Necdin Protects Motoneurons from Death Figure 1. Necdin is expressed in motoneurons during embryonic development and postnatal stage. (A) Sagital section of whole E12.5 mouse embryo immunostained with a Necdin specific antibody (in red). Section is stained with DAPI (in blue). sc, spinal cord; pt, pyramidal tract; cb, cerebellum; ot, optic tract and soa, supraoptic area. (B) Western blot analysis of Necdin levels in the ventral horn of the lumbar spinal cord at indicated stages. Actin served as a loading control. In absence of the paternal copy of the Necdin gene (the maternal copy is silent due to imprinting mechanism) or in homozygous animals deleted for both copies, Necdin expression is not detected. P, Post-natal day. (C) Immunohistochemical analysis of Necdin expression (in red) in the spinal cord at different stages. Transverse sections were counter-stained with DAPI (in blue). (D) Necdin expression (in green) is mainly restricted to motoneurons, as identified by Islet-1/-2 immunostaining (in red). Later (C), its expression is extended to other cells (E.13.5, E16.5 and P1). Scale bar: A: 500 mm and C, D: 100 mm. doi:10.1371/journal.pone.0023764.g001 brachial, thoracic, lumbar and sacral region [30], no significant difference was observed in the total number of cleaved caspase-3 positive cells in the brachial, thoracic or sacral regions of the spinal cord in wildtype embryos compared to mutant embryos (Figure 3D). In addition, we determined the number of lumbar motoneurons in 11-day-old Necdin-deficient and wildtype mice. We observed a significant difference of about 27% between Necdin+/+ and Necdin+/2 surviving motoneurons (Necdin+/+: 1492 (1387, 1558), n = 3; Necdin+/2: 1080 (1050, 1199), n = 3; P,0.05). This difference is similar to the one observed at E17.5. These results suggest that Necdin has a prosurvival function during early stages of lumbar motoneuron development. The increased motoneuron death in Necdin-deficient spinal cord explants does not depend on neurotrophic support Our next goal was to investigate whether Necdin acts by ‘‘potentiating’’ the action of trophic factors (first hypothesis) or by interfering with extrinsic signals (second hypothesis), mediated by PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e23764