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Markers associated with neuron-specific Ube3a imprinting during neuronal differentiation of mouse embryonic stem cells

Cytotechnology volume 70, pages 45–53 (2018)Cite this article

Abstract

Understanding gene expression in the brain requires allele-specific transcriptome analysis because of the presence of neuron-specific imprinted genes, which are expressed in a neuron-specific and parent-of-origin-specific manner. Ube3a is a neuron-specific imprinted gene with an expression pattern that changes from biallelic to maternal only (Ube3a imprinting) during differentiation. Because Ube3a imprinting occurs only in neurons, it has the potential to be a marker to assess the quality of neurons produced by in vitro neuronal differentiation of embryonic stem cells (ESCs). For the analysis of Ube3a imprinting, genetic polymorphisms between the two alleles are necessary to identify the parental origin of each. However, ESCs derived from commonly used inbred mouse strains have no genetic polymorphisms. To overcome this problem, we examined 10 markers of neurogenesis to determine whether they were associated with Ube3a imprinting. We measured the relative expression levels of these 10 gene markers and assessed the Ube3a imprinting status of 54 neuron samples differentiated under various in vitro conditions. Then we divided the samples into two groups depending on their Ube3a imprinting status and selected markers statistically associated with Ube3a imprinting. The identified markers included the antisense noncoding transcript of Ube3a and a mature neuron marker Mtap2, consistent with the markers we used empirically in our previous study to assess the quality of differentiated neurons. These findings provide new quality control criteria for differentiated neurons, and could also be applied to human ESCs and induced pluripotent stem cells.

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References

  • Albrecht U, Sutcliffe JS, Cattanach BM, Beechey CV, Armstrong D, Eichele G, Beaudet AL (1997) Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nat Genet 17:75–78. doi:10.1038/ng0997-75

    CAS  Article  Google Scholar 

  • Araki K, Takeda N, Yoshiki A, Obata Y, Nakagata N, Shiroishi T, Moriwaki K, Yamamura K (2009) Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain. Mamm Genome 20:14–20. doi:10.1007/s00335-008-9160-7

    CAS  Article  Google Scholar 

  • Bouhon IA, Kato H, Chandran S, Allen ND (2005) Neural differentiation of mouse embryonic stem cells in chemically defined medium. Brain Res Bull 68:62–75. doi:10.1016/j.brainresbull.2005.08.022

    CAS  Article  Google Scholar 

  • Bouhon IA, Joannides A, Kato H, Chandran S, Allen ND (2006) Embryonic stem cell-derived neural progenitors display temporal restriction to neural patterning. Stem Cells 24:1908–1913. doi:10.1634/stemcells.2006-0031

    CAS  Article  Google Scholar 

  • Chamberlain SJ, Brannan CI (2001) The Prader–Willi syndrome imprinting center activates the paternally expressed murine Ube3a antisense transcript but represses paternal Ube3a. Genomics 73:316–322. doi:10.1006/geno.2001.6543

    CAS  Article  Google Scholar 

  • Eitoku M, Suganuma N, Kiyosawa H (2016) Comparison of two types of non-adherent plate for neuronal differentiation of mouse embryonic stem cells. Cytotechnology. doi:10.1007/s10616-016-9968-z

    Google Scholar 

  • Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet MC, Friocourt G, McDonnell N, Reiner O, Kahn A, McConnell SK, Berwald-Netter Y, Denoulet P, Chelly J (1999) Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 23:247–256.

    CAS  Article  Google Scholar 

  • Ishikawa E, Kosako H, Yasuda T, Ohmuraya M, Araki K, Kurosaki T, Saito T, Yamasaki S (2016) Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1. Nat Commun 7:12756

    CAS  Article  Google Scholar 

  • Johansson BM, Wiles MV (1995) Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol Cell Biol 15:141–151

    CAS  Article  Google Scholar 

  • Kato H, Hiraki-Kamon K, Eitoku M, Kiyosawa H, Neeley CYO (2015) Exploring standards for industrializing human iPS cells. Pharm Bioprocess 3:199–213. doi:10.4155/pbp.15.9

    CAS  Article  Google Scholar 

  • Kim KK, Adelstein RS, Kawamoto S (2009) Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J Biol Chem 284:31052–31061. doi:10.1074/jbc.M109.052969

    CAS  Article  Google Scholar 

  • Kohama C, Kato H, Numata K, Hirose M, Takemasa T, Ogura A, Kiyosawa H (2012) ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner. Hum Mol Genet 21:1391–1401. doi:10.1093/hmg/ddr577

