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Fig. 1.
Caspase-9 mRNA and activated caspase-9 increase in metamorphic tadpole brains. (A) Activation of C9 was monitored by Western blotting on brain extracts of tadpoles at different stages of metamorphosis, by using an active C9 specific antibody. The 34-kDa band, corresponding to conversion of pro-C9 to active C9, as predicted from cleavage sites in the procaspase sequences (17), is seen from stage NF55 (onset of metamorphosis) up to stage NF63 (end of metamorphosis), peaking at stage NF60 (climax of metamorphosis). The immunoblot was repeated by using independent samples. Equal amounts of proteins were loaded per lane and subsequently confirmed by Ponceau-S staining (P). (B–G) Spatial distribution of C9 mRNA in Xenopus brains at stage NF63 with ISH, by using an antisense digoxigenin-labeled probe on coronal sections (B, D, and F). Strong localized expression of C9 mRNA is seen in the ventricular zone of the anterior (B), medial (D), and posterior (F) brain. Control adjacent sections were hybridized with a sense probe (C, E, and G), showing no labeling. ISH was done twice on independent groups of tadpoles showing identical results. VL, 3V, and 4V: lateral, third, and fourth ventricles. (Scale bars: B–G, 50 μm.) (H) Quantitative-PCR analysis of C9 and XR11 expression after T3-treatment of perchlorated tadpoles. Fold inductions were normalized against untreated tadpoles. Statistical analysis for PCR data were done comparing the means of δCT values ± SD, by using Student's t test (n ≥ 3): Differences were extremely significant for C9 (P < 0.001) and significant for XR11 (P < 0.05).
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Fig. 2.
Cell proliferation increases at metamorphic climax in brains of transgenics overexpressing DN9 and/or XR11. Cell proliferation was followed in Nβt-driven DN9-RFP (DN9xDN9), XR11-GFP (XR11xXR11), and double-transgenic XR11-GFP/DN9-RFP (XR11xDN9) tadpoles by using a PH3-antibody and compared with WT or Nβt-driven RFP and GFP transgenic controls at different stages of metamorphosis. (A) DAPI- labeled sagittal section of a brain showing the three areas analyzed: VL, 3V, and CB. Proliferating cells were counted in the cell layers bordering ventricles for each area. (B) PH3-positive cells were counted at stage NF55 (onset of metamorphosis), around the VL in the anterior brain (VL NF55), the 3V (3V NF55), and the CB (CB NF55). Note that mitosis was enhanced only in VL and CB for XR11xDN9, but overall proliferation at this stage did not differ between transgenics and WT. (C) PH3-positive cells were counted at stage NF61 (climax of metamorphosis), in the same areas as for stage NF55. Note that mitosis was strongly enhanced in all areas for all transgenics compared with WT. (D) PH3-positive cells were counted at stage NF66 (end of metamorphosis), in the same areas as B and C. Note that mitotic numbers only show a slight, but significant, increase in VL for XR11xDN9 and in CB for DN9xDN9 and XR11xDN9. Means ± SEM are given; n > 6 in each group; not significant (ns), P < 0.5; not quite significant (nqs), P < 0.1; â, P < 0.05; ââ, P < 0.01; âââ, P < 0.001. RPr, recessus preopticus; RI, recessus infundibuli; OT, optic tectum.
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Fig. 3.
DN9 expression increases active cell proliferation in the ventricular zone of transfected brain. SGT in brain was carried out at stage NF57 by using CMV-driven DN9-GFP or GFP constructs, and proliferation was compared between DN9 and controls at 3 and 15 days after transfection by using the PH3-antibody. (A–F) Mitotic cells were observed on para-sagittal sections at 15 days after transfection for control (B and magnification, C) and compared with DN9 (E and magnification, F), showing more proliferative cells in DN9-transfected brain. DAPI counterstaining shows the same anterior brain region compared with control (A) and DN9 transfections (D). Note at higher magnification the greater concentration of positive cells for DN9 (arrows) lining the ventricle. (G) PH3-positive cells were counted for the VL in the anterior brain, showing a strong increase in mitosis in brains transfected with DN9 vs. controls. Each experiment was repeated twice, giving similar results. Means ± SEM are given; n > 6 in each group; ∗∗∗, P < 0.001. (Scale bars: A-F, 100 μm.)
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Fig. 4.
