, and ectopic mitosis. (A) Depletion of Barhl2 or Casp3 generates neural tube hyperplasia. Xenopus embryos were injected with (a) MObarhl2-60 (60 ng), (b) MOcasp3-40 (40 ng), (c) bcl-XL cRNA, or (d) hbcl2 cRNA. Representative sections of st 26 embryos at the diencephalic level are shown dorsal side up. Cell nuclei are stained with BB. The white line indicates the midline of the neural tube and the limits of the neuroepithelium. The left side is the injected side. (B and C) Neuroepithelial cells all proliferate until st 26, and some neuroepithelial cells cease division after this point. The BrdU LI was measured after (B) 8 h starting at st 24 until st 26/27 or (C) 18 h starting at st 26/27 until st 30 of BrdU exposure in telencephalic and diencephalic sections. Each embryo was assessed independently. (B) Between st 24 and 27, 99.7% of telencephalic (n = 3) and 98.9% of diencephalic (n = 3) neural tube cells had incorporated BrdU and undergone at least one cell cycle. (C) Between st 26 and 30, only 84% of telencephalic (n = 3) and 88% of diencephalic (n = 3) neural tube cells had divided, arguing that some neuroepithelium cells had exited the cell cycle during this period. (D and E) Ectopic mitosis in Barhl2- and Casp3-depleted st 26 embryos. Representative sections of st 26 embryos at the diencephalic level, shown dorsal side up and immunostained for P-3H (red), merged with BB (blue) images. |
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Fig. S5. Barhl2 or Casp3 depletion phenotypic defects. (A and B) Disorganization of the neuroepithelium. Representative IHC for β-catenin (red; membrane staining) together with P-3H (red; nuclear staining) merged with BB (cyan) images on diencephalic sections of st 26/27 Xenopus embryos injected with MO- barhl2 (A) or MOcasp3 (B). The (Aa) control and (Ab) injected sides of representative sections are shown. Inj, injected side. Arrows indicate part of the apical surface where β-catenin staining is abnormal; arrow end indicates mitotic nuclei. (C and D) Increase in Wnt signaling in the prosomere P2 alar plate ccnd1 expression profiles at st 33 or 30 analyzed by ISH. The ct (Ca) and injected (Cb) sides of one representative dissected neural tube flat-mounted and (Cc) a section at the diencephalic level are shown. The percent of embryos exhibiting the phenotype is indicated. (Ca) ct and (Cb and Cc) Barhl2-depleted (89%, n = 19); (Da) ct and (Db and Dc) Casp3-depleted neural tubes (82%, n = 39). The white rectangle delineates the prosomere P2 alar plate. The black arrow indicates lo- calization of the optic stalk. |
