Cell Death and Differentiation - Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing

Original Paper
Cell Death and Differentiation (2011) 18, 383–395; doi:10.1038/cdd.2010.109; published online 24 September 2010

Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing
Edited by JC Marine

B Zimmer1, P B Kuegler1, B Baudis1, A Genewsky1, V Tanavde2, W Koh2, B Tan2, T Waldmann1, S Kadereit1 and M Leist1

1Doerenkamp-Zbinden Chair for In Vitro Toxicology and Biomedicine, University of Konstanz, Konstanz D-78457, Germany
2Bioinformatics Institute, 30 Biopolis Street, No. 07-01, Singapore 138671, Singapore
Correspondence: M Leist, Doerenkamp-Zbinden Chair for In Vitro Toxicology and Biomedicine, University of Konstanz, Box 657, Konstanz D-78457, Germany. Tel: +49 7531 885037; Fax: +49 7531 885039; E-mail: Marcel.Leist@uni-konstanz.de

Received 6 May 2010; Revised 14 July 2010; Accepted 27 July 2010; Published online 24 September 2010.


Abstract
As neuronal differentiation of embryonic stem cells (ESCs) recapitulates embryonic neurogenesis, disturbances of this process may model developmental neurotoxicity (DNT). To identify the relevant steps of in vitro neurodevelopment, we implemented a differentiation protocol yielding neurons with desired electrophysiological properties. Results from focussed transcriptional profiling suggested that detection of non-cytotoxic developmental disturbances triggered by toxicants such as retinoic acid (RA) or cyclopamine was possible. Therefore, a broad transcriptional profile of the 20-day differentiation process was obtained. Cluster analysis of expression kinetics, and bioinformatic identification of overrepresented gene ontologies revealed waves of regulation relevant for DNT testing. We further explored the concept of superimposed waves as descriptor of ordered, but overlapping biological processes. The initial wave of transcripts indicated reorganization of chromatin and epigenetic changes. Then, a transient upregulation of genes involved in the formation and patterning of neuronal precursors followed. Simultaneously, a long wave of ongoing neuronal differentiation started. This was again superseded towards the end of the process by shorter waves of neuronal maturation that yielded information on specification, extracellular matrix formation, disease-associated genes and the generation of glia. Short exposure to lead during the final differentiation phase, disturbed neuronal maturation. Thus, the wave kinetics and the patterns of neuronal specification define the time windows and end points for examination of DNT.

Keywords: stem cell; development; neurotoxicity; neuron; astrocyte; electrophysiology

Abbreviations: CNS, central nervous system; DNT, developmental neurotoxicity; DoD, day of differentiation; ESC, embryonic stem cells; GO, gene onthology; mESC, murine embryonic stem cell; N, gene onthology neuronal differentiation; NPC, neural precursor cell; RA, all-trans retinoic acid; Shh, sonic hedgehog; GMEM, Glasgow's modified Eagles medium; FBS, fetal bovine serum; PFA, paraformaldehyde; PBS, phosphate-buffered saline

full-text:
Cell Death and Differentiation - Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing