Directed expression of Gata2, Mash1, and Foxa2 synergize to induce the serotonergic neuron phenotype during in vitro differentiation of embryonic stem cells

Stem Cells. 2011 Jun;29(6):928-39. doi: 10.1002/stem.640.

Abstract

Investigation of serotonergic neuronal activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Serotonergic neurons derived from embryonic stem cells (ESCs) could provide a platform for such studies and provide models for use in drug discovery. Here, we report enhancement of serotonergic differentiation using a genetic approach. Expression of Gata2 increased the yield of serotonergic neurons. Enhancement was only achieved when Gata2 was expressed under the control of the tissue-specific promoter of the transcription factor Nkx6.1. High levels of Gata2 expression in ESCs compromised pluripotency and induced non-neuronal differentiation. Combined directed expression of Gata2, proneural gene Mash1, and forkhead transcription factor Foxa2 further enhanced serotonergic neural differentiation, resulting in a 10-fold increase in serotonin content. These neurons were also capable of depolarization (KCl, 30 mM)-induced elevations of intracellular Ca(2+) . The presence of sonic hedgehog during differentiation produced a further modest increase in numbers (1.5-fold). Transgene expression did not influence the number of tyrosine hydroxylase positive neurons in the cultures after 20 days, implying that Gata2, Mash1, and Foxa2 modulate in vitro differentiation at a time beyond the decision-point for dopaminergic or nondopaminergic commitment. This study demonstrates that the directed expression of specific transcription factors enhances serotonergic neuron differentiation in vitro and highlights the importance of transgene expression at the right stage of ESC differentiation to effect the generation of a desired neural subtype.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Cell Differentiation*
  • Cloning, Molecular
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / metabolism
  • GATA2 Transcription Factor / genetics
  • GATA2 Transcription Factor / metabolism*
  • GATA3 Transcription Factor / genetics
  • GATA3 Transcription Factor / metabolism
  • Genes, Reporter
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Hedgehog Proteins / pharmacology
  • Hepatocyte Nuclear Factor 3-beta / genetics
  • Hepatocyte Nuclear Factor 3-beta / metabolism*
  • Homeodomain Proteins / genetics
  • Mice
  • Neurons / metabolism*
  • Peptide Elongation Factor 1 / genetics
  • Phenotype
  • Promoter Regions, Genetic
  • Protein Biosynthesis / drug effects
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Serotonin / biosynthesis*
  • beta-Lactamases / biosynthesis
  • beta-Lactamases / genetics

Substances

  • Ascl1 protein, mouse
  • Basic Helix-Loop-Helix Transcription Factors
  • Foxa2 protein, mouse
  • GATA2 Transcription Factor
  • GATA3 Transcription Factor
  • Gata2 protein, mouse
  • Gata3 protein, mouse
  • Hedgehog Proteins
  • Homeodomain Proteins
  • Nkx6-1 protein, mouse
  • Peptide Elongation Factor 1
  • Recombinant Fusion Proteins
  • Shh protein, mouse
  • Hepatocyte Nuclear Factor 3-beta
  • Green Fluorescent Proteins
  • Serotonin
  • beta-Lactamases