Overexpression of DN9 and/or XR11 during metamorphosis modifies brain morphology in transgenic animals. (AâH) Representative brains seen in dorsal (AâD) or lateral (EâH) views are shown for WT (A and E), XR11xXR11 (B and F), DN9xDN9 (C and G), and XR11xDN9 (D and H) tadpoles. Note that commissures at the junctions between olfactory bulb or medial brain with the anterior brain (compare arrows) are more marked in control than in transgenics. Note also the larger CB in transgenics vs. WT (compare arrowheads). (I) Schematic representation of the forebrain of WT and each transgenic seen in E, F, G, and H, respectively (lateral views). The form of the forebrain shows a progressive compaction, XR11xXR11 and DN9xDN9 corresponding to an intermediate state and XR11xDN9 to the most marked compaction. (J) Different measurements were done for transgenic and WT brains, width of the forebrain (H1), width of the midbrain (H2 and H4), and width of the optic tectum (H3), and ratios were calculated with respect to brain length from the rostral tip to rear of optic tectum (L). All ratios H/L given for each measure were significantly increased in all transgenics in comparison with WT. Means ± SEM are given; n > 6 in each group; â, P < 0.05; ââ, P < 0.01; âââ, P < 0.001.
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Fig. 5. Correlation between caspase-9 expression, apoptosis and proliferation at the onset and the end of brain metamorphosis. Spatial distribution of C9 mRNA by in situ hybridization (ISH C9), using an antisense digoxigenin-labeled probe on coronal sections, was correlated with distribution of cell death (TUNEL-apoptotic cells) and cell proliferation (PH3-mitotic cells), throughout brains of stage NF57 Xenopus tadpoles (A: onset of metamorphosis) and at stage NF63 (B: end of metamorphosis). DAPI labeling annotated with stereotaxic nomenclature is given for each section from the anterior part of the brain (Upper) to the posterior part of the brain (Lower). Note that at stage NF63 there is a strong localized expression of C9 mRNA in ventricular areas, but also a widespread less-intense expression throughout most of the brain. A similar overall but less intense C9 mRNA brain distribution is found at stage NF57, whereas C9 mRNA expression in the ventricular areas at this stage is much less than at stage NF63. Localization of the majority of TUNEL-positive cells generally maps with the C9 distribution at the two stages observed, whereas proliferation is mainly associated with the ventricular zones. Interestingly, at stage NF57, proliferation is marked in ventricular areas when C9 expression is weak, whereas at stage NF63, proliferation decreases when C9 is strongly expressed in the ventricular areas. (Scale bars, 200 mm for all images, except boxes with magnification.) Sol, solitary tract; Ols, superior olivary; VI, abducens nucleus; VII, motor facial nucleus; VIII, vestibulocochlear nucleus; Te, tegmental nucleus; Pb, parabrachial nucleus; Cb, cerebellum; Poa, anterior preoptic nucleus; MeA, medial amygdala; Ls and Ms, lateral and medial septum; Lp, Mp and Dp : lateral, medial and dorsal pallium; Str, striatum; Sp, subpallium; LV, 3V and 4V : lateral, third and fourth ventricles.
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Fig. 5. Correlation between caspase-9 expression, apoptosis and proliferation at the onset and the end of brain metamorphosis. Spatial distribution of C9 mRNA by in situ hybridization (ISH C9), using an antisense digoxigenin-labeled probe on coronal sections, was correlated with distribution of cell death (TUNEL-apoptotic cells) and cell proliferation (PH3-mitotic cells), throughout brains of stage NF57 Xenopus tadpoles (A: onset of metamorphosis) and at stage NF63 (B: end of metamorphosis). DAPI labeling annotated with stereotaxic nomenclature is given for each section from the anterior part of the brain (Upper) to the posterior part of the brain (Lower). Note that at stage NF63 there is a strong localized expression of C9 mRNA in ventricular areas, but also a widespread less-intense expression throughout most of the brain. A similar overall but less intense C9 mRNA brain distribution is found at stage NF57, whereas C9 mRNA expression in the ventricular areas at this stage is much less than at stage NF63. Localization of the majority of TUNEL-positive cells generally maps with the C9 distribution at the two stages observed, whereas proliferation is mainly associated with the ventricular zones. Interestingly, at stage NF57, proliferation is marked in ventricular areas when C9 expression is weak, whereas at stage NF63, proliferation decreases when C9 is strongly expressed in the ventricular areas. (Scale bars, 200 mm for all images, except boxes with magnification.) Sol, solitary tract; Ols, superior olivary; VI, abducens nucleus; VII, motor facial nucleus; VIII, vestibulocochlear nucleus; Te, tegmental nucleus; Pb, parabrachial nucleus; Cb, cerebellum; Poa, anterior preoptic nucleus; MeA, medial amygdala; Ls and Ms, lateral and medial septum; Lp, Mp and Dp : lateral, medial and dorsal pallium; Str, striatum; Sp, subpallium; LV, 3V and 4V : lateral, third and fourth ventricles.
