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Fig. 1. Barhl2 or Casp3 depletion influence on neural tube hyperplasia, cell-cycle length, and neuroepithelium architecture. (A) barhl2 is expressed in the prosomere P2. ISH or double ISH on dissected neural tubes. (a) At st 26, barhl2 is expressed at the epichordal–prechordal border in the alar and basal plates of the neural tube. (b) At st 31 and 32, barhl2 expression overlaps with that of wnt3A (compare b and c). (c) The barhl2 rostral border of expression coincides with the rostral border of the ZLI (black arrow). (d) At st 33, barhl2 is expressed in the prospective diencephalon in the prospective pallium (1), the medial part of the pretectum (2), and the dorsal mesencephalon and future cerebellum (3). The pineal gland is indicated with a red star. (B) casp3 is expressed in the neural plate (st 15) and neural tube (st 26). RT-PCR analysis. chordin and Ncam are mesodermal and neuroectodermal markers, respectively. XEF-1α and the plasmid encoding Casp3 were used as positive controls, and water was the negative ct. (C) Average ratio of the number of nuclei on the MO/RNA-injected side of the neural tube relative to that on the ct side. Embryos were injected with MObarhl2, MOcasp3, bcl-XL, or hbcl2 cRNA. The dotted line indicates a ratio of 1. MObarhl2ct (n = 3); MObarhl2-40 (40 ng; n = 6); MObarhl3-60 (60 ng; n = 7); MOcasp3ct (n = 3); MOcasp3-20 (20 ng; n = 5); MOcasp3-40 (40 ng; n = 8); bcl-XL (n = 5); hbcl2 (n = 4). The differences in Rinj/ct between MObarhl2ct and MObarhl2-60 (P ≤ 0.033), MObarhl2ct and MObarhl2-40 (P ≤ 0.06), and MOcasp3ct and MOcasp-40 (P ≤ 0.036) are significant. (D–G) BrdU labeling index (LI). (D and E) Representative st 26 diencephalic sections immunostained for BrdU (green) merged with bisbenzimide (BB; blue). BrdU LI was measured in st 26 forebrain neuroepithelium in (F) Barhl2- and (G) Casp3-depleted sections. (F) MObarhl2-injected embryos: telencephalon (ct vs. inj; n = 5, P ≤ 0.41) and diencephalon (ct vs. inj; n = 9, P ≤ 0.0001). (G) MOcasp3-injected embryos: telencephalon (ct vs. inj; n = 5, P ≤ 0.141) and diencephalon (ct vs. inj; n = 9, P ≤ 0.0001). ct, ct side; inj, injected side. (H and I) BrdU cumulative analysis. In st 26 diencephalic neuroepithelium, the cumulative BrdU incorporation rate measured as the slope of the linear regression function is higher in the injected side (red) vs. the ct side (blue) of (H) MObarhl2-injected (0.130 vs. 0.094; n ≥ 4 for each time point) or (I) MOcasp3-injected (0.1344 vs. 0.098; n ≥ 4 for each time point) embryos. H, hours. The average cell-cycle length, as described by Tc value, decreases in both MObarhl2- (Tc = 6.9 ± 0.64 h) and MOcasp3-injected (Tc = 7.1 ± 0.64 h) cells compared with noninjected cells (Tc = 8.7 ± 0.65 h and Tc = 8.9 ± 0.65 h), respectively. The changes in Tc observed among different types of cells are not affected by Ts, the duration of the S phase, which is always evaluated at 1.54 ± 0.28 h. (J) Mitotic index. MObarhl2 (ct vs. inj; n = 8, P ≤ 0.008) and MOcasp3 (ct vs. inj; n = 8, P ≤ 0.001). (K) Ratio of ectopic mitotic figures. Ratio of P-3H–positive cells outside the ependymal zone (at least two cellular diameters) relative to the total number of mitotic cells. MObarhl2 (n = 8, P ≤ 0.0001) and MOcasp3 (n = 7, P ≤ 0.0001).
