Molecular and cellular analysis of skeletal muscle and neuronal development in a necdin-null mouse model of Prader-Willi syndrome

Abstract

Prader-Willi syndrome (PWS) is a recurrent microdeletion syndrome characterized by severe obesity, hyperphagia, hypotonia, and developmental delay, and is caused by the loss of expression of four protein-coding genes and set of small nucleolar RNAs on chromosome 15. NDN, encoding the protein necdin, is one of these genes, and a large body of literature supports the theory that necdin is important for the differentiation and survival of neurons. Given that necdin is also abundant in developing muscle and that hypotonia is a cardinal feature of PWS, I hypothesize that necdin promotes normal skeletal muscle development. I provide two lines of evidence demonstrating that loss of necdin impairs muscle development in mice. First, necdin interacts with the inhibitor of muscle differentiation EID-1 to relieve inhibition of MyoD-dependent transcription by sequestering this protein in the cytoplasm in over-expression assays. Unexpectedly, the presence of necdin increases EID-1 protein abundance in transfected cells and endogenous EID-1 is less abundant in Ndn-null embryonic mouse tissue compared to controls. Finally, conversion from MyoD+ to Myosin Heavy Chain+ cells is impaired in limb bud cultures from Ndn-null embryos, consistent with the hypothesis that loss of necdin impairs muscle differentiation by failing to relieve EID-1-dependent transcriptional inhibition. Second, loss of necdin impairs polarization of muscle progenitors in vitro and in vivo due to failed activation of the actin-myosin cytoskeleton, and reduces the proportional area of forelimb extensor muscles in Ndn-null mice at birth. This conclusion is supported by defective centrosome re-orientation due to impaired nuclear rearward movement and failed Cdc42 activation in Ndn-null mouse embryonic fibroblasts (MEFs), impaired myosin activation in Ndn-null MEFs and cortical neurons, and excessive branching and failure of hippocampal neurons to polarize with respect to a growth factor. Additionally, PWS patient fibroblasts display centrosome re-orientation defects and impaired myosin activation identical to Ndn-null MEFs, indicating that loss of necdin produces a similar phenotype in both mice and humans. These results provide strong evidence that necdin is critical for both the migration and primary differentiation of skeletal muscle, and validates the Ndn-null mouse as a model for hypotonia in PWS.