Human in vitro reporter model of neuronal development and early differentiation processes

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro </it>reporter models using doublecortin promoter sequences.</p> <p>Results</p> <p>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, βIII-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.</p> <p>Conclusion</p> <p>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.</p

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could induce the expression of the DCX-promoter-EGFP reporter (A-H). NTERA-2cells inducing the expression of EGFP upon retinoic acid treatment (I), also expressed DCX (J) and frequently Map2 (K). Parallel upregulation of the EGFP reporter and DCX mRNAs (M) and proteins (N) upon retinoic acid differentiation could also be detected in NTERA-2 clones, but not in HeLa clones. Induction of a neurogenic differentiation program following transient transfection of mouse Ngn2 also resulted in a significant induction of DCX-promoter-luciferase reporter in NTERA-2 clones, but not in HeLa clones (O).<p><b>Copyright information:</b></p><p>Taken from "Human in vitro reporter model of neuronal development and early differentiation processes"</p><p>http://www.biomedcentral.com/1471-2202/9/31</p><p>BMC Neuroscience 2008;9():31-31.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270856.</p><p></p

Human in vitro reporter model of neuronal development and early differentiation processes-3

ERA-2 cells (A . G) and D283 cells (B . H) showed a morphological response to retinoic acid. PC12 cells treated with NGF developed a complex network of cellular processes (I inset). Scale bar in L = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Human in vitro reporter model of neuronal development and early differentiation processes"</p><p>http://www.biomedcentral.com/1471-2202/9/31</p><p>BMC Neuroscience 2008;9():31-31.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270856.</p><p></p