Transcriptional profile of NeuroD expression in a human fetal astroglial cell line.
NeuroD1, a member of the basic helix-loop-helix (bHLH) protein family, is a transcription factor that plays a pivotal role in terminal differentiation of neural progenitors. The primary objective was to generate an early transcriptional profile triggered by NeuroD1 to guide future studies on mechanisms of neuronal differentiation. The human NeuroD1 coding region was amplified from human fetal brain RNA using specific primers and cloned into a CMV expression vector (CT-GFP-TOPO/pcDNA3.1). Transfection of a fetal glial cell line with this construct resulted in expression of NeuroD1 in 13-15% of the cells. Markers typical of early neuronal development were observed by immunocytochemical staining in a small proportion of transfected cells. To enrich the population of NeuroD1-expressing cells, fluorescence-activated cell sorting (FACS) was used to purify and collect the NeuroD1/GFP+ cells. Total RNA was extracted from the pair of cultures (NeuroD1/GFP vs. control plasmid/GFP) and processed for gene expression studies. A final gene list was composed from those probe sets that were either increased or decreased in the NeuroD1-expressing cells in three independent experiments (p < 0.001). Each gene was investigated further for possible roles in neurogenesis and a subset of 177 genes was chosen based on the following characteristics: a) genes that are potential NeuroD1 dimerization partners, b) genes that modulate other bHLH transcription factors, c) genes related to development, and d) genes associated with neural induction, outgrowth, and terminal differentiation. DNA microarray analysis of NeuroD1 expression in an astroglial cell line produced a "snapshot" transcriptional profile that will be useful in deciphering the complex molecular code that specifies a neuronal fate.
Kamath
Siddharth G
SG
Department of Neurology, University of South Florida College of Medicine, Tampa, FL 33612, USA.
Chen
Ning
N
Enkemann
Steve A
SA
Sanchez-Ramos
Juan
J
D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005333:Fetus; D015870:Gene Expression; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C093970:NEUROD1 protein, human