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Fig. 2. Barhl2 and Casp3 depletion increases β-catenin and Wnt activity. (A and B) Western blot analysis. Proteins (10 μg) extracted from st 26 heads of embryos injected with GFP (ct), MOcasp3, or MObarhl2. α-actin (42 kDa) is used as a loading ct. (A) Barhl2- and Casp3-depleted head extracts exhibit a significant increase in both β-catenin and active β-catenin levels (95 kDa) as indicated. (B) Active β-catenin detected in MOcasp3- or MObarhl2-injected head extracts is enriched in nuclear head extracts (N) compared with that in whole-cell head extracts (W). Sam68 is used as a marker of proper nuclear proteins extraction. (C) β-catenin mislocalization. Representative IHC images for β-catenin (red) merged with BB (blue) on diencephalic sections of st 26/27 embryos injected with MObarhl2 or MOcasp3. Arrows indicate the part of apical surfaces where β-catenin staining is disrupted. Inj, injected side. (D and E) Analysis of Wnt activity. axin2 expression profiles at st 30 analyzed by ISH. The ct (a) and injected (b) sides of one representative dissected neural tube flat-mounted or (c) a section at the diencephalic level are shown. The percent of embryos exhibiting the phenotype is indicated. (Da) ct and (Db and Dc) Barhl2-depleted (80%, n = 15) and (Ea) ct and (Eb and Ec) Casp3-depleted neural tubes (78%, n = 18). The white rectangle delineates the prosomere P2 alar plate. The black arrow indicates localization of the optic stalk.
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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. S1. casp3 and barhl2 are coexpressed in the stage (st) 26 prosomere P2. (A–C) ISH with casp3 antisens (A and C) or casp3 sens (B) probes on st 26 dissected neural tubes. (A and B) At st 26, casp3 is mostly expressed in the telencephalic prospective pallium, in the diencephalic and mesencephalic alar plates of the neural tube, and in the pineal gland. (C) Enlargement of the prosomere P2 area of A. barhl2 expression domain is indicated with a black line. (D) RT-PCR analysis on RNA purified from telencephalic (TEL) or diencephalic (DIEN) st 26 neural tube parts. Foxg1 and barhl2 are pallial TEL and DIEN markers, re- spectively. Histone H4 and the plasmid encoding Casp3 were used as positive controls, and water was a negative control (ct).
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Fig. S2. From st 20 to 33, apoptotic nuclei are uniformly distributed in the anterior neural tube, and Bcl-XL overexpression protects anterior neural tube cells from apoptosis. (A) Apoptosis patterns. (a and b) TUNEL-positive cells occurred mainly along the dorsal closure line of the neural tube during neurulation (st 18– 20; 70%, n = 122). (d, e, and g) From st 20 to 33, cells died in the anterior part of the neural tube but were not confined to specific areas (n ≥ 150 for each stage). (d ) WT representative embryos at st 24. Representative dissected neural tubes at (e) st 26–27 and (g) st 33. bcl-XL cRNA was injected into one dorsal blastomeres of four-cell embryos together with GFP as a tracer. TUNEL staining was performed on these embryos at (c) st 18–20, (f) 26 and 27, and (h) 33. (a–c) Dorsal view, anterior up; (b) an enlargement of WT st 19 embryos. (d) Anterior view, dorsal up. (e–h) Lateral view, anterior to the left. Re, retina; cg, cement gland.
(B) Bcl-XL overexpression prevents apoptosis. (a–c) bcl-XL cRNA was injected into two dorsal blastomeres at the four-cell stage. TUNEL staining of (a) control or (b) bcl-XL– injected representative dissected neural tubes is shown at st 26. (c) Comparison of the percentage of TUNEL-positive embryos (defined as having more than 30 apoptotic nuclei) at st 20 and 26 in control embryos (ct) and in embryos injected in the two dorsal blastomeres at the four-cell stage with bcl-XL cRNA (inj). Each experimental batch (n ≥ 60) was assessed independently. (d and e) bcl-XL cRNA was injected into one dorsal blastomere at the four-cell stage. (d) TUNEL staining of a representative bcl-XL–injected dissected neural tube is shown at st 26 (dorsal view, anterior to the left). (e) Average numbers of TUNEL-positive cells on the control or injected sides at st 26 of bcl-XL–injected anterior neural tubes (forebrain to midbrain; n = 14, bcl-XL–injected vs. ct, P ≤ 0.002).
