Necdin Protects Motoneurons from Death Figure 3. Increased apoptotic motoneuron cell death in the lumbar regions of mutant embryos compared to wildtypes. A wave of motoneuron developmental cell death is known to occur normally in the lumbar region between the embryonic stages E11.5 and E14.5. (A) Illustration of TUNEL and activated caspase-3 labeling on whole mount spinal cord, focused on the lumbar region at E13.5. (B) Quantification of TUNEL positive cells in the lumbar whole mount spinal cord reveals a significant increase of TUNEL positive cells in mutant (Necdin+/2)(n = 5) compared to wildtype (Necdin+/+) embryos (n = 5). (C) Quantification of activated caspase-3 positive cells on serial transverse sections of the lumbar spinal cord at E13.5 show a significant increase of activated-Casapse-3 positive cells/section in mutant (Necdin+/2) mice (n = 5) compared with the wildtype (Necdin+/+) embryos (n = 5). (D) Quantification of activated caspase-3 positive cells at different levels (brachial, thoracic, lumbar and sacral regions) of the spinal cord was done on whole mount spinal cord immunolabeled with anti-cleaved caspase-3 antibody (n = 3). Data in (B) and (C) are plotted in box-and-whisker format. Values in (D) represent means 6 standard deviation (S.D). Scale bar, 100 mm, (*P,0.05 and **P,0.01). doi:10.1371/journal.pone.0023764.g003 selective Hb9 promoter (Hb9::GFP), we demonstrated that the TNFR1 positive cells were motoneurons (Figure S4A and Figure S5). It is noteworthy that we did not observe any TNFR1-positive cells in the lumbar region at E11.5, prior PCD (Figure S4B). This observation is consistent with a role of TNFR1 signaling in the death of lumbar motoneurons, which mainly take place between E12.5 and E13.5. Overall our observations suggest that Necdin might modulate the death promoting activity of TNFR1. Increase of motoneuron death in the absence of Necdin is linked to the TNFR1 pathway In order to investigate whether in the absence of Necdin TNFR1 signaling leads to an increase in motoneuron death, we used cultures of embryonic motoneurons isolated from E12.5 spinal cords. Consistent with the previous in vivo and ex vivo experiments, we found that in the absence of NTFs the percentage of surviving Necdin-deficient motoneurons was significantly lower than the percentage of surviving wildtype motoneurons cultured under the same conditions (Necdin+/+: 46.7 (44.4, 51), n = 3; Necdin+/2: 31.5 (21, 34.3), n = 3; P,0.05) (Figure 6A). We next measured the axonal growth in the presence of NTFs and found no difference between Necdin-deficient and wildtype motoneurons (Figure 6B,C). These data suggest that in the presence of neurotrophic support, Necdin-deficient and wildtype motoneurons are indistinguishable. Then, we checked the efficiency of TNFa at promoting cell death of mutant and wildtype motoneurons (Figure 7A). Motoneurons were cultured in presence of NTFs PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e23764