    CAS  Article  Google Scholar 

  • Landers M, Bancescu DL, Le Meur E, Rougeulle C, Glatt-Deeley H, Brannan C, Muscatelli F, Lalande M (2004) Regulation of the large (approximately 1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn. Nucleic Acids Res 32:3480–3492. doi:10.1093/nar/gkh670

    CAS  Article  Google Scholar 

  • Le Meur E, Watrin F, Landers M, Sturny R, Lalande M, Muscatelli F (2005) Dynamic developmental regulation of the large non-coding RNA associated with the mouse 7C imprinted chromosomal region. Dev Biol 286:587–600. doi:10.1016/j.ydbio.2005.07.030

    Article  Google Scholar 

  • Lee JE (1997) Basic helix-loop-helix genes in neural development. Curr Opin Neurobiol 7:13–20

    Article  Google Scholar 

  • Lee MK, Tuttle JB, Rebhun LI, Cleveland DW, Frankfurter A (1990) The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. Cell Motil Cytoskelet. 17:118–132. doi:10.1002/cm.970170207

    CAS  Article  Google Scholar 

  • Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201–211

    CAS  Google Scholar 

  • Numata K, Kohama C, Abe K, Kiyosawa H (2011) Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription. Nucleic Acids Res 39:2649–2657. doi:10.1093/nar/gkq1201

    CAS  Article  Google Scholar 

  • Rougeulle C, Glatt H, Lalande M (1997) The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain. Nat Genet 17:14–15. doi:10.1038/ng0997-14

    CAS  Article  Google Scholar 

  • Rougeulle C, Cardoso C, Fontes M, Colleaux L, Lalande M (1998) An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nat Genet 19:15–16. doi:10.1038/ng0598-15

    CAS  Article  Google Scholar 

  • Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, Kono T, Shioda T, Hochedlinger K (2010) Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465:175–181. doi:10.1038/nature09017

    CAS  Article  Google Scholar 

  • Vu TH, Hoffman AR (1997) Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain. Nat Genet 17:12–13. doi:10.1038/ng0997-12

    CAS  Article  Google Scholar 

  • Walker TL, Yasuda T, Adams DJ, Bartlett PF (2007) The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells. J Neurosci 27:3734–3742. doi:10.1523/jneurosci.5060-06.2007

    CAS  Article  Google Scholar 

  • Yamasaki K, Joh K, Ohta T, Masuzaki H, Ishimaru T, Mukai T, Niikawa N, Ogawa M, Wagstaff J, Kishino T (2003) Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a. Hum Mol Genet 12:837–847

    CAS  Article  Google Scholar 

  • Yu A, Geng H, Zhou X (2006) Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR. BMC Genom 7:143. doi:10.1186/1471-2164-7-143

    Article  Google Scholar 

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Acknowledgements

This work was financially supported by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (KAKENHI 24115703 and 26290063) and a JSPS Grant-in-Aid for Young Scientists (KAKENHI 16K19273). The authors thank Dr. K. Araki for embryonic stem cell lines and Ms. H. L. Le, M. Okabayashi, and Mr. H. Makino for technical assistance.

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Author notes

  1. Hidenori Kiyosawa

    Present address: Department of Life Science, Chiba Institute of Technology, Tsudanuma, Narashino, Chiba, 275-0016, Japan

Affiliations

  1. Department of Environmental Medicine, Kochi Medical School, Kochi University, Oko-Cho Kohasu Nankoku, Kochi, 783-8505, Japan

    Masamitsu Eitoku, Narufumi Suganuma & Hidenori Kiyosawa

  2. Division of Functional Histology, Department of Functional Biomedicine, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, 791-0295, Japan

    Hidemasa Kato

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  1. Masamitsu Eitoku
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  2. Hidemasa Kato
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  3. Narufumi Suganuma
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  4. Hidenori Kiyosawa
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Correspondence to Hidenori Kiyosawa.

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Eitoku, M., Kato, H., Suganuma, N. et al. Markers associated with neuron-specific Ube3a imprinting during neuronal differentiation of mouse embryonic stem cells. Cytotechnology 70, 45–53 (2018). https://doi.org/10.1007/s10616-017-0126-z

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  • DOI: https://doi.org/10.1007/s10616-017-0126-z

Keywords

  • Ube3a imprinting
  • Embryonic stem cell
  • In vitro neuronal differentiation
  • Mus musculus molossinus
  • Mtap2
  • Noncoding RNA
  • SNP ratio quantification