Human in vitro reporter model of neuronal development and early differentiation processes.
BACKGROUND
During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop in vitro reporter models using doublecortin promoter sequences.


RESULTS
Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, betaIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression.


CONCLUSIONS
Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.
Couillard-Despres
Sebastien
S
Department of Neurology, University of Regensburg, Universitätsstr, 84, 93053 Regensburg, Germany. sebastien.couillard-despres@klinik.uni-regensburg.de
Quehl
Eike
E
Altendorfer
Katrin
K
Karl
Claudia
C
Ploetz
Sonja
S
Bogdahn
Ulrich
U
Winkler
Juergen
J
Aigner
Ludwig
L
D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D000089622:Doublecortin Domain Proteins; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000502:physiology*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D009479:Neuropeptides / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D051792:Basic Helix-Loop-Helix Proteins; D000089622:Doublecortin Domain Proteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D014212:Tretinoin; C102938:Neurog2 protein, mouse