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

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 This was again superseded towards the end of the process by shorter waves of neuronal maturation that yielded information on specification, extracellular matrix formation,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.

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

“…Indeed, cortical neurons derived from TAp73 −/− mice show a reduction in the levels of the neurotransmitters glutamate and GABA. Because neuronal differentiation is a complex biological process that is regulated by intrinsic pathways [46][47][48][49][50][51] and extrinsic signals, 52,53 it will be of interest to see whether these other metabolic effects of TAp73 also affect this biological process.…”

Monocrotophos in Gandaman village: India school lunch deaths and need for improved toxicity testing

“…While there is no deus ex machina to solve all these challenges for the experimental toxicologist, the advent of pluripotent stem cells (PSCs) provides significant improvements (Baquié et al 2012;Leist et al 2008), enabling the impact of potentially toxic pesticides to be studied on virtually all types of cells without recourse to immortalized cell lines. Within the last 5 years, the European ESNATS consortium has been developing pluripotent stem cell-based toxicity tests, and first results demonstrate that such test systems may indeed become powerful tools (Krug et al 2013;Bolt 2013;Vojnits et al 2012;Kern et al 2013;Meganathan et al 2012;Kuegler et al 2010;Zimmer et al 2011a, b;Balmer et al 2012). The consortium has shown that complex functional endpoints can be transferred to in vitro test systems Kadereit et al 2012;Zimmer et al 2012).…”

Test systems of developmental toxicity: state-of-the art and future perspectives

“…Nevertheless, an even deeper understanding of the established test systems is urgently needed. For example, neuronal differentiation in the ESNATS test systems is characterized by tightly coordinated waves of gene expression (Schulz et al 2009;Zimmer et al 2011;Gaspar et al 2012). This feature of the differentiating stem cells recapitulates expression waves of the developing central nervous systems in vivo.…”