Transcriptome Profiling of Spinal Muscular Atrophy Motor Neurons Derived from Mouse Embryonic Stem Cells Miho Maeda 1,4 , Ashlee W. Harris 1 , Brewster F. Kingham 5 , Casey J. Lumpkin 1,4 , Lynn M. Opdenaker 6 , Suzanne M. McCahan 2,3,7 , Wenlan Wang 1,2,4{ , Matthew E. R. Butchbach 1,2,4,7 * 1 Center for Applied Clinical Genomics, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, Delaware, United States of America, 2 Center for Pediatric Research, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, Delaware, United States of America, 3 Bioinformatics Core Facility, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, Delaware, United States of America, 4 Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America, 5 Sequencing and Genotyping Center, University of Delaware, Newark, Delaware, United States of America, 6 Center for Translational Cancer Research, University of Delaware, Newark, Delaware, United States of America, 7 Department of Pediatrics, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America Abstract Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2 +/+ ;mSmn 2/2 ) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs. Citation: Maeda M, Harris AW, Kingham BF, Lumpkin CJ, Opdenaker LM, et al. (2014) Transcriptome Profiling of Spinal Muscular Atrophy Motor Neurons Derived from Mouse Embryonic Stem Cells. PLoS ONE 9(9): e106818. doi:10.1371/journal.pone.0106818 Editor: Christoph Winkler, National University of Singapore, Singapore Received April 29, 2014; Accepted August 1, 2014; Published September 5, 2014 Copyright: ß 2014 Maeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The raw sequence data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE56284. Funding: This project was supported by an Institutional Development Award (IDeA) Networks of Biomedical Research Excellence (INBRE; grant number P20GM103446 to W.W. and M.E.R.B.) and Centers of Biomedical Research Excellence (COBRE; grant number P20GM103464 to M.E.R.B., S.M.M. and W.W.) programs of the National Institute of General Medical Sciences of the National Institutes of Health as well as the Nemours Foundation (M.E.R.B. and W.W.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: butchbach@nemoursresearch.org { Deceased. Introduction Spinal muscular atrophy (SMA) is an autosomal recessive, early- onset neurodegenerative disorder characterized by the degenera- tion of a-motor neurons (MNs) in the anterior horn of the spinal cord which leads to progressive muscle weakness and atrophy [1]. SMA is a leading genetic cause of infant death worldwide with 1 in 5000–10,000 children born with the disease [2,3] and a carrier frequency of 1:25–50 [4–7]. SMA results from the loss or mutation of the SMN1 (survival motor neuron 1) gene on chromosome 5q13 [8]. There is an inverted duplication of SMN1 in humans called SMN2 [9,10]. The duplication of SMN1 only occurs in humans. Within SMN2, there is a C-to-T transition in an exonic splice enhancer of exon 7 that results in the vast majority (about 80–90%) of SMN2 mRNAs to lack exon 7 (SMND7). SMND7 is not fully functional and prone to degradation [11,12]. SMN2 can, however, provide some fully functional SMN protein. The copy number of SMN2 modifies the severity of SMA phenotype in humans [13–20]. Although the genetics underlying SMA are known, the mechanisms leading to the disease are poorly understood. SMN is a ubiquitously expressed protein that facilitates the assembly of ribonucleoproteins (RNPs) [21]. The assembly of U- type small nuclear RNPs (snRNPs) is developmentally regulated in many cell types [22]. Knockdown of snRNP assembly in zebrafish results in degeneration of motor neuron axons [23]. snRNP assembly is defective in SMN-deficient SMA cells [24]. However, this function of SMN in U-type snRNP assembly is required by all PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e106818