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Fig. 6. Transgenic XR11xDN9 tadpoles show decreased neuronal apoptosis in brain. Apoptosis was followed by TUNEL staining at the climax of metamorphosis (stage NF61), in brains of wild type tadpoles (WT; A, B, D, and E) as compared to double transgenics overexpressing both the Bcl2-like protein XR11 and the C9 dominant negative DN9 (XR11xDN9; C and F). Labeling of apoptosis using a fluorescein-linked probe was observed on two different para-sagittal sections in the anterior brain (A and D), counterstained with DAPI to visualize the totality of the cells. Apoptotic labeling was strong in WT tadpoles, particularly in the ventral forebrain (B and E and magnification B' and E'), whereas only a few apoptotic cells (arrows) were observed in these same regions for double transgenics (C and F and magnification C' and F'). Each experiment was repeated twice on independent animals. BO, olfactory bulb; Sp, subpallium. (Scale bars, 100 mm in A-F').
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Fig. 7. Caspase-3 activity is decreased in the anterior brain of transgenic animals. Western blot analyses were compared, using a specific antibody against active caspase-3 (C3), on pooled extracts of either anterior or posterior brains of transgenic or WT tadpoles at the end of metamorphosis (stage NF66). Representative Western blots are showed for a WT animal (A), two different DN9xDN9 transgenics (B), and two different double transgenics XR11xDN9 (C), where C3 activity (C3), normalized against b-tubulin (b-tub), has been compared between the anterior (ant.) and the posterior (post.) part of the brain. For the control animals, no difference is seen between the posterior and the anterior part of the brain, whereas for the transgenic animals DN9xDN9 or XR11xDN9, there is less C3 activity in the anterior part of the brain compared to the posterior part. Note that the decrease in C3 activation for the transgenics can be strong (Left) or weak (Right). Immunoblots were repeated from n > 5 independent samples in each group.
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Fig. 8: Transfection with DN9 increases cell survival in brain. Somatic gene transfer (SGT) into the brain of stage NF57 tadpoles was carried out with the CMV-driven DN9-GFP or GFP constructs. Each plasmid was coinjected with the pcDNA3-LUC reporter plasmid, and cell survival was followed either by GFP fluorescence observation or by measuring luciferase activity in brain extracts at different time points. (A-D) In toto observation of GFP expression in brain at 3 days (A and C) and 2 weeks (B and D) posttransfection shows an increased number of cells that express the transgene at these two observed times for DN9 animals (C and D) compared with controls (A and B). (E) Luciferase expression was monitored at 24 h, 3 days, and 2 weeks posttransfection. At all time points, DN9 transfection resulted in a significant increase in luciferase expression compared with controls. Moreover, with respect to the relative ratios (DN9 vs. control) given at the three time points monitored, we showed that they increased 3-, 4.5-, and 8.5 -fold at 24 h, 3 days, and 2 weeks, respectively. Experiment was repeated twice, giving similar results. Means ± SEM are given; n = 10 in each group; ***P < 0.001. (Scale bar, 500 mm in A-D.)
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Fig. 9. Schema of comparative levels of caspase-9 activation and proliferation in DN9 transgenic and WT animals during metamorphosis. C9 activation (circles) is taken from Fig. 1. Cell proliferation levels in the lateral ventricle are taken from our mean VL values obtained at stages NF55, NF61, and NF66 for WT (triangles) and DN9xDN9 (squares) animals (Fig. 2). Note that cell proliferation in WT decreases at metamorphic climax, when C9 activity is high. Inversely, when C9 is inactivated by DN9, proliferation peaks at metamorphic climax. All values are given using arbitrary units, to permit comparison of their individual variations during metamorphosis.
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