Nestin is known as a specific marker for neural stem cells and regulated by the enhancer located on the nestin 2nd intron of nestin gene. Nestin 2nd intron is highly conserved among species and crucial for nestin expression in central nervous system (CNS) progenitor cells. The transgenic mice that constructed to express green fluorescence protein (GFP) with nestin enhancer have been developed and have great advantage to represent live neural stem cells during research in vivo and vitro.
In this study, we characterized GFP expressing the neural stem cells or progenitors in vitro culture. We also investigated that Ngn1, which is a basic helix-loop-helix (bHLH) transcription factor that is expressed in neuronal precursors during development of the nervous system, can promote nestin expression by nestin 2nd intron enhancer. E-box 2 in 637bp of nestin 2nd intron were thought to be strong candidates for bHLH factors during the early neurogenesis. Ngn1 increased the E-box 2 mediated reporter gene activity. To evaluate the functional mechanism of E-box mediated nestin gene expression in vivo, we generated transgenic (Tg) mice with the 637bp fragment of nestin 2nd intron containing the wild or mutated E-box2. Wild type tg mice successfully exhibited CNS-specific LacZ expression. We also found that LacZ positive cells are proliferating neural progenitors or intermediate progenitors, which is expressed with Ki67 and Tbr2, and can differentiate into vGLUT2 positive glutamatergic neurons in developing cortex. These results indicate that pro-neural Ngn1 promotes nestin expression through nesitn 2nd intron E-box 2 and Ngns mediated LacZ positive cells can differentiate into glutamatergic neurons.
