Article Figures & Data
Figures
- Figure 1.
Generation and characterization of FADD-DN mice. A, Scheme of the FADD-DN construct: the DED of human (h) FADD was deleted and replaced by an N-terminal AU-1 tag, whereas the death domain (DD) was kept. The gene is under the control of a human β-actin promoter. B, C, Expression of FADD-DN in adult (B) and E11 (C) tissues was shown by Western blot analysis, using a mouse monoclonal anti-human FADD antibody. In B and C, a positive control (+) corresponding to thymus from lck promoter-FADD-DN transgenic mouse is shown. Expression under theβ-actin promoter is surprisingly restricted, primarily to the heart and the nervous system. D, Expression of FADD-DN and endogenous FADD in E12.5 spinal cord shown by Western blot analysis using a rabbit polyclonal anti-FADD antibody recognizing both human and murine forms. WT, Wild-type.
- Figure 2.
Survival properties of mutant Fas mice motoneurons in vitro. Trophic deprivation-induced death of cultured motoneurons is delayed but not abolished by mutations affecting Fas. lpr/lprmice(A),Fas-/-mice(B), and FADD-DNmice(C)are shown. The percentage of motoneuron survival was calculated as a ratio between the number of cell scultured in the absence versus presence of NTFs (DIV). The number of living motoneurons at 12 hr was defined as 100%. (asterisk indicates Student'sttest,p<0.05). Motoneurons from mutant Fas mice are resistant to exogenous activation and combined treatment with anti-Fas antibody, and TNFα shows no additive effect on motoneuron death induction. lpr/lpr (D), Fas-/- (E), FADD-DN (F, G), and wild-type (H) mice are shown. Treatment with anti-Fas antibody (D–F) and TNFα (G, H) was performed 16 hr after cell plating in the continued presence of NTFs, and surviving motoneurons were counted 2d after. Counts are expressed as a percentage of the surviving motoneurons with NTFs alone.
- Figure 3.
The Fas death pathway is activated after early postnatal (P3) facial nerve transection. A, Facial motoneurons are sensitive to Fas-induced death when isolated from E13.5 wild-type mice and are resistant when isolated from Fas mutant mice. Facial nuclei were localized and dissected after a protocol developed by Jean Livet, Vilma Arce, and Odile Delapeyrière (personal communication). Motoneurons were purified, cultured, treated, and counted as in Figure 2. B–G, Facial motoneurons survive better from axotomy in Fas mutant mice. Motoneurons were counted in cresyl violet-stained sections throughout the facial nucleus of P10 mice whether axotomized or not at P3 (mean ± SEM; *p < 0.001; n = 5 for each genotype). D–G, Cresyl violet staining of comparable serial sections from contralateral (contra) and axotomized (axo) facial nuclei of lpr/lpr (D, E) and C57BL/6 control (F, G) mice 1 week after lesion (P10). B, G, Total number of facial motoneurons (B) and rescue index (G) defined as the ratio between the number of motoneurons in the axotomized nucleus and the number of motoneurons in the contralateral one of the same animal inlpr/lprcompared with C57BL/6 control mice, and in FADD-DN mice compared with wild-type littermates (mean± SEM; *p < 0.001; n = 5 for each genotype). Dashed lines in D–G indicate the border of the facial nucleus. Scale bar: (in G) D–G, 100 μm.
Tables
Mouse genotype TUNEL+ profiles (E12.5 thoracic spinal cord) C57BL/6 598 ± 33 (n = 3) lpr/lpr 616 ± 26 (n = 3) Fas+/- 602 ± 64 (n = 3) Fas-/- 594 ± 52 (n = 3) Number of TUNEL + motoneurons in the thoracic part of E12.5 spinal cords of lpr/lpr and Fas-/- mice with their respective controls. Values correspond to mean ± SD.
