Arts-1, NucB2 and Necdin in cultured E12.5 embryonic motoneurons (Figure S5 and Text S1, Supporting materials and methods). Because of the limited quantity of material obtained from primary culture of motoneurons, we were not able to perform immunoprecipitation assays with the different partners. In order to reveal in those motoneurons a functional role of the Arts-1-NucB2 complex in extracellular TNFR1 release, we then performed a proximity ligation assay (PLA) using the Duolink system to visualize potential physical interactions (10–30 nm) between TNFR1 and Arts-1, TNFR1 and NucB2 and TNFR1 and Necdin [47,48] (Figure S5D–I). Necdin-NucB2 and NucB2-Arts1 pairs were not tested due to the overlapping species specificities of the available antibodies. Analysis of PLA signal was performed using confocal imaging and by counting the number of dots. We detected a high number of PLA signals for the TNFR1-Arts-1 pair, comparable with the positive control (Figure S5D,G,I). A lower number of dots was detected for the TNFR1-NucB2 pair (Figure S5E,I) and only few dots were counted for the TNFR1-Necdin pair (Figure S5F,I). These data suggest a physical interaction between TNFR1 and Arts1 and potentially TNFR1 and NucB2. These preliminary data suggest that the TNFR1/Arts-1/NucB2 complex might also exist in developing motoneurons, but we cannot demonstrate that Necdin interacts with NucB2 since appropriate antibodies for such an experiment are not available. In our hypothesis, Necdin would influence the release of TNFR1 into the extracellular space and/or the quantity of TNFR1 at the cell surface in motoneurons. Necdin and the motor deficit in Prader-Willi syndrome PWS results from a loss of paternal expression of several contiguous imprinted genes located in the 15q11-q12 region. Recent studies support a major role of the SNORD116 locus in the PWS phenotype, with three patients showing an overlapping microdeletion [49,50,51], encompassing the SNORD116 genes. However, the question about the mechanism(s) by which these deletions, including the SNORD116 locus, cause the physiopathol- ogy observed in these patients remains elusive. Necdin-deficient mice present defects reminiscent of specific PW symptoms and interestingly, at the cellular level, a same defect of polarization of the cytoskeleton is described in fibroblasts of PW patients and in mouse embryonic fibroblasts of Necdin deficient mice [25]. This observation reinforces the idea that the lack of Necdin is indeed responsible for some PW symptoms. It would be interesting to evaluate whether levels of Necdin in patient’s tissues are altered by SNORD116. The causes of the severe hypotonia, in early infancy, and of motor deficiency in later life, observed in PWS [52], are unresolved but relate to impairments in the nervous and muscular system and body composition. Our results reveal an important role of Necdin during motoneuron development, strongly suggesting that the lack of Necdin is involved in motor deficiency in PWS. The expression of NECDIN in human embryonic spinal cord is in accordance with this hypothesis [2]. Since the TNFa-TNFR1 pathway is involved in injury-induced apoptotic death of adult motoneurons, our study raises the question on the role of Necdin in motoneuron death following nerve injury or in neurodegener- ative diseases. Interestingly, in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) expressing the G93A mutation of superoxide dismutase-1, a modulation of Necdin expression has been documented. Necdin was found to be upregulated at the presymptomatic stage and downregulated at the end stage, suggesting that Necdin might contribute to the neurodegenerative process in ALS [53]. Necdin Protects Motoneurons from Death Materials and Methods Ethics statement All breeding and experiments were carried out in accordance with the European and National guidelines for the care and use of laboratory animals (Council Directive 86/6009/EEC). Breeding and genotyping of Necdin-deficient mice Necdin-deficient mice were generated as previously described [6]. We used a mouse colony generated on the C57BL/6J background (more than 10 backcrosses). Because Necdin is an imprinted gene, only paternally expressed, we crossed heterozy- gote 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+/2). The age of the embryos was determined at 9:00 am by the presence of a vaginal plug in the pregnant mothers and considered as embryonic day 0.5 (E0.5). For motoneuron cultures, embryos were kept in Hibernate E (Invitrogen, Carlsbad, CA, USA) at +4uC during their genotyping. All embryos were genotyped by PCR with the following primer sets: Nec F, 59-TCTCATGCTTGAACTGCA- 39 and Nec B, 59- CAGGTAATTCTGCTGGAC-39; a 1503 bp and a 228 bp fragment were generated, which correspond to the wildtype or mutant allele respectively. PCR conditions were: 94uC 1 mn then 35 cycles: 94uC 20 sec, 56uC 20 sec, 72uC 45 sec. Immunohistochemistry Embryos or explants were collected, fixed and sectioned using a cryostat (10 or 12 mm thick) as previously described [27]. Sections were blocked for 1 hour at room temperature in phosphate buffered saline (PBS) containing 10% heat-inactivated goat or donkey serum, 0.5% Triton X-100 and incubated overnight at +4uC with primary antibodies diluted in PBS containing 10% heat-inactivated goat serum and 0.1% Triton X-100. The primary antibodies were: rabbit polyclonal anti-Necdin (07-565; Millipore, Bedford, MA, USA; 1:500), mouse monoclonal anti-Islet-1 and Islet-2 (2D6 and 4D5; Developmental studies Hybridoma Bank of Iowa University; 1:500 and 1:100 respectively), rabbit polyclonal anti-cleaved-caspase-3 (9661, Cell Signaling Technology, Beverly, MA, USA; 1:500), rabbit polyclonal anti-Unc5 (ab39654, Abcam, 1:500), mouse monoclonal anti-TNFR1 (sc-8436, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500). Sections were washed 3 times in PBS and incubated for 1 hr at room temperature with secondary antibodies diluted in 5% Goat serum, 1% BSA, 0.3% Triton X-100 in PBS. Biotinylated secondary antibodies and the ABC complex from the Vectastain kit (Vector Laboratories, Burlingame, CA, USA), were used for detection. Alternatively, fluorochrome-conjugated secondary antibodies were used (Jackson Immunoresearch laboratories, West Grove, PA, USA). Sections were examined on a Zeiss Axioplan 2 microscope with an Apotome module. TUNEL assay To identify cells undergoing apoptosis, the terminal deoxynu- cleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) technique was used. Whole-mount TUNEL was performed essentially as previously described [30]. Spinal cords were fixed, dehydrated, and rehydrated through graded ethanol concentrations into PBS and then stained with the ApopTag kit (Oncor Gaithersburg, MD, USA). Spinal cords were incubated in ApopTag equilibration buffer for 5 min at room temperature and transferred to the working strength TdT enzyme solution for 12 hr at 4uC, followed by 2 hr at 37uC. The reaction was stopped by incubating the spinal cords in ApopTag stop PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e23764