Authors: KAMATH, SIDDHARTH G.¹; CHEN, NING²; ENKEMANN, STEVE A.³; SANCHEZ-RAMOS, JUAN⁴

Source: Gene Expression, Volume 12, Number 2, 2005 , pp. 123-136(14)

Publisher: Cognizant Communication Corporation

Abstract:

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.

Keywords: Transcription factors; Neuronal differentiation; Gene expression

Document Type: Review article

Affiliations: 1: Departments of Neurology, University of South Florida College of Medicine, Tampa, FL 33612 2: Departments of Neurosurgery, University of South Florida College of Medicine, Tampa, FL 33612 3: Departments of Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, FL 33612 4: Departments of Neurology, University of South Florida College of Medicine, Tampa, FL 33612, Departments of Neurosurgery, University of South Florida College of Medicine, Tampa, FL 33612, James Haley VA Hospital, University of South Florida Colleg

Publication date: 2005-02-01

More about this publication?

• The Molecular and Cellular Biology area of Gene Expression covers all aspects of the gene including it structure, functions, and regulation in prokaryotes, eukaryotes, and viruses; molecular and cell biological aspects of cell growth and development, chromatin structure and function. These include topics such as DNA replication, DNA repair, gene transcription, transcriptional control, RNA processing, posttranscriptional control, oncogenes, molecular mechanisms of action of hormones, molecular mechanism of cellular differentiation, growth and development, protein synthesis, and posttranslational control.
  The Molecular and Cellular Neuroscience area of Gene Expression covers all aspects of gene expression as described but is devoted exclusively to the nervous system in health and disease. Topics include studies of neurogenesis, development, aging, and neurodegeneration. Complex neural systems, motor control, special senses, and higher cortical function, when viewed from the perspective of gene expression, are appropriate for the journal. Research related to molecular mechanisms of drug tolerance, dependence, and withdrawal are solicited. Manuscripts on state-of-the-art methods and protocols for molecular profiling of neuronal structure and function are welcome.

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