deletion of Necdin leads to the loss of lumbar motoneurons. This loss is due to an increase death of motoneuron during the wave of naturally-occurring cell death, which is predominantly dependent on the accessibility to neurotrophic support. Interestingly, we show that Necdin-deficient motoneurons present an increased suscepti- bility to neurotrophic factor (NTF) deprivation, which involves the activation of the tumor necrosis factor alpha (TNFa)/TNF receptor (TNFR) signaling pathway. Overall, our findings strongly suggest a developmental cause of motor deficit observed in PW patients. Results Targeted deletion of Necdin in mice leads to hindlimb motor deficits Because Necdin is an imprinted gene, only paternally expressed, we crossed heterozygote males (Necdin2m/+p) with wildtype C57BL/6J females, in the generated litters, half the embryos were control (Necdin+/+) and half were functionally Necdin-deficient (Necdin+m/2p). Previous phenotypical studies revealed alterations of the sensory system in Necdin-deficient mice but also suggested a motor deficit [27]. Indeed, we observed that from birth, Necdin pups and young mutant (10-day-old) mice display obvious problems in moving and flexing their hindpaws (Figure S1), although this deficit cannot be quantified at this age. Although, this motor problem tends to diminish over time, we were able to confirm that adult Necdin- deficient mice have decreased motor performance in an accelerating rotarod test compared to age-matched wildtype mice (Figure S1 and Text S1, Supporting materials and methods). These results show that Necdin-deficient mice have a motor deficit starting at birth, suggesting a developmental problem. Necdin is expressed in post-mitotic motoneurons during development and at postnatal ages We previously described an expression of NECDIN gene in the large ventral horn neurons of the spinal cord from 10-week-old human embryo [2]. Then, in order to investigate the mechanisms underlying motor deficit observed in Necdin mutant mice, we first studied the expression pattern of Necdin in the spinal cord at different developmental and post-natal stages. Using Necdin specific antibodies, we observed Necdin expression along the rostro-caudal axis of the spinal cord at embryonic day 10.5 post- co¨ıtum (E10.5), which becomes obvious at E12.5 (Figure 1A, S2A). Necdin expression in the spinal cord is maintained throughout embryonic development and is present after birth (Figure 1B,C). Necdin is distributed in a homogeneous way in all motoneuron columns. With regard to the Necdin phenotype, we focused our attention on the lumbar region and showed that at E10.5 and E12.5 Necdin is exclusively expressed in the ventral horn of the spinal cord (Figure 1C,S2A). Furthermore, at these stages, Necdin- immunoreactive cells were identified as motoneurons as demon- strated by co-immunolabeling with Islet-1/-2 (Figure 1D) and Hb9 (Figure S2A). These double labeling experiments also confirmed that all the developing post-mitotic motoneurons express Necdin. From E13.5, Necdin expression was increased in the medio- ventral part of the spinal cord, and at E16.5 and P1, we observed an extension of this pattern to the central and dorsal region (Figure 1C). We confirmed using in situ hybridization that the expression of Necdin mRNA was consistent with the developmental expression profile of the protein (Figure S2B,C). Thus, Necdin appeared to be expressed first in developing post-mitotic motoneurons and progressively in others cell types. Necdin Protects Motoneurons from Death Loss of lumbar specified motoneurons in Necdin- deficient mice during embryonic development We next looked for cellular defects in motoneuron development in Necdin-deficient mice. We studied two embryonic stages: E11.5, when the pool of developing postmitotic motoneurons is established [28,29,30] and E17.5 at the end of the period of programmed cell death (PCD), when all embryonic motoneurons are specified. At E11.5, we observed a similar number of Islet-1/-2 positive neurons between wildtype and mutant embryos (Necdin+/+: 109 (106, 119), n = 4; Necdin+/2: 113 (108, 119), n = 4; non significant, n.s) (Figure 2A,B). In contrast we performed a quantitative analysis of the Islet-1/- 2 positive neurons in the ventral horns of E17.5 lumbar spinal cords, we found a significant reduction of 31% of Islet-1/-2 positive cells in Necdin-deficient compared to wildtype spinal cords (Necdin+/+: 16 (15, 18), n = 4; Necdin+/2: 11 (8, 12), n = 4; P,0.05) (Figure 2C,D). We showed that this reduction of Islet-1/-2 positive neurons was specific to motoneurons since the number of the medio-ventral sub-population of Islet-1/-2 positive interneurons, did not differ between Necdin-deficient and wildtype mice at E17.5 (Figure S3) (Necdin+/+: 29 (29, 30), n = 4; Necdin+/2: 28 (27, 30), n = 4; n.s). The loss of motoneurons observed in the lumbar region of Necdin-deficient mice at E17.5, but not at E11.5, stages suggests that the genetic deletion of Necdin does not influence the generation of post-mitotic motoneurons, but rather might act during the period of naturally-occurring cell death that takes place between E12.5 and E15.5 in lumbar spinal cord. Necdin deficiency leads to an increase of lumbar motoneuron death during the period of programmed cell death We then asked whether Necdin plays a role in the survival of motoneurons during the period of PCD. Towards this goal, we first compared the number of apoptotic cells, between wildtype and Necdin-deficient embryonic spinal cords using a longitudinal whole-mount terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling analysis (TUNEL) [30]. At E11.5, very few apoptotic nuclei were detected in wildtype and mutant mice. At E12.5, apoptotic nuclei were present in controls and Necdin mutants (data not shown). At E13.5, when a peak of normal motoneuron PCD occurs [30], a significant increase of about 50% of the total number of TUNEL-positive cells was observed in Necdin-deficient spinal cord (Necdin+/+: 229 (194, 251), n = 5; Necdin+/2: 484 (442, 505), n = 5; P,0.05) (Figure 3 A,B). At E15.5, when PCD is nearly complete [30], TUNEL staining was reduced to few cells in the wildtype as well as in mutant spinal cords (data not shown). Using double staining, we then confirmed that, in both wildtype and mutant spinal cords, TUNEL-positive nuclei were Islet-1/-2 positive. Interestingly Islet-1/-2 staining revealed that the TUNEL signal was not restricted to specific pools of motoneurons in both Necdin-deficient and wildtype embryos (data not shown). To confirm that Necdin deficiency leads to increased neuronal death during the period of PCD, we quantified the number of cleaved caspase-3 positive cells, at E13.5, in Necdin- deficient and wildtype lumbar spinal cords. Consistently, we observed a significant 41% increase of cleaved caspase-3 positive cells in Necdin-mutant mice compared to wildtype mice (Necdin+/+: 5.3 (5.1, 6.4), n = 5; Necdin+/2: 8.9 (8.8, 10.7), n = 5; P,0.05) (Figure 3A,C). Therefore, the loss of lumbar motoneurons observed in Necdin mutants at E17.5 could be attributed to an increase in cell death during the period of developmental cell death. Notably, at E13.5, when the peak of PCD occurs in the PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e23764