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

Department of Neurology, University of Regensburg, Universitätsstr, 84, 93053 Regensburg, Germany.
BMC Neuroscience (Impact Factor: 2.67). 02/2008; 9(1):31. DOI: 10.1186/1471-2202-9-31
Source: PubMed


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.
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.
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.

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    • "It was demonstrated that RA induces NT2/D1 cells toward neuronal differentiation (21) and these cells represent an appropriate in vitro model system to study early steps of neuronal differentiation. Our previous work showed that induction of NT2/D1 cells by RA during first 48 h is accompanied by upregulation of SOX3 gene expression (13). "
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    ABSTRACT: SOX3 is one of the earliest neural markers in vertebrates, playing the role in specifying neuronal fate. In this study we have established first functional link between CREB and human SOX3 gene which both have important roles in the nervous system throughout development and in the adulthood. Here we demonstrate both in vitro and in vivo that CREB binds to CRE half-site located -195 to -191 within the human SOX3 promoter. Overexpression studies with CREB or its dominant-negative inhibitor A-CREB indicate that this transcription factor acts as a positive regulator of basal SOX3 gene expression in NT2/D1 cells. This is further confirmed by mutational analysis where mutation of CREB binding site results in reduction of SOX3 promoter activity. Our results point at CREB as a positive regulator of SOX3 gene transcription in NT2/D1 cells, while its contribution to RA induction of SOX3 promoter is not prominent.
    Full-text · Article · Nov 2013 · BMB reports
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    • "Even though the use of specific promoters can selectively target specific cell populations, such transgenic systems remain confined to preclinical research. Furthermore, given the large size of the promoters to detect neural stem and progenitor cells respectively (Yamaguchi et al., 2000; Karl et al., 2005; Couillard-Despres et al., 2006, 2008b; Ladewig et al., 2008). Besides the transgenic models, the delivery of retroviral vectors encoding fluorescent proteins to neurogenic regions has also been fruitfully exploited (Carleton et al., 2003; van Praag et al., 2005; Toni et al., 2008). "
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    ABSTRACT: Adult neurogenesis, i.e., the generation of new neurons in the adult brain, presents an enormous potential for regenerative therapies of the central nervous system. While 5-bromo-2'-deoxyuridine labeling and subsequent histology or immunohistochemistry for cell-type-specific markers is still the gold standard in studies of neurogenesis, novel techniques, and tools for in vivo imaging of neurogenesis have been recently developed and successfully applied. Here, we review the latest progress on these developments, in particular in the area of magnetic resonance imaging (MRI) and optical imaging. In vivo in situ labeling of neural progenitor cells (NPCs) with micron-sized iron oxide particles enables longitudinal visualization of endogenous progenitor cell migration by MRI. The possibility of genetic labeling for cellular MRI was demonstrated by using the iron storage protein ferritin as the MR reporter-gene. However, reliable and consistent results using ferritin imaging for monitoring endogenous progenitor cell migration have not yet been reported. In contrast, genetic labeling of NPCs with a fluorescent or bioluminescent reporter has led to the development of some powerful tools for in vivo imaging of neurogenesis. Here, two strategies, i.e., viral labeling of stem/progenitor cells and transgenic approaches, have been used. In addition, the use of specific promoters for neuronal progenitor cells such as doublecortin increases the neurogenesis-specificity of the labeling. Naturally, the ultimate challenge will be to develop neurogenesis imaging methods applicable in humans. Therefore, we certainly need to consider other modalities such as positron emission tomography and proton magnetic resonance spectroscopy ((1)H-MRS), which have already been implemented for both animals and humans. Further improvements of sensitivity and neurogenesis-specificity are nevertheless required for all imaging techniques currently available.
    Full-text · Article · May 2011 · Frontiers in Neuroscience
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    • "In the adult CNS, expression of DCX is mainly detected in the adult dentate gyrus of the hippocampus and in the subventricular zone/rostral migration stream/olfactory bulb axis (SVZ/RMS/OB) [4,6-8]. Based on the close association between DCX expression and neurogenesis [5], we previously generated transgenic mice, to monitor neurogenesis in vitro and in vivo, in which reporter genes were driven by the DCX promoter [9-13]. "
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    ABSTRACT: Lack of appropriate tools and techniques to study fate and functional integration of newly generated neurons has so far hindered understanding of neurogenesis' relevance under physiological and pathological conditions. Current analyses are either dependent on mitotic labeling, for example BrdU-incorporation or retroviral infection, or on the detection of transient immature neuronal markers. Here, we report a transgenic mouse model (DCX-CreERT2) for time-resolved fate analysis of newly generated neurons. This model is based on the expression of a tamoxifen-inducible Cre recombinase under the control of a doublecortin (DCX) promoter, which is specific for immature neuronal cells in the CNS. In the DCX-CreERT2 transgenic mice, expression of CreERT2 was restricted to DCX+ cells. In the CNS of transgenic embryos and adult DCX-CreERT2 mice, tamoxifen administration caused the transient translocation of CreERT2 to the nucleus, allowing for the recombination of loxP-flanked sequences. In our system, tamoxifen administration at E14.5 resulted in reporter gene activation throughout the developing CNS of transgenic embryos. In the adult CNS, neurogenic regions were the primary sites of tamoxifen-induced reporter gene activation. In addition, reporter expression could also be detected outside of neurogenic regions in cells physiologically expressing DCX (e.g. piriform cortex, corpus callosum, hypothalamus). Four weeks after recombination, the vast majority of reporter-expressing cells were found to co-express NeuN, revealing the neuronal fate of DCX+ cells upon maturation. This first validation demonstrates that our new DCX-CreERT2 transgenic mouse model constitutes a powerful tool to investigate neurogenesis, migration and their long-term fate of neuronal precursors. Moreover, it allows for a targeted activation or deletion of specific genes in neuronal precursors and will thereby contribute to unravel the molecular mechanisms controlling neurogenesis.
    Full-text · Article · Dec 2010 · BMC Neuroscience
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