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Fig. S3. MO against Casp3 protect Xenopus embryos from barhl2-induced apoptosis. Two MOs, MOcasp3I and MOcasp3II, were designed to inhibit endog- enous Casp3 activity together with a control MO, MOcasp3ct. We used three different means to evaluate the capacity of the Casp3 MO to specifically block endogenous Casp3 activity. (A) MOcasp3I and MOcasp3II bind their complementary sequences and inhibit translation in embryos. The 5′ end of the casp3 gene containing the MOcasp3I and MOcasp3II sequences was subcloned in-frame upstream of the EGFP cDNA. The 5′-gtttggatcagatcgggttttggtagccaagATGgaa- gaatcccagaatggt-3′ sequence was subcloned in the ClaI site of the vector pCS2-EGFP to generate the MO-EGFP expression vector. Western blotting analysis showed that translation of this construct containing the 5′ end of the casp3 gene fused upstream of the EGFP gene was completely inhibited by coinjection with each specific MO, whereas coinjection with the control MO had no effect. (A) Western blot analysis against EGFP with 3-μg protein extracts from Xenopus st 13 embryos. Translation of the MO-EGFP fusion protein (lane 1) is inhibited by coinjection with MOcasp3I (lane 2) or MOcasp3II (lane 3), whereas MOcasp3ct has no effect (lane 4). α-actin (42 kDa) was used as a loading control. (B and C) MOcasp3I and MOcasp3II protect Xenopus embryos from apoptosis induced by Xbarhl2 overexpression. The protective effect of MOcasp3I and MOcasp3II against Barhl2-induced apoptosis was shown by ELISA to detect cytoplasmic histone- associated mono- and oligo-nucleosomes, which are specifically released during apoptosis (B) or by Western blotting (C). (B) Kinetics of Barhl2-induced ap- optosis between st 8 and st 14 in the presence or absence of MOcasp3. Embryos were injected in one dorsal blastomere at the two-cell stage with EGFP, barhl2 cRNA together with EGFP, barhl2 cRNA together with MOcasp3I, MOcasp3II, or MOcasp3ct and collected at the indicated developmental stages. Cell death was measured by an ELISA for cytoplasmic histone-associated mono- and oligo-nucleosomes that are specifically released during apoptosis. The apoptosis en- richment factor (EF) was calculated using GFP-injected embryos as a control. From st 10.5 on, a steady increase in the apoptosis EF can be observed in barhl2- injected embryos (EF = 8 at st 14). Coinjection of barhl2 cRNA with MOcasp3I or MOcasp3II induces a significant delay in the onset of apoptosis (EF = 4 at st 14), whereas embryos injected with barhl2 cRNA together with MOcasp3ct behave like embryos injected with barhl2 cRNA alone (EF = 7.8 at st 14). (C) Western blot analysis of 15 μg protein extracted from the same batches of embryos at st 13: lane 1, barhl2 (50 pg) and EGFP (50 pg); lane 2, barhl2 (50 pg) and MOcasp3I; lane 3, barhl2 (50 pg) and MOcasp3II; lane 4, barhl2 (50 pg) and MOcasp3ct. A nonspecific band was used as a loading control.
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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.
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ig. S6. otx2, Barhl2, and casp3 transcripts are present in anteriorized ACs. RT-PCR analysis. casp3 and barhl2 transcripts are present in st 20 anteriorized ACs prepared in different conditions. Otx2 is used as an anterior neuroectodermal marker, Histone H4 is a positive control, and water is a negative control.
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Fig. S7. A model for the genetic pathway for the regulation of β-catenin activity by Barhl2 and Casp3
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